Solid phase system for ligand assay

A method for conducting a ligand assay in an inert porous medium wherein a binding material is immunologically immobilized within the medium, which includes the steps of immunologically immobilizing a binding material within a finite zone of the medium, applying an analyte to the zone containing the immobilized binding material, applying a labeled indicator to the zone which becomes immobilized within the zone in an amount which can be correlated to the amount of analyte in the zone, applying a solvent to substantially the center of the zone to chromatographically separate the unbound labeled indicator from the zone, and measuring the amount of labeled indicator remaining in the zone.

BACKGROUND OF THE INVENTION 
1. Field of Invention 
The present invention relates to a solid phase system for ligand assay. 
More particularly, the invention relates to an immunological method for 
analyzing biological fluids wherein analytical reactions are conducted in 
an inert porous medium. 
2. Discussion of Prior Art 
Variations of natural immunological reactions have been found very useful 
as analytical techniques. These reactions have been most useful in 
clinical laboratory procedures, but their use is not limited to clinical 
applications. Because of the specificity of the reactions, they are of 
particular advantage in analyzing complex biological fluids. Often, 
conventional chemical analyses are not capable of differentiating complex 
molecules in a biological fluid. Such fluids can be analyzed for a variety 
of components such as drugs, enzymes, hormones, etc. by contacting the 
fluid with an appropriate antibody. Likewise, analyses for specific 
antibodies can be conducted by contacting the fluid with an appropriate 
antigen. Other natural binding proteins are also quite useful in some 
types of assay procedures. 
Unfortunately, the antibody-antigen reaction is generally not directly 
measurable, therefore, techniques have been devised for its indirect 
measurement. For instance, an antibody, antigen, or binding protein 
(collectively referred to as a ligand) may be labeled by various means. 
The amount of bound labeled ligand can thus be correlated to the 
concentration of the analyte in the biological fluid. Conventional labels 
include radioactive tags, e.g., .sup.125 I or tritium, enzymes, 
chromophores, fluorophores and enzymes cofactors and effectors. In the 
case of radioactive tags, the concentration of the labeled ligand is 
usually determined by placing the compound in a scintillation counter. 
Enzymes may be measured by reacting the labeled ligand with a substrate, 
which by the action of the enzyme, releases a chromogenic or fluorogenic 
substance that can be measured by conventional techniques. Ligands labeled 
with enzyme cofactors or effectors can be detected similarly by their 
effect on enzyme action on a substrate. Compounds labeled with 
chromophores may be directly measurable, e.g., by fluorescence, 
ultraviolet spectroscopy or other spectroscopic means. 
Immunochemical assays generally fall into one of two classifications. In 
the competitive assay, a limited quantity of binding material is contacted 
with a solution containing the analyte and a known concentration of a 
labeled analyte. The labeled and unlabeled analyte compounds compete for 
the binding sites on the binding material. By reference to a calibration 
curve, the amount of labeled analyte bound to the binding material can be 
correlated with the concentration of the analyte in the test solution. A 
second type of immunological assay, the sandwich assay, involves 
contacting a binding material with a solution containing the analyte to 
cause the analyte to bind to the binding material. This complex is then 
contacted with a solution of a labeled binding material, generally an 
antibody, which reacts with the bound analyte. The amount of bound labeled 
binding material is thus directly proportional to the amount of bound 
analyte. 
In all of the described methods, an essential step is to separate the 
unbound labeled material from the bound labeled material. A technique 
widely employed for such separation is to immobilize one of the reactants. 
For instance, an antibody may be adsorbed onto a solid support such as a 
test tube wall. After labeled material and analyte become bound to the 
immobilized antibody, the solid support is rinsed free of unbound labeled 
material. A variety of solid supports have been proposed for this purpose. 
Such supports include test tube walls, plastic cups, beads, plastic balls 
and cylinders, paper, and glass fibers. 
