Note: Descriptions are shown in the official language in which they were submitted.
WO90/10227 PCT/US90/01028
-1- 20~77~2
M~T~OV ~ND DIA~NOSTIC TEST RIT FOR
DETECTION OF ~N~I-CARDIOLIPIN
BACRGRO~ND OF T~E I~VENTIO~
There exists three types of anti-phospholipid autoantibodies.
The first anti-phospholipids were detected during the World
War II mass screening for syphilis. In a number of patients
with systemic lupus erythematosus ~SLE), the test ~.or syphilis
yielded a false positive. ~his false positive test for
syphilis eventually led to the discovery of the three types of
anti-phospholipid antibodies responsible for biological false
positive reactions (BFP): VDRL (Venereal Disease Research
Laboratory), lupus anti coagulant, and antl-cardiolipin
antibodies. Although correlation studies show that all three
types of anti-phospholipid antibodies associated with similar
pathologies, the. three antibodies are not identical and each
is indicative of differing medical risks.
Research has been performed on all o~ the anti-phospholipids;
unfortunately the tests developed for VDRL and lupus anti-
coagulant have major limitations. Therefore, clinical testing
and immunological studies have focused on the development of
radioimmunoassays and ELISAs for detection of the third anti-
phospholipid antibody, anti-cardiolipin. The anti-cardiolipin
antibody has been linked with recurrent cerebral thrombosis,
rec~rrent arterial ~hrombosis, recurrent abortion,
thrombocytopenia J chorea, epilepsy, and idiopathic pulmonary
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WO90/10227 ~ ~7 ~ ~2 -2- PCT/US90/0~028
hypertension. These medical problems have been identified in
a subset of SLE patients, which have a common syndrome
identified as the Anti-Cardiolipin Syndrome.
The reasons for the strong association between thrombosis and
anti-cardiolipin antibodies has not been determined. Two
hypotheses have been tendered: (l) a reaction between the
phospholipids in endothelial cell membranes which effect the
release of prostacyclin; or (2) an action of anti-cardiolipin
antibodies ~aCL) against the phospholipids în the platelet
membrane. Regardless of the mechanisms which link aCL and
thrombosis, the relationship between high levels o~ aCL and
further occurrence of thrombosis is well established.
Although the biological mechanisms which trigger aCL to react
causing the clinical manifestations of aCL syndrome to appear
are not fully understood, the clinical significance of
monitoring aCL is substantial. High levels of aCL appear to
correlate well with renewed disease activity. ACL levels
should be monitored in SLE patients undergoing anti-coayulant
therapy, in patients with previous thrombosis, in patients
under 45 who have had myocardial infarctions, in patients with
venous thrombos~s, or placental infarctions or recurrent
incidence of intra-uterine death and spontaneous abortions,
and in patients electing to take oral contraceptives to
; determine th~ statuC o~ their aCL levels.
WO90/10227 PCT/US90/01028
~3~ ~77~2
The development of a stable, reproducible anti-cardiolipin
antibody test has become an international project. In April
of 1986, scientists from thirty laboratories in Britain, the
U.S.A., France, Italy, New Zealand, the Netherlands, and
Sweden all participated in an international study to evaluate
anti-cardiolipin (aCL) tests. At least three types of (aCL)
tests have been developed: a diagnostic kit based on the
ELISA method (by Cheshire Diagnostics, Cheshire, UK), a solid
phase radioi~munoassay method (Harris, et. al., The Lancet,
1211-1214, [Nov. 26, 1983~), and a sandwich ELISA technique
(E.N. Harris, et. al., Clin. Exp. Immunol. 68, 215-225
[1987]). Anti-phospholipid syndrome can cause a patient to
have a high level of IgG aCL alone, or a high level of IgM
aCL; therefore, these methods are designed to measure the
concentration of both IgG and IgM anti-cardiolipins.
A variety of different techniques have been developed in the
search for a stable, reproducible, speedy method of detecting
IgG and IgM aCL. Although each method has shown some promise,
none of the methods has ade~uately met the requirements
necessary to be a suitable assay method. Examples of the c
various limitations of the prior techniques follow.
Firstly, RIA techniques have been developed for anti-
cardiolipin concentration measurements. RIAs provide
sensitive and accurate measurements of high and medium levels
of IgM and IgG concentrationsO However, RIAs are plagued with
the hazards and expense associated with radioactive material.
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W09OtlO227 PCT/US90/01028
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one specific RIA develped by E.N. Harris for the detection of
anticardiolipin antibodies is 400 times more sensitive than
the preceding test, which was the precipitaltion method used in
the Venereal Disease Reference Laboratory test. Although this
RIA was more sensitive than the previous test, it
unfortunately had drawbacks of its own. The run-time of this
test is twenty-two hours, which makes this test impractical in
clinical laboratories.
Secondly, aCL ELISAs have been developed for routine
diagnostic testing in clinical laboratories. Unfortunately,
ELISAs have not been able to detect low concentrations o~ IgG
or IgM anti-cardiolipin antibodies. The scientific community
has attempted to standardize results to avoid the lack of
sensitivity in the low levels of positive (S. Loizon, 1985
Chem. Exp. Immunol. 62, 738-745). This lack of sensitivity
has lead to a host of false negative test results, which
renders these tests unsuitable for diagnosis or monitoring of
a subclass o~ patients.
An ELISA for aCL has been produced as a diagnostic kit by
Cheshire Diagnostics Limited. A disadvantage is that this
test kit has a shel~ e of only 12 weeks from the date of
~anufacture, and problems with variations in results, as
evidenced by the kits Interpre~tation o~ Results section which
requires a repeat of tests with results in the IgG and IgM
bordering positive range. As a consequence, to overcome the
~O90/10227 ~ 7 7 4 2 PCT/US9~/0102~
limitation of insensitivity to low levels of aCL, repeat tests
must be run increasing the CGSt to the patient and increasing
the work time expended by lab personnel. However, the test
kit's run time of 2 l/2 hours is substantially less than most
other aCL assays. Development of a stable, speedy ELISA
capable o~ accurately measuring low levels of anti-cardiolipin
antibodies is essential to surmount the problems of the prior
methods.
It is therefore an objective of the present invention to
provide compositions, methods, and articles for the detection
of aCL antibodies at low, medium, and high levels of
concentration. It is the further objective of th1s invention
to overcome the aforementioned length of run time, and
stability and sensitivity disadvantages inherent in other
methods of detection of anti-cardiolipin antibodies. It is
furthermore an objective of this invention to provide a novel,
highly adaptable, readily utilizable means ~or quantitative
detection of anti-cardiolipins, and further to provide test
kits which use such a technique for the purpose of clinical
detection, or any other purpose associated with human or
animal medical testing.
