Note: Descriptions are shown in the official language in which they were submitted.
1()~4931
BACKGR_UND OF THE INVENTION
There are a multitude of techniques employed for the quantitative
determination of an unknown quantity of a sample derived from a biological
fluid (e.g., serum or urine). Techniques based upon radioactive labelled sub-
stances include indirect ~competitive binding, saturation or displacement) and
direct (sandwich and immunoradiometric) radioimmunoassays. Other known techniques
are based on fluorochrome and enzyme la~els. After completion of the above
reactions, the labelled substances which have been bound must be separated from
the unreacted labelled substance which includes free and non-specifically bound
substance. This separation step in liquid form can be inefficient, unreliable
and inconvenient.
A number of techniques have been proposed to solve this problem by use
of diagnostic reagents on solid surfaces which combine with the labelled sub-
stance. In each of these systems, the solid substrate containing the bound
labelled substance is separated from the liquid phase containing the free
labelled substance and is thereafter washed to remove the residual free and the
non-specifically bound labelled suastance.
In one technique for the detection of antigen, reagents are coated
upon plastic test tubes or inserts by the physical adsorption of antibodies
specific to the sample substances to be tested. Such techniques are disclosed
in United States Patents 3,646,346 and 3,826,619. Such physical adsorption is
difficult to control because of the non-uniformity of plastic surfaces as well
as imprecisIon in the coating techniques. See, e.g,, the work entitled
"Radioimmunoassay ~ethods", European Workshop, September 15-17, 1970, Edinburgh
(Editors Kirkham et al). At page 441, the authors state that the amount of
binding is between 20% and 70% using various coated polys~tyrene tubes in a
particular system, which led to the abandonment of this technique. Furthermore,
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10~4t~31
in suhsequent immunochemical reactions, a significant amount of "non-specific"
binding of the labelled substance to the solid surface occurs. This binding,
of a hydrophobic or ionic nature, is weaker than the immunochemical bond. Hence,
vigorous and repeated washings required to effectively remove solution residual
and non specifically bound labelled substance frequently disrupts the weaker
physical coated bond resulting in the loss of physically bound diagnostic re-
agent. The results of assays employing such coated surfaces are thus relatively
imprecise and non-reproducible because of these kinds of misclassification
errors. ~Envall et al, Biochim. Biophys. Acta, 251 (1971) 427-434, at p. ¢30).
Another disadvantage of physically coating as with antibodies is that the anti-
bodies in that form are relatively unreactive with large antigens. It is believed
that this is due to steric hindrance effects.
In the technique of Bennich et al, United States Patent 3,720,760,
immunological substances are more firmly secured by attachment to porous micro-
reticular swellable polymer of the type sold under the trademark "Sephadex".
Such material is lndicated as being preferably in particulate form. Such micro-
reticular materials have the inherent disadvantage of being difficult to wash
their interstit;~al void spaces free of non-specifically bound and entrained free
labelled substance. This results in undesiraBly high background readings. Also,
it is difficult to rinse the interparticulate void spaces sufficiently free of
residual free labelled suBstances, generally requiring several displacement
~ashes. Each washing step requires a centrifugal and careful decantation separ-
ati`on to avoid misclassificat~on to minimize inadvertant spillage of particles
during the washing procedure.
This technique is unsuitable in fluorometric and colorimetric surface
systems because the particles cannot be accurately viewed. This is because
loose microret~cular beads used ~n radioactive assays produce an ill-defined and
-- 3 --
10~ ~9.~1
clif-ficult to control surface for uniform illumination and viewing.
