CA1106281A - Detection of antigen associated with hepatitis by "sandwich" method - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A sensitive direct immunoassay system is provided for the detection of an antigen associated with hepatitis in body fluids. An antibody which reacts with a hepatitis antigen or antigens and which is uniformly bonded to an insoluble member com-prising a polymeric material having irregular surfaces, is incubated with a test sample. During this first incubation, a portion of an antigen present in the test sample will combine with the antibody immobilized on the insoluble member. The antibody bonded member, to which antigen is attached, is then washed and incubated with an enzyme tagged antibody reagent comprising immunochemically reactive hepatitis antibody conjugated with a functional enzyme such as alkaline phosphatase.
During the second incubation, the tagged antibody reacts with antigen fixed to the insoluble member in the first incubation.
Thus, a "sandwich" is formed of an insoluble member- antibody-antigen- enzyme tagged antibody.
After the second incubation, the member is washed again to remove unreacted enzyme-antibody conjugate reagent. The member is then exposed to a substrate which is induced by the now immobilized enzyme to produce a detectable end product. The enzyme-antibody conjugate will be fixed in the second incubation only if antigen was present in the sample. The amount of enzyme tagged antibody fixed is proportional to the amount of antigen or antigens present in the test sample up to the maximum capacity of the test. The concentration of the end product, and hence the amount of antigen or antigens, is determined by a spectro-photometer which measures the optical absorption of light by the end product. This readout is then compared against a standard value for both antigen negative and antigen positive samples.
All reagents for the test may be conveniently fur-nished in the form of a kit which has an extended shelf life and greatly simplifies the procedure.

Description

~62~1 . .
This invention relates, in general, to a method for detecting the presence of antigens associated with hepatitis.
In particular, it relates to an immunoassay for antigens associated with hepatitis involving the use of an enzyme-tagged antibody which reacts with an antigen to detect the presence of hepatitis.
Hepatitis, which means "an inflammation of the liver", is due to an infection or obstruction of the bile channels.
There are thought to be two varieties of viral hepatitis, one hav-ing a longer incubation period than the other. In the past, when a patient contracted hepatitis and had a known parenteral exposure, the hepatitis was termed "serum hepatitis". If the patient did not have a known parenteral exposure and he con-tracted hepatitis orally it was called "infectious". However,~
it has been documented that in addition to having overlapping incubation periods, "infectious hepatitis" can be contracted parenterally, and, the so-called "serum hepatitis" can be contracted orally. Thus, although there would appear to be two forms of hepatitis caused by at least two distinct agents, the terms "serum hepatitis" and "infectious hepatitis" should not be used to distinguish them. Accordingly, it has been suggested that the terms "hepatitis A" be used to designate the form most closely resembling "infectious hepatitis", and, the term "hepatitis B" be used to designate the form most closely resembling "serum hepatitis".
The examples appearing in this specification are directed to the detection of the antigen or antigens associated with hepatitis type B. Patients who contract the form most 30 closely resembling serum hepatitis, no matter how contracted, ~ .

