Note: Descriptions are shown in the official language in which they were submitted.
WO 90/10~29 2~ 3~ Pcr/~s90/o1029
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IRETHOD AND DIAGNOSTIC TEST Kl~ FOR~;.', ;~;
DET1::TION OF AUTOIMMWNE ANTIBODY
BACKGROUN~ OF TIIE iNVENTlON
The immune system is the body's defense mechanism against foreign substances and5 invading microorganisms. The underlying operating principle of the immune system is a
self/nonself recognition pattern. If the invader organism is recognized as not being part of
the "se1f", then a defensive immune response is mounted against it. In the case of
autoimmune diseases (See Figure 7) the immune system fails to properly recognize "sel~" and
mounts a defense immune response against its own norrnal body components. Figure 7 is
10 a list of autoimmune diseases and the antigens associated with them.
Antibodies generated by the immune system to diverse tissue and cellular components have
been used to diagnose and monitor autoimmune disease activity. These antibodies include
anti-dsDNA, anti-RNP (ribonucleoprotein3, anti-DNP (Deoxynucleoprotein), anti-Cardiolipin,
anti-histones (symptomatic of drug induced lupus), anti-Sm (Smith), anti-ENA (extractable
15 nuclear antigen), which are often found in patients with Systemic Lupus Erythematosus (SLE),
anti-RNP, and anti-ENA are also symptomatic of Mixed Connective Tissue Disease (MCTD).
Other antibodies, such as anti-RNA (ribonucleic acicl), anti-Scl-70 (Scleroderma-70) are often
found in patients with Progressive Systemic Sclerosis (PSS); and anti-SS-A and B (Sjogren's
Syndrome Antibody A and B) which are frequently found in patients with Sjogren's Syndrome.
~o In SLE (a type of autoimmune disease) one of the antibodies produced reacts with DNA that
is found widely distributed in cell nuclei in a multitude of body tissues. Formation of
antibodies to double-stranded or native deoxyribcnucleic acid tanti-dsDNA), is relatively
specific to SLE. Although other disorders, such as Mixed Connective Tissue Disease
(MCTD), Drug Induced Lupus (DIL)1 Rheumatoid Arthritis, Sclerodermal PSS, and Sjogren
2~ Syndrome, produce similar clinical manifestations as SLE high levels of anti-dsDNA are
seldom associated with these disorders. Therefore, detecting anti-dsDNA is useful in
specifically diagnosing SLE. Anti-dsDNA levels correlate well with the disease activlty of the
patient; thus making It a good monitoring tool.
Also in SLE one of the antibodies produced reacts with Sm antigen that is tound widely
30 distributed in cell nuclei in a mul!itude of body tissues. Formation of antibodies to Sm
antigen is r~latively specHiG to SLE. Detecting anti-Sm antibodies which are specific for SLE
is useful in diaynosing SLE. Anti~Sm antibodies are frequently accompanied by another
antibo~y anti-RNP. Antibodies directed against the RNP antigen are found in the absence
:
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wo 90~1022g ' i'` ! -' ~ PCI'/US90/01029
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of anti-Sm antibodies in MCTD, which is an aoverlap syndrome~ that combines features of
SLE, PSS, and Polymyositis. Therefore, detecting anti-Sm/RNP antibodies is useful in
diagnosing two different autoimmune diseases, SLE and MCTD.
Many different techniques and different diagnostic kits have been developed in the search for
a standardized, accurate, rapid, and stable method for the detection of anti-dsDNA, anti-Sm,
and anti-Sm/RNP antibociies. Most of the methods have been somewhat successful, but due
to unacceptably high levels of cross-reactivity (such as with single-stranded DNA [ssDNA]~,
the slow run time of the assays, and the short shelf iife of the kit components, no method is
fully adequate. i=xamples of the limitations of the prior methods follow.
A number of techniques have been developed to detect antibodies to dsDNA, including
immunofluorescent assays (FIA), radioimrnunoassay (RiA), and en~yme-linked
immunosorbent assays (ELISA).
Firstiy, the RIA technique has been developed in a variety of formats to measure levels of
anti-dsDNA in sera. A RIA diagnostic test kit (using the Farr technique) has been developed
1~ for commercial sale to clinical laboratories. This test precipitates the bound labelled DNA
which is then retained ~or counting. This test is sensitive to high levels of anti-DNA but has
Iess specificity in lower levels of anti-dsDNA activity, thus resulting in false negatives.
Unfortunately, this test has a run time of approximately two hours and fifteen minutes, and
a limited stable shelf life. Furthermore, this test kit has the same draw-backs which are
inherent in all RlAs; the expense associated with radioactive rnaterial, which also have a
!imited sheif life and can be potentially dangerous.
Secondly, Crithidia luciliae immunofluorescencs tests have been developed as diagnostic test
, kits ior the detection of anti-dsDNA. This test kit is based on a staining method associated
with the kinetoplast of the protozoan. A positive reaction is demonstrated by detection of
specific fluorescence in the kinetoplast of most cells. The kinetoplast, within the protozoan,
contains many other components besides dsDNA which can cross react with anti-dsDNA or
other antibodies, thus rendering false positive results. This test is extremely subjective, as
it i9 based on an individual's abiiity to recognize a positive fluorescent pattern. Fur~hermore,
this rnethod, which has a run time of approximately two hours, has a lack of sensitivity in low
levels of concentration of anti-isDNA resulting in false negatives. The false negative and false
positive results make this method more useful when used together with other tests in clinical
practice.
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Thirdly, the immunological community has developed diagnostic test kits for the
determination of anti-dsDNA levels in the standard ELISA format. Although a variety of these
kits have been ~eveloped, the same limitations of stability, cross reactivity with ssDNA, and
length of run time plague each assay. Many of these kits claim a low level of cross r~activity
with ssDNA which should render good sensitivity to low, medium, and high levels of anti-
dsDNA; however, as evidenced by the kit instructions, there is a lack of sensitivity in the sera
sample ~,vhich are borderline positives. As a consequence, these kits cannot be us~d to
accurately monitor patients who are borderline positives. In fact, most kits require that low
positives must be retested to assure confidence in the results, thereby increasing the cost to
~0 the patient. Development ol an assay which is sensitive to all levels of anti-dsGNA can only
be achieved by reducing non-specific binding by sueh components as ssDNA.
A number of techniques including immunodiffusion or Ouchterlony analysis have been
developed to detect antibodies to Sm antigen and Sm/RNP antigen often called ENA(Extractable nuclear antigens). Examples of the limitations of the prior methods follow.
Firstly, the immunodiffusion technique has a long incubation time, typically 24 hours or more.
This time span is of course inconvenient for both the patient and the lab personnel.
Secondly, the results gathered from Ouchterlony analysis are subjective. This results in
different lab technlcians reporting varied results based on individual data interpretation.
The imrnunological community has developed diagnostic test kits for the determination of
!0 anti-Sm antibody levels in the standard ELISA format. These ELlSAs have shown certain
limitations in stability, cross reactivity, and length of run time.
it is therefore an objective of the prasent invention to provide compositions, methods, articles,
and a diagnostic tast kit for thc selective adsorption, or affixing of dsDNA, Sm antigen, er
Sm/RNP antigen, or any af the various antigens to the autoimmune antibodies previously
listed, to a pre-coated plate for the effective detection of the specific autoimmune antibodies
present in sera or plasma. It is a further objective of this invention to overcome the
aforemantioned sensitivity, subjectivity, stability, and shelf life disadvantages inherent in many
of the previously dascribed assays. It is, moreover, also an objective of this invention to
provid~ in the ~orm of a kit a noval, readily utilizabie maans for quantitative and qualitative
O detection of anti-dsDNA, anti-Sm or anti-Sm/RNP. This will provide a method and apparatus
for the purpose of c!inical detection of anti-dsDNA, or tha anti-Sm antibody, or the anti-
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Srn/RNP antibody, or any of the listed autoimmune antibodies listed, or the detection of the
specific antibody for any other purpose associated with human or animal medical testing.
It is believed that with little modification the present invention can be used to detect anti-
histone, anti-RNA, anti-SS-A, anti-SS-B, anti-Scl-70, and anti-DNP.
BRIEF SUMNIA~Y OF THE INVENTION
The present invention includes novel compositions, novel methods and articles for the direct
selective absorption, adsorption or attachment, by whatever mechanism, of the anti-dsDNA,
or the anti-Sm or the anti-Sm/RNP antibody, or any of the listed autoimmune antibodies, from
the body fluid sample to the dsDNA, the Sm-antigPn, or the Srn-RNP antigen, or possibly for
~10 any of the autoimrnune antigens coated onto any suitable solid support, such as test tubes,
plates or wells (hereinafter referred to as wells or microwells~, for the purpose of quantitative
and qualitative identification. The present invention utilizes immobilized native DNA, Sm, Sm-
RNP, or possibly for any listed autoimmune antigen from whatever source, which has an
affinity for attachment of anti-dsDNA, anti-Sm, or anti-Sm/RNP, or the other antibodies of the
listed autoimmune antibQdies.
This invention utilizes a sandwich ELISA format which includes pre-coated wells made of any
suitable material such as plastic, glass, etc., and the various reagents and antibodies
necessary to run a highly sensitive, 45 minute assay for the presence of anti-dsDNA, or for
the presence of anti-Sm, or for the presence of anti-Sm/RNP.
The various reagents and the method of coating these reagents to the rnicrowells have been
employed advantageously in the practice of the present invention to formulate a test which
has minimized or eliminated the problems associated with previous assays. The coating
protocol has advantageously utilized methylated Bovine Serum Albumin (mBSA) for two main
purposes; namely, the mBSA provides a positively charged surface which enhances the
adherence of the antigen such as dsDNA, Sm, and Sm/RNP to the polystyrene wells, and
second to eliminate the binding of anti-histone antibodies, which can create false positive
results.
The use of mBSA as a coating prior to the application of DNA antigen for use in solid phase
assays for anti-dsDNA was flrist explored by R. Rubin (J. Immunology 63, 359-366 l1983]).
In the practice of this invention, protamine sulfate as well as other functionally equivalent
substltutes have been found to be capable of forming a positiveiy charged surface; however,
WO 90/10229 ;~ 4C~ P(~/US90/0~iO29
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due to the reactivity of these coatings with other components such as ssDNA and anti-histone
antibodies, mBSA is preFerred. It was highly surprisin~ to find that a coating of mBSA on a
solid support underlying the DNA antigen, or the Sm antigen, or the Sm-RNP antigen
provides the pre-coated wells with a consistently high level of reproducibility even over a
5 period of one year.
Methylated Bovine Serum Albumin is utilized for the previously mentioned reasons, the
second of which is directly related to the specificity and sensitivity of these varlous assays.
The lack of good correlation between ELlSAs, RlAs and Crithidia luciliae staining procedures
and other tests in some cases appears to be due to binding by anti-histone antibodies which
10 gives the ELISA an elevated antibody reading. The mBSA pre-coating appears to have
alleviated this area of non-specific binding.