Anotller technique which has been proposed is described in an article en-
titled "Solid Phase Disc Radioimmunoassay of 11uman Growth Hormone" by Catt et al,
J. Lab. ~7 Clin. Med., (1966) lOO, 31c. Small discs of substituted graft copoly-
mer sold under the trademark "Protapol DIJl", are coated with antibody. As set
forth in the above Kirkham et al work, at pages 294 and 295, this technique
yields poor replication, i.e., there is a very wide scatter between the replicates
at any given point, and sometimes the differences are very large. The discs re-
quire repeated handling and each time they are touched by a solid object the
chance for loss of bound labelled substance or contamination by free labelled
substance or other background contributing contaminants is considerable. Like
the above particle technique, the disc would not be accurately viewed in fluoro-
metric or colorimetric systems and would be difficult to manipulate into proper
viewing relationship with an optical instrument.
In view of the foregoing, there is an apparent need for a solid phase
analytical technique in ~hich the surface is easy to wash, separations are easy
to make, which is efficient and reproducible, and which can be presented easily
for accurate viewing.
SUMMARY OF THE INVENTION AN~`OBJECT~
In accordance with the present invention, there is provided a body suit-
able for use in the fluorometric detection of a aiologically derived sample, said
body comprising handle means connected to at least one nonparticulate, nonswell-
able, impermeable, continuous, flat surface region defining an area of at least
one square millimeter and a di~agnostic reagent bound to at least one biologically
derived sample labelled with a fluorochrome, s-aid diagnostic reagent being co-
yalently attached to said surface region. The surface with the attached reagent
(e.g., antibody) is reac~ed with the sample substance (e.g., antigen). In one
competitive technique, the labelled substance (e.g., fluoro-
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:
9:~1
chrome-labelled antigen) is simult~meously reacted with the sample substance.
In a sandwich technique, the labelled substance (e.g., fluorochrome-labelled
antibody) is subsequently reacted. In another type of competitive technique,
the attached reagent and sample substance are the same, and they both are simul-
taneously reacted with labelled substance, (e.g., fluorochrome labelled antibody).
In any of these techniques, the reaction of the surface and sample is acceler-
ated by stirring with the handle. Then, the surface is conveniently separated
from the sample and transported by the handle of the body to a detection station
for measuring the amount of labelled substance without need to touch the surface.
In one embodiment, the body includes at least two attached diagnostic
reagent regions in which one contains a standard region of a predetermined
quantity of the labelled substance. In another em~odiment, the body is polyhedral
and has a different diagnostic reagent on each of a plurality of its faces.
Also, a plurality of diagnostic reagents may be attached in a single region in
random dispersion and reacted with at least two different sample substances and
labelled s-ubstances which emit different signals.
It is an object of the invention to provide a body and method for use
~in the labelled quantitative detection of a biologically derived sample which
overcomes the aforementioned was~ing, separation, and handling pro~lems of solid
phase techniques of the prior art.
It is another object of the invention to provide a body of the fore-
going type with a handle ~hich facilitates non-touchable ~andling of the solid
surface and eliminates the need to centrifuge and decant.
It is a particular object of the invention to provide a body and
-method of the foregoing type which can be employed for reproducible precise
sample detection wi~th low bac~ground noise and minimal misclassification error.
It is a particular oBject of the invention to provide a device and
-- 5 --
~109~9~1
method of the foregoing type which is par~icularly adapted to precise and re-
producible viewing in an optical system such as a fluorometer or colorimeter.
Further objects and features of the invention will be apparent from
the following description of its preferred embodiments taken in conjunction with
the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic view of one embodiment of the body of the
invention viewed by a fluorometric system.
Figures 2 and 3 are different embodiments of the body of the system.
DETAILED DESCRIPTION OF THE PREFERRED EMB~DIMENT~
, .,
The present invention relates to a body suitable for use in the
labelled quantitative determination of a biologically derived sample substance.