~1~62~1 1 often have these antigens in their blood. At this point, it should be noted that there is no reliable assay for determining the presence of an antigen associated with hepatitis A or a hypothesized hepatitis C. Thus, the examples in this specifi-cation are directed to the detection of the presence of those antigens associated with hepatitis B. However, there is no reason why the process of the present invention could not be used to detect the presence of antigens associated with other types of hepatitis once their antigens have been identified.
Contraction of "serum hepatitis" or hepatitis B
creates a serious clinical problem that cannot be ignored.
Because of the severity of this problem, a variety of test methods for the detection of hepatitis have been developed.
These included Micro-Ouchterlony, immunodiffusion, complement fixation, immunoelectro-osmophoresis, haemagglutination and haemagglutionation inhibition, electron microscopy, and solid phase radioimmunoassay. See British Medical Bulletin, 1972, Vol. 28, No. 2 (Viral Hepatitis) pages 138 - 141 for a brief description of each.
Immunoelectro-osmophoresis or counterelectrophoresis (CEP) provides a rapid, simple method for the detection of the hepatitis antigen and its antibody. However, this technique is somewhat less sensitive then, for example, complement fixation. Its principle advantage is that tests can be completed within two hours. However, because of its low sensitivity level, CEP is no longer approved by the Food and Drug Administration.
The application of radio-immunoassay (RIA) for routine diagnostic purposes is believed to be somewhat limited, not only because of the relatively complex, specialized, and expensive equipment necessary for conducting the test, but also ~3LG6281 1 because of the strict precautions required in handling radio-active isotopes. Isotope tagging presents a serious potential health hazard, requires monitoring, and Atomic Energy Commission licensing (for user and manufacturer), and presents waste disposal problems. Nevertheless, this technique is now rather well established for immunoassay.
Immunological methods depend, of course, upon a primary characteristic of all antibodies and antigens, i.e., their ability to react with a specific complementary antigen or 10 antibody. Thus, if an antibody is added to a serum containing its antigen, the antibody and antigen will complex and may precipitate from the solution. In most of the above-mentioned test methods, the presence of antigens in human sera is detected by making use of this simple fact.
Labeled antibodies have been used previously for identifying various antigens. If an antibody known to be specific for a particular antigen is isolated from the globulin portion of serum or plasma of a host animal which has been stimulated to produce that antibody, it can be labeled or tagged by known means. By conjugating the antibody with a labeling agent, e.g., a physically detectable substance such as a radio isotope, as above-mentioned, or fluorescent chemicals,`
the presence of the anitbody can be detected. Thus, when used diagnostically, if the counterpart antigen is present in some prepared test sample, the labeled antibody will attach itself to that antigen, and the presence of the antigen can be confirmed through detection of the labeled antibody in the sample.
A labeled antibody, for diagnostic purposes, should be made sufficiently specific so that it will react only with those antigens whose detection is desired and without cross-reaction 62~
1 with other closely related antigens which may have quite dis-similar or insignificant consequences. Thus, it is apparent that both the source and the manner of preparation of the anti-body is quite critical in any immunoassay.
One manner of detecting hepatitis associated antigen, as earlier mentioned, involves solid phase radio-immunoassay.
Such a precedure is disclosed in United States Patent No. 3,867,517.
As disclosed therein, in the performance of the assay, a tube well, or insert for use therewith, of molded polystyrene 10 is first coated with antibody. This is accomplished by incubating the member to be coated with an antibody solution.
Afterwards, the unknown sample is incubated with the coated well or insert to react the antibody with antigen present in the sample. The well is then washed and incubated with an antibody labeled with the radioactive isotope I-125. It is then again washed to remove any unbonded labeled antibody. Thus, in the event any antigen is present in the test sample, a sandwich is formed from the polystyrene well (or insert), the antibody, the antigen, and the I-125 tagged antibody. The radiation emitted from the I-125 tagged antibody is then counted and compared against a control.
It has also been disclosed that a disc of poly-tetrafluoroethylene onto which is grafted a substituted poly-styrene, e.g., isothiocyanostyrene, might be useful in performing radioimmunoassay. This polystyrene is an insoluble material having specifically designed surfaces of protein-reactive groups which may be used to covalently bond proteins to provide a reagent useful in bioassay procedures.
Recently, an important alternative to labeling anti-bodies with radio-isotopes or fluorescent chemicals has been 11~;36~

1 developed. This involves labeling or tagging an antibody with an enzyme. Such a procedure is described in U.S. Patents 3,654,090 and 3,791,932. In Clinica Chemica Acta, 48 (1973) 15-18, an enzyme-linked immunoassay for alphafetoprotein by either the competitive or the sandwich procedure is disclosed.
Compared to radio-immunoassay, enzyme labeling offers several important advantages. For example, every enzyme tagged molecule in the final mixture participates in the readout. On the other hand, only a very small proportion of isotopic atoms 10 in the final mixture undergo decay during readout to participate in the assay. In enzyme tagging, every tag repeatedly parti-cipates in readout, by attacking many substrate molecules to form a detectable end product, e.g., up to 100,000 times per minute, hence greatly enhancing sensitivity. An isotope tagged atom decays only once during readout, after which it is lost from participation. An enzyme tagged reagent has a long shelf life; whereas, the isotope tagged reagent is constantly decaying, and presents serious shelf life problems. While there is minimal health hazards associated with using an enzyme 20 tagged reagent, serious potential health hazards are encountered with isotope tagging. Lastly, an immunoassay involving enzyme tagging can use simple, relatively inexpensive equipment for readout. The success of isotope immunoassay, in contrast, is dependent on the efficiency of detecting decay, and hence on the quality of very expensive detection equipment.
Although the finding of an antigen associated with hepatitis in one's blood may not be the equivalent of obtaining a clinical history of hepatitis, investigations have revealed a high incidence of hepatitis infection when a patient has received blood which tests positively for antigens associated with ~281 1 hepatitis. Since decisions on whether to use particular blood units available from a blood center must often be made in a realtively short time period, a sensitive,rapid, easy to perform screening test ~or hepatitis, without need for expensive equipment, is of extreme importance. Although the various tests used in the past for the detection of the antigens associated with hepatitis have been satisfactory to a degree, they are all attendant with one or more disadvantages.
Each year hepatitis causes thousands of deaths and hospitalizations. It has long been thought that the key to bringing the disease under control would be a technique for screening blood which could be made available world-wide and could be conducted simply and routinely. While, as indicated above, there are now a number of techniques available, because they are relatively insensitive, require a lengthy and detailed procedure, require the use of sophisticated equipment not readily available in most hemotology labs, or require the use of reagents which are highly unstable and thus cannot be main-tained at-hand, they have not satisfied the demands of the ideal test.