A second factor which contributes to the lack of sensitivity and non-specHicity is the cross
reactivity. This is particularly a problem when detecting antibodies to dsDNA, because of the
cross reactivity hetween ssDNA and anti-dsDNA. To eliminate the potential binding of the
~15 anti-dsDNA antibodies to ssDNA the microwells which have been coated with dsDNA were
treated with an endonuclease, S, nuclease, which is an enzyme specific for the breakdown
degradation of ssDNA and has no affect on dsDNA. Although other anti-dsDNA tests utilize
S, nuclease ffor example see A.G. Tzioufas, Clinical and Experimental Rheumatology 5,
247-253 [1987]) their sensitivity levels are still substantially lower than acceptable levels for
~2û patients with borderline positives. The use of S, nuclease in a digestion buffer with an acid
pH, combined wi~h the use of a mBSA pre-eoating eliminates ssDNA activity and anti-histone
activity providing a highly sensitive assay which renders few ~alse positives. The S, nuclease
is of course only necessary in the detection of anti-dsDNA antibodies and not in anti-Sm or
anti-Sm/RNP antibody detection.
The shelf life and the reproducibility of results from the ELISA is associated not only with the
mBSA pre-coating, and the S, nuclease treatment (in the anti-dsDNA kit), but also with the
next two steps of forming the pre-treated wells; and tha addition of a hydrolyzed casein
blocker and the drying.
A casein-type blocker has been used in various ELISA techniques ~Robert F. Bogt; J.
Imrnunological Methods, 101, 43-50 ~1987l) to block non-specific hinding to ptastic through
a protein-plastic interaction. It is an unexpected realization that a coating of hydrolyzed
casein blocker maintained a consistent inhibition of non-specific binding over an extended
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WO 90/10229 `~ ; PCl'/US9~/0102~
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period of time, when the wells were stored at 4 degrees C in a sealed plastic bag.
Although the direct mechanisms by which the drying process and the blocker increases the
stability and shelf life of the wells is not fully understood, it is obvious that the storage time
is increased by these processes. Little to no ssDNA, or loss of Sm or Sm-RNP antigen
activity is generated by this method during the storage condltions in a period of up to one
year.
A variety of differing blocking agents could be utilized which are ~unctionally equivalent to or
chemically relatsd to the casein blocking agent. For example, BSA and porcine thyroglobulin,
dried milk, whole goat serum, etc., however the most preferable is the hydrolyzed casein
0 (commercially available from Sigma) due to its high level of inhibition of non-specific binding
and its storage stability.
'
This invention's use of mBSA, the S, nuclease treatment (in the anti-dsDNA klt), combined
with the casein blocker and the drying process have unexpectedly resulted in novel assays
whlch are characterlzed by a low level of cross-reactivity, a high level of Inhibition of non-
specific bindlng, and a long shelf life.
The pre-coated wells are then used to detect the presence of anti-dsDNA antibody, anti-Sm
antibody, or the anti-Sm/RNP antibody in the sample. The plasma or serum samples are
prepared with a sarnple diluent and are then assayed for their components by an
immunoassay technique, the ELISA and the fluorescent immunoassay (FIA) formats being
0 the preferred methods, though it is possible to perform a RIA or a luminescent assay with little
modification.
:,
The assays depicted in the following examples have an approximate run time of 45 minutes.
Th~ wells, when exposed to the samples, are provlded with approximately 15 minutes at
;~ room temperature to allow the blnding of the antlbody to the antigen to go to completion.
5 Then the labelled goat anti-human antibodies are exposed to the wells and a similar 15
minute incubation at room temperature is provided for. If the enzyme is utilized a substrate
` can ba added (although this is not necessary) and 10 minutes is allotted for the production
of the color. If a fluorescent marker is used on the goat anti-human antibody then no
substrate is needed, therefore the run time is shortened by 10 minutes reducing it to 35
minutc-.
WO 90/10~29 ~5~3~3 PCI'/US90/01029
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Subsequent qualitative and quantitative detection of the antibody is relatively simple if the
forrnat is either an ELISA or a FIA. Numerous enzyme-conjugated antibodies and fluorescent-
labelled antibodies specific for any of the immuno~3lobulin classes can be utilized in this
invention. The quantitation of the antibodies present is accomplished by the related
instruments. The ELISA technique utilizes a spectrophotometer, and the FIA technique
utilizes a microfluorometer. Use of the ELISA techniques were flrst described by Engvall and
Perlman ([1971] Irnmunochemistry 8, 871-874 and [1972} J. Immunology 109, 129-135), and
The Enzyme Linked Immunosorbent Assay (ELISA) by Voller, A., Bidwell, D.E., and Bartlett,
A., (1979) Dynatech Laboratories, Inc., Alexandria, Virginia, both of which are, in their totality,
; 10 incorporated herein by re~erence.
BRIEF DESCRIPTION OF THE GF~APH
Figure 1 - This graph depicts a standardized curve based on arbitrary units/ml. This curve
was generated by using the ELISA ~ormat of this invention. The Center for Disease Control
(CDC) ANA Human Reference Serum #1 has an antigen binding capacity of .59 micro grams
1~ DNA bound per ml of serum. This is equivalent to 100 AU per ml.
Figure 2 - This graph depicts a standardized curve obtained ~rom the data in Example 3.
Figure 3 - This graph depicts a standardized curve based on arbitrary RE~DS~ units/ml. This
curve was generated by using the ELISA format of this invention. The Center for Disease
Control (CDC~ ANA Human Reference Lot #82-001 #5 Serum ffor Sm antigen) was run on
an immunodiffusion assay with median titer of 1:64. This standard was serially diluted with
normal sera and graphed. The 1:1 or neat dilution was assigned arbitrarily a value of 400
RE~DS units.
:
Figure 4 - This graph dspicts the range of negative and positive human sera control values
expected for the anti-Sm tast kit.
:,
2~ Figure 5 - This graph depicts a standardked curve based on arbitrary RE~DS ED units/ml. This
curve was generated by using the ELISA format of this invention. The Center for Disease
Control (CDC;) ANA Human Reference Lot ~82-001 ~5 Serum (for Sm antigen) and the ANA
Human Referencs Lot #82-011 #4 Serurn ffor RNP antigen) was run on an immunodiffusion
assay with median titer of 1:64. An equal volume of reference sera #4 and ~5 are combined
to 1Orm the standard. This .standard was serially diluted with normal sera an~ graphed. The
.
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WO 90/10229 ' PCT/llS90/01029
~ 8 -
1:1 or neat dilution was assigned arbitrarily a value of 140C RE~DS units.
Figure 6 - This graph depicts the range of negative and positive hurnan sera control values
expected for the anti-Sm/RNP test kit.
Figure 7 - This charl shows autoimmune disease and their associated antigens which could
possii~ly be detected by the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions are supplied for the purpose of clarifying the invention and are not
intended to limit the scope of the invention:
Methylated Bovine Serum Albumin Soiution: Unless otherwise specified, is intended to mean
a solution of 1 milliliter of water or PBS with 20 micrograms of methylated bovine serum
Albumin (mBSA) dissolved in it. A substitute for mBSA is protamine sulfate or any other
chemical or chemical process capable of producing a slighily positively charged coating
which is evenly distributed over the surface of the microtitre well. (It is believed that the anti~
histone test may not ~mploy methylated bovine serum coating on the microtiter plate.)
~5 PBS Solution: A .01 molar solution ot buffer containing 1.43 9 potassiurn phosphate, dibasic,
" .~5 g potassium phosphate, monobasic, and ~.5 g sodium chloride in one liter of water. The
' pH is 7.3 +/- .1.
. ,
~, dsDNA Solution: Purified dsDNA solution from cali~ thymus was utilized. Altcrnative sources
~î of DNA include, but are not limited to, native linear DNA from E. Coli, and circular DNA from
~0 plasmid, virus, crithidia, and synthetic polynucleotides (poly (dA.dT)).
Sm Solution: Puriiied Sm antigen solution from calf thymus was utilized. Alternative source
can be employed.
Srn/RNP Svlution: Purified Srn/RNP antigen solution from calf thymus was utilized.
Alternativ~ source can be empioyed.
~,5 ~N~C~ tion: 15 rniliigrams of hydrolyzed casein blocker (Sigma), 2 mi
glyercol, 10 grams sucro~e was dissolved in TEN buffer sufficient to brin9 the final volume
,
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WO 90/1022~ i a'l~'C~/US90/01029
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to 100 mls. The solution is adjusted to pH 7.3 +/- .1,
(Sm and SmlRNP) Caseln Blocker Solution: 25 milligrams of hydrolyzed casein blocker
(Sigma), 2 ml glyercol, 10 grams sucrose was dissolv~ed in TEN buffer sufficient to bring the
final volume to 100 mls. The solution is adjusted to pH 7.3 +/- .1.
TEN Buffer: Is made by adding 6.1 g TRIS, .38 g EDTA, 8.B g NaCL, 3.8 mL of concentrated
HCL to 900 rnL deionized water. Adjust pH to 7.3 and add deionized water sufficient to give
iOOO milliliter total volume.
Anti-dsDNA Ant bodv: Circulating autoantibodies directed against dsDNA.
Anti-Smith Antibodv: Circulating autoantibodies directed against Sm antigen.
Io Anti-Sm/RNP Antibody: Circulating autoantibodies directed against Sm/RNP antigen.
Anti-histone Antibody: Circulating autoantibodies directed against histone antigen.
Anti-DNP Anhbodv: Circulating autoantibodies directed against DNP antigen.
~: Circulating autoantibodies directed against ENA antigen.
Anti-~NA: Circulatiny autoantibodies directed against RNA antigen.
Anti-Scl-70: Circulating autDantibodies directed against Sci-70 antigen.
Anti-SS-A: CirculaUng autoantibodies directed against SS-A antigen.
:`
: Circulating autoantibodies directed against SS-B antigen.
,~ S, Nuclease: An endonuclease (enzyme) which is capable of digestion of ssDNA antigen.
;, oub ~ Ar L~Q~LISA or F A: A solid support is coa~ed with material which
detects and blnds the antibody of interest to the coated surface. To render a signal, a
second conjugated aniibody with an affinity for tha previously bound antibody is exposed to
the coated surface. This antibody binding to the original antibody makes the sandwich. If
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the sandwich assay is an ELISA then the second antibody is conjugated with an enzyme and
substrate is used to produce a color. If the assay is an FIA then the second antibody is
marked with a fluorescent tag ancl a substrate is unnecessary.
Buffer for S, Nuc~ea$e Digestion: 95 mls of acetate/acetic acid buffer, pH 4.6, is mixed with
5 mls glycerol, .29 ~ NaCL, .û29g ZnS04, for a final volume of 100 mls.
. ~
S, Nuclease i3uffer Solution: Buffer for S1 nuclease digestion plus 100 units (Sigma) S1
nuclease per ml of buffer. One unit of S, nuclease is defined as: Causing 1.0 microgram
of ssDNA per minute to become perchloric acid solubl~ at pH 4.6 and 37 degrees C.