BRI~F 8~RY_OF ~HB I~V~TIO~
The present invention provides no~el compositions and novel
~ethods ~or performing a sandwich assay for the detection of
I~M or IgG (aCL) antibody, for the purpos o~ identification,
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WO90/10227 PCT/US90/01028
2`~ 2 -6-
or quantitation. The present invention utilizes in its
broadest sense, anti-phospholids antibodiles, which have a
particular affinity for association with cardiolipin. As
described herein, the anti-cardiolipin antibodies in human
serum are employed to directly a~fix to the pre-coated
cardiolipin, and furthermore are specifically selected to have
an affinity for an enzyme conjugated goat anti-human IgM, or
an enzyme conjugated goat anti-human IgG antibody, which
produce, throu~h enzymatic action with the substrate, a
colored by-product which is detectable and q~antitatable using
standard photometric instrumentation. This color change,
which is generally measured as optical density, is in direct
relationship to the concentration of IgG or IgM present in the
sample solution. (~he anti-human antibody can be obtained
from a variety of differing animal species.)
The REAADSR test kit is a sandwich ELISA that employs pre-
coated suitable solid support, such as test tubes, plates or
wells (hereinafter referred to as wells or microwells). The
coating which is advantageously utilized to allow adherence of
the cardiolipin to the side walls and to the bottom of the
wells is methylated bovine serum albumin (mBSA). The mBSA
provides a positively charged surface which enhances the
adherence of the cardiolipin to all surface areas of the well.
While methylated serum preparations have frequently been
utilized in anti-DNA ELISA methods, it is unique and highly
surprising to find that a coating of methylated bovine serum
on a solid support affixes cardiolipin in an even
WO90/10227 PCT/US90/010~8
~7~ 2~ 177~2
concentration throughout thP well without the typical problem
of the formation of globules of cardiolipin on the bottom of
the well. In the practice of this invention protami~e sulfate
as well as functionally equivalent substitutes have been found
to be capable of forming a positively charged surface;
however, due to cross reactivity of some of these substrates,
mBSA is the preferred coating. The utili~ation of a
methylated bovine serum underlying the cardiolipin antigen
coating provides for pre-treated wells which have a shelf life
of six months to one year; furthermore, the wells also provide
a high level of reproducibility of results.
The reproducibility of results from the present invention is
associated with the mBSA and also with the next two steps of
forming the pre-treated wells; the drying and the addition of
a hydrolyzed casein blocker. A casein-type blocker has been
used in various E~ISA techniques (Robert F. Bogt [1987] J.
Immunological Methods, lOl, 43-50) to prevent through a
protein-plastic interaction non-specific binding to plastic.
It is an unaxpected realizatior1 that a microwell coated with
hydrolyzed casein blocker properly dried and stored at 4
degrees C in a sealed plastic bag maintains a consistent
inhibition o~ non-specific binding over an extended period of
time.
Although the direct mechanisms by which the drying process and
the blocker increase the stability and shelf life of the
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WO90/10227 PCTtUS90/~1028
~c~ -8-
microwells is not fully understood, it is clear that the
storage time is increased by these two processes.
A variety of differing blocking age~ts could be utilized which
are functionally equivalent to or chemically related 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 (commercially
available by Sigma) due to its high level of inhibition of
non-specific binding and its storage stability.
The pre-treated wells are then used to detect the presence of
anti-cardiolipin antibody in the samples. The plasma or serum
samples are prepared with a sample diluent and are then
assayed by an immunoassay technique, the ELISA and the
fluorescent immunoassay (FIA~ formats being 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. The wells when exposed to
the samples are provided with approximately 15 minutes to
allow the binding process to go to completion. Then the
labelled goat anti-human antibodies are exposed to the wells
and a similar 15 minute incubation at room temperature is
provided. If the anzyme format is utilized, the substrate is
added and lO minutes is allotted for the production of the
color. If a fluorescent marker was used on the anti-human
W090/l0227 PCT/~S90/~1028
_9_ -
2~77 42
antibody then no substrate i5 needed, therefore the run time
is shortened by 10 minutes reducing it to 35 minutes.
Subsequent qualitative and quantitative detec:tion of the anti-
cardiolipin antibody is relatively simple if the format is
either an ELISA or a FIA. Although horserad.ish peroxidase was
used in the examples, numerous enzyme-conjugated antibodies
and fluorescent marked antibodies specific for any o~ the
immunoglobulin classes can be substituted to perform the
assay. The quantitation of the anti-cardiolipin 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
first described by Engvall and Perlman (~1971] Immunochemistry
8, 871-874 and tl972] 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 o~ which are, in their
totality, incorporated herein by reference.
DBTAI~ED DE~CRIP~ION OF T~E INV~NTION
The following definitions are supplied for the purpose o~
clarifying the invention and are not intended to limit the
scope of the invention:
Methylated _8OV ne _Serum Albumin Solution: Unless otherwise
sp~cified, is intended to mean a solution with 20 micrograms
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WO90/10227 PCT/US90/01028
2~Ll;~7'~ -lo-
of methylated bovine serum ~lbumin (mBSA) dissolved in water
at a ratio of 1 ml of distilled water to 20 micrograms of
(mBSA). A substitute for mBSA is protamine sulfate or any
other chemical or chemical process capable of producing a
slightly positively charged coating which is evenly
distributed over the surface of the microtitre well.
PBS Solution: A .01 molar solution of buffer containing 1.43
g potassium phosphate, dibasic, .25 g potas~ium phosphate,
monobasic, and 8.5 g sodium chloride in one liter of water.
The pH is 7.3 +/- .1.
Cardiolipin ,(~rom beef heart~_Solution: Purified cardiolipin
(Sigma) is dissolved in 100% denatured ethanol at 20
micrograms cardiolipin per ml of solution. Alternative
sources of antigen includes, but are not limited to,
phosphatidylserine, phos-phatidylcholine, phosphatidylethano-
lamine, phosphatidylglycerol, phosphatidylinositol.
Casein ,B,loc,k,er Sol~ut,ion: 2 ml glyercol, 10 grams sucrose and
15 milligrams of hydrolyzed casein added to TEN buffer for a
total volume of 100 ml. Adjust pH to 7.3 +/- .1.