SUMMARY OF THE INVENTION

The invention broadly involves a direct immunoassay for antigens associated with hepatitis involving the "sandwich"
principle. In the performance of the assay, antigen to be detected is sandwiched between antibody layers which react with it. One antibody layer is labeled with an enzyme. The other layer is covalently bonded to an insoluble member. The enzyme is exposed to a chemical substrate which will undergo a chemical change to produce a reaction product in the presence of the enzyme catalyst. The presence of hepatitis is determined .

~1~362~1 1 by determining the presence of the reaction product.
In accordance with another aspect of the invention, a hepatitis detection kit or set is provided. The three main reagents of the test set are the insoluble polymeric solid having antibodies reactive with antigens associated with hepatitis bonded thereto, enzyme tagged hepatitis antibody reagent, and an enzyme substrate capable of being chemically changed under the catalytic influence of the enzyme to form a detectable end product. In the preferred embodiment, the enzyme of the con-jugate is an alkaline phosphatase and the enzyme substrate isp-nitrophenylphosphate. The test set may also contain control sera including sera negative, weakly positive, and strongly positive for hepatitis antigens, a solution of a horse globulin additive for the test sample for minimizing the frequency of non-specific reactions between the test sample and the insoluble polymeric solid, a buffer designed to maintain the pH of the enzyme substrate solution in the optimum range for reaction, and a plurality of vials of a size calculated to promote contact between the insoluble solid and the small quantities of reagents used in the incubations. When alkaline phosphatase and p-nitrophenylphosphate are used as the enzyme-enzyme substrate system, the buffer is an aqueous solution 0.02 8M in Na2C03 and 0.001 M in Mg In another aspect of the invention, an immunologically active purified hepatitis antibody conjugated, i.e., chemically linked, with a functional en2yme such as alkaline phosphatase is provided for use in the immunoassay.
In still another aspect of the invention, a disc-like insoluble member or matrix is provided for use in the immunoassay.
The matrix has a plurality of groups reactive with proteins grafted uniformly to its surface layer. Purified hepatitis il~6281 1 antibody is covalently bonded to the reactive groups to provide an exterior layer of hepatitis antibody.
In the immunoassay, the insoluble member is placed in a flat-bottomed vial and just covered with a liquid test sample.
For the test to be reproducible, it is important that the antigens in the test sample be exposed to the entire surface of the antibody coated discs. It has been discovered that use of a flat or planar surfaced disc resulted in loss of sensitivity.
~ Accordingly, the two opposed surfaces of the disc of the invention are distorted to an irregular configuration to reduce the disc surface-vial bottom contact. In one preferred embodi-ment, the opposed surfaces are rendered waffle-like by passing the disc through a press prior to attachment of the antibody.
Accordingly, it is a primary object of the invention to provide a hepatitis detection kit and a procedure for its use such that the presence of antigens associated with hepatitis~
may be rapidly, simply, accurately, and routinely determined in body fluids.
Another object of the invention is to provide a unitized test set which is adapted for the performance of a sensitive, reproducible immunoassay of antigen& associated with hepatitis on a routine basis by relatively unskilled persons.
Another object of the invention is to provide such a test set which is designed to minimize procedural errors in the performance of the immunoassay and which contains all the necessary reagents, reaction containers, etc. in a form designed to optimize the accuracy and sensitivity of the test.
Another object of the invention is to provide a test set containing laboratory equipment designed to standardize the various steps performed during the assay.