Serum: Is intended to mean the fluid component of any body fluid remaining after cells and
coaguiable proteins such as fibrin which may be present in such body fluidic components
have been removed by appropriate physical, chemical, or physicochemical means. Typically,
this term refers to the residual watery fluid remaining after clotting of blood and removal of
the clot, but in its broad sense is intended to include the fluidic component of cerebrospinal
fluid, urine, interstitial fluid, cellular cytoplasm, and the like.
Sample Di!uent: A 1 liter solution has 100 mls of native bovine serum, 1.42 grams of
potassium phosphate (dibasic), .26 9 of potassium phosphate (monobasic), 1 gram of
~I sodium azide, and 8.6 grams of sodium chloride dissolved in 900 mls of water. If an ELISA
is run, then 1 ml of stock green dye is added to the solution. If ths FIA format is used then
the dye is unnecessary. The solution is then filtered through a .2 micron filter and stored at
û 4 degrees C.
..,
Diluted ldsDNA3 Sample Solution: 10 microliters of sera dissolved in 500 microliters of
sample diluent (in dsDNA).
- Diluted (Sm and Sm~RNP) Sample Solution: 10 microliters of sera dissolved in 490
. microliters of sample diluent.
S ~Y93tLe~: A phosphate buffer, and protein stabilizer, plus .02% thimerosal adjusted
to a pH of 7.5 (commercially available from Medix i3iotech Inc.) into which is added a
protease inhibitor, aprotinin, (commercially available from Miles Pentex) at .01% of the volume
ol the buffor.
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WO 90/10229 ~ ' PCr/US90/nl029
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(dsDNA) Workina ConJugated Antibody Solution: 1 volume of concentratecl conjugated
antibodies/3000 volumes of conjugate ~iluent. The dilution is subject to change based on
the concentration Isvel of the conjugated antibody (dsDNA).
(Sm and Srn/RNP) Workina_onjugated Antibody Solution: 1 volume of concentrated
conjugated IgG antibodies/400G volumes of conjugate diluent, and 1 volume IgM to 1500 vol.
The dilution is subject to change based on the concentration level of the conjugated antibody.
Coniugated Antibodies: For an ~LISA, antibodies were chemically conjugated with
horseradish peroxidase. For FIA, antibodies were chemically conjugated with Fluorescein
Isothiocyanate.
,
Imm nogLobu!in: Any member of the gammaglobulin fraction of serum possessing the ability
to bind another agent.
j .~
Antiaen: Molecules ffrom whatever source, nature or man-made) which induce an immune
- , - reaction when recognized by the host's immune system.
-- Antibodv: A class of serum proteins which specifically bind to an antigen which induced the
formation of the antibody.
,' ~
Imrnuno~lobulin Classes: Antibodies separated by electrophorectic rnobility specifically, IgG
and IgM.
Substrat~ Solution: To quantitate the horseradish peroxidase, 100 microliters of buffered
. (3,3',5,5'~ Tetramethylbenzidine/ hydrogen peroxide (commercially available from Kirkegaard
Perry) was used.
Labelleci Antibodies: Any antibody substance which has been covalently or otherwise
oornbined with a molecule or ion for the purpose of selectively identifying that group of
~, antibodles. Such adduct molecules or ions include enzymes, fluorescen~ substances,
,1 tadionuclides, and the like.
` ''15 Labelled Antigens: Any antigen substance which has been covalently or otherwise combined
with a molecule or ion for the purpose of selectiveiy identifying ~hat group of antigens. Such
adciuct molecules or ions include enzymes, fluorescent substances, radionucli~es, and the
. .
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WO 90~10229 ~ PC~`/IJS90/01029
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like.
: OpticaL De sity (OD~ Absorbance: A number which refers to the color absorbance of a
sample. Optical density is related to the percent cf light transmitteci through the sample by
the following formuia: OD = 2-log(percent transmittance).
Test Kit tor Anti-dsDNA
,t.
~; The preferred ernbodiment of the method and apparatus for the detection of anti-dsDNA in
sera is a diagnostic test kit. The optimized kit contains:
.
5 vials (200 microliters) Assay Calibrators for anti-DNA containing these levels of anti-dsDNA
i ~ ~ activity: 120, 60, 30, 15, 7.5, 3.8 A.U./rnl (quantitative format only)
,, .
1 vial (30 ml) Sample Diluent - green solution: contains 0.1% sodium azide.
1 vial Human Negative Serum Control (about 3 A.U./ml).
,
1 vial High Human Serum Positive Controls - about 100 A.U./ml with antigen binding capaeity
of 59 x 10-4 microgram of DNA.
1 vial Moderate Human Serum Positive Control - about 45 A.U./rnl.
12 coated 8-well Microwell Strips with frame holder.
.,
: 1 vial (15 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase
conjugated anti-human IgG, and IgM.
~, ,
1 bottle (8 mi) TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
~ ~ .
1 bottle (8 rnl) TMB Substrate Solution B - contains hydrogen peroxide.
,,
0 1 botUe (12 ml) Stop Reagent: contains 2.5 N H2S04. (1.0 N HC1 can be used as a
. substitute)
1 packet Phosphate Buffered Saline (PBS) - reconstitutes to 2 liters ot 0.01 M PBS, pH 7.4.
c, ~
~,
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Wo 90/10229 2~5(~34~ Pcr/us9o/olo2~
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Plate Template
This kit for the measurement of anti-dsDNA in serum samples has been designed for use in
a clinical laboratory. To determine the AU/ml of the anti-dsDNA present in the sample the
kit includes calibrators and controls for generating a standard curve. The selection of the
ievels of anti-dsDNA activity in these controls and c:alibrators can be varied without affecting
perFormance of the assay.
Optimization of the process has yielded kits with pre-coated test wells and reagents, which
produce low levels of variation between assays and within assays. Furthermore, these
reagents and test wells are stable for extended periods of time. This kit has been optimized
. 10 to have a 45 minute run time at room temperatur0, which is significantly shorter than other
test kits, presently rnarketed for anti-dsDNA detection.
What ~ollows is a description of a preferred embodiment of the pre-coated wells, and the
: method of coating the wells of the present invention, along with the preferred msthod for
preparation of and ulilization of the various elements of an anti-dsDNA diagnostic test kit.
Step 1 - Affixation of the Coatina on the Microwells: Methylated Bovine Serum Albumin
(mBSA) is dissolved in distilled water or PBS at a ratio of ~0 ug/ml. An aliquot of 100
micrograms of this prepared solution is placed in each microwell, to produce a surface which
is slightly positiveiy charged. The mBSA servesi two important functions; one is to provide
a stable evenly coated rnicrowell surface, and second is to inhibit non-specific binding such
as anti-histone activity. Ten mls of mBSA solution will coat 96 microtiter wells, such as a
Dynatech immulon 2, Dynatech Immulon 4, or Nunc Maxisorp. The coated wells are
incubated overnight at 4 degrees C.
~,
Unbound mBSA solution is shaken from the w911s, and they are rinsed with PBS solution, pH
7.3, and drained thoroughly.
'. .
Next, the ligand or antigen is pre,i~ared and exposed to the receiving surfaces of the
microwalls. Purified dsDNA (calf thymus) is dissolved into .01 I\A PBS, pH 7.4 in a ratio of 5
,~ micrograms/ml (weight per volume). Th~ buffered ligand is then dispensed into each
rnicrowell, 100 microliters of buffered ligand per well. The bindin9 is enhanced by an
~` incubation period ot 1B-24 hours at 4 degrees C. The excess solution is shaken from the
~,. .
.,;j;. .
WO 90/10229 ~ ~ ` PCl'tUS90/01029
rr~ ~
- 14 -
3~
wells.
The next coating is applied to eliminate the cross reactivity with ssDNA. A buffer is prepared
for S1 nuclease digestion. To prepare the buifer .03M acetic acid was titrated together with
.03M sodium acetate in a ratio of 1.l part acetic acid to 1 part sodiurn acetate to a final pH
of 4.6. 95 mls of the prepared acetate/acetic acid buff~r is mixed with 5 mls of glycerol, .29
g NaC1, .029 g ZnS04, for a final volurne of 100 rnls. Added to this buffer was 100 units of
S, nuclease (Sigma) per ml of digestion buffer solution to form a S, nuclease buffered
solution which is dispensed in 100 microliter increments into each individual receiving well.
Thus, 10 units of S, nuclease is contacted with each individual well. This solution is of
suHicient concentration to digest the unwanted ssDNA in the previously coated ligand material
aiter a two hour incubation period at 37 degrees C. S, nuclease eliminates ssDNA which is
a source of false positives in many assays. A~ter the incubation period the wells are invertecl
to remove excess solution, then the wells are thoroughly rin~ed twice with P8S solution. The
wells are again inverted on a paper towel and allowed to drain.
The next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots
of 200 microliters. This step decreases the non-specific binding that can occur due to
protein-plaslic interaction. The hydrolyzed casein used in the blocker solution can be
commercially obtained from Sigma. The casein biocker solution is prepared by mixing 2 rnl
glycerol, 10 ~ sucrose, and 15 mg of hydrolyzed casein, and adding sufficient TEN buffer to
~0 make 100 ml of solution. The wells containing casein blocker solution are again incubated
at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15
minutes. Then the wells are uprighted and allowed to dry at room temperature for at least
24 hours.
:
This compietes ~he process of coating the wells and each diagnostic kit is then supplied with
-5 96 coated wells. The shelf life of coated microwells when stored at 4 degrees C in a seaied
plastic bag is up to one year.
St~p 2 - Adherin~ AnU-dsDNA to the Prepared Wells From Step 1: Tha sample diluent is
supplied in the kit as a 30 ml green solution. To prepare a 1000 ml solution of Sample
Dilusnt, 100 milliliters of nativ~ bovine serum, 1.42 9 of Potassium Phosphate (dibasic), .26
1 9 of Potassium Phosphate (rnonobasic), 1 gram of sodium azid0, and 8.6 9 of sodium
-~' chloride, and 1 ml of stock green dye are dissolved in deionized water sufficient to make 1000
milliliters of solution. This solution is then filtered through a .2 micron filter.
.7
.
; "
WIO 90/10229 2~ ) PCr/US90/01029
5 ~
The Sample Diluent acts as a blocking agent similar to the casein blocker which was
previously coated on the wells. The principle blocking component in the Sample Diluent is
the native bovine serum which acts to inhibit the binding of any BSA-reactive antibodies to
the mBSA coating on the surtace of the well.
Prior to contacting the body fluid with the prepared l~late, the serum is diluted by adding
aliquots of sera to the sample diluent in a 1:50 ratio (volume of serum:volume of sample
diluent), although this exact dilution is not critical and depends upon the nature of the body
fluid and the assay techniqu0s employed. To form the dihlted sample solution the body fluid
is aliquoted in 10 microliter proportions into 500 microliters of Sample Diluent. In an
individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the
anti-dsDNA is enhanced by 15 minutes incubation at room temperature.
