Immunoadsorbent polymeric material and method of making same

Gamma globulins from antisera having a titer so low in selected antibodies that the antibodies adsorbed on a surface of polymeric material are ineffective for use in immunoassay, are cross-linked, then adsorbed on the polymeric surface to provide an immunologically active coating. Binding proteins incapable of effective adsorption on polymeric surfaces are cross-linked with gamma globulin, then adsorbed on a surface of polymeric material capable of adsorbing gamma globulin to provide a specific binding protein coating. Kits containing test tubes having such coatings are used in radioimmunoassay, in enzyme immunoassay, and in fluoroimmunoassay.

This invention relates to immunoassay and competitive protein binding 
procedures and to a device for use in such procedures and pertains more 
specifically to a method and apparatus for radioimmunoassay, enzyme 
immunoassay, and fluoroimmunoassay of antigens. 
It has previously been proposed in Catt U.S. Pat. No. 3,646,346 to provide 
a solid water-insoluble polymeric material, preferably in the form of a 
test tube, having its surface coated by adsorption of an antibody to the 
protein to be assayed, the coated tube then being brought into contact 
with an aqueous sample containing the protein to be assayed and also with 
a known quantity of the same protein which is radioactively labeled, 
finally separating the polymeric surface from the aqueous sample and 
determining, by measuring the emitted radiation, the amount of labeled 
protein which has been bound to the coated polymer. The solid phase 
antibody coated polymeric material can be made and used in the Catt 
procedure only when the antiserum containing the antibody to be adsorbed 
is of moderately high titer; moreover, the coated polymeric material and 
procedure cannot be used with some binding proteins such as hog gastric 
intrinsic factor (a binding protein for vitamin B-12) which are not 
adsorbed on polymeric surfaces sufficiently even when available in high 
titer solutions. 
It has been proposed by Avrameas et al., Immunochemistry, Vol. 6, pages 
53-66 (Pergamon Press, 1969) to insolubilize antigens or antibodies by 
cross-linking them with glutaraldehyde and to employ the insolubilized 
proteins thus produced, either in column procedure or in batchwise 
operation, for the isolation of antigens or antibodies. It has also been 
proposed by Avrameas, loc. cit., pages 43-52 to couple enzymes to human 
immmunoglobulin-G and serum albumin as well as to sheep and rabbit 
antibody with glutaraldehyde and to use the products, which retain a 
substantial part of their immunological and enzymatic activity, for the 
intracellular detection of antigens and antibodies and for 
characterization of antibodies after immunoelectrophoresis. It has in 
addition been proposed by Barrett et al. at the Ninth International 
Congress on Clinical Chemistry, 13-18 July, 1975, Toronto, to bring a 
plastic tube into contact with glutaraldehyde to provide reaction aldehyde 
residues on the surface of the plastic, and then to bring the plastic into 
contact with antibody to cause the antibody to be coupled to the 
glutaraldehyde. 
There has now been found a method of making a solid immunoadsorbent for use 
in immunoassay procedures which comprises cross-linking the gamma globulin 
content of low titer antiserum, bringing the cross-linked gamma globulin 
into contact with a surface of a solid polymeric material capable of 
adsorbing gamma globulin to form a coating of cross-linked gamma globulin 
adsorbed on said surface. There has further been found a method of making 
a solid immunoadsorbent for use in immunoassay procedures which comprises 
cross-linking a binding protein with gamma globulin to form a cross-linked 
material, then bringing the cross-linked material into contact with a 
surface of a solid polymeric material capable of adsorbing gamma globulin 
to form a coating of cross-linked material adsorbed on said surface. The 
solid polymeric material preferably is in the form of a plastic test tube 
as described in Catt et al. U.S. Pat. No. 3,646,346 having an inner 
surface of solid polymeric material capable of adsorbing gamma globulin, 
this adsorbed coating consisting essentially of cross-linked gamma 
globulin or of binding protein cross-linked to gamma globulin. The coated 
plastic tube is preferably supplied as part of a kit; the kit also 
contains a supply of labeled tracer and a standard, as described, for 
example, in Catt U.S. Pat. No. 3,646,346. 