As is defined herein, the term "sample substance" is one which is contained in a
biological fluid, Sample substances include antigens, antihodies, hormones~
enzymes, drugs, infectious agents and other su~stances. The sample substance is
reacted with a diagnostic reagent covalently attached to a continuous solid sur-
face. As defined herein, "diagnostic reagent" is one ~hich is attached to a
surface and specifically reacts directly or indirectly with the sample substance,
The diagnostic reagent is further reacted with a labelled substance for quantit-
atlve measurement. T~e term ~'la~elled substance" is- defined as a substance
whi~ch includes a-material detectiBle by a detection device and which is specif-
ically reactive w;t~ the sample substance or diagnostic reagent. The sample
substance itself sometlmes may be autofluorogenic ~e,g., tetracycline or enzym-
atic~ e.g., trypsin) or can be made so i`-situ. Labels include fluorochromes,
radionucli~des and enzymes.
The la~elled substances may ~e reacted with the sample substance before
Qr after isolation, or may b-e linked to the solid surface ~ith homologous labelled
materials ~e.g., antigen or antibody, one of which has ~-een labelled).
T. 6 ~
10949~1
For simplicity of description, the present specification will first
describe a typical fluorometrically labelled system of the sandwich type. ~he
procedure may be summarized as including the following steps:
(1) diagnostic reagent (e.g., antibody) is covalently attached to a
surface region of a solid body;
~ 2) the attached diagnostic reagent is reacted with a sample sub-
stance (e.g., antigen) and bound to the surface region;
(3) the surface region is washed to remove unbound sample and solvent;
(4) the surface region is reacted with labelled substance (e,g.,
fluorochrome-labelled antibody);
(5) the surface region is again washed; and
(6) the body is- transported to a detection station to measure the
amount of labelled substance,
Referring to step one, the diagnostic reagent, antibody, is covalently
attached to a nonparticulate, nonswellable, impermeable, continuous surface
; region of the body. Such covalent attachment may ~elperformed directly between
the diagnostic reagent and a surface reactive with the same. Examples of such
surfaces include polymers such as polymethylmethacrylate, polystyreneJ polyamide
(nylon) and polysaccharides. Diagnostic reagents having carboxylic acid and
amino groups are reactive ~ith such surfaces~
In an indirect coupling technique J groups reactive with the above
groups serving as bridges ~etween the polymer surfaces and the diagnostic re-
agent may either be present in the basic polymer or coupled thereto by known
chemical reactions. Such coupling reagents include amino groups, hydroxyl groups,
mercapto groups, amido groups, and carboxyl groups. SuitaBle coupling techniques
bet~een the polymer and diagnostic reagent are set forth in Bennich et al,
United States Patent 3J720J760.
~ 7 -
10~
Other covalent linkages may be performed indirectly with ceramic sub-
strates by use of intermediate coupling agents as set forth in Weetall Patent
3,652,761. Although that patent describes the coupling to microreticular sur-
faces, the techniques are also applicable to the continuous impermeable non-
particulate surface of the present body.
The so-called direct sandwich technique is performed as follows. A
coupling reagent is first attached to the substrate. In one specific coupling
technique, a polymeric surface, e.g., an acrylic, may be reacted with a coupling
agent or spacer arm in a carbodiimide catalyzed nucleophilic suastitution type
reaction. Thereafter, the diagnostic reagent, e.g., antibody, is similarly co-
valently attached to the coupling agent.
In the following step, the body surface with covalently attached anti-
body is contacted ~ith a fluid suspected of containing sample suhstance, e.g.,
antigen, reactive with the anti~ody. If present, the antigen reacts with and
comBines to the anti~ody on the surface during an incubation period. It is highly
advantageous to agitate the reactive surface during incubation, This provides
a si~nifIcantly faster reaction time, In addition, it has been found to increase
the reproduc~bility of the experimental results, For this purpose, as set forth
hereinafter, the body is-provided with a handle which can be used to stir the
reactive surface region either mechanically or manually.
In the next step, after incubation with the sample s:erum containing
antigen, the surface is simply s-eparated from the solutlon and the surface is
washed with a suitaale solvent such as aqueous phosphate auffer or distilled
water.