In U.S. Pat. No. 3,888,629, June 10, 1975, K. D. Bagshawe discloses a 
reaction cell and an immunoanalytical method, in which antibody is 
immobilized in a matrix pad of absorbent material such as a glass fiber 
pad. The reference discloses an antibody impregnated sheet of the 
absorbent material. Disks or pads of the material are then punched from 
the mat and placed in a reaction cell. Solutions of the analyte and other 
reactants are placed on the pad where the immunological reaction occurs. A 
buffer is then filtered through the pad to wash out unreacted labeled 
material. Because of surface tension and capillary action, liquid does not 
easily pass through the pad; therefore, an absorbent material is placed 
under the pad to facilitate filtration. 
To quantitate the reaction conducted in the Bagshawe cell, the pad must be 
removed from the cell and either placed in a gamma counter (in the case of 
a radioimmunoassay) or placed in some type of indicator solution (in the 
case of an enzymeimmunoassay). 
A need exists for a rapid, quantitative solid phase ligand assay, which can 
be conducted entirely on a solid matrix. Advantageously, the method would 
not require special reaction cells, and the separation of unreacted 
labeled material could be effected cleanly without filtering large amounts 
of solvents through the matrix. The amount of binding material deposited 
in the reaction zone on the solid matrix should be accurately 
controllable. Such accurate control has not heretofore been realized in 
prior art methods. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a method is disclosed for 
conducting a ligand assay in an inert porous medium wherein a binding 
material is immobilized within said medium, the improvement which 
comprises: 
(a) immobilizing a binding material within a finite zone of said medium; 
(b) applying to said zone under binding conditions an analyte to which said 
binding material is specific; 
(c) applying to said zone a labeled indicator under conditions which allow 
said labeled indicator to become immobilized within said zone in an amount 
which can be correlated to the amount of analyte in said zone; 
(d) applying to substantially the center of said zone a stream of solvent 
in a quantity sufficient to effect a radial chromatographic separation of 
unbound labeled indicator from said zone; and 
(e) determining the amount of labeled indicator remaining in said zone. 
DETAILED DESCRIPTION OF THE INVENTION 
The method of the present invention is conducted in a porous solid matrix. 
Such matrix is in sheet form and may be made of any suitable inert 
material which does not deleteriously react with any of the reactants, the 
products, or the solvents. The term, inert, when referring to the matrix, 
means that the matrix does not chemically react with any of the materials 
applied thereto, does not dissolve, does not react immunologically with 
such materials, and has negligible nonspecific attraction for such 
materials. Thus, the matrix, or more precisely, the interstices within the 
matrix, simply acts as a vessel or site for reactions to occur. 
The interstices or pores within the matrix are small enough so that the 
reaction fluid is retained within the matrix by capillary action. The 
matrix is advantageously a mat of compressed fibers, such as a mat of 
glass or synthetic fibers or a porous paper mat. The matrix may, however, 
be constructed of other porous materials such as sintered glass, ceramics, 
synthetic spongy materials, etc. A glass fiber mat is the preferred matrix 
because of its inertness. Other materials such as paper tend to have a 
greater non-specific attraction for the materials applied thereto and may 
require pretreatment to inactivate reactive sites. 
The present invention is useful for a wide variety of biological assays. 
For instance, blood or urine may be quickly and accurately analyzed for 
therapeutic drugs, natural or synthetic steroids, hormones, antibodies and 
the like. Therapeutic drugs which may be determined by this method include 
digoxin, dilatin, phenobarbital, theophylline, gentamycin, guinidine, 
propranolol, and the like. Steroids, such as cortisol, aldosterone, 
testosterone, progesterone and estriol may also be monitored conveniently 
by the present method. The method may be useful for determining serum or 
urine levels of hormones such as the thyroid hormones, triiodo-thyronine; 
the peptide hormones, insulin, coricotropin, gastrin, angiotensin, and 
proangiotensin; the polypeptide hormones, thyrotropin, luteotropin, and 
somatotropin, and the like. The binding materials for such assays are 
generally antibodies specific for the analyte of interest. Such antibodies 
may be prepared by conventional methods, well known by those skilled in 
the art. Typically, antibodies are prepared by innoculating an animal, 
such as rabbit, goat, horse, donkey, sheep, chicken, or human with the 
antigen of interest, and harvesting the antibodies from the blood of the 
animal. In addition to antibodies, the binding material may be a 
non-antibody binding protein, such as vitamin B-12 intrinsic factor, 
thyroxine binding globulin, folate binding protein, or sex hormone binding 
protein. 