In the drawing, 
The FIGURE is a graphical representation of the increase in binding 
capacity of a coated tube achieved by one embodiment of the present 
invention. 
A kit such as that mentioned above is useful in both qualitative and 
quantitative immunoassay procedures for determining proteins, drugs, 
steroid hormones and other physiologically active materials which bind 
with their corresponding antibodies or binding proteins. The cross-linking 
step makes it possible to prepare useful solid immunoadsorbents from 
antisera of such low quality that they cannot be used effectively for 
adsorption of a selected antibody itself on the surface of polymeric 
material; the cross-linking step also makes it possible to prepare solid 
immunoadsorbents useful in immunoassay procedures from solutions of 
binding proteins which are incapable by themselves of sufficient 
adsorption on the polymeric surface, even from solutions of high titer, to 
prepare useful solid inmmunoadsorbents. 
The amount or extent of cross-linking which is desirable varies depending 
upon the identity and concentration of the particular antiserum involved. 
In general, the extent of cross-linking desired for optimum results is 
from two to five times the molecular weight of the starting material. That 
is, the molecular weight of the cross-linked product is from two to five 
times the molecular weight of the uncross-linked antibody or binding 
protein. The time required for the desired extent of cross-linking may 
vary from a few minutes to several hours depending upon the identity of 
the proteins involved, and the nature of the cross-linking procedure 
employed. Cross-linking is generally carried out at a pH well below 8, 
whereas adsorption of the cross-linked product occurs to a substantial 
extent principally at a pH from 8 to 10, the optimum pH for adsorption 
depending upon the particular selected antibody or binding protein 
involved. Consequently, the cross-linking can be completed before bringing 
the antiserum or solution of binding protein into contact with the surface 
of the polymeric material, or if desired, the proteins can be cross-linked 
while in solution in contact with the surface of the solid polymeric 
material, after which the pH may be raised to pH 8-10 by addition of a 
suitable buffer to cause adsorption to occur without removing the solution 
from contact with the polymeric material. 
Cross-linking can be effected by simple heating of the antiserum or of the 
mixture of binding protein with gamma globulin to a temperature of at 
least 55.degree. C., preferably 70.degree.-75.degree. C. for a few 
minutes. Such heating causes cleavage of intramolecular disulfide linkages 
which occur in gamma globulins and frequently in binding proteins to 
generate reactive species which cross-link with each other. 
Cross-linking can also be effectively carried out by mixing the antiserum 
or solution of binding protein and gamma globulin with sodium periodate, 
which causes cleavage of gem diols of those proteins which contain sugar 
residues to generate dialdehydes which in turn cross-link with each other. 
Similar results can be obtained by employing a carbodiimide reagent to 
activate endogenous carboxylate functions of the protein molecules which 
then react with amino functions of other protein molecules to effect 
cross-linking by formation of amide linkages. 