1~6%81 1 Still another object of the invention is to provide a test set containing standard control samples, negative, weakly positive, and strongly positive for hepatitis associated antigen, with which the test samples may be compared.
Another object of the invention is to provide a hepatitis antibody-enzyme conjugate for use with the immunoassay wherein both the antibody and the enzyme retain their respective desired chemical characteristics.
A further object is to provide such a conjugate which can be stored for extended periods without losing its immunological or catalytic characteristics and which is highly reactive with antigens associated with hepatitis.
Still another object of the invention is to provide hepatitis antibody conjugated with an enzyme which is capable of catalyzing a reaction to produce an end product at a high reaction rate, the concentration of the end product being precisely detectable using a photometric detector.
Another object of the invention is to provide a conjugate as disclosed above which is highly purified, and thus contributes heavily to promoting sensitive and accurate immuno-assays of antigens associated with hepatitis.
Another object of the invention is to provide an insoluble matrix useful as described in the above-mentioned test which has a high concentration of purified, hepatitis associated antibody distributed uniformly over its entire surface.
A further object is to attach hepatitis antibody to a solid such that it will not be dislodged by mechanical or chemical forces to which it may be subjected during use. In this regard, it is imperative to effect the attachment such that the antibody remains functional, i.e., capable of participating in its immunological reaction and immunochemically unaltered _ g _ 1~62t31 by its attachment.
A further object of the invention is to provide an improved shape for an insoluble antibody coated solid which is easy to handle during immunoassay and which is designed to expose a constant and a greater portion of its coated surface to the solutions used during incubations.
It is a further object of the present invention to provide a process for determining the presence of antigens as-sociated with hepatitis in a sample comprising the following steps:
(a) incubating the sample with an antibody immobilized on an insoluble member, the antibody being reactive with the ant-igens associated with hepatitis, the incubation enabling a bond to be formed between the antibody and an antigen present in the sample to produce an insoluble member having an antigen bonded thereto in the event antigens associated with hepatitis are present in the sample;
(b) separating the insoluble member from any unbonded substances;
(c) incubating the insoluble member with a solution con-taining tagged antibody reactive with an antigen associated with hepatitis, the tagged antibody being tagged with an enzyme capable of effecting a reaction of a substrate to produce a detectable reaction product, the incubation being conducted to enable the tagged antibody to bond to any antigen bonded in step (a) to the antibody on the insoluble member;
(d) separating the insoluble member from the enzyme tagged antibody solution to remove any unbonded tagged anti-body therefrom;

(e) exposing the insoluble member to a substrate solution 11~362~31 which the enzyme of the tagged antibody reacts upon to enable a chemical change in the substrate to take place and to produce a detectable reaction product; and (f) detecting any reaction product present in the sol-ution to determine the presence of antigens associated with hepatitis.
These and other objects and features of the invention will be apparent to those skilled in the art from the following description of a preferred embodiment.

BRIEF DESCRIPTION OF THE DRAWING
Fig. 1 is a flow chart summarizing the steps of the process of the present invention;
Fig. 2 is a perspective view of the preferred insol-uble member used in the test set and the process of the present invention;
Figs. 3, 4 and 5 depict three incubation stages of the immunoassay of the present invention; and Fig. 6 is a schematic representation showing the "sandwich" structure developed during positive immunoassay of a test sample tested in accordance with the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT
At the outset the invention is described in its broadest overall aspects with a more detailed description fol- -lowing. The method of detecting hepatitis according to the present invention is carried out in four distinct stages as is illustrated in Fig. l.
The first stage involves reacting the immobilized anti-body on the insoluble member l0 (see Fig. 2) with an antigen present in the test serum as is shown in Fig. 3. This reaction - 10a -11~6281 immobilizes antigen so that on reaction in the next stage with an enzyme tagged antibody, as is shown in Fig. 4, the tagged antibody is also immobilized. As is shown in Fig. 5, the enzyme is exposed to a suitable substrate which is reacted upon by the enzyme to produce changes in colour. The colour change acts as an indication of the presence of antigens associated with hepatitis. The last stage involves reading the degree of colour and comparing the value obtained with a standard of control.
The hepatitis detection test set of the instant invention which contains all materials needed in the test out-lined above is designed for 100 tests. Obviously, larger or smaller sets may be manufactured by proportionally increasing or decreasing the quantities of reagents, etc. disclosed herein-after. The kit is distributed, in, e.g., a box, containing reagents and controls to be stored at 2-8C until used. In addition, vials sufficient for conducting 100 tests are provided.
One hundred to one hundred and five polymeric discs, having waffle-like surfaces, as shown in Fig. 2, and having a layer of antibody reactive with hepatitis antigens bonded to their exterior and lyophilized are supplied. The exact nature of these insoluble solid discs and their method of manufacture are disclosed hereinafter.
The second reagent in the set comprises a sample of hepatitis associated antibody-alkaline phosphatase conjugate.
The exact nature of this reagent, the method for its preparation and alternative useful reagents are disclosed hereinafter.
This reagent is supplied as a solution ready for use.
A third reagent supplied in the test set is 400 mg of p-nitrophenylphosphate enzyme substrate. This compound is stable in its powdered form, but, when dissolved in buffer to 11~62~31 1 form a 1 mg/ml solution, becomes relatively unstable. Con-sequently, the solution must be prepared just prior to use.
The other materials used duxing the procedure include a quantity of horse globulin, sample diluent, various buffer solutions, and various wash solutions. The exact nature and method of preparation of these reagents and materials are indicated below.