TEN Bu,ffer: Is made by adding 6.1 g TRIS, .38 g EDTA, 8.8 g
NaCl, 3.8 ml of concentrated HCL to 900 ml deionized water.
Adjust pH to 7.3 and add deionized water ~ufficient to give
1000 milliliter total volume.
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WO90/10227 PCT/US90/01028
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Anti-~hos~holipid Antibody: Circulating autoantibodies
directed against complex lipid antigens such as cardiolipin.
Double AntibodY Sandwich ELISA or FIA: An assay utilizing a
solid support that is coated with material which detects and
binds the-antibody of interest to the coated surface. To
render a signal, a second conjugated antibody with an affinity
for the previously bound antibody is exposed to the coated
surface. The binding of the conjugated antibody to the
original antibody makes the sandwich. If the sandwich assay
is an ELISA then the second antibody is conjugated with an
enzyme and a substrate is used to produce a color. If the
assay is an FIA then the second antibody is marked with a
fluorescent tag and a substrate is unnecessary.
Serum: Is intended to mean the fluidic component of any body
fluid remainillg after cells and coagulable 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 a~ter clotting of blood and
removal of the clot, but in its broad sense is intended to
include the ~luidic component of cerebrospinal fluid, urine,
interstitial fluid, cellular cytoplasm, and the like.
Sample Diluent- A liter solution containing 100 mls of native
bovine serum, 1.42 g of potassium phosphate (dibasic~, .26 g
of potassium phosphate (monobasic), 1 g of sodium azide, and
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WO90/10227 ~ 12- PCT/US90/01028
8.6 g of sodium chloride dissolved in 900 mls of water. l ml
of stock green dye is added to the solution, if a FIA format
is used then the dye is unnecessary. The solution is then
filtered through a .2 micron filter.
Sam~le Diluent Solution: lO microliterc of sera or plasma
dissolved in 500 microliters o~ sample diluent.
Con~uaate Diluent: A phosphate bu~fer, and protein
stabilizer, plus .02% thimerosal adjusted to a pH of 7.5,
(commercially available from Medix) into which is added a
protease inhibitor, aprotinin, (commercially available from
Miles P~ntex) at .01% of the volume of the buffer.
Workin~ _Con~u~ated Antibody Solution: l volume of
concentrated conjugated antibodies/3000 volume o~ conjugate
diluent. The dilution is subject to change based on the
concentration level o~ the conjugated antibody.
Coniuqated Antibodies: For an ELISA, antibodies were
chemically conjugated with horseradish peroxidase. For a FIA,
antibodies were chemically conjugated with Fluorescein
Isothiocyanate.
Immunoqlobulin. Any member of the gammaglobulin fraction of
serum possessing the ability to bind another agent.
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WO90/10~27 PCTtUS90/01028
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Antibod~: A class of serum proteins which specifically bind
to an antigen which induced the formation of the antibody.
Antiqen: Molecules ~rom whatever source nature or man-made)
which induce an immune reaction when recognized by the host's
immune system.
Immunoalobulin Classes: Antibodies separated by
electrophoretic mobility specifically IgG and IgM.
Substrate Solution: To quantitate the conjugated antibody, a
buffered solut.ion containing (3,3',5,5') Tetramethylbenzidine/
hydrogen peroxide, (commercially available from Kirkegaard
Perry) was used. The substrate solution will vary according
to the enzyme used or the test format used.
Labelled Antibodies: Any antibody substance which has been
covalently or otherwise combined with a molecule or ion for
the purpose of selectively identifying that group of
antibodies. Such adduct molecules or ions include enzymes,
fluorescent substances, radionuclides, and the like.
Labelled Antiaens: Any antigen substance which has been
covalently or otherwise combined with a molecule or ion for
the purpose of selectively identifying that group of antigens.
Such adduct molecules or ions include enzymes, fluorescent
substances, radionuclides, and the like.
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WO90~10~27 PCTIUS90/01028
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Optical Density ~OD) or Absorbance: A number which refers to
the colox absor~ance of a solution. Optical density is
related to the percent of light transmitted through the
solution by the following formulate: OD = 2-log(percent
transmittance).
The preferred embodiment of the method and apparatus for the
detection of anti-cardiolipins in sera is a diagnostic test
kit. The optimized kit contains:
1 vial (50 ml) Sample Diluent - green solution: contains 0.1
sodium azide.
1 vial (0.25 ml) Human Negative Control. Contains 0.1% sodium
azide.
4 vials (0.25 ml) Human Positive Controls - anti-cardiolipin
activity on the label. Contains 0.1% sodium azide.
(Controls included for GPL and MPL).
5 vials (0.25 ml) of Calibrator sera~ standardized against
Nigel Harris' re~erence preparations.
12 pre-coated B~well Microwell Strips with frame holder.
1 vial (8 ml) Conjugated Antibody Working Solution
containing horseradish p roxidAse-conjugated anti-human
IgM.
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WO90/10227 PCT/US90/01028
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1 vial (8 ml) Conjugated Antibody Working Solution
containing horseradish peroxidase-conjugated anti human
IgG.
1 bottle (8 ml) TMB Substrate Solution A - containing
3,3',5,5', tetramethylbenzidine in buffer.
1 bottle (8 ml) TMB Substrate Solution B - containing hydrogen
peroxide
1 bottle (12 ml) Stop Reagent: contains 2.5N H2SO4 ** 1 N
HCL may also be used as stop reagent.
1 packek - Phosphate Buffered Saline ~PBS) - reconstitutes to
2 liters of O.O1 M PBS, pH 7.4.
Plate templat~
Two sets of Calibrator sera have been included in order to
generate a standard curve, one set for IgM antibodies (MPL),
and one set for IgG antibodies (GPL). These have been
standardized against the Reference Sera proposed by Dr. Nigel
Harris. The amounts supplied and the levels of anti-
cardiolipin activity in the controls and calibrators can be
varied without affecting the performance of the assay.
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WO90/10227 PCT/US9~/0102X
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%~77~2
This kit is designed to be used in clinical laboratories to
measure l,e~,els o~ anti-cardiolipin in approximately 45
minutes. The results of the kit's measurement are adequately
reproducible within and between assays. Due to the ~tability
of the pre-coated wells and of the various reagents used in
the assay, the shelf life of the kit is greater than six
months. What follows is a description of a preferred
embodiment of the pre-coated wells of the present invention,
along with the preferred method for preparation of and
utilization of the various elements of the kit to detect anti-
cardiolipins in the sample of body fluid.