The desired cross-linking can also be achieved by mixing with the antiserum 
or with the mixture of binding protein and gamma globulin, a difunctional 
reagent capable of forming covalent bonds with proteins as described, for 
example, by Wold, Methods Enzymol., Vol. 11, page 617 (1967) and by Means 
et al., Chemical Modification of Proteins, Holden-Day, Inc., San 
Francisco, Calif. (1971). The difunctional reagents which are useful 
usually contain functional groups which react with amino functions such as 
those that occur in lysine or other amino acid residues of proteins. The 
difunctional reagents which can be used include those having the general 
structure Y-B-Z where B may be any aliphatic or aromatic hydrocarbon 
residue of sufficient length to permit efficient interaction to take place 
with two or more binding proteins or immunoglobulins, and where Y and Z 
may be 
##STR1## 
--CH.sub.2 X, or --N.dbd.C.dbd.O where X is halogen, preferably bromine or 
iodine, and where Y and Z may be any compatible pair of different groups 
or may be identical. The hydrocarbon residue may contain other functional 
groups to provide such desirable properties such as water-solubility or 
selective cleavability. Other difunctional reagents which may be used for 
cross-linking include the bis-diazotized aromatic amines which react with 
amino acids in proteins containing aromatic rings, and there may also be 
used dithiols which can undergo disulfide interchange with the disulfide 
bridges present in the proteins without disrupting the binding site of the 
binding protein or gamma globulin, and anhydrides of dicarboxylic acids 
such as adipic anhydride which react with amino groups. The difunctional 
reagents of choice for the purpose of the present invention are alkyl 
dialdehydes, such as the dialdehyde made by reacting cyclohexanediol-1,2 
with sodium periodate; glutaraldehyde is particularly preferred, optimum 
results being obtained when the glutaraldehyde is present at a 
concentration of 0.001 to 0.1 molar in the antiserum or mixture of binding 
protein and gamma globulin to be cross-linked. 
The desired cross-linking can be carried out in a glass or other container, 
after which the cross-linked antiserum or cross-linked mixture of binding 
protein and gamma globulin can be brought into contact with the surface of 
the solid polymeric material, or, as pointed out above, the cross-linking 
may be carried out in contact with the polymeric material at a pH below 8, 
after which the pH is raised to 8-10 to cause adsorption on the polymer 
surface to occur. There may be used as the polymeric material any of those 
materials described in Catt U.S. Pat. No. 3,646,346, the polymeric 
material preferably being in the form of a test tube or similar container 
made of such conventional polymeric materials as polystyrene, 
polyethylene, nitrocellulose, copolymers of acrylonitrile with styrene and 
the like, polypropylene being preferred. 
There may be included in the cross-linked serum or cross-linked mixture of 
binding protein and gamma globulin additional ingredients which serve to 
stabilize the coating, including reducing agents such as sodium 
borohydride or sodium cyanoborohydride to inactivate residual 
cross-linking agents such as dialdehydes; ascorbic acid or other 
antioxidants; and sodium azide or other inhibitors of bacterial action. 
Adsorption onto the surface of the polymeric material of the cross-linked 
gamma globulins from the antiserum or of the cross-linked binding protein 
and gamma globulin from a solution thereof can be accomplished simply by 
maintaining the antiserum or solution together with a suitable buffer at 
pH 8-10, in contact with the previously cleaned and dried polymeric 
surface for a period of time ranging from minutes to many hours at room 
temperature; elevated temperatures up to 40.degree. C. may be employed but 
ambient temperature (room temperature) is usually more convenient. After 
the desired adsorption has occurred to provide a coating of cross-linked 
gamma globulins or cross-linked binding protein and gamma globulin on the 
polymeric surface, the coating solution is removed and the surface washed 
with a buffer to remove any unadsorbed material, after which the coated 
polymeric material is dried at room temperature. The coated polymeric 
material is stable during storage in the dry state over a period of many 
months. 
To carry out an immunoassay, the serum sample to be assayed is brought into 
contact with the coated surface of polymeric material by placing the 
sample, for example, within a coated plastic test tube along with a known 
amount of labeled antigen in buffer. Competition between unlabeled antigen 
in the sample and the labeled antigen determine the amount of activity 
which is bound to the coating of the tube. After completion of the binding 
reaction, the liquid phase, which contains the unbound antigen, both 
labeled and unlabeled, is removed from the tube, after which the amount of 
labeled antigen which is bound can be determined in the usual manner and 
compared with a standard. When the antigen is labeled with a radioactive 
atom, conventional counters can be used to determine the amount of 
radiation emitted by the tube. When the antigen is labeled with an enzyme 
as described, for example, by Avrameas, Immunochemistry, Vol. 6, pages 
43-52 (Pergamon Press, 1969), the amount of labeled antigen can be 
determined by measuring enzymatic activity of the tube. When the antigen 
is labeled with a fluorophore, for example by reacting the antigen with 
anthroyl carboxylic acid or with 1-dimethylaminonaphthalene-5-sulfonic 
acid or with a fluorescein derivative, e.g., fluorescein isothiocyanate, 
the amount of labeled antigen can be determined by measuring fluorescence 
as described by Dandliker et al., Immunochemistry, Vol. 10, 219 (1973) or 
by Aalberse, Clin. Chim. Acta, Vol. 48, 109 (1973). However, the labeling 
is accomplished, the labeled material serves as a tracer. 