A. Preparation of Antibody In order to practice the present invention, it is necessary to obtain antibody that will react with an antigen or antigens associated with hepatitis. It should be noted that such an antibody exists, and thus, the present invention is not intended to be limited to the use of any particular antibody.
A reactive antibody may be prepared by purifying bleeding from a host animal which has been injected with a known sample of antigen. An antibody which is reactive with an antigen associated with hepatitis may, in general, be prepared by the process disclosed in British Patent No. 1,387,625.
The preparation of hepatitis antibody depends first of all on obtaining blood which is known to be positive for antigens associated with hepatitis. Consequently, blood units obtained from various sources must first be evaluated to determine their suitability for preparing the immunospecific purified antibody of the invention.
A blood bag segment, containing blood which is believed to be positive for hepatitis antigens, is held at 2-8C in an upright position to allow the blood cells to settle to the lower half. The plasma is separated from the cells and a titer is run on the undiluted plasma and a sample diluted 1:16 in normal saline against a standard antibody by the well-known technique ~62~31 1 of counterlectrophoresis (CEP). When both the undiluted plasma sample and the 1:16 diluted sample are positive, the blood unit is considered acceptable for use in the preparation of the purified antibody of the invention as described hereinafter.
Portion of antigens isolated from blood units are used for either stimulating antibody production in a host animal or purifying the antibody produced by that animal. The antigens must be subjected to a preliminary isolation process prior to either of the above uses.
The plasma is transferred to a sterile vacuum container and clotted by adding a 5M solution of a CaC12 on the basis of 0.75 ml CaC12 per 200 ml of plasma. This solution is then in-cubated at 37C in a water bath for one hour or until a clot forms. After a firm clot forms, the plasma is frozen at -20 C
and allowed to thaw at 2-8C to allow clot retraction. The serum is separated from the clot and filtered if necessary, then is ready for preparation of a hepatitis antigen pellet for immunization or for use in lmmunoabsorbent columns.

(1) Preparation of Antigen Pellet for Immun ation Hepatitis positive sera which have been subjected to the above process are centrifuged at 10,000 rpm for 30 minutes at 4C. The supernatant of this centrifugation is distributed into ultra centrifuge tubes and centrifuged in, for example, a Beckman L2-65B ultra-centrifuge, at 40,000 to 50,000 rpm for 4 - 20 hours at 4C. The supernatant in each tube is removed and discarded; the pellet, which contains antigens, is given - a preliminary rinse with normal (.15M) saline.
A small volume of normal saline is then added to each centrifuge tube and the contents are subjected to sonication to break up the pellet. The suspensions in the sonicator tubes ~1~62~31 1 are then pooled and redistributed equally into clean tubes which are filled with normal saline. This solution is again centrifuged in the Beckman L2-65B centrifuge at 40,000 to 50,000 rpm for 4 - 20 hours at 4C, as mentioned above.
The procedure in the preceeding paragraph may be repeated 5 or more times.
The pellet material, after removing the supernatant from each centrifuge tube, is pooled in the minimal volume of normal saline. A sample is assayed against a standard hepatitis antibody. If the pellet titers at 1:25 or higher by CEP, it can be used for immunization. The pooled antigen pellet may be divided into 3 ml aliquots and frozen at -20C for future use.

(2) Production and Preliminary Purification of Hepatitis Antibody A sample of the antigen pellet, prepared as described above, is added to an equal volume of FREUND'S complete adjuvant the morning of the immunization and an emulsion is prepared in accordance with procedures well known to those skilled in the art. The antigen is then injected into a host animal, e.g., a horse, in accordance with techniques known per se, to produce hepatitis antibody. The immunized horses are bled or subjected to plasmaphoresis according to conventional techniques. Alter-natively, or in addition, preparations without adjuvant can be used with other routes of immunization.
These bleedings must be treated to isolate the hepatitis antibody in anticipation of the final immunoabsorption purification step. Broadly, this preliminary purification is accomplished in three steps. First, plas~a from the host animal is recalcified. Second, the serum is mixed with a sufficient amount of normal human plasma (NHP) to precipitate antibodies other than those associated with hepatitis by inducing insoluble 1~36~
1 antigen-antibody complex formatibn. The absorbed antiserum is assayed for hepatitis antibody using CEP. Third, -the antibody reactive with antigens associated with hepatitis is precipitated with ammonium sulfate. This material can he frozen until used.

(3) Pre~ tion of Charcoal Immunoabsorbent Column The preparation of the purified antibody which is used to produce the reagents of this in~ention is accomplished by subjecting the henatitis antibody produced as disclosed above to an immunospecific extraction process. For a general discussion of this procedure, reference should be made to sritish Patent No. 1,387,625 to Bradish et al., March 19, 1975, entitled "Immunospecific Separation of Antigens and Antibodies" ~
In general, this purification process as utilized in the present invention, takes advantage of the ability of anti-bodies reactive with hepatitis antigens to complex with these antigens to the exclusion of other extraneous antibodies and proteins which are inevitably present in the antibody sample extracted from the bleedings.