Step l: Preparatlon o~ the Pre-Coated Microtiter Wells: 20
ug/ml methylated bovine serum albumin is dissolved in water.
This solution is used to render the surface of the microtitre
wells slightly positively charged. To affix m8SA to the
surface of the wells, lO0 microliters of the prepared solution
is placed in wells that have been rinsed with deionized water
and thoroughly drained. Thus, 2 microgram~ of mBSA is placed
in each individual well. Examples of microwells that have
been used are Dynatech Immulon 2, Dynatech Immulon 4, or Nunc
Maxisorp. The wells are incubated at room temperature for two
hours, and when removed the excess solution is shaken from the
plate and the wells are inverted to drain thoroughly.
Next, the ligand or antigen is diluted and coated to the
receiving sur~ac of the wells. Cardiolipin is added to lO0~
denatured ethanol with the finai concentration of cardiolipin
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WO90/10227 PCT/US90/01028
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in the resultant solution being 20 micrograms~ml weight per
volume. lO0 microliters of this s~lution i5 placed in contact
with each well and the solution is allowed to completely
evaporate at room t~mperature. This evaporation process lasts
about 18 to 24 hours.
The casein blocking step decreases th2 non-specific binding
that can occur due to protein-plastic interaction~ Hydrolyzed
casein can be commercially obtained from Sigma, and the
blocking solution is prepared by mixing 2 ml glycerol, lO g
sucrose, and 15 milligrams of hydrolyzed casein, and adding
sufficient TEN buffer to make 100 ml of solution. The pH is
adjusted to 7.3 +/- .1. Next, 200 ul of caseln blocking
solution is dispensed into each well, and the wells are
incubated at 4 degrees C overnight. Following incubation the
wells are inverted, shaken to remove excess solution, and
allowed to drain for 15 minutes. The wells are turned upright
and allowed to dry at room temperature for at least 24 hours.
This completes the coating of the microtiter wells and each
kit is then supplied with 96 coated wells. The shelf life of
the pre-treated wells stored at 4 degrees C in a sealed
plastic bag is up to one year.
':
vell~ Sample Diluent is supplied in the kit as 50
~1 of a green solution. To prepare a 1000 ml solution of
sample diluent lO0 milliliters o~ native bovine serum, l.42`g
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WO90/10227 P~T/US90/01028
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of Potassium Phosphate (dibasic3, .26 g of Potassium Phosphate
(monobasic~, 1 gram of sodium azide, and 8 0 6 g of sodium
chloride, 1 ml of stock green dye are dissolved in
demineralized water -sufficient to make 1000 milliliters of
solution. This solution is then filtered through a .2 micron
filter and stored at 4 degrees C. Prior to contacting the
body fluid with khe prepared wells, the serum is diluted by
adding an aliquot of serum to the Sample Diluent in a 1:50
ratio (volume of serum:volume o~ sample diluent), although the
dilution is not critical and depends upon the nature of the
body fluid and the assay techniques employed.
100 microliters of the diluted sample is then placed into the
appropriate wells. Adherence of the anti-cardiolipin ~rom the
body fluid to the pre-coated wells is enhanced by room
temperature incubation for fifteen minutes.
Following incubation to achieve antibody adherence, the excess
solution is shaken from the wells removing nonbound antibodies
that are present in the sample. This is done since there are
many more free antibodies than there are bound to the
cardiolipin on the wells and residual free antibodies would
elevate background absorbance values. The wells a~e then
washed four times with phosphate-buffered saline, and drained.
Step 3 Assay for Anti-Cardioli~in_Antibodies A~fixed to the
Plate: Standard enzyme-linked assay techniques, previously
described, are used for this assay, althouyh any suitable
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WO90/10227 PCT/~S90/0~028
2~4~742
means of detection such as radioactive labeling, Eluorescence,
or the like can be employed. For the examplas described
hereinafter, anti-human IgG and IgM induced in goats was used
to ascertain whether anti-cardiolipin IgG and IgM antibodies
were present. These antisera were linked to horseradish
peroxidase, an enzyme which yields 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
described hereinafter, the substrate was (3,3',5,5')
Tetramethylbenzidine and hydrogen peroxide.
The kit contains two 8 ml vials of previously diluted
conjugated antibody, one contains anti-human IgM antibody
conjugated to horseradish peroxidase, and the other contains
anti-human IgG similarly conju~ated. ~o prepare the conjugate
diluent used to dilute the conjugated antibody a phosphate
buffer with protein stabilizer and .02% thimerosal solution at
pH 7.4 (commercially available from Medix) was mixed with
protease inhibitor (commercially available from Miles Pentex)
at a .01% ratio of inhibitor to volume of buffer. This
diluent solution enhances the stability of the conjugated
antibody. ~he working conjugated antibody solution is
prepared at a ratio of 1:3000; one part of concentrated
conjugated IgM or IgG antibodies is aliquoted into 3000 parts
conjuyate diluent. This dilution ratio will vary depending on
concentration ffl concentrated conjugated antibody~
WO90/10227 PCT/US90/01028
2~ ~7 ~`2- -20-
The enzyme conjugated goat anti-human antibody solution,
prepared as described, is then added to each well in lOO
microliter increments. Binding of these antibodies to the
anti-cardiolipin is permitted for at least 15 minutes at room
temperature. The wells are emptied of their contents, and
washed ~our time~ with PBS and allowed to drain.
The presence of a labeled antibody, as previously described,
is determined by incubating the plates with a solution of
(3,3',5,5') Tetramethylbenzidine and buffered hydrogen
peroxide. This solution is supplied in the kit in two 8 ml
vials; one contains (3,3',5,5') Tetramethylbenzidine; the
other bottle contains hydrogen peroxide. The separate vials
are necessary due to the interaction between the two
solutions. The two solutions are mixed in a one ko one ratio
just prior to use, and 100 microliters of the mixed solution
is dispensed into each microwell. The reaction is permitted
to continue for l0 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 100 microliters of 2.5 normal sulfuric acid to
each well and the intensity o~ color (the optical density, or
OD or abscrbance) is read by a spectrophotometric device such
as as a Dynatech MR600 or the like.