The following specific examples are intended to illustrate more fully the 
nature of the present invention without serving as a limitation upon its 
scope.

EXAMPLE 1 
The gamma globulin fraction of a rabbit antiserum to thyroxine 
(tetraiodothyronine, T4) is isolated by treating one ml. of the serum for 
18 hours at 4.degree. C. with an equal volume of 80% saturated ammonium 
sulfate, pH 7.1. The precipitated gamma globulin fraction is spun down in 
a refrigerated centrifuge at 3000 rpm. for 30 minutes, then diluted in 
2000 ml. of an isotonic 0.01M phosphate buffer pH 6.0. To this is added 
400 ml. of 0.02M glutaraldehyde cross-linking reagent in distilled water. 
This mixture is allowed to react for 20 minutes at 37.degree. C. and is 
then diluted for coating by the addition of 2000 ml. of 1M phosphate 
buffer pH 8.5., to give a final dilution of cross-linked gamma globulin of 
1/4400. One ml. aliquots of this coating solution are introduced into the 
bottom of clean, dry polypropylene plastic tubes. After incubation for 1 
hour at 37.degree. C., the tubes are aspirated and washed twice with an 
isotonic 0.01M tris buffer pH 7.4 containing 0.1% gelatin to remove any 
unadsorbed cross-linked gamma globulin. The tubes are dried at ambient 
temperature (22.degree.-25.degree. C.) and stored in sealed plastic bags. 
A series of assays was run in these coated tubes using 1 ml. of an isotonic 
0.01M tris buffer pH 7.4 containing 0.1% gelatin, quantities of inhibitor 
T4 ranging from 41 pg to 10 ng per tube and 14,000 cpm of .sup.125 I 
labeled T4 as radiolabeled tracer followed by room temperature incubation 
for 3 hours. At the minimum quantity of added T4, 59% of the added tracer 
was bound to the coated tube, decreasing amounts of tracer being bound at 
higher levels of added T4 as shown in the drawing. 
Analysis of the data by a modification of the procedure of Pinckard et al., 
Handbook of Experimental Immunology, page 498 (Great Britain 1967) gives 
an apparent affinity constant (K) of 4.5 .times. 10.sup.9 M.sup.-1 and the 
amount of T4 antibody bound to the tube (Ab.sub.o) of 1.8 .times. 
10.sup.-10 M. 
When the cross-linking reagent is omitted, the precipitated gamma globulin 
fraction being simply diluted 1/1000 in a buffer 0.01M in tris, 0.01M 
sodium chloride and 0.01M sodium azide, pH 8.5, and the diluted gamma 
globulin (one ml. aliquot) is incubated for 1 hour at 37.degree. C. in 
polypropylene tubes, then washed as described above to remove unadsorbed 
gamma globulin, the coated tubes display much lower immunoactivity when 
tested in the same way by a series of assays under the same conditions as 
described above. Only 34% of the added tracer is bound at the minimum 
quantity of added tracer, as shown in FIG. 1, and the displacement curve 
has a lower slope; the apparent affinity (K) of the coated tube was 
calculated as 1.9 .times. 10.sup.9 M.sup.-1 and the amount of immobilized 
T4 antibody (Ab.sub.o) as 1.4 .times. 10.sup.-10 M. 
These results show that cross-linking produces approximately the same 
amount of T4 antibody as does the untreated gamma globulin fraction when 
the latter is at more than four times the concentration, with maximum 
binding approaching twice that of the untreated gamma globulin fraction. 