Following the affixation of the anti-dsDNA to the coated wells, the wells are thoroughly
washed four times with PBS to remove the free unbound antibodies that are present in the
sample, which if allowed to remain, would elevate the backgrourld absorbance.
Slep 3 - Assay for the Anti-dsDNA Affixed to the Wells: Standard enzyme-linkëd
immunoassay techniques, previously described, are used for this assay, although any suitable
means of detection such as radioactive labeling, fluorescence, or the like can be ernployed.
For the examples described hereinafter, anti-human IgG and anti-human IgM induced in goats
were used to ascertain whether anti-dsDNA IgG and igM antibodies were present. Please
?O note that other species of animal can be used to produce anti-human antibodies. These
antisera were linked to horseradish peroxidase, an enzyme which yieids a colored product
whenever one of its substrates is present together with hydrogen peroxide. The substrate
should be chosen to be consistent with the enzyme conjugated to the antibody. For the
examples ciescribed hereinafter, th~ substrate was (3,3',5,5') Tetramethylbenzidine and
hydrogen peroxide.
The kit contains one 15 rnl vial of conjugated antibody solution with anti hurnan I~M and IgG
antibody conjugat~d to horseradish peroxidase. To prepare a working conjugated antibody
solution, a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4
(commercially availabls frorn Medix) was mixed with aprotinin, a protease inhibitor
0 (commercially available from Miles Pentex) at a .01% ratio of inhibitor to voiume of buffer.
;, This diluent enhances the stability of the conjugated antibody. The solution is mixed at a
, .
.. ~ . - . , ~. . . , ................. , , - , ......... . .
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WO ~0/10229 ~ ~; Pcr/VS~)/01029
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- 16-
ratio of 1/3000; one part of concentrated conjugated IgM and IgG antibodies is aliquoted into
3000 parts conjugate diluent. The ratio of concentrated conjugated antibody to conjugate
diluent is subject to wide latitudes of dilutions based on the manufacturer's concentration of
conjugated antibody used in the assay.
Next 100 microliters of the enzyme conjugated goat anti-hurnan antibody working solution,
(prepared as described) is added to each microtiter well. Binding of these antibodies to the
anti-dsDNA is permitted for at least 15 minutes at room temperature, then the rnicrotiter wells
are emptied of their contents, washed four times with PBS, and allowed to draln before the
next step.
.
The presence of a label and antibody, as previously described, is determined by incubating
the wells with a solution of buffered (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
- This solution is supplied in the kit in two 8 ml via!s; one contains (3,3',5,5')
Tetramethylbenzidine; the olher vial contains hydrogen peroxide. The separate vials are
necessary due to the interaction between the two solutions. The two solutions are mixed in
-i~ a one to one ratio just before use, and 100 rnicroliters of the mixed solution is dispensed into
each microwell. The reaction is permitted to continue for 10 minutes at room temperature,
or until sufficient color appears to be read on the spectrophotometric device used. The
reaction is subsequently stopped through the addition of an equal volume of 2.5 normal
sulfuric acid, and the intensity of color (the optical density, "OD", or absorbance) is read by
a spectrophotometric device such as a Dynatech MR600 or ~he like.
As with any enzyme-linked immune assay, the resultant color of the reaction product is
,; proportional to the number of conjugated antibodies which have bound to the anti-dsDNA.
For most cases, the number of bound conjugated antibodies is linearly related to the number
of anti-dsDNA antibodies. Hence, as the amount of anti~dsDNA bound to the wells increas0s,
~5 so does the optical density, or absorbance of the enzyme reaction.
Test K~t ~or Anti~Sm or ~nU-Srt~ NP
.,
This prefetred embodiment of the method and apparatus for the detection of anti Sm
anUbodies in a sera is a diagnostic test kit. Similarly ~he preferred embodiment of the method
`', and apparatus for the detection of anti-Sm/RNP antibociies in sera is a diagnostic test kit, with
~0 the following components (qualitative format).
~'
.', .
,: . ... .. :, . . . . . . .. . .
WO 90/102~9 ~ P~/US~0/01029
1 vial (40 ml) Sample Diluent - green solution- contains 0.1% sodium azide.
1 vial Human Negative Serum Control (about 100 microliters).
1 vial High Human Serum Positive Controls (about 100 microliters).
12 coated 8-well Microwell Strips with frame holer.
. . .
1 vial (12 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase
conjugated anti-human IgG, and IgM.
1 bottle (8 ml) TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
1 bottle ~8 ml) TMB Substrate Solution B - contains hydro~en peroxide.
1 bottle (12 ml) Stop Reagent: contains 2.5 N H2S04. (1.0 N HC1 can be used as a substitute) (not supplied).
1 packet Phosphate Buffered Saline (PBS) - reconstitutes to 2 liters of O.O i M PBS, pH 7.4.
Plate Temp!ate
This kit for the measurement of anti-Sm antibodies in serum samples has been eiesigned for
use in a clinical laboratory. To determine the RE~DS units/ml of the anti-Sm antibodies or
the anti-Sm/RNP antib~dies present in the sample kit can include calibrators and controls for
generating a standarci curve. The selection of the levels of anti-Sm or anti-Sm/RNP antibody
activity in these controls and calibrators can be varied without affecting performance of the
assay.
What foilows is a description of a preferred embodiment of the pre-coatecl wells, and the
2Q method of coating the wells of llhe present invention, along with the preferred method for
preparation of and utilization of the various elements of the anti Sm or the anti-Sm/RNP
antibody diagno~tic test kit. The differerlce bet~veen the anti Sm and the anti Sm/RNP kits
~' is only the concentration of antigen coated to the microwells.
Step 1:~ion s;)f the oating,on~Q~y~: Mathylated Bovine Serum Albumin
.
,.
,, ... , .. ~ . ,. .. , .. . - . ,
WO 90/10229 ';; PC.`T/I)S91)/01029
~i~33~
(mBSA) is dissolved in distilled water or PBS at a ratio of 20 ug/ml. An aliquot of 100
microliters of this prepared solution is placed in each rnicrowell, to produce a surface which
is slightly positively charged. The mBSA serves two important functions; one is to provide
a stable evenly coateci microwell surface, and second is to inhibit non-specific binding such
5 as anti-histone activity. Ten mls of mBS~ solution will coat 96 microtiter wells, such as a
Dynatech Imrnulon 2, Dynatech Immulon 4, or Nunc Maxisorp. The coateci wells areincubated overnight at 4 degrees C.
Unbound mBSA solution is shaken from the wells and drained thoroughly.
Next, the ligand or antigen is prepared and exposed to the receiving surfaces of the
microwells. Purified Sm antigen or the purified Sm/RNP antigen (calf thymus) is dissolved
into .01M PBS, pH 7.4 in a ratio of 5 units/ml, and for Sm/PNP 1 unit/ml. The buffered
ligand is then dispensed into each microwell, 100 microliters of buffered ligand per well. The
binding is enhanced by an incubation period of 18-24 hours at 4 degrees C.
';
The next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots
of 200 microliters. This step decreases the non-specific binding that can occur due to
protein-plastic interaction. The hydrolyzed casein used in the blocker solution can be
commercially obtained from Sigma. The casein blocker solution is prepared by mixing 2 ml
glycerol, 10 g sucrose, and 25 mg of hydrolyzed casein, and adding sufficient TEN buffer to
make 100 ml of solution. The wells containing casein blocker solution are again incubated
-~!0 at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15
minutes. Then the wells are uprighted and allowed to dry at room temperature for at least
24 hours.
This compietes the process of coating the wells and each diagnostic kit is then supplied with
96 coated wells. The shelf life of coated mierowells when stored at 4 degrees C in a sealed
;5 plastic bag is up to one year.
Step 2: Adhering Anti-Sm or Anti-SrnLRNP antibodies to the Prepared Wells From Step 1:
The sample diluent is supplied in the kit as a 40 ml green solution. To prepare a 1000 ml
`1 solution of Sample Diluent, 100 milliliters of native bovine serum, 1.42 g of Potassium
Phosphate (dibasic), .26 g ot Potassiurn Phosphate (monobasic), 1 gram of sodium azide,
0 and 8.6 9 of sodium chloride, and 1 ml of stock green dye are dissolved in deionized water
sufficient to make 1 OOû milliliters of solution. This solution is then filtered through a .2 micron
. ~
. ~,, .. ,,, , . ~. . . ..
,. . ~ . .: . . , ~ ..
. . ~ - . , : :,
-'~ ' ' .' " ~ ' ' ':' . '
WO 90/10229 ;~ 3D~ PCr/U~90/0102~
~ ` .. .. .. .
- 19 -
fllter.
The Sample Diluent acts as a blocking agent similar to the casein blocker which was
previously coated on the wells. The principle biocking component in the Sample Diluent is
the native bovine serum which acts to inhibit the bincling of any BSA-reactive antibodies to
the mBSA coating on the surface of the well.
Prior to contacting the body fiuid with the prspared ~late, lhe serum is diluted by adding
aliquots of sera to the sarnple diluent in a 1:50 ratio (volume of serum:volume of sample
diluent), although this exact dilution is not critical and depends upon the nature of the body
fluid and the assay techniques employed. To ~orrn the diluted sample solution the body fluid
is aliquoted in 10 microliler proportions into 490 microliters of Sample Diluent. In an
individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the
anti-Sm or anti-Sm/RNP antibody is enhanced by 15 rninutes incubation at room
temperature.
Following the affixation of the anti-Sm or anti-Sm/RNP antibody to the coated wells, the wells
j 15 are thoroughly washed four times with PBS to remove the free unbound antibodies that are
present in the sample, which if ailowed to remain, would elevate the background absorbance.
.
Step 3: Assax for the Anti-Sm or Anti-SmlRNP antibod~/ Affixed to the Wells: Standard
enzyme-linked immunoassay techniques, previously described, are used for this assay,
although any suitable means of detsction such as radioactive labeling, fluorescence, or the
like can be employed. For the examples describecl hereinafter, anti-human IgG and anti-
h~man IgM induced in goats were used to ascertain whether anti-Sm IgG and IgM antibodies
or anti-Sm/RNP IgG and IgM antibodies were present. Please note that other species of
animal can be used to produce anti-human antibodies. These antisera were linked to
horseradish peroxidase, an en~yme which yields a colored product whenever one of its
substrates is present together with hydrogen peroxide. Tho substrate should be chosen to
be consistent with the enzyme conJugated to the antibody. For the examples described
hereinafter, the substrate was (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
The kit contains one 12 ml vial of conjugated antibody solution with anti-human IgM and IgG
antibody conjugated to horseradish peroxidase. To prepare a working conjugat d antibody
solution, a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4
(commercially available from Medix~ was mixed wilh aprotinin, a protease inhibitor
'
.,
,~
,. , . .: : .. . . . ..
WO ~0/102~g PCI'/US~0/~)1029
(commercially available from Miles Psntex) at a .01% ratio of inhibitor to volume of buffer.