In the following step, the surface is contacted ~ith a labelled sub-
stance, antibody solution laaelled with a fluorochrome. The solution is again
incubated for a sufficient time to complete the reaction between the labelled
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11~9~93~
antibody and antigen. As with the incubation of sample, it is advantageous to
agitate the solution during this incubation to decrease the reaction time as
well as improve tlle reproducibility of the detection.
In the next step, the solution containing unbound labelled antibody
is separated from the solid surface containing bound labelled antibody.
Subsequently, the reacted solid surface is thoroughly washed to remove
residual free and nonspecifically bound antibody which may remain on the solid
surface. The efficiency of this washing step is extremely important in obtain-
ing accurate results. Thus, the surface is thoroughly ~ashed wlth a suitable
rinsing solution such as aqueous phosphate buffer or distilled water.
T~is ~ashing step establishes the importance of the initial covalent
at~achment of the dLagnostic reagent ~ith the solid surface in the first step.
If, for example, such reagent ~ere merely coated as by adsorption on the solid
surface, a portion of it together with accompanying labelled substance can be
~ashed away during thi~s vigorous washing procedure.
After washing, the body containing the labelled suastance is trans-
ported to a detection station to measure t~e amount of the same.
By way of background, one standard technique for binding sample sub~
stances to substrate is called by immunologists the "Sandwich Technique". In
2~ the conventional case, an antibody to the specific disease whose presence is
being determined, is coated to the surface. A typical case uses the test for
~'Australian Antigen" in the blood which is regarded as prima facie evidence of
hepatitis infection, The anti~ustralian antigen-antibody is coated in a film
to the plastic substrate. The serum of the su~ject suspected of carrying the
antigen is reacted with the anti~ody. If the antigen is present, it binds im-
munologically to the anti50dy, ~fter a rinse with ~ater to remove all unbound
material, more antibody with a fluorescent label is added, It binds to the
.~ 9,
~.09~931
antigen if it is present. ~en a final rinse removes all unbound labelled anti-
body. Such a technique is described in Bratu, United States Patent 3,826,618
with respect to RIA. In the present invention, the antibody is first covalently
attached to the surface. In a f]uorometric system, the fluorescence, if any,
emitted from the surface is detected by a fluorometer.
In another type of direct technique, the diagnostic reagent, e.g.,
antibody, is first covalently attached to the substrate as set forth above.
Thereafter, the sample containing antigen, prevlously labelled as with fluorogen
is contacted with the surface for immunological reaction between the antigen and
antibody. The surface is then read ~y a fluorometer. This system is illustrated
in Example 1, supra.
In another example of a sandwich technique, the body is covalently at-
tached with antigen - a preparation of Treponema pallidum, for instance, if it is
desired to determine antibodies for syphilis in the subject. The serum of the
subject is incubated with the body, then rinsed. Then labelled antihuman anti-
Bodies (harvested from goat or rabbit) are added, incubated and rinsed. If
human antibody for treponemae was present in the serum~ then it adhered to the
body by immuno-reaction and, in turn, captured the labelled antiBodies from the
goat! for example, hence, a quantitative reading of titre for syphilis can be
obtained, This is a modification of the standard accepted FTA-ABS Test (fluor-
; escent treponemal antiBody-adsorption) whereby the body system of this invention
enables accurate quantitation.
Any ~nfectious disease producing antibodies would be amenable to assay
by this technique and ~ncludes such diseases of public health interest as:
syphilis-, gonorrhea, "strep" throat infection, dysentry, salmonella infection,
typhoid, rabies, serum hepatitis, tnfluenza types, etc. This invention can also
~e useful in quality control in the food and pharmaceutical ~ndustries.