A~ with any enzyme linked immune assay, the resultant color of
the reaction product is proportional tv the number of
conjugated antibodies which have bound to the anti-
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WO90/~0227 PCT/~S90/01028
-21- 2 ~ ~77 ~2
cardiolipin. For most cases, the number of bound conjugated
antibodies is linearly related to the number of anti-
cardiolipins. Hence, as the amount of conjugated antibodies
~ixed on the film increases, so does the optical density, or
absorbance o~ the enzyme reaction.
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WO90/10227 PCT/US90/01028
-22-
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CARDIOLIPIN
EXAMPT.~ 1
In a preferred embodiment polystyrene microtiter wells were
coated with a ~ilm of methylated bovine serum albumin (mBSA)
by the following procedure:
1. 20 ug/ml of methylated bovine serum al~umin in powdered
form (available commercially from Sigma) was dissolved in
water.
2. The methylated bovine serum albumin solution was placed
in the microwells in aliquots of 100 microliters per
well, and incubated at room temperature for two hours.
3. After the incubation, the solution was emptied ~rom the
wells and they were inverted to drain.
4. 20 microgram/ml of cardiolipin from beef heart
(commercially available from Sig~a) was dissolved in 100
denatured ethanol.
5. 100 microliter of the cardiolipin solution was placed in
each microwell, and incubated at room temperature ~or 18-
;24 hours until the ethanol was complet~ly evaporated.
:
'- . , : '', . ~.
-
WO90/~0227 PC~/US90/01028
-23-
2~77~2
. 100 ml of casein blocker solution is comprised of 2 ml
glycerol, 10 g of sucrose and 15 millic~rams of hydrolyæed
casein diluted to 100 ml by the addition of TEN (Tris,
EDTA, NaCL) buffer. The casein blocker solution was
buffered to a pH of 7.3 +~- .1. 200 microliters of
blocker solution was dispensed into each microwell.
7. The wells containing casein blocker solution were
incubated overnight at 4 degrees C, then the blocker
solution was shaken from the wells and they were inverted
and allowed to drain for 15 minutes.
8. The microwells were then uprighted and allowed to dry at
room temperature for at least 24 hours.
9. The microwell plates were stored at 4 degrees C in sealed
plastic bags, or used in the next step.
The cardiolipin coated wells were then used to determine the
presence of anti-phospholipid antibody in serum or plasma
samples drawn from individuals with:
1. no apparent pathology; (normal)
2. anti~Cardiolipin positive plasma;
3. positive RPR (rapid plasma xeagin) syphilis plasma.
WO90/10227 ~ PCT/VS90/01028
The sera and plasma had been drawn from patients known to have
systemic lupus erythematosus. An anti-~cardiolipin assay,
using the cardiolipin roated wells, as previously described,
with a sample from one o~ the three sample groups, was
performed by the procedure summariæed below:
l. A lO00 ml s~lution of Sample Diluent was comprised of lO0
mls of native bovine sexum; l.42 g of potassium phosphate
dibasic, .26 g of potassium phosphate monobasic, l gram
of sodium azide, and 8.5 g of sodium chloride, 1 ml of
stock green dye, and 900 mls of distilled water. The
diluent solution is then filtered through a .2 micro
Nalzene filter and stored at 4 degrees C.
2. Sera were prepared at a l to 50 ratio of sample to Sample
Diluent.
3. lO0 ul of the diluted samp}e or calibrator was
transferred to the microwells in duplicate.
4. The wells were incubated at room temperature for 15
minutes. Following incubation the wells were washed four
times with PBS and inverted to drain.
5. One set of duplicate microwells were exposed to a mixture
containing horseradish peroxidase (HRP~ conjugated goat
anti-hu~an IgM antibodies ~hereinafter referred to as M
conjugated~ and conjugate diluent; the r~maining wells
, . ~ , . . .
. . .
' . . . :.
WO90/10227 PCl`/US9OtO1028
-25- 2~77~
were exposed to a mixture of HRP conjugated goat anti-
human IgG antibodies (hereinafter referred to as G
conjugate) and conjugate diluent. lOO ul of either
conjugate solution was dispensed to eac:h microwell.
6. The wells were then in~ubated at room temperature for 15
minutes, to allow attachment of the conjugate. After
incubation, the wells were washed four times with PBS to
remove the unbound enzyme conjugated antibodies. The
wells were inverted between each wash to empty the xcess
fluid. After the final wash the wells were inverted to
drain excess fluid.
7. Each well was assayed for horseradish peroxidase activity
by adding lOO microliters of (3,3/~5,5~)
Tetramethylbenzidine/ buf~ered hydrogen peroxide solution
to each microwell. The wells were allowed to incubate at
room temperature for lO minutes, after which lOO ul of
2.5 N H2So~ (l.O N HCL can be substituted) was added to
terminate the color reaction. The presence of anti-
cardiolipin was detected by the presence of color. The
color was quantitated at 450 nm using a Dynatech MR600
plate reading spectophotometer which was calibrated
against a water blank. Reagent controls were wells which
were not contacted with sample.
D~T~C~IO~ 0~ C~RDIO~IPI~ IgG
SOUEC~ GPL OD
Cali~rator l 6 .08
Calibrator 2 l8 .17
WO90/10227 PCT/US90/01028
~ 26-
Calibrator 3 35 .29
Calibrator 4 70 .52
Calibrator 5 105 .68
Refer to Figure 1
Source Mean Value GPL OD
Normal Serum < 2 .05
(positive) anti CL 46
moderate positive plasma .36
antibody plasma
RPR positive plasma 45 .42
DETECTION OF CARDIOLIPIN IgM
Source MPL OD
Calibrator l 5 .08
Calibrator 2 l0 .ll
Calibrator 3 20 .2
Calibrator 4 50 .45
Calibrator 5 l00 .73
Refer to Figure 2
Source Mean Value MPL OD
Normal Serum < 3 .03
(positive) anti CL 80 high positive plasma .85
antibody plasma
RPR positive patient 59 .55
In each test sample, IgG and IgM aCL levels are reported in
GPL and MPL units respectively. One GPL unit is defined as
the cardiolipin binding activity of l ug/ml of an affinity
purified Ig& aCL preparations from a standard serum. one MPL
unit is defined as the cardiolipin binding activity of 1 ug/ml
of an affinity purified IgM aCL preparation from a standard
serum. The unit~ have been established by Dr. Nigel Harris.
SUBS~Tl~ 1E $HEET
.
~ : : . . .