This diluent enhances the stability of the conjugated antibody. The solution is mixed at a
ratio of 1/~000 for IgG and 1/1500 for IgM; one part of concentrated conjugated IgM and IgG
antibodies is aliquoted into 4000 and 1500 parts conjugate diluent, respectively. The ratio of
concentrated conjugated antibody to conjugate diluent is subject to wide latitudes of dilutions
based on the manufacturer's concentration of conjugated antibody used in the assay.
. Next 100 microliters of the enzyme conjugated goat anti-human antibody working solution,
(prepared as described) is added to each microtiter well. 8inding of these antibodies to the
anti-Sm or anti-Sm/RNP antibody is permitted for at least 15 minutes at room temperature,
then the microtiter wells are emptied of their contents, washed four times with PBS, and
allowed to clrain before the next step.
The presence of a label and antibody, as previously described, is determined by incubating
the wells with a solution of buffered (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
This solution is supplied in the kit in two 8 ml ~ials; one contains (3,3',5,5')Tetramethylbenzidine; the other vial contains hydrogen peroxide. The separate vials are
necessary due to the interaction between the two solutions. The two solutions are mixed in
a one to one ratio just before use, and 100 microliters of the mix~d solution is dispensed into
each microwell. The reaction is permitted to continue for 10 minutes at room temperature,
or until sufficient color appears to be read on the spectrophotometric device used. The
reaction is subsequentty stopped through the addition of an equal volume oi 2.5 normal
sulfuric acid, and the intensity of color (the optical density, ~OD", or absorbance) is read by
a spectrophotometric device such as a Dynatech MR600 or the like.
As with any enzyme-linked immune assay, the resultant color of the reaction product is
proportional to the number of conjugated antibodies which have bound to the antibody. For
2~ most cases, the number of bound conjugated antibodies is linearly related to the number of
bound antibodies. Hence, as the amount of anti-Sm or anti-Sm/RNP antibody bound to the
wells Increases, so does the optlcal density, or absorbance of the enzynne reactlon.
:.
.,
.~ , . ~ . . . . .
,,~, , :
.
Wo 90/10229 ~3~ rcr/us90/0l029
- 21 -
E~MPLE 1
Protocol for Pr~Co~tin~ Microwells
with Purlfled dsDNA
Polystyrene wells were coated with (native) double stranded deoxyribonucleic acid (dsDNA)
by the following procedure:
1. Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was
dissolved at a ratio of 20 micrograms/ml in distilled water or PBS.
2. 100 microliters of the mBSA solution was placed in each microwell1 and incubated
overnight at 4 degrees C.
.
10 3. The excess coating solution was shaken from the plate after incubation. The plate
was rinsed once with a solution of .01 M PBS, pH 7.3 to remove unbound mBSA. Thewells were inverted to drain thoroughly.
4. 5 ug/ml of purified dsDNA was diluted in PBS, .01M, pH 7.3, and 100 microliters of
the diluted dsDNA solution was dispensed into each microwell and allowed to
-16 incu~bate for 18-24 hours at 4 degrees C.
5. ~ A~er ineubation, the excess dsDNA solution was shaken from the wells and 100
microliters of S-1 nuclease in digestion buffer was added to each well.
. .
6. To form S-1 nuclease buffer solution, .03M sodium acetate and .û3M acetic acid in a
1.1 to 1 ratio at pH 4.6 was pr~pared. Then 95 mls of this buffer was mixed with 5 mls
~ ~ of ~Iycerol, .29 9 NaC1 and .029 9 ZnS04 for a flnal volume of 100 rnls at pH 4.6.
S-1 nuclease (Sigma) was dissolved into this solution at a concentration of 100
units/ml buffer. (One unit of S-1 nuclease is defined as causing 1.0 microgram of
ssDNA per minute to beeome perchloric acid soluble at pH 4.6 and 37 degrees C.)
7. 100 microliters of S 1 nuclease buffer solution was contacted with each well for two
: 35 hours at 37 degrees C. ~ter incubation, the wells were emptied, rinsed twic~ with
1~ PBS, and draineci thoroughly.
.
8. ~ Casein blocker solution (pH 7.3) was dispensed in 200 microliter increments into each
,, .
~.:
W~ 90/10~2~ PC~/US90/01029
2~ 33~o ` f~
- 22 ~
well. The casein bloek~r solution consists of 2 ml glycerol, 10 g sucrose, 15 mgcasein and sufficient TEN buffer to bring the volume to -00 ml.
9. The wells coated with the blocker solution were incubated overnight at 4 degrees C.
After incubation, the solution was shaken from the wells and the wells were inverted
for 15 minutes. Then the weils were uprighteci and allowed to dry 24 hours at room
temperature.
;
The coated dsDNA wells were then used to determine the presence of anti-dsDNA in serum
samples obtained from individuals with:
1. no apparent pathology (normal);
2. Systemic Lupus Er~nhematosus.
These sera were drawn frorn patients with known pathological status. Described hereinafter,
is a method of performing an anti-dsDNA assay, using the dsDNA coated wells in a sandwich
ELISA format.
1. Standards, Sera from patients with no apparent pathology and patients with SLE, a
i~ high positive human serum control (100 AU/ml), a moderate positive human serum
control (45 AU/ml), and a negative hurnan serum control ~3 AU/ml) were diluted with
Sample Diluent in a 1:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample
Diluent was a solution consisting of 100 mls of native bovine serum in a phosphate
buffered saline consisting of 1.42 9 of potassium phosphate (dibask~,), .26 g potassium
phosphate (monobasic), 8.6 9 sodium chloride, .1% sedium azide, 1 ml stock greendye, and distilled water added to bring the volume to 1000 mls. The Sample Diluent
was then filtered through a .2 micron filter.
2. 100 microliters of the Sample Diluent solutions were placed in the designated wells
at room temp~rature for 15 minutes to allow completion of the antigen-antibody
binding process. Following incubation, the wells were emptied and washed four times
` with PBS solution.
3. The welis were expo~ed to a working conjugateci antibody solution eonsistint~ of 1 part
per volume horseradish peroxidase conjugated IgG and IgM speoific antibodies, and
, . . . f . ~ ............ .. . . . . .
.~ . . : ~ , . . , . ..... . . , . , :, .. ; . . .
. . ' . ` !
~, . ~' ' ' . . ' . . I '~
WO 90/10~29 PCI/lJS90/01029
- ~3 -
3000 part per volurne conjugate diluent (Medix) consisting of phosphate buffer, protein
stabilizer, and .02% thimerosai, to which was added .01% by volume a protease
inhibitor (commercially available from Miles Pentex).
4. 100 microliter of the working conjugated antibody solution was placed in eachmicrotiter well and allowed to incubate at room temperature for 15 minutes. After
incubation, the wells were rinsed four times with PBS solution to remove unboundconjugated antibodies.
5. Each well was assayed for horseradish peroxidase activity by mixing equal volumes
(3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and
~10 dispensing 100 microliters of this substrate solution into each well. The presence o~
the anti-dsDNA was detected by a blue color appearing after the 10 minute room
temperature incubation. 100 microliters per well of 2.5 N sulfuric acid terminated the
reaction producing a yellow color. The yellow color was quantitated at 450 nm using
a Dynatech MR600 plate reading spectrophotometer.
Deteotion of anti-dsDNA (IgG & IgM)
Source Mean O.D.
; CDC Ref S~d. 1.0~
anti DNA pos 1.13
pos SLE #1 .54
pos SLE #2 1.75
` pos anti nucleolar .10
normal serum .09
.
1 . :
~, .
WO9(1/lOZ~ 2~- PCr/US90/0102
EXA U9PLE
Adsorp~ion ot anti-dsDNA f:rom Serum
by Uncoated Polystyrene Wells
To demonstrate the beneflcial effect of the coating tr0atment, polystyrene wells were left
5 totally uncoated but exposed to the sera and the standards as described in the previous
example. The wells were then exposed to horseradish peroxide conjugated IgG and IgM
antibodies, and assayed lor peroxidase activity. As expected, c?nly slight selectivity was
exhibited by untreated wells and high amounts of non-specific binding led to very high
background absorbance.
G.D. Coated O.D.
Source AU/ML?~as in txample 1 Uncoated Wells
Calibrator
120.0 1.38 1.28
2 60.0 1.05 1.08
,15 3 30.0 .55 .77
- 4 15.0 .37 .57
7.5 .29 .67
- 6 3.8 .22 .50
Normal Serum 1 8.0 .28 .84
Normal Serum 2 9.0 .29 .97
Normal Serum 3 3.0 .19 .65
I ligh Positive SLE
112.0 1.34 .54
. 2 50.0 .89 .51
3 120.0 1.96 1.67
Low Positive SLE
, ~
~ 1 13.0 .35 1.76
*AU/ml vaiues calculated In previous experiment.
~ .
:, :
. I
,,~
~ ~ . : . ' ' ' '
wa:) 90/I~û229 27~ 3~ ; P~/U~90/û1029
r .
. . .
- 2i~ -
EXAiV'PLE 3
A Diagnostie Test Kit Assa,y Procer''ure
The kit contains pre-coatf?d microwells and,
1 vial (30 ml) of Sample Diluent (green solution containing 0.1% sodium az3de);
5 6 vials (200 ul each) of Assay Standards for anti-dsDNA, containing these levels of anti-
dsi~NA activity 120, 60, 30, 15, 7.5, 3.8 AU/ml (anti-dsDNA activity 100 AU/ml of this assay
calibrator has an antigen binding capacity of 59 x 10-4 ug of DNA),
1 vial of high positive hurnan serum control (about 100 AU/rnl),
1 vial of moderate positive human serum control (about 25 AU/ml),
1 vial of negativZs human serum eontrol (about 3 AU/ml). Also included in the diagnostic kit
was a vial (15 ml) of conjugated antibody working solution Z~iontaining horseraciish peroxidase
conjugated antihuman IgG, and IgM
1 bottle (8 ml) of TMB Substrate; solution A containing 3,3',5,5', tetramethylbenzidine,
1 bottle (8 ml) of TiYiB Substrate; Solution B containinZ~, hydrogen peroxide. When rnixed
~15 with equal parts Solution A will form a substrate capable of generating a colored product.
i
1 bottle (12 ml) of Stop Reagent containing 2.5 N H2S04 (1 N HCL can be substituted),
1 packet of Phosphate Buffere,d Saline (PBS) which reconstitutes to 2 liters of 0.01 M PBS,
pH 7.3, which is utilized as a wash solution
Plate Template.
., .
~0 The various reagents of th~ kit were utili~ed to perform the assay. The directions for the
, methoci of assaying for anti~dsDNA were included in the kit and these directions were
followed precisely during this experiment.
, .
The piate templates were labelled for sample placement in the microwells. A 1:50 dilution of
:
, .
:.::
2~3~ P~/US90/0102~
- 26 -
the standards, controls and patient sarnples was prepared in Sample Diluent (green solution).
10 ul of sample was added to 500 ul sample diluent in a one volurne to 50 volume sample
dilution. 1:50 dilutions of the assay standards and controls were diluted in the same manner.
100 ul of each diluted sample, control or standard was added to the appropriate mierowell(s).