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~09~931
Also, as another applicationemploying the predescribed techniques, it
is often of value not only to diagnose the presence of disease (antigens) but
determine the body's protective immunity to disease (antibody titre) as a result
of deliberate innoculation or vaccination. As an example, a physician innocu-
lates a child with D.P.T. vaccine to provide immunity to diptheria, pertussis or
whooping cough, and tetanus, He then assumes that all 3 have "taken". A test of
the serum for antibody for each would, in fact, determine if all 3 antigens
were effective in creating sufficient immunizing antibodies. Present technology
does not allow this simple screening procedure.
When the method of the present invention is used in the so-called
competitive technlque, the foregoing systems are modified generally as follows.
Instead of coupling diagnostic reagent (antiBody)-sample ~antigen)-laBelled sub-
stance (antibody), the laBelled substance and sample of the same type (e.g.,
antigen) are added simultaneously to compete with each other for diagnostic re-
agent ~e.g., antibody~ covalently attached to the solid surface, After this
incubation or contact time, the solid sur~ace is washed with the same advantages
as~the sandwich technique.
Illustrations of the system of this invention where antigens or anti-
bodies in the strict sens~e are not involved but where protein-binding procedures
are still employed include such combinatlons as thyroxine-binding globulin ~TBG)
and thyroxine (T4), intrinsic factor and vitamin B12, and bovine lactoglobulin
and folic acld, for example.
In all cases, one of the pair ~a first type of protein) may be coval-
ently attached to a solid surface and may also bind with and remove from blood
serum the other member of the palr (a second type of substance capable of binding
to the protein of the f~rst type~, competing wlth fluorochrome-laBelled molecules
of its own type. These examples and the generalized use are strictly analogous
- 11 -
10~931
to the antibody-serum antigen-labelled antigen systems described in several of
the immunological reactions.
In the present specification, diagnostic reagent comprises one of the
above pair and the biologically derived substance includes the other two. The
reaction product of the pair of substances are "bound" to each other.
In another type of competitive technique, a known quantity of antigen
is covalently attached to the surface and is contacted with specific labelled
antibody simultaneously with a solution of the same type of antigen of unknown
quantity. An RIA technique employing the above technique with a noncovalently
bound surface in which radioactively labelled antibody in solution is measured
is described in a paper by J. S, Noodhead, et al, entitled "The Immunoradiometric
Assay and Related Techniques", Br, Med. Bull. 30:44 (1974). In the present
system, the antigen is covalently attached to the surface, the labelled antibody
; on the surface is measured, and a fluorescent label is preferred.
Variations can include the use of a variety of fluorescent labels such
as lissamine-rhodamine B, D.A,N.S. (l-dimethylamino-napthalene-5-sulfonic acid)
orthophthaladehyde, and fluorescamine, which are frequently used in fluorescence
microscopy. The first two possess an orange or red emission spectra rather than
the yellow green or fluorescein and the second two possess a blue or green
emission spectra. The only variation in the fluorometer ~ould ~e the change in
exitation and emission filters used~ as well as the change in the fluorescent
label on the antibodies in the reagent kit.
The present technique is also applicable to the use of enzyme labelled
systems. Gne such system is described in an article by Pesce et al entitled
"Use of Enzyme=Linked Antibodies to Measure Serum Anti-DNA Antibody in Systemic
Lupus Erthyematosus", ~l~in"`Chem. 20~3, 353-359 ~1974), The described system
differs from the one described herein in that the diagnostic reagent, DNA, is
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1()9~9~1
adsorled to a test tube support. ~11ereafter, serum containing DNA antibodies is
reacted with the coated tube followed by reaction with an anti-human gamma
globulin peroxidase enzyme conjugate with the coated tube. Then a colored react-
ion product is developed by action of peroxidase on the substrate which is color-
imetrically measured by conventional techniques.
The follo~ing description will make particular reference to the body
employed for use in the above techniques, It includes two important character-
istics; a handle means and at least one nonparticulate, nonswellable, imperme-
able, continuous surface region capable of covalent attachment to a diagnostic
reagent. The handle means may be the side of a test tube or a portion of a solid
object. A particularly preferred handle means is a handle specifically formed
to facilitate mechanical or manual handling of the body as for stirring or the
like.