EXAMPLE 2 
One ml. of a rabbit anti-Angiotensin I-serum (previously shown not to be 
useful in the Catt procedure) is treated for 18 hours at 4.degree. C. with 
an equal volume of 80% saturated ammonium sulfate, pH 7.1. The 
precipitated gamma globulin fraction is spun down in a refrigerated 
centrifuge at 3000 rpm for 30 minutes and redissolved in 250 ml. of an 
isotonic 0.01M phosphate buffer, pH 6.0. To the diluted gamma globulin 
preparation is added 50 ml. of 0.1M glutaraldehyde in distilled water, 
prepared by weighing out 2 gms of commercial 25% glutaraldehyde and 
diluting to 50 ml. 
The mixture is incubated for 20 minutes at 37.degree. C. with occasional 
stirring. After the incubation, an additional 250 ml. of isotonic 0.01M 
phosphate buffer pH 6.0 and 500 ml. of a 0.8M phosphate buffer pH 8.5 are 
added to raise the pH for efficient coating and to dilute the cross-linked 
gamma globulin for proper coating. One ml. portions of the coating 
solution are carefully put into clean, dry polypropylene plastic tubes and 
allowed to stand undisturbed for 18 hours at ambient temperature, 
22.degree.-25.degree. C., after which the coating solution is removed by 
aspiration and the tube is given two washes with an isotonic 0.01M tris 
buffer pH 7.4 containing 0.1% gelatin and 0.01M sodium azide. The tubes 
are dried at the same ambient temperature as above and stored in plastic 
bags. 
The angiotensin I antibody coated tubes prepared by this invention can be 
used in a radioimmunoassay for angiotensin I according to the following 
procedure. One ml. of a 0.1M tris buffer adjusted to pH 7.4 with glacial 
acetic acid is added to the coated tube. One hundred .mu.l of patient 
plasma to be assayed is added, as well as 20,000 counts per minute of 
angiotensin I-5-(3-.sup.125 I tyrosine). The reaction mixture is incubated 
at ambient temperature (22.degree.-25.degree. C.) with occasional shaking 
for 3 hours, when the reaction is terminated by aspirating the liquid 
phase. Separation of bound and free tracer is accomplished by this 
aspiration. Competition between unlabeled hormone in the patient plasma 
and the .sup.125 I labeled hormone determines the amount of labeled 
material bound to the immobilized angiotensin I antibody. 
The total immunoactivity of the coated tube can be determined by following 
the foregoing procedure for radioimmunoassay but omitting the patient 
plasma. Samples of the coated tubes prepared as described in this example 
are found by this procedure to bind from 30% to 60% of the amount of the 
added radiolabeled angiotensin I. In contrast, when the glutaraldehyde 
cross-linking agent is omitted, being replaced by an equal volume of 
phosphate buffer, the procedure of this example produces a coated tube 
having a total immunoactivity less than 10% of the added radiolabeled 
angiotensin I, an amount so low that the coated tube could not be used for 
quantitative immunoassay procedures. On the other hand, pretreating the 
plastic tubes with glutaraldehyde, followed by introducing uncross-linked 
samples of angiotensin I antibody at the same 1/1000 dilution as above 
resulted in tubes which bound only 20% of the added radiolabeled 
angiotensin I. 
EXAMPLE 3 
One ml. of a rabbit anti-Angiotensin I serum is treated for 18 hours at 
4.degree. C. with an equal volume of 80% saturated ammonium sulfate, pH 
7.1. The precipitate is spun down in a refrigerated centrifuge for 30 
minutes at 3000 rpm and redissolved in 100 ml. of an isotonic 0.01M 
triethylamine buffer pH 8.5. To this solution is added 100 ml. of 0.0625M 
dimethyl suberimidate cross-linking reagent in the same buffer and the 
reaction mixture is incubated for one hour at 37.degree. C. Dilution of 
the reaction mixture with 800 ml. of 0.01M sodium phosphate, 0.01M sodium 
azide buffer, pH 9.0, yields one liter of coating solution. 