5 The wells were allowed to incubate ior 15 minutes at room temp~rature. To perform the rinse
step the contents of PBS packet was added to 2 lilters of reagent grade water, and the
solution was mixed well until all the crystals were dissolved. Then th~ PBS buffer was used
- ~ to wash the wells four times. The microwells were inverted between each wash to empty the
fluid, after which the wells were drained and blotted on absorbent paper to remove residual
~10 wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
100 ul of working conjugated antibody (red solution) was added to each well, incubated for
15 minutes at room terrlperature, and then the wells were again washed ~our times with PBS
colution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TMB Substrate Solution A and TMB Substrate Solution B were combined
to form the color generating substrate. The kit instructed that if properly combined this
substrate solution would be colorless and it was colorless. Next a 100 ul of the colorless
working substrate solution v~as added to each well and the wells were incubated for ten
rninutes at room temperature.
~0 Next 100 ul stop reagent was added to each well to end the enzyme reaction, and the O.D.
of each well was read at 450 nm against a water blank. A standard curve graph was
- generated by p!otting AU/ml of standards against O.D. of standards, and sample results were
obtained from this standard curve.
Source O.D. AU/ml
;25 Calibrator 1 .2 3.8
Calibrator2 .3 7.5
Calibrator 3 .4 15.0
Calibrator 4 .57 30 0
Calibrator 5 .88 60.0
Calibrator 6 1.24 120.0
Refer to Graph 2
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WO 90/10229 2~ 34~ r~us90/0l029
t: ~
- 27 -
Controls O. D. AU~
high positive 1.11 104.0
moderate positive .55 24.0
negative .14 2.0
O.D. AUlm~
normal human sera #1 .14 2.0
norrnal human sera #2 .04 1.0
normal human sera #3 .18 3.0
SLE positive #1 .53 23.0
SLE positive #2 1.36 130.0
SLE positive #3 1.48 14Z.0
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WO 90/10229 ;~Cl S ~34~ ' PCT/US90/01029
- 2E~ -
EXAMPLE 4
Accelerated Stabliity Study
DNA coated microwells were treated with S,-nuclease using the optimized protocol, then
duplicate microwells were stored at 37 degrees C and 30 degrees C, and each set of wells
~` 5 was tested at various time intervals using anti-dsDNA and anti-ssDNA monoclonal antibodies.
The results of this experiment are shown in the follovving table:
' ~ Real Time Equivalent Time
In Days~ In Mont,,hs**, % ssDN,A Activity
O 0.0 3-4%
1 0 1 1 .6 3-~%
' ~ 2 3.0 ~-3%
7.5 3-4%
~, 7 1 0.5 4-5%
, g 13.5 3-4%
13 19.5 3-4%
',
~incubated at 37 degreés C
~; : **if incubated at 4 degrees C
,,1
,'~ The dsDNA activity in these microwells remained unchanged during the accelerated stability
study. The results obtained with the microwells stored at 30 degrees C were similar to those
,70 presented above and confirmed that the stability of the dsDNA coated on the microwells
~1 using this protocol was equivalent to more than one year without the generation of significant
amounts of ~sDNA.
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WO 90/10229 Z~ 5~ ¢3 PCr/US90/01029
29
E3CAMPLE 5
Pro~ocol for Pr~Coatin~ Microwell~
with Purified Smith Antigen
Polystyrene wells were coated with Sm antigen by the following procedure:
1. Methylated Bovine Serum Albumin (Sigma) (hereinafter ciesignated as mBSA) was
dissolved at a ratio of 2û micrograms/ml in distilled water or PBS.
2. 100 microliters of the mBSA solution was placed in each microwell, and incubated
overnight at 4 degrees C.
3. The excess coating solution was shaken from the plate after incubation. The wells
were inverted to drain thoroughly.
;
4. 5 units/ml of purified Sm antigen vvas diluted in PBS, .OlM, pH 7.3, and 100microliters of the diluted Sm antigen solution was dispensecl into each microwell and
allowed to incubate for 18-24 hows at ~ degrees C.
5. Casein blocker soluUon (pH 7.3) was dispensed in 200 microliter increments into each
s well. The casein blocker solution consists of 2 ml glycerol, 10 g sucrose, 25 mg
casein and sufficient TEN buffer to bring the volume to 100 ml.
:
6. Tha walls coated with the blocker solution wcre incubated overnight at 4 degrees C.
After incubation, the solution was haken from the wells and the wells were inverted
for 15 minutes. Then the wells were uprighted and allowed to dry 24 hours at room
:i~o ternpera~ure.
Tha coated Sm antigen wells ware then used to determin~ th~ presence of anti-Srn antibody
in seven CDC reference sera for anti-nuclear antibodies. Thle r~sults show that only anti-Sm
antibodies rsact in the assay.
(
Describsd hereinafter, is a method of performing an anti-Sm antibody assay, using the Sm
~5 antigen coated walls in a s~ndwich ELISA format.
`, .
1. Standards, seven rieferance sera samples, a negative human serum control
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WO 90/102~ . PCr/US90/01029
- 30 -
RE~DS/ml, a high positive human s~rurn control (RE~DS/ml), a moderate positive
human serum control (RE~nS/ml), and a low positive hurnan serum control
(RE~DS/ml) were ciiluted with Sample Diluent in a 1:50 ratio; 1 part serum to 50 parts
Sample Diluent. The Sample Diluent was a solution consisting of 100 mls of native
bovine serum in a phosphate buffered saline consisting of 1.42 g of potassium
phosphate (dibasic), .26 g potassium phosphate (monobasic), 8.6 9 sodium chloride,
.1% ~odium azide, 1 ml stock green dye, anai distilled water added to bring the volume
to 1000 mls. The Sample Diluent was then fiHered through a .2 rnicron filter.
2. 100 microliters of lhe Sample Diluent solutions were placed in the designated wells
~10 at room temperature for 15 minutes to allow completion of the antigen-antibody
binding process. Following incubation, ths wells were emptied and washed fourtimes
with PB5 soiution.
3. The wells were exposed to a working conjugated antibody colution consisting of 1 part
per volume horseradish peroxidase conjugated Igt; and IgM specific antibodies, and
4000 and 1500 part per volume conjugate diluent, resp0ctively, ~Medix) consisting of
phosphate buffer, protein stabili~er, and .02% thimerosal, to which was added .01%
by volume a protease inhibitor ~commercially available from Miles Pentex).
4. 100 microliter of the working conjugated antibody solution wac placed in each~, microtiter well and allowed to incubate at room temperature for 15 minutes. After
~o incubation, the wells were rinsed four times with PBS solution to remove unbound
~i conjugated antibodi~s.
.
5. i_ach w~ll was assayed for horseradish peroxidase activity by mixing equal volumes
(3,3',5,5') Tetramethylbenzidine (K~rkegaard Perry) and hydrogen peroxide solution and
. disp0nsing 100 rnicroliters of this substrate solution into each well. The presence of
~5 the anti-Sm antibodies was detected by a blue color appearing after the ~iO rninute
- ~- room temperature incubation. i 00 microliters per well of 2.5 N sulfuric acid terminated
the reaction producing a yellow color. The yellow color was quantitated at 450 nm
- using a Dynatech MF16û0 plate r0ading spectrophotom~ter.
Resuits are calcuiated by the following procedure:
~0 1. Calculatc tha mean absorbance values for the duplicates of the controls. The
duplicate absorbance values of the positive control should be within 10% of ~he mean
.
WO 90/1û229 ~ .PCr/VS90/01~2
- 31 -
absorbance value.
~.
2. The mean absorbance reading of the reagent blank should be less than 0.05.
Readings greater than 0.05 may indicate possible contaminated of the Substrate
Solution.
3. Calculate the cut-off absorbance values for the assay, and write them in the appropriato blanks on the results graph.
.
a. The mean absorbance (O.D.) of negative control x 3.2 = negative cut-off
absorbance, which is equal to 20 RE~DS units of anti-Sm activity. Sarnples
with O.D. values below the cut-off have less than 20 RE~DS units of anti-Sm
~10 activity, and should be scored as ~negative~. Samples with O.D~ values equal
to or higher than the cut-off have greater than 20 R~DS units anti-Sm activity,
and should be scored as ~positive~. Samples with O.D. values close to the cut-
off may warrant further testing (see Figure 4~.
The results were calcuiated by determining a cut-off O.D. The cut-off O.D. was determined
~1~ by running 200 normal sera samples and taking the mean of the O.D.s plus three standard
d~viations. This cut-off was equal to 3.2 x the O.D. of the standardized negative control.
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PC~I'/IJS90/01029
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- 3~ -
Detection of antl-~m antibo~i~s (IgG & ~gM)
SampLe O.D. St~tus
normal cut-off 0.1 12
Iow positive serum 0.21 positive
medium positive serum 0.40 positive
high positive serum 0.89 positive
anti Sm
1.22 positive
anti-~NP 0.112 negative
anti-SS-A 0.02 negalivs
. anti-SS-B 0.04 negative
anti-Sel-70 0.06 negative
anti-nucleolus
0.04 negative
.
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WO 90/102~9 . ` Pl-r/US~)0/0102~
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- 33 -
EXAMPLE 6
~ D~agnostic Tes~ Kit Assaly Procedure
The kit contains pre-coated microwells (qualitative format) and,
1 vial (40 ml) of Sample Diluent (green solution containing 0.1% sodium azide);
1 vial ~100 ul) of high positive human serum control (about 20 28 RE~DS/ml),
1 vial (100 ul) of calibrator (optional),
.~
1 vial ~100 ul) of negative human serum control (about ~.2 RE~DS/ml). The negative cut-
off value is 20 RE~DS units. To calculate O.D.s into RE~DS units, the negative value 6.2 is
divided by the mean value O.D. it produces; the resuHant number is multiplied by the O.D.s
of the samples to give RE~DS units.
1 vial ~12 mi) Conjugated Antibody Working Solution - containing horseradish peroxidase
eonjugated anti-human I~G and igM,
1 bottle (8 ml) of TMB Substrate, solution A containing 3,3i,5,5', tetramethylbenzidine,
1 bottle (8 ml) of TMB Substrate; Solution B containing hydrogen peroxide. When rnixed
with equal parts Solution A will form a substrate capable of generating a colored product.
.
1 bottle ~12 ml) of Stop Reagent containing 2.5 N H2SO4 (I N HCL can be substituted) (not
- necessarily supplied in kit),
1 packet of Phosphate Buffered Saline (PBS) which reconstitutes to 2 liters of 0.01 M PBS,
pH 7.3, which is utilized as a wash solution
Plate Template.
The various reagents of the kit were utilized to perforrn the assay. The direetions for the
~, method of assaying for anti-Sm antibodies were included in the kit and these directions were
~: followed precisely during this expçriment.
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Wo 90/10229 ~ i3~ Pcr/usgo/01029
~",-,
- 3~- .
The plate templates were labelled for sample placement in the microwells. A 1:50 dilution of
the controls and pa~ient samples was prepared in Sample Diluent (green solution) (no
calibrators were used in this example). 10 ul of sarnpl~ was added to 490 ul sample diluent
in a one volume to 50 volume sample dilution.