Referring specifically to Figure 1, a body is illustrated in conjunc-
tion with a fluorometric system, The body 10, has Been bound with a standard
fluorescent labelled substance, 11, i,e., of the same fluorescent substance as
is used to label the antibody containing surface 12 at the lower portion of cyl-
inder 10 and has a known titre as measured on the detection device which is em-
ployed in the test tube or assay~ in this instance, a fluorometer, Suitably a
blank space 13 is left around the surface of the cylinder 10, separating the
upper and lower layers 11 and 12, respectively. The lower layer 12 may contain
streptoccocal fluorescent-labelled antibody prepared in any of the foregoing
methods, The lower layer is immersed in the body liquid to determine if any of
the suspected antigen or antibody is present in the serum being tested.
A fiber optical cable 14 conducts ultra-violet light from a light
source 16, The light then passes through a suitable gelatin filter 18 which en-
sures that only light of the excited wavelength reaches the body and strikes the
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'10~4931
body surface whereupon it excites fluorescence of the fluorochrome labelled sub-
stance. Two fiber optical cables 19 and 20 are provided with respective filters
19a and 20a. One such cable 19 conducts fluorescent light from the standard
fluorescent coating to a photomultiplier tube 21, and the other such cable 20
conducts emitted fluorescence from the lower fluorescent layer 12 to the photo-
multiplier tube 21. A chopper wheel 22 operated by motor 23 revolves and alter-
nates the flow of light from each coating 11 and 12 to tube 21. In this manner,
a direct comparison is obtained between the standard and test portions.
In the embodiment of Figure 2, a paddle shaped body 26 includes a
handle 27, a stem 28, and a wide, flat head 29 at the other end, the head 29
bearing a layer 30 of sample. In this embodiment, a fluorometer is illustrated
in which two fiber optic cables 31 (for excitation light) and 32 ~for emitted
fluorescent light2 are parallel to each other.
In the embodiment of Figure 3, a multiple test body is illustrated.
The body includes a cube 33 at the end of handle 34. The cube 33 can present
four faces-, two faces 36 and 37 being visible, Each face has a different sample
layer, This body is~particularly adapted for fluorometry. Thus, four different
fluorescent labels can be provided, and the fluorometer may have a filter wheel
with four selected wavelength regions to isolate energy going to photomultiplier
tube 21. As- the operator rotates the tube 33 ~or other multifaceted polyhedron
body~, each test can be read in sequence. A handle can similarly be attached
to a cylinder, sphere or other substrate. Other means of moving the substrate
upon which di`fferent bi`ologically-derived substances are layered may be employed
to vary the surface exposed to the fluorometer.
Broadly stated, the Banded cylinder of Figure 1 and the cube of Figure
3 constitute two forms of the u$e of multiple areas coated on the substrate
adapted for rapid multiple quant~tative determination, especially of the fluoro-
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;
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~0'~ ~93~
metric tyl~c. Any shape of substrate may be employed so long as it includes afirst area of sample substrate labelled as with a fluorochrome and at least one
other area of such labelled substance. Such different areas may include a
standard area of predetermined quantity of the same type of substance as the
fluorochrome-labelled substance. In this manner, two different areas, such as
bands 11 and 12 of Figure 1, may be viewed to provide a direct comparison of the
standard and test portions.
In another embodimentJ a single surface region may include a plurality
of labelled substances in random dispersion chemically attached to the sub
strate. At least two of the labels for the sample are fluorochromes which emit
fluorescence responsive to different wavelengths of light, These may be employed
in cases where it is desirable to detect the presence of more than one sample
substance.