A first step of the present method is to immobilize a binding material 
within a finite zone of the matrix. Immobilization can be accomplished by 
any convenient method, such as adsorption, evaporative deposition from a 
volatile solvent solution, covalent bonding between the binding material 
and the matrix, or immunological immobilization. Covalent bonding may, for 
example, involve bonding the binding material to a matrix through a 
coupling agent, such as cyanogen bromide or glutaraldehyde, as described 
by Grubb, et al., U.S. Pat. No. 4,186,146 (Sept. 18, 1979). Immunological 
immobilization is preferred for the practice of the present invention. To 
immunologically immobilize the binding material within the matrix, the 
binding material can be reacted with an antibody to such material in a 
solution absorbed in the reaction zone. In practice, the immunological 
immobilization may be accomplished by applying to a point on the matrix a 
small volume, e.g., 2-100 .mu.l, of a mixture of a solution of the binding 
material and an antiserum or a solution thereof containing an antibody to 
the binding material. By capillary action, the mixture diffuses out from 
the point of application to encompass a finite zone of the matrix. The 
matrix is then incubated under conditions of temperature and humidity for 
a period of time sufficient to effect an immunological reaction between 
the reactants. The reaction conditions will vary depending upon the 
reactants being used, but generally a temperature of from about 
-10.degree. C. to about 60.degree. C., preferably from about 0.degree. to 
about 25.degree. C. is employed, and an incubation time of from about 1 
minute to about 24 hours, preferably from about 3 minutes to about 18 
hours has been found sufficient to effect the reaction. 
For quantitative applications of the present method, the amount of binding 
material immobilized within the reaction zone should be accurately 
controlled. One of the advantages of the present method over those 
described in the prior art is that very accurate control can be achieved 
through the use of accurate volumetric micropipettes and solutions of 
known concentrations. The antiserum is generally employed in an amount to 
insure that substantially all of the binding material is immobilized. 
Alternative methods of immobilizing the binding material in the matrix 
include applying solutions of binding material and antibody to the 
reaction zone, either sequentially or simultaneously, and incubating to 
effect the immunological reaction. The matrix sheet may also be 
impregnated with the antibody by applying a solution thereof uniformly to 
the sheet and then drying the sheet. As the solution of binding material 
is spotted on the matrix, the antibody in the reaction zone is redissolved 
and reacts immunologically with the binding material. 
Those skilled in the art will appreciate that a sheet of matrix may have 
one reaction zone or several. The sheet may be dried after immobilization 
of the binding material, and stored for prolonged periods before being 
utilized in an assay procedure. Sheets having multiple reaction zones may 
have several spots of the same binding material or may comprise different 
binding materials, for different assays. 
Solutions of analyte and labeled indicator are applied to the reaction zone 
containing the immobilized binding material; and after incubation under 
appropriate conditions, the immunological reaction occurs resulting in 
analyte and labeled indicator being bound in the reaction zone. Whether 
the analyte is applied prior to, concurrently with, or subsequent to the 
application of the labeled indicator depends upon the type of assay being 
conducted. For instance, in a competitive assay for an antigen, such as 
the drug digoxin, the binding material is an anti-digoxin antibody. The 
analyte, which may be a patient serum, and the labeled indicator, which is 
suitably labeled digoxin, are applied to the reaction zone substantially 
concurrently. In a sandwich assay, e.g., for IgG, the labeled indicator 
would be a labeled anti-IgG antibody, and could be applied to the reaction 
zone before, after or simultaneously with the reaction between the analyte 
and the binding material. 