WO90/10227 PCT/US90/01028
-27-
2~47742
~XAMPLE 1'~0
To demonstrate the importance of the cardiolipin coating, a
polystyrene well was uncoated and the test was run under the
procedure described in Example 1. After the wells were
assayed with horseradish peroxidase conjugated antibodies,
there was only a slight selectivity observed, and a very high
background was observed, in contrast to the background and
selectivity of the coated wells.
Uncoated
Calibrator~MPL Coated Well~ O.D. Wells O D.
lO0 MPL .85 .72
50 MPL .50 ~4
20 MPL .20 .19
10 MPL .12 .13
5 MPL .07 .lO
Uncoated
Calibrator~GPL Co~ted ~ells O.D. Well~ O.D.
105 GPL .93 .85
70 GPL .76 .76
35 GPL 44 53
18 GPL .28 .36
6 GPL .13 .29
Uncoated
~ourGe~PL Co~tea Well~ O.D. Wells O.D.
~ 7
Normal Serum.04 ,09
Positive #1 .65 .50
Positiva #21.1} .~
WO90/~0227 PCT/US90/01028
-28-
t~ 4~
Vncoated
8Ource GPL ~oated_Well~ O.D. Wells O.~.
Normal Serum .0~ .25
Positive #1 .63 .39
Positive #2 .53 .44
EXANPLE THREE
Test Performed with the Diagnostic Test Kit
The kit contained 96 pre-coated microwells with affinity for
anti-cardiolipin and:
:.
1 vial of (30ml) Sample Diluent (green solution) - containing
0.1~ sodium azide, to be used for diluting sera samples,
calibrators and controls.
1 vial of (0.25 ml) Human Negative Control.
2 vials of (0.25 ml) Human GPL Positive Control (two moderate
positive controls.
2 vials of (Q.25) ml Human MPL Positive Control (moderate
positive control and high positive control).
5 vials of (0.25 ml) calibrators for GPL.
5 vials of (0.25 ml) calibrators ~or MPL.
- , ,
. :
': ' ' . : ' ,
WO90/10227 PCT/US90/01028
-29- 20~77~2
l vial of (8 ml) Conjugated Antibody Working Soluti4n
containing horseradish peroxidase con-jugated anti-human
IgM; and a second similar vial of (8 ml) Conjugated
Antibody Working solution containing horseradish
peroxidase conjugated anti-human IgG.
One bottle of (8 ml) of TMB Substrate solution A containing
3,3',5,5', temtramethylbenzidine, and one (8 ml) bottle
of Peroxidase Substrate Solution B containing hydrogen
peroxide. Solution A and B are combined to form a
substrate capable of generating a colored product.
l bottle of (12 ml) Stop Reagent containing 2.5N H2SO4; ~lN
HCl may also be substituted);
A packet of Phosphate Buffered Saline ~PBS). This
reconstitutes to 2 liters of 0.0l M PBS, pH 7.4.
l packet of Phosphate Buffered Saline (PBS) which
reconstitutes to 2 lit~rs of 0.0l M PBS, pH 7.3, which is
utilized as a wash.
l Plate template.
The plate template was labelled for sample placement in the
microwells. A 1:50 dilution of the controls, calibrators and
patient samples were prepared in sample diluent (green
.. , . . .. - ,
,
WO90/10227 PCT/U~9~/01028
30-
2~7~ ~
solution). 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, control or calibrator was added
to appropriate microwells. The plate was allowed to incubate
for 15 minutes at room temperature. To perform the rinse step
the contents of the PBS packet was added to 2 liters of
reagent grade water, and the solution was mixed well, until
all the crystals were dissolved. Then the buffer was used to
wash the wells four times. The microwells were inverted
between each wash to empty the fluid, and the wells were
blotted on absorbent paper to remove residual wash fluid. Per
the klt instructions, the wells were not allowed to dry-out
between washes.
100 ul of conjugated antibody (red solution) was added to each
well, incubated 15 minutes at room temperature, and then the
wells were again washed four times with PBS solution. Next,
the wells were blotted on absorbent paper to remove residual
wash fluid.
The working substrate was prepared ~ust before using according
to the kit instruction. Equal volumes of TMB Su~strate
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. 100 ul of working substrate solution
,
.
WO90/1~227 PCT/US90/01028
2~77~2
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
enzyme reaction and the O.D. of aach well was read at ~50 nm
against a water blank. The results were then calculated by
plotting GPL or MPL calibrator values against O.D.s on linear
graph paper. Sample results are read from the resulting
"standard curve".
Normal Ranqes and Deqree of Positivity:
The following estimates have been established by Dr. Nigel
Harris:
Normal = < 5 GPL and ~ 3 MPL
Low Positive = ~ 15 GPL and < 6 MPL
Mod Positive = 15-100 GPL and 6-60 MPL
High Positive = > 100 GPL and > 60 MPL
DETECTION OF CARDIOIIPIN IgG and IgM
Refer to Figures 3 and 4
Control results fell within predetermined ranges.
Mean Mean
Controls Value ~PL O~D. ValuQ MPL OoD~
Normal 2 .7 2 .02
~oderate positive ~6 .76 16 .32
Moderate positive 78 .99 81 .~6
High positive 78 .99 81 o96
SlJBS~lT~T~ SHE~
WO 90/10227 PCr/US90/01028
32-
~ean ~ean
~;our~e Value GPL O . D, alus MPL O . D.
Normal human serum 3 . 08 2 . 02
aCL ~high positive) 92 1.15 70 . 87
aCL (moderate positive) 44 . 73 17 . 34
:
, ~
.
W09OtlO227 PCT/U~90/01028
~33~ 2 0~77 ~2
EXAMP~B 4
Samples wexe drawn from 52 patients that tested VDRL positive.
The test was run in accordance with Example 3. Approximately
17% of the samples showed positive for anti-Cardiolipin
antbodies. The low positive IgG was determined to be 23 GPL,
and the low positive IgM was determined to be 11 MPL. After
converting the O.D. data, the following values were obtained:
Patient GPL MPL
1 5.5 4.7
2 7.~ 1.3
3 23.1 6.7
4 2.9 6.7
16.0 8.0
6 9.3 3.3
7 65.2 .6.0
8 6.5 3.3
9 39.9 12.7
9.3 7.3
11 7.8 10.7
12 4.6 4.7
13 18.3 12.0
14 12.9 2.0
28.5 6.0
16 13.2 16.0
17 14.8 4.0
18 12.6 6.7
19 9.5 4.0
6.8 7.3
21 10.3 2.7
22 3.3 2.7
23 4.1 4.0
24 7.7 6.0
5.5 5.3
26 19.9 8.0
27 7.1 1.7
28 8.7 5.8
29 7.7 5.0
4.2 2.9
31 15.5 10.8
32 ~.1 6.7
33 13.4 9.6
34 7.4 4.2
3.7 ~.3
36 31.6 6.7
... , ~ .