100 ul of each diluted sample and control was added lo lhe apprnpriate microwell(s). The
wells were allowed to incubate for 15 minutes at room temperature. To perform the rinse step
the contents of PBS packet was added to 2 liters of reagent grade water, and the solution
was nnixed well until all the crystals were dissolved. Then th0 PBS buffer was used to wash
the wells four times. The microwells were inverted between each wash to empty the fluid,
after which the wells were drained and blotted on absorbent paper to remove residual wash
fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
- 100 ul of working conjugated antibody (red solution) was added to each well, incubated for
15 minutes at room temperature, and then the wells were again washed four times with PBS
solution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TMB Substrate Solution A and TMB Substrate Solution B were combined
to ~orm the coJor generating substrate. The kit instructed that if properly eombined this
substrate solution would be colorless and it was colorless. Next a 100 ul of the colorless
working substrate solution was added to each well and the wells were incubated for ten
minutes at room temperature.
Next 100 ul stop reagent was added to each well to end the en~yme reaction, and the O.D.
of each well was read at 450 nm against a water blank. The final resulting data can be read
as O.D.s. To determine if the sample is positive for anti-Sm antibodies, the sample O.D. is
eompared to the cut-off C).D. value calculated for the assay (see Figure 4). For semi-
~5 quantitative results the O.D. value can be converted to R~DSunits by rnultiplying the sample
O.D. by the quantity (6.2 RE~DS units/negative control O.D.). 6.2 FlE~DS units is the anti-
Sm activity of the negative c:ontrol, which was standardized against the CDC referenee serurn.
The cut-off Is 20 RE~DS units.
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WO 90/~()229 ~ PCI/U~;~0/011J2~
(,
- 35 -
'I
SLE ~era Sa~ Q~ RE~DSUnits
0.16 16.5
2 0.10 10.3
3 0.07 7.2
4 0.09 9.3
0.12 12.4
6 0.10 10.3
7 0.12 12.4
8 0.08 8.
10 ~ 009 93
0.12 12.4
11 0.19 19.6
12 0.18 18.5
13 0.27 27.8*
5 14 0.35 36.0*
0.29 ~9.9*
16 1.17 121.0
17 0.15 15.4
18 0.35 36.0*
2019 0.13 13.4
:: :
*positive result
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WO 90/10229 ~ PCr/US90/01029
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36 -
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EXAI\APLE 7
Adsorptlon ot anti Sm antibodi~s From Serl~m
by ~he Test Kit Invention and a Competitive Kit
To demonstrate the sensitivity of the anti-Sm test kit samples from SLE patients were run in
a competitive kit and were run in the test kit in accordancs with the present invention under
the protocol demonslrated in Example 6, with the ~xception that only the negative serum was
used to produce a cut-off level. The test was run as ia quaiitative test showing only negative
or positive results. 19%-40% of SLE patients are expected to have anti-Sm antibodies
present. With all the included samples (the data is only representative of a portion of the
'O data), approximately 28% of the sera samples were positive according to the present
invention and only 21% were positiv0 according to the competitive kit. Note the present
invention detects both IgG and IgM and the competitive kit only detects IgG.
The resulting O.D.s were obtained. The cut-off value for the present invention was 0.33 O.D.
The cut-off value of the competitive kit was the given by the kit instructions.
;
SLE Sera O.D. From O.D. From
Samples Invention Status Competitive Kit Status
0.59 positive .94 positive
2 0.15 negative .05 negative
3 0.36 positive .12 negative
4 0.19 negative .08 negative
0.48 positive .61 positive
6 0.29 negative .13 negative
7 0.24 negativa .07 negative
8 0.21 negative .08 negative
9 0.21 negative .22 negative
0.19 negative .OB negative
11 0.25 negative . 1 0 negative
0.19 negative .13 n~gative
13 0.28 negative .14 negative
~;3 14 0.29 negative .16 negative
V.08 negative .05 negative
18 0.86 positive 1.10 positive
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Wo 90/10229 ~ Pcr/vsso/olo29
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- 37 -
XANiPLE 8
Accelerated Stability Study
Sm antigen coated microwells were treated with the optimized protocol, then duplicate
rnicrowells were stored at 37 clegrees C and 4 degre~es C, and each set of wells was tested
on days 1, 4, 6, 8, and 12, using a high positive anti-Sm sera, a moderate positive anti-Sm
sera, and a negative anti-Sm sera. The results of this experiment are shown in the following
. table:
'.' .
O.D.s O.D.s O.D.s O.D.s O.D.s
Dav 1 Day 4 Da.~ Day ~ Day 12
.~
;~0 High anti-Sm activity
4 1.12 1.14 1.12 1.23 1.00
37C (high) 1.15 1.05 1.08 1.09 1.12
-~,
Moderate~ anti-Sm
- l, activity
`?~ 4 0.92 0.93 0.78 0.93 0.95
37~ 0.94 0.92 0.86 0.90 0.80
- Low anti-Sm activity
4 (negative) 0.07 0.07 0.05 0.06 3.05
, 37 0.06 0.09 0.0~ O.o~ 0.05
0 *(1:2 dilution of the hi~h anti-Sm sera)
~j The Sm antigen activity in these rnicrowells remained unchanged during tha accelerated
~', stability study. The results obtained with tha microwells stored at 37 degrees C wera similar
l to those presented abov~ and confirmed that the stability of the Sm anti~en coated on the
`~ microwells using this protocol was equivalent to more than one year shelf life.
.
.
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WO 90/10229 , PClr/US~0/01029
2t~ i34L0 - 3B -
EXAMPLE 9
Protocol for Pr~Coating Microw~lls
with Purified Sm/RNP ;intlgen
Polystyrene wells were coated with Sm/RNP antigen by the following procedure:
5 1. Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was
dissolvad at a ratio of 20 micro~rams/ml in ciistilled water or PBS.
2. 100 microliters of th~ mBSA solution was placed in each microwell, and incubated
- overnight at 4 degrees C.
3. The excess coating solution was shaken from the plate after incubation. The wells
.0 were inverted to drain thoroughly.
: ~`
. 1 unit/ml of purified Sm/RNP antigen was diluted in PBS., .01M, pH 7.3, and 100
rnicroliters of the diluted Sm/RNP antigen solution was dispensed into each microwell
and allowed to incubate for 18-24 hours at 4 degrees C.
. :
5. Casein blocker solution (pH 7.3) was ciispensed in 200 microliter increments into each
. 5 well. The casein blocker solution consists of 2 ml glycerol, 10 g sucrose, ~5 mg
.~ casein and sufficient TEN buffer to bring the volume to 100 ml.
6. The wells coatecl with the blocker solution were incubated overn,.ght at 4 degrees C.
After incubation, the solution was shaken from the wells and the wells were inverted
for 15 minutes. Then the wells were uprighted and allowed to dry 24 hours at room
o temperature.
Th~ coated Sm/RNP antigen wells were than used to determine the presence of anti-
Sm/RNP antibody in serum samples obtained from individuals with Progressive SystemicSclerosis (PSS).
i~j Described hereinafter, is a rnethod of performing an anti-Sm/RNP antibody assay, using the
;:5 Sm/RNP antigen co~teci w~lls in a sandwlch ELiSA format.
,' :
WO 9~/1022~ PCrtUS90/01029
2~ i34~) ,
1. Sera from patients with PSS, a high positive human serum control (RE~DS/ml), and
a negative human serum control ~RE~DS/ml) were diluted with Sample Diluent in a
~:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample Diluent was a
solution consisting of 100 mls of native bovine serum in a phosphate buffered saline
consisting of 1.42 g of potassium phosphate (dibasic), .26 g potassium phosphate(monobasic), 8.6 9 sodium chloride, .1% sodium azide, 1 ml stock green dye, and
distilled water added to bring the volume to 1000 mls. The Sample Diluent was then
filtered through a .2 rnicron filter.
2. 100 microliters of the Sample Diluent solutions were placed in the designated wells at
room temperature for 15 minutes to allow completion of the antigen-antibody binding
process. Following incubation, the wells were emptied and washed four times with
PBS solution.
3. The wells were exposed to a working conjugated antibody solution consisting of 1 part
per volume horseradish peroxidase conjugated IgG and IgM specific antibodies, and
4000 and 1500 part per volume conjugate diluent, respectively, (Medix) consisting of
phosphate buffer, protein stabilizer, and .02% thimerosal, to which was added .01%
by volume a protease inhibitor (commercially available from Miles Pentex).
,
- 4. 100 microliter of the working conjugated antibody solution was placed in each
;~ microtiter weli and allowed to incubate at room temperature for 15 minutes. After
~o incubation, the wells were rinsed four times with PBS solution to remove unbound
conjugated antibodies.
5. Each well was assayed for horseradish peroxidasa activity by mixing equal volumes
(3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and
dispensing 100 mlcroliters of this substrate solution into each well. Th0 presence of
2S the anti-Sm/RNP antibodies was detected by a blue color appearing after the 10
minute room temperature incubation. 100 rnicroliters per well of 2.5 N sulfuric acid
terminated the reaction producing a yellow color. The yellow color was quantitated at
45û nm using a Dynatech MR600 plate reading spectrophotometer.
R~sults
1. Calculate the mean absorbance values for the duplicates of the controls. The
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WO !~0/10229 PCI'/lJS90/~1029
- 40 ~
duplicate absorbance values of the positive control should be within 10% of the mean
absorbance value.
2. The rnean absorbance reading of the reagent blank should be less than 0.05.
Readings greater than 0.05 may indicate possible contamination of the substrate
solution.
3. Calculate the cut-off absorbance values for the assay:
a. The mean absorbance (O.D.) of negative control x 2.8 = negative cut-off
absorbance, which is equal to 17.5 RE~DS units of anti-SM/RNP activity.
Samples with O.D. values below the cut-off have less than 17.5 RE~DS units
~0 of anti-SM/RNP activity, and should be scored as "negative". Sarnples with
O.D. values equal to or higher than the cut-off have greater than 17.5 RE~DS
units anti~SM/RNP activity, and should be scored as ~positive~. Samples with
O.D. values very close to the cut-off may warrant further testing (see Figure 6).
':
The results were calculated by determining a cut-off O.D. The cut-off O.D. was determined
by running 200 normal sera samples and taking the mean of the O.D.s plus three standard
deviations. This cut-off was equal to 2.8 x the O.D. of the standardized negative control. The
cut-off for the flrst 40 samples was 0.11 negative O.D. x 2.8 = 0.31 O.D. The cut-off for the
second 40 samples was 0.09 x 2.8 = 0.25 O.D. See Figure 6.