The concentrations of analyte and labeled indicator are controlled so that 
the amounts of such compounds deposited in the reaction zone are 
appropriate for the particular type of reaction being employed. Such 
concentrations can be determined in accordance with relationships known in 
the art. For instance, in a competitive assay, the total amount of analyte 
and labeled indicator applied to the reaction zone is substantially equal 
to or greater than that required to bind to all of the binding sites on 
the binding material. The total concentration of binding sites of the 
analyte and labeled indicator generally constitute from about 0.1 to about 
10, preferably from about 0.5 to about 1.5 times the concentration of 
binding sites on the binding material. In a sandwich assay, the 
concentration of the analyte will be such that it can be substantially 
completely bound in the reaction zone. To accomplish substantially 
complete binding, generally there is an excess of binding material in the 
reaction zone with respect to the analyte. The binding material is 
advantageously employed at a concentration of from about 1 to about 100, 
preferably about 1 to 10, times that required to bind all of the binding 
sites of the analyte. The labeled indicator should then be employed in a 
quantity sufficient to react with the bound analyte. Thus, the labeled 
indicator is employed in an amount from about 1 to 2, preferably 1 to 1.5, 
times the amount required to react with all of the bound analyte. 
The analyte and labeled indicator are applied to the reaction zone in 
solution in such a manner that they diffuse throughout substantially the 
entire reaction zone. Such application is advantageously accomplished by 
pipetting small volumes of solutions to substantially the center of the 
zone. 
If desired, matrix sheets can be prepared for routine use which have 
binding material and labeled indicator already applied thereto. The 
binding material may be applied as described above. The labeled indicator 
may then be applied to the surface of the matrix, e.g., by a printing 
process. Advantageously, a barrier layer is applied to the surface prior 
to application of the labeled indicator to prevent premature reaction of 
the labeled indicator with the binding material. The barrier layer is a 
thin coating of a material which is water soluble. Thus, when the analyte 
is applied to the reaction zone, the barrier layer dissolves and allows 
the labeled indicator to diffuse into the reaction zone. 
The matrix sheet is incubated under conditions which enable the analyte 
and/or the labeled indicator to be bound to the binding material. A 
principal advantage of the present invention is that such reactions 
typically occur very rapidly at room temperature. Generally, incubation 
temperatures of from about 5.degree. C. to about 60.degree. C. are 
employed with a preferred temperature in the range of from about 
15.degree. to about 40.degree. C. The immunological reactions are usually 
sufficiently complete in from about a few seconds to about 30 hours, 
depending on the particular reaction being conducted. 
After the reactions have occurred, the unbound labeled indicator is 
separated from the reaction zone. In accordance with the present 
invention, the unbound labeled indicator is quickly and quantitatively 
separated from the reaction zone by a chromatographic procedure. A stream 
of a solvent, in which the labeled indicator is soluble, is applied to 
substantially the center of the reaction zone. The solvent may be water or 
a buffer solution in which such compounds are conveniently dissolved. As 
the solvent migrates radially out from the center of the reaction zone, 
unbound reactants are chromatographically separated from the bound 
reactants. A small quantity of such solvent effectively separates the 
unbound reactants from the reaction zone. Such reactants, if visible, 
would appear as a ring around the reaction zone, and the degree of 
separation is dependent on the volume of solvent zone, and the degree of 
separation is dependent on the volume of solvent used and the R.sub.f 
values for the reactants. Small volumes have been found effective for good 
separation, thus providing a quick, economical, and reliable assay 
procedure. Typically, solvent volumes of from about 10 .mu.l to about 150 
.mu.l, preferably 25 to 100 .mu.l are employed. The solvents may 
conveniently be applied to the reaction zone with a pipette or hypodermic 
syringe. 
When the present method is being utilized as an enzyme-immunoassay, a 
solution containing an enzyme substrate may be used for the dual function 
of applying the substrate and as a chromatographic solvent. Such a 
procedure reduces the total number of steps and thus reduces the time 
required to conduct an assay. 
After the ligand assay reaction has been completed and the unbound labeled 
indicator has been separated from the reaction zone, the reaction zone is 
observed, either visually or with the aid of appropriate instruments, to 
determine the magnitude of the signal generated by the labeled indicator. 