One ml. aliquots of the coating solution are carefully introduced into 
clean, dry polypropylene plastic tubes and allowed to stand undisturbed 
for 18 hours at ambient temperature (22.degree.-25.degree. C.). Aspiration 
of the coating solution terminates the coating process. After washing, as 
previously described, the tubes are dried and stored in sealed plastic 
bags at room temperature. 
Comparison of these coated tubes with tubes coated with angiotensin 1 
antibody without cross-linking using the radioimmunoassay procedure of 
Example 1 (except that a dilution of 1/1000 was used for both cross-linked 
and uncross-linked angiotensin I antibody) yields values of K=2 .times. 
10.sup.8 M.sup.-1 and Ab.sub.0 =1.3 .times. 10.sup.-9 M for cross-linked 
angiotensin I antibody and K=1.5 .times. 10.sup.8 M.sup.-1, Ab.sub.0 =4.1 
.times. 10.sup.-10 M for uncross-linked angiotensin I antibody. In the 
case of the cross-linked antibody, from 30% to 60% of the radiolabeled 
tracer is bound at minimal addition of unlabeled hormone, but only 10- 15% 
in the case of the uncross-linked angiotensin I antibody under the same 
conditions. 
EXAMPLE 4 
Hog gastric intrinsic factor is a binding protein for vitamin B-12 which, 
unlike rabbit gamma globulin, apparently does not adsorb to plastic 
surfaces in amounts sufficient to prepare a useful solid phase immobilized 
adsorbent. Howver, it is possible to prepare a solid phase immobilized 
vitamin B-12 adsorbent by using rabbit gamma globulin covalently bound to 
the binding protein. The modified gamma globulin will adsorb to plastic 
and carry with it the binding protein. 
Five hundred microliters of an isotonic 0.01M phosphate buffer, pH 6.0, 
containing rabbit gamma globulin at a dilution of 1:200 and 100 .mu.g of 
intrinsic factor is introduced into the bottom of a clean, dry 
polypropylene plastic tube. To this is added 100 .mu.l of 0.2M 
glutaraldehyde in distilled water. This mixture is allowed to react for 10 
minutes at room temperature. Five hundred microliters of a 1M pH 8.5 
phosphate buffer is added to raise the pH for more efficient coating. The 
tube is then coated for 1 hour at 37.degree. C., aspirated and washed 
twice with an isotonic 0.01M tris buffer pH 7.4 containing 0.1% gelatin to 
remove unadsorbed binding protein-antibody conjugate. After drying at 
ambient temperature the tubes are stored in sealed plastic bags. In this 
example, cross-linking and coating are carried out in the same tube. 
One ml. of a glutamic acid-potassium cyanide-gelatin buffer containing 5400 
cpm of .sup.57 Co labeled vitamin B-12 tracer is incubated in the binding 
protein-gamma globulin conjugate coated tube, prepared as described above, 
at 4.degree. C. for 18 hours. On aspiration, it is found that 32% of the 
added labeled tracer is bound to the tube. Deletion of any one of the 
three critical components in the coating solution, i.e., glutaraldehyde, 
intrinsic factor and rabbit gamma globulin results in tubes that bind 
indistinguishably from background, or about 2% of added tracer. 
EXAMPLE 5 
A 5 .mu.l aliquot of an antiserum to Vitamin B-12 is dissolved in 0.5 ml. 
of 0.1M pH 6.0 potassium phosphate buffer. To this is added 0.5 ml. of a 
0.4M solution of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide 
metho-p-toluenesulfonate in the same buffer. The reaction mixture is 
incubated for one hour at 37.degree. C. to allow activation of endogeneous 
carboxylate functions and their subsequent reaction with amino function to 
give cross-linking via intermolecular amide bonds. 