To detect the above randomly dispersed labelled substances of differ-
ent wavelengths, a fluorometer may be equipped with a filter wheel so that sever-
al different wavelengths can be selected for several particular fluorescent
labels, e.g., (1) fluorescein isot~iocyanate, (yellow-green~; (2) lissamine
rhodamine B~200 (deep orange~; (3) D,A.N.S, (l~dimethylamino-napthalene-5-
sulfonic acid; red), (4) ortho-phthaladehyde (blue green); and (5) fluores-
camine ~lue-green) and when each label is attached to a different antibody for
three microorgan~sms of interest in urinary tract infections (B. coli9 Pseudomon~
as, and Staphy~lococcusl, then one, two or all three may be simultaneously de-
termined and identified.
Fluorescent label s~ubstances include sodium fluorescein isothiocyanate
or other suitable substances. This particular fluorochrome having an excitation
at 460 nanometers and emission at 520 nanometers is advan~ageous.
,
.
1.0~t'1931
The surface region of the body is characterized by certain physical
and chemical properties. The primary chemical property, set forth above, is its
ability to covalently attach with the particular diagnostic reagent. With respect
to the physical properties, such surface is nonparticulate, nonswellable, im-
permeable and continuous.
The nonparticulate nature of the body surface region distinguishes it
from microbeads or the like, It should have a sufficient surface area to presenta microscopic single surface to the detector to enable integration of the fluor-escent light or radioactivity from large aggregates of labelled substances there-
by reducing sampling errors from sample to sample, Suitably such area is at
least 1 millimeter square. The use of such loose particles, often ~tsed in radio-
active assays, produce an ill-defined and difficult to control surface for uniform
excitation and precise viewing o~ fluorescence.
Another characteristic of the surface is that it is impermeable to
liquid. This distinguishes from gel-like microreticular beads. When such beads
are employed for this purpose as set forth in the background, it is extremely
difficult to wash their interstitial spaces free of nonspecifically bound and
entrapped label reagent. By the use of impermeable surfaces, the washing pro-
cedure is faster and more efficient.
Another characteristic of the surface region is that it be continuous.
This feature is related to impermeability. By formation of a continuous non-
porous surface, the aforementioned difficulties of washing are avoided. It
should be understood that a surface treated with acid or other reagent to assistcoupling is-within the scope of the invention even though the surface may be
roughened. Any porosity, permea~ility, or dIscontinuity of such a roughened sur-face is negl~gible in comparison to the microreticular particles of the prior art.
- 16 -
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,
- : .
'`'` ' ,
- , : , :
~ ' : ` .: ~ : ` '
iC~ 3~
Another feature of the surface region is that it be nonswellable.
Th;s contrasts to the three-dimensional gels such as Sephadex which are difficult
to wash free of unbound reagents and to control dimensionally.
Example 1
This example illustrates a fluorescent measurement of an antibody-
antigen reaction in which an antigen in solution is fluorescently labelled and
reacted with an antibody immobilized on a surface. In this so-called "direct"
technique, the fluorescence of the surface is proportional to the antigen concen-
tration in solution.
Acid soaked polyamide strips were coated with Anti-Streptolysin 0 by
immersing them for 30 minutes in a slowly stirred solution of Anti-Streptolysin 0
diluted 1:8 in saline solution while 1% of a coupling agent, glutaraldehyde was
slowly added. After water washing and drying the covalently attached strips were
exposed to various concentrations of Streptolysin 0 Toxin in distilled water.
Two ml of each concentration were first reacted ~ith 0,2 ml of fluorescamine
solution ~40 mg in lOQ ml acetone) to affect labelling of the antigen. The
strips were then a~ded and stirred for 30 minutes, removed7 water rinsed, and
allowed to dry. They were then placed in the instrument, excited at a wavelength
of 375 nm and fluorescence measured at a wavelength of 475 nm. The following
results were obtained,
` Streptolysin O Toxin DiIutionFluorescent Signal
1:5 280
l:lQ 155
1~20 105
1:40 45
1:1000 5
Blank o
- 17 -
10~ 3~
E~ample 2
~lis example illustrates a fluorescent measurement of bacteria in which
bacteria in suspension are bound to a surface and reacted with fluorescently
labelled antibody, in a so-called "sandwich" technique. The fluorescence of the
surface is proportional to the bacterial concentration of the sample.