A column is prepared by packing prewashed, sorbent carbon into a glass or plastic tube by using conventional techniques. A pool of antigen is prepared from at least six individual serum specimens to obtain a diverse mixture of hepatitis antigens. The pool is then adjusted to a protein concentration of between 1 and 2 mg protein per ml solvent, based on UV absorption.
To attach the antigen onto the carbon, this diluted solution is introduced at a flow rate within the range of 300 to 1,000 ml per hour. Seventy five mg protein should be added per gram of charcoal in the column. The effluent from the column is ~1 2~

1 collected in 500 ~1 aliquots, each of which are checked for protein content. The column is considered saturated with the antigen when the effluent has a proteln content approximately equal to that of the starting material. The bed is washed by flowing phosphate buffered saline (PBS) through the column until the effluent shows no appreciable detectable absorption at 280 nm.
To elute any loosely attached protein, the charcoal bed is flushed with freshly prepared 5M sodium iodide solution containing 200 mg per liter of sodium thiosulfate. Afterwards, the sodium iodide solution is flushed from the column by running a sufficient volume of PBS therethrough. After a final washing of the bed with PBS containing 1 mg per ml sodium azide (preservative), the column can be stored at 2-8C until ready for use.

(4) Preparation of Antibody for Use in Enzyme Conjugate and Insoluble Solid The antibody, purified as described above, is freed of ammonium sulfate and diluted with PBS on the basis of 1 part antibody to 2 parts buffer. The column is set up and situated so that fractions can be collected. The antibody solution is added continuously to the column with a flow rate of about 200 ml/hr. Effluent is collected and tested for protein and hepatitis antibody content to determine when the column is saturated with antibody. After saturation is achieved, the ~ -column bed is washed with PBS to remove loosely absorbed protein.
At this point, antigens immobilized on the charcoal column have formed a bond with the antibodies reactive with them. `~
Other extraneous proteins and antibodies, nonspecific to the absorbed antigens having passed through the coLumn and have been separated from the antibody.

28~ ( To break this antibody-antigen bond, and to elute the purified antibody, a 5M solution of NaI prepared immediately prior to use is introduced into the column. The volume of NaI
solution used should be sufficient to remove all antibody bound to the column. With the flow rate of the column set at least 200 ml per hour, the eluate is collected in fractions of appro-priate volumes. The total amount collected should be at least equal to the volume of sodium iodide solution added.
As each fraction of the purified antibody is collected, 1~ it is subjected to a dual filtration; first, through a 0.45 p membrane, and second, throuyh a 0.22 ~u membrane. The filtrate is diluted 1:3 using distilled water at 2-8C, e.g., 200 ml of filtrate is added to 400 ml of distilled water. These diluted antibody fractions are then added to, for example, an AMICON*
concentrator equipped with an XM-50*membrane, and the fractions are concentrated.
As a last purification step, the concentrated, purified, antibody is dialyzed. Following dialysis, the antibody is removed and centrifuged. The supernatant is dialyzed for at least 24 hours against 0.01M sodium phosphate solution, as com-pared to against PBS in the first dialysis. After completion of this final dialysis, protein concentration of the antibody is measured.
The antibody is then assayed for activity against the standard antigen according to the CEP technique to determine antibody content and, if found acceptable, is lyophilized and stored until used.

B. Preparation of_Anti ody-Enzyme Conjugate -~

Calf intestinal alkaline phosphatase is mixed with a solution of the reconstituted antibody in a ratio of 3:1, enzyme * Trade Marks - 17 -1~62t31 1 t:o an-tibody, -to a final concentr--tion c;reater than 10 mg of to-tal protein per ml o~ solution in PBS (pEI 7.4). Th~ solution is dialyzed thoroughly to remove NE~4 ions.
The dialyzed antibody enzyme mixture is then centri-~uged to remove any insoluble material. The pro-tein content of the supernatant is adjusted to 10 mg/ml by adding the PBS-Mg solution. To this solution, 8% glutaraldehyde is added on the basis of 1 ml gluteraldehyde solution per 10 ml antibody-enzyme solution. After stirring slowly for 3.5 to 20 ~ minutes, during which time the antibody and enzyme are chemically linked by the glutaraldehyde, the solution of conjugate is dialyzed against PBS containing O.OOlM Mg to remove the glutaraldehyde.
The dialyzed material is then centrifuged and the - supernatant is diluted by addition of an aqueous solution of 0.05M in tris(hydroxymethyl) aminomethane (pH 8) buffer, 1%
normal human albumin (crystalline), .02% NaN3, and .OOlM in MgC12.