This signal might be a measure of radioactivity in the case of a 
radioimmunoassay, or a colorimetric, ultraviolet or fluorescence response 
in the case of an enzyme-immunoassay. Typically, colorimetric, 
ultraviolet, or fluorescent assays may be used for rate determinations, 
where the rate of formation (or disappearance) of the measured chromophore 
or fluorophore is compared to a calibration standard as as indication of 
concentration. Such measurements may be made directly from the porous 
medium, employing front surface fluorometers or reflectometers. In its 
simplest form, the present method may be used as a qualitative or 
semiquantitative test, wherein the reaction zone is observed visually, 
under visible or ultraviolet light, to determine whether a reaction has 
occurred or to obtain an approximate indication of the extent of the 
reaction.

The present invention is further illustrated by the following examples, 
which are not intended to be limiting. 
EXAMPLE I 
This example describes a competitive type of enzymeimmunoassay for serum 
digoxin in accordance with the present invention. 
I. REAGENTS 
A. Preparation of Anti-Digoxin Papers. 
1. Diluent Buffer--1.times.10.sup.-2 M Phosphate, 1.5.times.10.sup.-1 M 
NaCl, 1.times.10.sup.-2 M EDTA, pH 7.3, with 0.5% bovine serum albumin and 
0.1% NaN.sub.3. 
2. First antibody-Solution--16 microliters of a 1:100 dilution of rabbit 
anti-digoxin plus 10 microliters of normal rabbit serum was diluted to a 
final volume of 2000 microliters. 
3. Second antibody solution--1:100 dilution of goat anti-rabbit serum. 
4. Paper Matrix--Whatman type GF/F glass microfiber filter paper. 
To 175 microliters of first antibody solution was added 175 microliters of 
second antibody solution. The solution was mixed rapidly and immediately 
pipetted in 50 microliter aliquots onto the paper matrix. The anti-digoxin 
papers were incubated in a moist condition at 4.degree. C. for 18 hours. 
They were then washed with 100 microliters of 1.5.times.10.sup.-1 M NaCl 
containing 0.1% Triton X-100 which was applied to the center of the 
antibody spot diffusing out radially. Papers were dried under vacuum and 
stored at 4.degree. C. 
B. Preparation of Enzyme Labeled Digoxin. 
1. Digoxin--100 milligrams in 10 milliliters of methanol. 
2. Periodate--214 milligrams of sodium periodate in 10 milliliters of 100 
millimolar potassium phosphate pH 8.0. 
3. Alkaline Phosphatase (ALP)--from E. coli dialized against 100 mM Tris 
buffer pH 8. 
4. Sodium Cyanoborohydride--6 milligrams per milliliter in 500 millimolar 
phosphate pH 6.0. 
To 500 microliters of digoxin in methanol was added 500 microliters of 
periodate suspension. The materials were reacted with agitation for 1 hour 
in the dark at room temperature. The mixture was centrifuged to remove the 
periodate. The pH of the supernate was adjusted to 8 with potassium 
carbonate. The reaction was effective to oxidize the vicinal hydroxyls of 
the digitose, cleaving the ring and forming the dialdehyde. To the 
oxidized digoxin was added 200 microliters containing 2 milligrams of 
alkaline phosphatase. The mixture was incubated in the dark at room 
temperature for 1 hour. Octanol (1 drop) was added to retard foaming and 
the pH was adjusted to about 6 with formic acid. The reaction produces 
Schiff base bonds between the amine groups of the enzyme and the aldehyde 
groups of the digoxin. The excess aldehydes and the Schiff bases are then 
reduced by the dropwise addition of 300 microliters of sodium 
cyanoborohydride. The conjugate thus formed was purified by dialysis (3 
times) against saline and by gel filitration chromatography on a Sephadex 
G-25 column using 10 millimolar Tris, 1.5.times.10.sup.-1 molar NaCl, pH 
8.0 as eluant. 