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W090/10~9 Z05i~34~ Pcr/1590/~1029
-41
Detection of anti-Sm/RNP antibodies (IgG, ~ IgM~
(PSS! SeraSample ~ Status
0. 14 negative
2 0.16 negative
. 5 3 0.17 negative
4 0.17 negative
0.09 negative
6 0.10 negative
7 0.15 negative
~0 8 0.11 negative
9 0.19 negative
0.22 negative
11 0. 1 7 negative
` 12 0.19 negative
.5 13 0.18 negative
14 0.13 negative
0.17 negative
16 0.11 negative
, 17 0.15 negative
ilo ~ 1B 0.13 negative
-' 19 0.15 negative
0.15 negative
21 0.22 negative
Z2 0.18 negative
23 0.15 negative
i 24 0.15 negative
0.60 positive
26 0.1 1 negative
, ~7 0.16 negative
D 28 0.17 negative
', 29 0.13 negative
0.16 negative
31 0.18 negative
32 0.13 negative
, . .
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WO 90/10229 ` i `; P(:~US90/01029
3~ - 4~
~PSS) Sera Sample O.D. Status
33 0.?0 negative
34 0.24 negative
0.24 negative
36 0. 11 negative
37 0.19 negative
38 0.28 negative
39 0.12 negative
0.10 negative
41 0.18 negative
42 0.19 negative
43 0.1~ negative
~4 0.16 negative
0.08 negative
J 5 46 0.18 negative
47 0. 11 neyative
48 0.38 positive
49 0.09 negative
0.25 positive
~0 51 0.23 negative
52 0.19 negative
53 0.21 negative
54 0.~7 positive
0.17 negative
` '5 56 0.15 negative
- 57 0.21 negative
: 58 1.23 positive
sg 0.16 negative
- 60 0.33 positive
- '~ 61 0.12 neyative
62 0.11 negative
63 0.24 negative
.~ 64 0.11 negative
0.09 negative
~'5 66 0.22 negative
:~l 67 0.18 negativa
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WO 9~/10229 ~ 3'L~ P~l/U590/ûl029
~ :.
- 43
(PSS! Sera Sample O.D. Sta~
68 0.47 positive
69 0.09 negative
70 0.14 negative
71 0.18 negative
72 0.13 negative
73 0.20 negative
74 0.10 negative
0.17 negative
76 0.22 negative
77 0 07 negative
78 0.10 negative
79 o. ~ 7 negative
0.15 negative
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~ 44 - t
EXAMPLE 1 0
A Diagnostic Test Kit Assay Procedure
(Oualitative Format)
The kit contains pre-coated microwells and,
1 vial (40 ml) of Sample Diluent (green solution containing 0.1% sodium azide);
1 vial (100 ul) of high positive human serum control (about 100 RE~DS/rnl),
1 vial (100 ul) of calibrator (optional),
,
1 vial (100 ul) of negative human serunn control (about 6.3 RE~DS/ml). The negative cut-
off value is 17.5 RE~DS units. To calculate O.D.s into RE~DS units, the negative value 6.3
- ~ 10 is divided by the negative O.D. rnean value. The resultant number is muttiplied by the O.D.s
of the samples to give RE~DS units.
1 vial (12 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase
conjugated anti-human IgG and IgM,
~:;
1 bo~iile (8 ml3 of TMB Substrate; solution A comaining 3,3',5,5', tetramethylbenzidine,
i15 1 bome (8 ml) of TMB Substrate; Solution B containing hydrogen peroxide. When mixed
.; ~ with equal parls Solution A will form a substrate capable of generating a colored product.
1 bottle (12 ml) of Stop Reagent containing 2.5 N H2S04 (1 N HCL can be substituted),
~'
1 packet of Phosphate Buffered Saline (P8S) which reconstilutes to 2 liters of 0.01 M PBS,
pH 7.3, which is utilized as a wash solution
; 20 Plate Template.
The various reagents of the kit werc utilized to perform the assay. The directions for the
method of assaying for anti-Sm/RNP anlibodies were included in the kit and these directions
- ' were followed preoisely during this experiment.
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- 45 -
The plate templates were labelled for sample placement in the microwells. A 1:50 dilution of
the controls and SLE patient samples was preparecl in Sample Diluent (green solu~ion). 10
ul of sample was added to 500 ul sample diluent in a one volume to 50 volume sample
dilution.
100 ul of each diluted sample and controls was addled to the appropriate microwell(s). The
wells were allowed to incubate for 15 minutes at room temperature. To perform the rinse step
the contents of PBS packet was added to 2 liters ol reagent grade water, and the solution
was mixed well until all the crystals were dissolved. Then the PBS buffer was used to wash
the wells four times. The microwells were inverted between each wash to empty the fluid,
after which the wells were drained and blotted on absorbent paper to remove residual wash
fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
.
100 ul of working conjugated antibody (red solution) was added to each well, incubated for
- 15 rninutes at room temperature, and then the wells wer~ again washed four times with PBS
solution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TM8 Substrate Solution A and TMB Substrate Solution B were combined
to form the color generating substrate. The kit instructed that H properly combined this
substrate solution would be colorless and it was colorless. Next a 1ûO ul of the colorless
working substrate solution was added to each well and the wells were incubated for ten
rninutes at room tsmperature.
. ~
Next 100 ul stop reagent was added to each well to end the enzyme reac~ion, and the O.D.
of each well was read at 450 nm against a water blank. For semi-quantitative results the
conversion factor to convert O.D. of the sampla to RE~DS units was the quantity (6.3/mean
- O.D. of negative control). 6.3 RE~DSunits is the anti-Sm/RNP activity of the negative control
~5 which was standardi~ed against the CDC reference serum. In this example the conversior
factor was 6.3/.0~75 = 72.
:,
: Controls RE~DS~m
, cùt-off 17.5
negative 6.3
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WO !)0/10229 ' .~ ` .,PC~/US90/()1029
- 46
_LE Patient Sera O D. RE~DS Units
- 1 0.24 17.3
2 0.18 12.6
3 0.19 13.7
0.11 7.9
0.66 48.2*
6 0.15 11.2
7 0.16 11.5
8 0.84 60.51t
9 . 0.01 0.36
0.44 32.0*
11 0.31 22.3*
12 0.53 38.2*
13 1.03 74.2*
14 0.31 22.3*
1.49 107.0*
: ~6 0.52 37.1*
17 1.42 102.0*
, 18 0.53 38. *
~; 20 *positive results
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WO 90/10229 ~,3~ p~lUS90/01029
- 47 -
EXAMPLE 1 1
Adsorption ot anU-Sm/RNP ~ntibodies From Serum
To demonstrate the sensitivity of this assay for anti-SM and anti-RNP, the test kit was used
, in accordance with the protocol shown in Example 10. Nine CDC reference for nuclear
antigen were tested. All were negative except for the anti-Sm and anti-RNP sera which were
positive.
Sa,mple O.D.......... Stat,us
normal cut-off 0.12
anti-Snn 0.98 positive
10 anti-RNP 0.56 positive
anti-SS-A 0.03 negative
anti-SS-B 0.06 negative
', anti-Scl-70 0.08 negative
anti-nucleolus 0.06 negative
.15 anti-centromere 0.06 negative
- ` anti-dsDNA monoclonal 0.01 negative
anti-ssDNA monoclonal 0.01 negative
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- 48 -
EXAMPLE 12
Accelerated Stabllity Study
Sm/RNP antigen coated microwells were treated with the optimized protocol, then duplicate
microwells were stored at 37 degrees C and 4 degrees C, and each set of wells was tested
at on day 1, 4, 6, 8, and 12, using a high anti-Sm/RNP ssra sample, a moderate anti-
Sm/RNP sera sample, and a negative anti-Sm/RlYP sera sample. The results of thisexperiment are shown in the following table:
O.D.s O.D.s O.D.s O.D.s O.D.s
: Day 1 5~ Day 6 Day 8 Davl2
ES 22 4 1.39 1.26 1.35 1.42 1.33
; ~high) 37 1.36 1.20 1.34 1.40 1.26
ES 22 (1:2) 4 1.09 1.08 1.13 1.17 1.00
(moderate) 37 1.08 1.01 1.07 1.16 1.06
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negative 4 0.10 0.10 0.07 0.07 0.09
(low) 37 0.09 0.08 0.08 0.08 0.08
. . .
The significant Sm/P~NP antigen activity in these microwells remained unchanged during the
accelerated stability study. The Sm/RNP antigen coated on the microwells using this
protocol was equivalent to more than one year shelf life.
:,
The foregoing examples serve to illustrate the efficiency and utility of the methylated BSA to
20 provide a coating which Inhibits non-specific binding and provides a coating capable of
affixing antigen evenly over the solid support. Without being bound to the specific quantities
given in the definitional section, it is possibl0 to utilize a wide latitude of concentrations of
mBSA or other similar, functionally equivalent substitutes which have the capability to evenly
attach antigen to the support solid by providin~ a charged surface or by any other like
`25 mechanism to affix the and to continue to inhibit non-specific binding.
I Likewise, the blocker utilized in the preferred embodiment of this invention to stabiliz0 the
2~ shelf life and elirninate non-specific binding cannot be limited to the cornpounds or the
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WO 90/10229 i . . .PCI/US90/01029
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- 49 -
ingredients or the concentrations thereof listed in the definitional section. A variety of
functional equivalent blocking agents are known to those skilled in the art. A partial listing
of some materials which could be utilized to perform a similar function is found in ~Robert F.
Bogt; J. Immunological Methods, 101, 43-50 [1987]) and is hereby incorporated herein by
re~erence. A third element in the anti-dsDNA test kit which plays a function in elirninating non-
specific binding is the S, nuclease usad to degrade the ssDNA and thus to avoid any cross
reactivity between the anti-dsDNA and the undesired ssDNA. The described rnethod of
limiting cross reactivity is not iinnited to the definition given but the method can be performed
with varying concentrations of S, nuclease or other similar, endonuclease enzymes, or similar
0 functional equivalents which are capable of eliminating problems of cross reactivity without
adversely affecting the antigen present in the invention.
The treatment of the solid support which can be any of a variety of formats, i.e. test tubes,
plates, wells, etc., made of various suitable materials, i.e. glass, plastics, etc. with the
aforementioned technology affords many important and useful approaches to the detection
of the specific antibodies. The detection of antibodies need not be limited to the conjugation
of enzymes. Addition of fluorescent chemicals such as fluorescence or the like to the
antibody will impart fluorescence to the assay if the antibody is present. Similarly,
conjugation of the antibody with a radionuclide will impart radioactivity to the assay if the
antibodies are present- in the assay. Many other methods of detection of antibodies aiso
0 exist, and these methods will yield positive resul.s provided that the antibody exists in the
assayed sera and is affixed according to the methods descrlbed herein.
The test kit and the underlying coating and detection methods herebefore descrlbed are not
intended to be limited by the assay format described or by the volumes or the concentrations
or specific ingredients given for the various reagents, controls, and calibrators. It should be
understood that sirnilar chemicai equivaients or other functional equivalents of the
components found in the coatings, or in any of the various reagents, controis, and calibrators
can be utilized within the scope of this invention.
It is contemplated that the inventivo concepts herein described may have difFering
embodiments and it is intended that the appended claims be eonstrued to include all such
3 alternative embodiments of the invention except insofar as they are limited by the prior art~
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