In addition to alkaline phosphatase (EC 3.1.3.1), other enzymes are useable in the procedure of the present in-vention. Indeed, there is an almost limitless list of enzymes which can be covalently bonded to the antibody. Of the various ;~
enzymes, the following table indicates enzymes of particular interest.

1. alcohol dehydrogenase EC 1.1.1.1 2. glycerol dehydrogenase ~ EC 1.1.1.6 3. glyoxylate reductase ~ EC 1.1,1.26 4. L-lactate dehydrogenase - EC 1.1.2. 3

5. malate dehydrogenase ~ EC 1.1,1. 37

6. glucose 6-phosphate dehydrogenase ~ EC 1,1.1.49

7. mannitol l-phosphate dehydrogenase ~ EC l~lel~17

8. glucose oxidase - EC 1.1.3.4

9. galactose oxidase - EC 1.1. 3 ~ 9

10. L-amino acid oxidase ~ EC 1.4.3.2 - ~8 -,,,., ~
5''' ~7 ~ ' 2~31 1 11. D-amino acid oxidase - EC 1.4.3.3 12. polyphenol oxidase - EC 1.14.18.1 13. ascorbate oxidase - EC 1.10.3.3 14. catalase - EC 1.11.1. 6 15. peroxidase - EC 1.11.1. 7 16. cholinesterase- EC 3.1.1.7 17. phospholipase C - EC 3.1.4.3 18. ~-amylase - EC 3.2.1.1 19. lysozyme - EC 3.2.1.17 20. ~-galactosidase - EC 3.2.1.23 21. amyloglucosidase - EC 3.2.1.3 22. ~-glucuronidase - EC 3.2.1.31 23. carboxypeptidase A - EC 3.4.12.2 24. urease - EC 3.5.1.5 25. inorganic pyrophosphatase - EC 3.6.1.1 26. aldolase - EC 4.1.2.13 27. carbonic anhydrase - EC 4.2.1.1 28. histidase - EC 4.3.1.3 The enzyme that is used for tagging the antibody is selected with several considerations in mind. These consi-derations include the stability of the enzyme, the ease of assay of the enzyme, the ability of the enzyme to withstand the conditions of the covalent bonding of the antibody, the availabili-ty of the enzyme, and the cost of the enzyme.

C. Preparation of Antibody Coated Insoluble Member ~ . :
The next step in the preparation of the reagents isto covalently bond a portion of the purified antibody to an insoluble member. To effect this bonding, the insoluble member used must be provided with reactive groups or sites capable of reacting with the specific antibody used in the bioassay.
U.S. Patent No. 3,700,609 entitled "Graft Copolvmers", to G.W. Tregear et al., discloses an insoluble continuous polymeric substanae eompris7 ng a ~olymeric bac~bone onto which side chains Z~l 1 of another polymer or copolymer are grafted. sy suitable choice of the grafted polymer, it is possible to chemically link biolog-ical substances to the insoluble matrix. A product which is disclosed in the above patent is commercially available in a disc form under the tradename PROTAPOL DI/l from Imperial Chemical Industries of Australia and New Zealand (ICIANZ).
The PROTAPOL DI/l comprises a polytetrafluoroethylene backbone having isothiocyanopolystyrene groups grafted uniformly over its surface and is designed for use in radioimmunoassay.
The discs, as presently available, are approximately .01 inches thick and 0.5 inch in diameter.
In accordance with one important embodiment of the matrix of the present invention, as shown in Fig. 2, each disc 10 is provided with a waffle-like pair of surfaces 11 comprising a first series of linear ridges 12 and a second series of linear ridges 14 which form grids. Ridges 12 and 14 are preferably perpendicular to each other and hence define a plurality of square depressions 15. The sides of each ridge 12 and 14 taper upwardly from adjacent pairs of depressions 16 to form a line defining the top of the ridge. It should be noted that in order to facilitate the description of the invention, the ridges 12 and 14 are greatly exaggerated in the drawing.
The desired configuration of the disc is achieved by passing the disc through rollers having projections on the surface of the rollers designed to impart the desired configuration on the disc. As is obvious, the rollers are designed to provide a sufficient amount of pressure to disfigure the polymerlc material in the disc without actually puncturing the disc. This is important because the disc has a reactive layer on its surface.
Thus, penetration of the disc would expose interior portions ~36Z81 1 to which no antibody can be bonded. Exposure of the poly-tetrafluoroethylene layer would actually result in a disc which would have a lowex bonding capaci-ty.
The main consideration is to provide a disc matrix with surfaces which, when placed in a flat bottomed vial, will be substantially in complete contact with the test sample, i.e., there should be a minimum of surface-to-surface contact between the matrix and the bottom of the vial. In another important embodiment of the disc of the invention, the disc's surfaces are configured to have a field of high and low points.
The antibody, produced as disclosed above in the lyophilized form, is reconstituted by adding 100 ml of O.lM
NaHC03 (pH 9.6) for each 5.0 mg of antibody. In general, the procedure for attachmen-t involves contacting the waffled discs with the dilute solution at 2-8C for 8 to 16 hours, with agitation. Afterwards, the antibody solution is discarded and the discs are washed twice with successive volumes of O.lM
NaHC03, pH 9.6, phosphate buffered saline, and cold (2-8C) 0.3% bovine serum albumin in phosphate buffered saline with 0.5% TWEEN*20. After an additional washing with crystalline bovine serum albumin, and freezing over dry ice, lyophilization is carried out and the discs are stored at 2-8C until ready for use.
Although the description in this specification relates to the preparation of discs having hepatitis antibody bonded thereto, it should be apparent to those skilled in the art that the disc of the present invention is useful to immobilize an almost limitless number of proteins. For e~ample, the increased -contact between the test sample and the disc enables the disc to be used in tests which involve the bonding of the following * Trade Mark ~,J