This reaction mixture is then diluted in an isotonic phosphate buffer, pH 
9.5, such that the original antiserum is at a dilution of 1/5000. One ml. 
aliquots of this solution are introduced into clean dry polypropylene 
tubes and allowed to stand for 18 hours at ambient temperature 
(22.degree.-25.degree. C.) to allow the polymerized gamma globulin to coat 
the surface of the tube. After coating, the tubes are aspirated and washed 
with a 0.1% gelatin solution in isotonic 0.01M tris buffer pH 7.4 to 
remove unadsorbed cross-linked gamma globulin. 
When tubes coated in this maner are assayed with 10,000 cpm of .sup.57 Co 
labeled vitamin B-12 tracer for 30 minutes at 37.degree. C., 26% of the 
added tracer is bound. If tubes are coated with the same antiserum that 
has not been treated with carbodiimide, only 18% of the added tracer is 
bound. Thus, the cross-linking of the gamma globulin solution with 
carbodiimide increases binding by 44%. 
EXAMPLE 6 
Ten mls. of a 1/100 dilution in isotonic saline of an ammonium sulfate 
fractionated digitoxin antiserum adjusted to pH 9.0 with sodium carbonate 
is treated with 21.4 mg. of sodium periodate. The cleavage of 1,2-diol 
functions of the sugar residues of the gamma globulin molecules to form 
aldehydes and their subsequent reaction with the amino functions of other 
gamma globulin molecules occurs at ambient temperature 
(22.degree.-25.degree. C.) in 12-18 hours, leading to cross-linked gamma 
globulins. After a 90-fold dilution in 0.01M tris buffer, containing 0.01M 
sodium chloride and 0.01M sodium azide at pH 8.5, one ml. aliquots of the 
1/9000 diluted cross-linked gamma globulin are introduced into clean dry 
polypropylene tubes and allowed to coat for 15 minutes at 37.degree. C. 
After coating the tubes are aspirated and washed with a 0.1% gelatin in 
isotonic 0.01M tris buffer pH 7.4 to remove unadsorbed cross-linked gamma 
globulin. 
When tubes prepared in the above manner are assayed with 11,000 cpm of 
.sup.125 I digitoxigenin derivative tracer in 1.0 ml. of tris buffer for 
45 minutes at 37.degree. C., 35.2% of the added tracer is bound to the 
solid phase immobilized cross-linked gamma globulin. If the same ammonium 
sulfate fractionated antiserum, without treating with sodium periodate, is 
used to coat tubes at a dilution of 1/9000 under identical conditions, 
only 12.4% of the added counts are bound in 45 minutes at 37.degree. C. 
Thus, cross-linking the gamma globulins with sodium periodate allows one 
to produce tubes that bind 300% more tracer than tubes coated with native 
gamma globulin at the same dilution. 
EXAMPLE 7 
An aliquot of an ammonium sulfate fractionated digitoxin antiserum is 
diluted 1/50 in isotonic 0.01M phosphate buffer pH 7.4 and heated in a 
glass vial at 75.degree. C. for 5-20 minutes. After heating, the solution 
is diluted 80-fold in a 0.01M tris, 0.01M sodium chloride, 0.01M sodium 
azide pH 8.5 buffer, and one ml. aliquots are introduced into clean dry 
polypropylene tubes for 15 minutes at 37.degree. C. After this incubation, 
the tubes are aspirated and washed with a 0.1% gelatin in isotonic 0.01M 
tris buffer pH 7.4. 
When these tubes were assayed with 11,000 cpm of an .sup.125 I 
digitoxigenin derivative tracer in 1.0 ml. of an isotonic 0.01M tris pH 
7.4 buffer, 42-45% of added tracer was found to be bound to the solid 
phase immobilized cross-linked digitoxin antibody on separation of bound 
and free by aspiration of the free tracer. If the same fractionated 
digitoxin antiserum is not heat treated, but otherwise handled in an 
identical manner, only 34% of added counts are bound to tubes coated with 
this antiserum. Thus, heat treatment increased binding by about 30%.