.
, . ; . : .
: ,. ;,:
W~ 90/10227, P~/US90/~102
--3~
37 13.0 1.7
38 9.2 4.6
39 5.7 2.5
4.8 2.1
41 7.1 5.0
42 2.5 2.1
43 18.7 7.9
44 48 . 2 30 . O
8.~ 1.7
46 ~7.5 ~;.7
47 15.3 3.3
48 5.2 1.7
49 6.4 1.7
11.8 6.7
51 2.7 8.3
52 8.5 1.3
.
''~' , ' ' ' ~:
, ~.
.
WO90/10~27 PCT/~S90/01028
-~5-
2~7~2
EXAMPLE 5
The following test was run according to the protocol outlined
in Example 3. The samples were taken ~rom patients known to
have rheumatoid arthritis, thus some anti-cardiolipin
antibodies are expected to be present. A low positive was
determined to be 23 GPL and 11 MPL base~ on the calibrators
used. There were approximately 42% positive for IgG anti-
cardiolipin antibodies and 6% positive for IgM anti-
cardiolipin antibodies. The following data was generated
using the present invention.
Patient Iq~ O.D. GPL IaM O.D. MPL
1 .10 6.6 .0~ 3.1
2 .28 18.3 .12 4.7
3 .44 ~28.~ .16 6.2
4 .46 *30.1 .11 4.3
.52 *34.1 .18 7.0
6 .46 *~0.1 .19 7.4
7 .51 *33.4 .16 6.2
8 .12 7.9 .10 3.9
9 .12 7.9 .08 3.1
.68 *44.5 .18 7.0
11 .20 13.1 .09 3.5
12 .13 8.5 .10 3.9
13 .20 13.1 .013 S.1
14 .31 20.3 .48 18.7
.12 7.9 .16 ~.2
16 .15 ~.~ .11 4.3
.
: ' . . , . ' ~ : ' - ~ . , . "
'': ' ' .': : . ' : ' - '' ' ~ ,
.: ~ : : : . ::
WO90/10227 ~ 36- PCT/US90/010~8
17 .15 9.8 .04 1.6
18 .18 11.8 .20 7.8
19 .60 *39.3 1.21 *47.2
.36 *23.6 .17 6.6
21 .22 14.4 .11 4.3
22 .38 *24.9 .12 4.7
23 .4~ *32.1 .1~ 6.2
24 .21 13.8 .07 2.7
.30 19.7 .06 2.3
26 .023 15.1 .1~ 6.2
27 .21 13.8 .21 8.2
28 .19 12.4 .10 3.9
29 .15 9.8 .16 6.2
.31 20.3 .86 33.5
31 .20 13.1 .08 3.1
32 .15 9.8 .07 2.7
33 .30 19.7 .09 3.5
34 .56 *36.7 .12 4.7
.20 14.4 .12 4.7
36 .2~3 18.3 .11 4~3
37 .32 20.1 .14 5.5
38 .56 *36.7 .07 2.7
39 .28 18.3 .11 4.3
.27 17.7 .11 4.3
41 ~41 *30~0 ol2 5~1
4~ .34 *24.9 .5 ~21.1
43 .30 21.9 .~2 5.1
44 .41 *30.0 .12 5.1
. ` ' ' , ' '' ' ' ~- ~ .. '
, - . ~ .':
. ; . .
WO90/10~27 P~TIU~90101028
_37_ 2~77~2
.32 *23.4 .09 3.8
46 .32 *23.4 .10 4.2
47 .33 *24.1 .20 8.4
48 .54 *39.5 .14 5.9
49 .21 15.4 .12 5.1
.32 *23.4 .17 7.2
51 .46 *33.6 .08 3.4
52 .23 16.8 .07 2.9
53 .05 3.7 .02 .8
54 .64 *46.8 .20 8.4
.44 *32.2 .07 2.g
56 .23 16.8 .11 4.6
57 .14 10.2 .12 5.1
58 .22 16.1 .09 3.8
59 .34 *24.9 .07 2.9
.61 *44.6 .12 5.1
61 .32 *23.4 .12 ~1
62 .51 *37.3 .09 3.8
63 .12 8.8 .10 4.2
64 .30 2~.9 .10 4.2
.34 *24.g .18 7.6
66 .37 *27.0 .10 4.2
67 .42 *30.7 .12 5.1
~8 .~5 *40.2 .19 8.0
69 .15 11.0 .10 4.2
.36 *26.3 .39 1~.4
71 .~8 13.2 .16 6.7
72 .20 14.6 .16 6.7
' ~ ' ' ' ''' . .:
WO90/tO227 PCT/US90/01028
~ -38-
73 ` .06 4.4 .17 7.2
74 .30 21.9 .13 5.5
.48 *35.1 .10 4.2
76 .35 *25.6 .08 3.4
77 .22 16,1 .07 2.9
78 .15 11.0 .14 5.9
79 .21 15.4 .13 5.5
.16 11.7 .18 7.6
*Positive Results.
EXAMPLE 6
The following test was run according to the pro~ocol outlined
in Example 3. The samples were suspected of hav:ing anti-
cardiolipin antibodies as the patients were all SLE patients.
The low positive was determined to be 23 GPL and 11 MPL. Seven
patients were GPL positive and seven were MPL positive. The
followin~ data was gathered by the test kit procedure:
PATIENT IqG O D. GPL IaM O.D. MPL
1 O43 *26.4 .20 10.2
2 .11 7.9 .14 *12.4
3 .25 17.9 .04 3.6
4 .10 7.2 .0~ 8.0
~12 8~6 ol3 *11~6
6 ~07 5~0 ~13 *11~6
7 ol9 1306 ~13 *11~6
8 ~13 ~3 ~15 4~4
9 ~17 12~2 ~35 *31~1
' , , , . - ~ ~ :
. . : : . .
., ~ ~ ' . ' '
- ~ :
. .
W09~10227 2 ~ l1 7 7 ~ 2 PCT/usgo/olo28
-39-
lo .22 15.8 .13 ~11.6
11 .15 10.8 O07 6.