C. Preparation of Calibrator-Trace Mixtures 
1. The enzyme labeled digoxin was added to commercial normal human serum 
calibrators, containing azide preservatives, to form calibration-tracer 
mixtures having a free digoxin concentration of 0 to 6 nanograms per 
millimeter and a tracer digoxin concentration of 1.6 nanograms per 
milliliter. 
D. Preparation of Substrate Wash Solution 
1. A 10 milligram amount of 4-methylumbelliferyl phosphate was added to 100 
milliliters of 1.5 molar Tris, 1 millimolar magnesium chloride, pH 8.0. 
II. ASSAY OF DIGOXIN 
Three anti-digoxin paper spots were labeled for each calibrator-tracer 
mixture and each sample-tracer mixture. An aliquot of 50 microliters of 
the appropriate calibrator-tracer mixture or sample-tracer mixture was 
pipetted onto the center of the anti-digoxin paper spot. The mixture was 
quickly absorbed into the matrix. The reaction spot was incubated in a 
moist chamber at room temperature for 5 minutes. An aliquot of 100 
microliters of substrate wash solution was applied slowly (60 seconds) to 
the center of the spot. The wash solution diffused out radially carrying 
with it any digoxin-ALP not bound by the anti-digoxin immobilized within 
the matrix. When the diffusion of the substrate begins to slow, the 
reaction rate of the antibody bound digoxin-ALP is measured in an area 
about the center of the antibody spot within a radius of 4.5 millimeters. 
A delimited area (4.5 millimeter radius) is chosen for the monitoring of 
the bound enzyme substrate reaction so as to sample a homogenous area of 
the anti-digoxin reaction matrix (total radius of 6 millimeters). At the 
same time the free digoxin-ALP has been removed from the monitored area by 
the subtrate wash solution (13 millimeter radius). 
The enzymatic conversion of the non-fluorescent substrate, 
4-methylumbelliferyl phosphate, to the fluorescent product, 
4-methylumbelliferone, by the bound digoxin-ALP is measured in a suitable 
front surface fluorometer. 
EXAMPLE 2 
This example describes a competitive type of radioimmunoassay for serum 
digoxin in accordance with the present invention. 
I. REAGENTS 
A. Preparation of Anti-Digoxin Papers 
1. Diluent Buffer--1.times.10.sup.-2 M phosphate, 1.5.times.10.sup.-1 M 
NaCl, 1.times.10.sup.-2 M Na.sub.2 EDTA, pH 7.3, with 0.5% bovine serum 
albumin and 0.1% NaN.sub.3. 
2. Paper Matrix--Toyo type GA-100 glass microfiber filter paper. 
To 0.67 milliliters of buffer was added 100 microliters of normal rabbit 
serum, 20 microliters of 2% patent blue dye and 20 microliters of a 1:100 
dilution of anti-digoxin serum. The solution was mixed thoroughly. To this 
was added 200 microliters of goat anti-rabbit serum. The solution was 
mixed rapidly and immediately pipetted in 100 microliter aliquots onto the 
paper matrix. The anti-digoxin papers were incubated in a moist condition 
at 4.degree. C. for 18 hours. They were then washed with 2 aliquots of 200 
microliters each of 100 mM PO.sub.4 -saline which was applied to the 
center of the antibody spot, diffusing out radially. Papers were dried 
under vacuum and stored at 4.degree. C. 
B. Preparation of .sup.125 I Labeled Digoxin. 
A commercially available .sup.125 I-digoxin derivative was diluted to 915 
picograms per milliliter in 1.times.10.sup.-2 M phosphate, 
1.5.times.10.sup.-1 M NaCl, 1.times.10.sup.-2 M EDTA, pH 7.3, with 0.5% 
bovine serum albumin and 0.1% NaN.sub.3. 
C. Preparation of Calibrator-Tracer Mixtures. 
The .sup.125 I-digoxin solution was added to commercial normal human serum 
calibrators containing azide preservative, to form calibration-tracer 
mixtures having a free digoxin concentration of 0 to 6 nanograms per 
milliliter and a tracer digoxin concentration of 458 picograms per 
milliliter. 
D. Wash Solution. 
1.times.10.sup.-2 M phosphate, 1.5.times.10.sup.-1 M NaCl. 