A pure culture of Streptococcus beta-hemolyticus Type A was grown in
trypsin broth, autoclaved, centrifuged, and washed with phosphate buffer saline
~PBS). The cells were resuspended in PBS to concentrations of 107, 105, and 103
organisms per ml.
DEAE-cellulose strips ~ere coated with Strepococcus A Antiserum by
immersing them for 15 minutes in a stirred solution of antiserum diluted 1:16 inphosphate buffer while 0.5 ml of 50% of coupling agent, glutaraldehyde, was
slowly added. After ~ashing, the covalently attached individual strips were
incubated for 5 mlnutes in 5 ml of each of the concentrations mentioned above,
flowed by washing with PBS.
Next, the strips were incubated for 5 minutes in a solution of fluores-
cein isothiocyanate la~elled Streptococcus A antibody diluted 1:4 in phosphate
buffer, rinsed ~lth buffer and air dried. The strips were then placed in the
instrument and the following readings were obtained. -'
Streptococcal Concentration
Organisms per'ml ' - - _ Fluorescent Signal
840
105 305
122
While in this example, attachment was obtained by the cross-linking
of antiserum proteins into a strongly adhering film, covalent attachment directly
to cellulose derivatives such as amlno-cellulose are similarly possible.
~ 18
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: :
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109 1~31
E~amele 3
This example describes the fluorescent measurement of multiple surfaces
tllat have been e~posed to a common solution containing several substances for
wllich measurement is desired.
Three squares of cellulose were each reacted with a different immuno-
globulin antibody ~goat anti-human IgA, IgG, and IgM) using glutaraldehyde
coupling agent. The cellulose squares mounted on a plastic strip are placed in
a sample of serum diluted in suitable buffer. After incubation for ten minutes,
the strip is removed and the three coated squares rinsed with buffer. They are
then reacted with a solution containing equally active concentrations of fluor-
escein isothiocyanate labelled goat anti~human IgA, IgG, and IgM diluted in
suitable buffer and allo~ed to incubate another ten minutes. The strips are
again washed and each area is separately read in the instrument. The relative
concentrations of the various immunoglokulins are so determined,
Example 4
This example describes the fluorescent measurement of a common surface
exposed to a solution containing several substances for which individual measure-
; ments are desired.
A circular disc of polymethylmethacrylic acid containing a random mix-
ture of the same three different immunoglobulin antibodies as set forth aboveare exposed to a sample of serum diluted in suitable buffer. ~fter incubation
for ten minutes, the strip is removed and rinsed with buffer. It is then incub-
ated in a solution containing a mixture of goat antibodies to these immunoglobu-
lins, each antibody labelled wi~th a different fluorochrome~ i.e., fluor~scamine
labelled goat anti;I`gA, fluorescein isothiocyanate labelled goat anti-IgG) and
rhodamine labelled goat anti~IgM diluted in suitable ~uffer and incubated for
twenty minutes, The strip ~s removed and washed with bufer. It is then read
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~C~94931
sequentially in the instrument e~citing first at 390 nm and reading emission
at 485 nm, then at 480 nm and 520 nm, respectively, and finally at 520 nm and
595 nm, respectively. The concentrations of IgA, IgG, and IgM are so deter-
mined.
It will be understood that the above specific description and drawings
have been given for the purposes of illustration only and that variations and
modifications can be made therein without departing from the spirit and scope
of the appended claims.
To those skilled in the art to which this invention relates, many
changes in construction and widely differing embodiments and applications of
the invention will suggest themselves without departing from the spirit and
scope of the invention. The disclosure and the description herein are purely
illustrative and are not intended to be in any sense limiting.
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