12 .~8 *29.4 .01 .5
13 .09 ~.5 .02 1.8
14 .51 *31.3 .08 - .41
- .18 12.9 .05 4.4
16 .56 *40.2 .02 1.8
17 .37 *26.5 .03 2.7
18 .50 *35.9 .lO ~.9
19 .40 *2~.7 .04 3.6
.29 20.8 .34 . *30.2
*Positive Results.
EXAMPLE 7
The following test according to the protocol in Example 3 was
performed on 20 patients having progressive systemic
sclerosis. Some anti-cardiolipin antibodies were expected to
be present in the samples. Using 23 GPL and 11 MPL as the low
positive cut-off number, 4 samples were found positve for IgG
and 1 sample was positive for IgM. The following data was
generated by the kest kit:
.. :: . ~,: .................... ; ... . - . . . . -
,. .. . , : ., , ~ - . : .
. .. .. .
2~ P~T/US90/01028
PATIENTS ~qG 0.D. GPL IgM O.D. MPL
1 .10 6.7 .07 2.9
2 .09 6.0 .10 4.2
3 .15 10.0 .11 4.6
4 .17 ~1.3 .02 .08
.19 12.7 .03 1.2
6 .41 *27.3 .22 9.2
7 .32 22.7 .10 2.1
8 .34 21.3 .05 2.1
9 .07 4.7 .04 1.7
.10 6.7 .04 1.7
11 o58 *38.7 .35 *14.6
12 .10 6.7 .04 1.7
13 .80 *53.4 .08 3.3
14 .05 3.3 .02 .08
.32 21.3 .14 5.8
16 .17 11.3 .17 7.1
17 .15 10.0 .02 .~
18 .20 14.0 .08 3.3
19 .20 13.3 .09 3.7
.46 *30.7 .11 4.6
* Positive results
Example 8
.
~he prevalence of IgG and IgM a-Cl antibodies in a large
series of serum samples obtained from 149 systemic lupus
erthematosus ~SLE), 93 progressive sclerosis (PSS), 80
.
!
: ' ' .
~,.~ ' , . ~ '.
' . . ~ .
,: ' ' ' . ' ' :~ ,
, . . ..
... . . . .
W090/~0227 PCT/US90/01028
77 ~ 2
rheumatoid arthritis (RA), 24 osteoarthritis (OA) patients,
was tested by the test kit in accordance with the protocol in
Example 3. The results were compared to the results from the
test kit generated from 202 serum samples obtained from
healthy individuals. These patients were followed in
rheumatology clinics and the diagnosis of SLE, PSS, and RA was
based on ARA criteria. One hundred and two of the healthy
individuals were screened and selected by the absence of any
clinical manifestation related to the a-CL syndrome,
medication, pregnancy or recent infection. The test kit used
include controls and calibrators that are standardized against
reference samples from EN Harris. The results were expressed
as GPL vr MPL units. Each sample was assayed in dup:Licate and
the normal ranges were established by the mean value of the
unselected healthy individuals + 2 s.d. The prevalence rate
is reported as the percent of positive samples above the
normal range.
~ean GPLPrevalenceMean MPL Prevalence
Serum~/- 1 s.d. GPL (%L +t- 1 s.d. MPL ~
Healthy12 +/- 5.3 2 5 +/- 2.9 4
Healthy7 ~/- 3.0 5 6 ~/- 3.1 4
(selected)
SLE*16 -~/-10.8 21 6 ~/- 5.3 10
PSS15 +/-11.0 13 ~ +/- 3.0 3
R~**Zl +/-10.4 42 6 +/- 6.~ S
O~11 +/- 9.7 4 3 ~/- 1.7 0
*No signi~icant correlation between GPL (r = 0.19) or MPL (r =
0.04) with anti-dsDNA was ~ound.
**No signi~icant correlation between GPL (r = 0.01) or MPL (r
= 0.09~ with IgM-RF was found.
' ~ . : , ' '
.
.
WO90/10227 PCT/US90/01028
~a4~ 4~ -42-
In this study, the results indicate statistically significant
higher serum levels of IgG a-CL antibody activity in patients
with SLE (p < 0.00l), PSS (p < 0.025), and RA (p < 0.00l) when
compared to healthy individuals and OA patients. The highest
prevalence was found in patients with ~ and SLE and this
activity cannot be attributed to cross-reactivity ~ith IgM RF
or dsDNA antibodies. There was no statistical difference in
IgG or IgM a-CL serum levels in SLE patients with active
versus inactive disease. The selection process of the healthy
group appears to significantly lower the IgG a-CL mean value
(p < 0O00l). IgG a-CL serum levels seem to be the most
clinically significant in autoimmune disorders.
The foregoing examples serve to illustrate the efficiency and
utility of the methylated BSA to provide a coating which
inhibits non-specific binding and provides a coating capable
of affixing cardiolipin antigen evenly over the solid support.
Without being bound to the specific quantities given in the
definitional section, it is possible to utilize a wide
latitude of concentrations of mBSA or other similar,
functionally equivalent su~stitutes which have the capability
to evenly attach cardiolipin antigen to the support solid by
providing a charged surface or by any other like mechanism to
affix the cardiolipin and to continue to inhibit non-specific
binding.
,
WO90/10227 PCT/~S90/01028
~43~ 2~7742
Likewise, the blocker utilized in the preferred embodiment of
this invention to stabilize the shelf life and eliminate non-
specific binding cannot be limited to the compounds or the
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, lOl, 43-50 [1987]) and is hereby
incorporated herein by reference.
The treatment of the solid support which can be any of a
variety of formats, i.e. test tubes, plates, wellsj 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 anti-cardiolipin
antibodies. The detection of anti-cardiolipin 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 also exist, and these methods will
yield positive results provided that the antibody exists in
the assayed sera and is affixed according to the methods
described herein.
'' ~' ' . ~ ' ' '
: . . ' . , ~.
. : , . . ,., , - ~
'~ .: ~ ' ' .', ' ,': '
WO90/lOZ27 PCT/US9OfO10~8
~ -44-
The test kit and the underlying coating and detection methods
herebefore describ~d 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
similar chemical equivalents or other functional equivalents
of the components found in the coatings, or in any of the
various reagents, controls, and calibrators can be utilized
within the scope of this invention.
It is contemplated that the inventive concepts herein
described may have differing embodiments and it is intended
that ths appended claims be construed to include all such
alternative embodiments of the invention except insofar as
they are limited by the prior art.
. .
, .. ~ - . .~ . ,,, ., :