II. ASSAY OF DIGOXIN. 
Anti-digoxin paper spots were labeled for each calibrator-tracer mixture. 
An aliquot of 100 microliters of the appropriate calibrator-tracer mixture 
was pipetted onto the center of the anti-digoxin paper spot. The mixture 
was quickly absorbed into the matrix. The reaction spot was incubated in 
open air, on a wire rack at room temperature for 3 minutes. Two aliquots 
of 200 microliters each of wash solution was applied to the center of the 
spot. The wash solution diffused out radially carrying with any .sup.125 
I-digoxin not bound by the anti-digoxin immobilized within the matrix. A 
disc (radius 9 millimeters) was then punched from the center of the 
anti-digoxin paper spot. The discs were counted in a gamma scintillation 
counter for 1 minute each. The number of counts for each disc was found to 
be inversely proportional to the concentration of digoxin applied to the 
disc. 
EXAMPLE 3 
This example describes a sandwich type of radioimmunoassay for serum 
thyroid stimulating hormone (TSH) in accordance with the present 
invention. 
I. REAGENTS 
A. Preparation of anti-TSH papers 
1. Diluent Buffer--50 millimolar phosphate, pH 7.4, with 0.1% bovine serum 
albumin and 0.02% azide. 
2. Filter Paper Matrix--Toyo glass fiber paper type GA-100. 
To 2.25 milliliters of buffer was added 2.5 milliliters of a 1:100 dilution 
of goat anti-TSH serum and 250 microliters of rabbit anti-goat serum. The 
solution was mixed rapidly and immediately pipetted 100 microliter 
aliquots onto the paper matrix. The spots were incubated in a moist 
condition at 4.degree. C. for 18 hours. They were then washed 2 times with 
200 microliter aliquots of 100 millimolar phosphate buffer pH 7.3. The 
papers were dried under vacuum and stored at 4.degree. C. 
B. .sup.125 I-Anti-TSH Tracer. 
A commercial preparation of .sup.125 I labeled rabbit anti-TSH serum 
containing 58,000 counts per minute per 100 microliters. 
C. TSH Calibrators 
A commercial secondary standard material was diluted in charcoal treated 
normal human serum to concentrations of 0, 1.5, 6, 12, 25 and 50 
microunits per milliliter. 
D. Pre-Application of Tracer to Paper. 
To each anti-TSH spot was added 100 microliters of rabbit .sup.125 
I-anti-TSH. The spots were dried under vacuum. 
II. ASSAY OF TSH. 
One anti-TSH paper spot was labeled for each TSH calibrator. 100 
microliters of the appropriate calibrator was applied onto the spot. The 
papers were incubated in a moist chamber for 1 hour at 37.degree. C. 
Papers were then washed with 2 aliquots of 200 microliters each of 10 mM 
phosphate pH 7.4. A disc of 9 mm was cut from the center of each spot. All 
discs were counted in a gamma counter for 1 minute. The number of counts 
for each disc was found to be proportional to the concentration of TSH 
applied to the disc. 
EXAMPLE 4 
This example describes a sandwich type of enzyme immunoassay for human IgG 
in accordance with the present invention. 
I. REAGENTS 
A. Preparation of Anti-Human IgG Papers 
1. Diluent Buffer--0.01M Tris with 0.1% BSA and 0.5% sodium azide, pH 8.0. 
2. First Antibody Solution--2 microliters of goat anti-human IgG is diluted 
to a final volume of 2000 microliters. 
3. Second Antibody Solution--1:100 dilution of rabbit anti-goat serum. 
4. Paper Matrix--Whatman type GF/F glass microfiber filter paper. 
To 175 microliters of first antibody solution is added 175 microliters of 
second antibody solution. The solution is mixed rapidly and is immediately 
pipetted in 50 microliter aliquots onto the paper matrix. The anti-human 
IgG papers are incubated in a moist condition at 4.degree. C. for 18 
hours. Papers are stored at 4.degree. C. 
B. Preparation of enzyme labeled anti-human IgG--a modification of the 
method described by A. Murayamd et al. Immuno-chemistry 15: 523 (1978). 
1. Anti-human IgG--20 ml of rabbit anti-human IgG serum is mixed for 4 
hours at room temperature with 0.4 gms of Aerosil 380. The material is 
centrifuged and the supernate collected. The treated rabbit anti-human IgG 
serum is then diluted with equal volume of 0.1M ethylene diamine (EDA) 
buffer pH 7.0 and applied to a column of QAE Sephadex which has been 
equilibrated with the EDA buffer. The column is eluted with additional EDA 
buffer. The protein fractions which are eluted are pooled and concentrated 
to 20 mg/ml in an Amicon cell fitted with a type PM10 membrane. The 
material is then dialized against 0.01M K H.sub.2 PO.sub.4. 
2. Periodate--32.5 milligrams of sodium periodate are combined with 177.5 
milligrams of KCl and 3.0 ml of methanol. 
3. Alkaline Phosphatase (ALP)--from E. coli. The enzyme is dialized against 
0.01M phosphate buffer, pH 6.5. 
4. Sodium Cyanoborohydride--6 milligrams per milliliter in 0.5M phosphate 
pH 6.0. 
To a test tube wrapped in foil containing 45 .mu.l of purified rabbit 
anti-human IgG is added 80 .mu.l of periodate suspension. The pH is 
adjusted to between 4.0 and 4.5 with 1N HCl. The mixture is incubated at 
room temperature with constant mixing for 30 minutes. The mixture is 
centrifuged and the supernate collected. 20 .mu.l packed volume of G-25 
Sephadex equilibrated with 0.01M K H.sub.2 PO.sub.4 is added to the 
supernate. The Sephadex is mixed by inverting for 20 minutes at room 
temperature. The Sephadex is then centrifuged down and the supernate is 
collected. 16 mg of ALP is added to the supernate and the pH is adjusted 
to 6.5 with 0.5M K H.sub.2 PO.sub.4. The solution is mixed and then 
dialized against 0.01M phosphate buffer pH 6.5 for 18 hours at room 
temperature. To the dialized conjugate is added 50 .mu.l sodium 
cyanoborohydride. The mixture is allowed to react at room temperature for 
1 hour. The solution is then dialized against 0.01M Tris 0.15M NaCl pH 
8.0. 
The conjugate is applied to a Biogel A 1.5 column and the fractions 
containing both enzyme and antibody activities are collected. 
C. Preparation of human IgG calibrators. Human IgG is weighed out and 
dissolved in 0.01M Tris with 1.0% gelatin and 0.1% sodium azide, pH 8.0 to 
form calibrators having human IgG concentrations of between 0 mg/ml and 50 
mg/ml. 
D. Preparation of Substrate Wash Solution. A 10 milligram amount of 
4-methylumbelliferyl phosphate is added to 100 milliliters of 1.5M Tris, 
0.001M MgCl.sub.2, pH 8.0. 
II. ASSAY OF HUMAN IgG. 
Two anti-human IgG paper spots are labeled for each calibrator and each 
sample. Calibrators and samples are diluted 1:50 in 0.01M Tris, 0.15M NaCl 
pH 8.0. 
The diluted calibrators and samples are pipetted in 50 microliter aliquots 
onto the center of the anti-human IgG paper spot. The mixture is quickly 
absorbed into the matrix. The reaction spot is incubated in a moist 
chamber at room temperature for 5 minutes and the human IgG in the sample 
is bound by the antibody immobilized within the matrix. An aliquot of 50 
microliters of ALP-anti-human IgG conjugate is applied and is incubated at 
room temperature for 5 minutes. The conjugate binds to the human IgG bound 
within the matrix. An aliquot of 100 microliters of substrate wash 
solution is applied slowly to the center of the spot. The wash solution 
diffuses out radially carrying with it any ALP-anti-human IgG conjugate 
not bound within the matrix. When the diffusion of the substrate begins to 
slow, the reaction rate of the bound ALP-anti-human IgG is measured in a 
suitable fluorometer.