6Z~

1 proteins thereto: antibodies to drugs such as digoxin, opiates, and steroids; antibodies to natural products, for example insulin and other hormones; and specific enzymes to metabolities found in blood and other body fluids.

Exam~

The following procedure was used to prepare 8,000 discs, each of which were first treated with the press to produce the desired configuration as described. A batch of 8,000 discs requires 40 mg of hepatitis antibody, i.e., 5 mg per 1,000 discs.
The protein content of the reconstituted hepatitis antibody is adiusted to .05 mg/ml in a final volume of 800 ml in O.lM
NaHCO3 (pH 9.6). The entire 800 ml of buffered antibody is then added to a 1,000 ml screw-cap bottle provided with a leak proof liner containing the 8,000 discs, and the bottle is rotated for 16 hours, e.g., overnight, at 2-8C to slowly tumble the discs through each rotation cycle. Afterwards, the liquid is poured from the bottle and discarded and the discs are transferred to a wide-mouth 2 liter flask.
The discs are washed twice with successive 1 liter volumes of cold t2-8C) O.lM NaHCO3, pH 9.6, following which the buffer is removed. The-discs are then washed again, this time using two successive 1 liter volumes of cold buffer (O.OlM
sodium phosphate, 0.15M NaCl, pH 7.4). After removing residual buffer, the discs are washed for a third time, using two successive one liter volumes of cold bovine serum albumin solution tO.3%).
The discs are finally washed with two successive 1 liter volumes of a solution of cold crystalline bovine serum albumin (pH 8) at a concentration of 2 mg/ml. This step is performed to provide a protein environment for the protein on

11~36281 1 the disc. The cliscs, aEter removing the residual wash, are then transferred to dishes or trays (9 x 9"), each of which is lined with a sheet of filter paper and each of which contains 200 ml of -the crystalline bovine serum albumin solution. When the transfer is complete, a sheet of filter paper is used to cover them. suffer is thoroughly removed. The discs are then quick frozen for 30 minutes on dry ice.
The contents of the tray are then lyophilized and the dry discs are removed and stored in stoppered containers.
0 (1) Preparation of Control Serum In order to obtain meaningful data from the immuno-assay, it is necessary to provide negative and positive control sera for proper comparison with a given test sample. The preparation of these controls is disclosed in detail below.
The negative control is made from human plasma which has been tested and found negative for hepatitis associated antigen, by, for example, the radioimmunoassay technique.
To each unit that is clearly negative for hepatitis antigen, 5M CaC12 is added to induce clotting, on the basis of 0.75 ml of CaC12 solution per 200 ml of plasma. This plasma is then incubated at 37C in a water bath until a clot forms. The clotted plasma units are then frozen at -20C and stored for at least 12 hours. The plasma units are then allowed to thaw at 2-8C and the serum is collected. If the serum appears excessively turbid, it may be desirable to clarify it by centrifugation, e.g., at 9,000 rpm for 30 minutes at 2-8C.
Twenty grams of silica, e.g., AEROSIL-380, is added per liter of serum and mixed for 2 hours at room temperature to remove lipoproteins and stabilize the serum. The mixture is then centrifuged and the precipitate is discarded. If desired, * Trade Mark '"'`'1 1 s ~ ,, 11~62~31 f the silica can be removed by filtrat:ion through appropriate filter media.
The supernatant is then further processed by filtration through, for example, MILLIPORE or HORM membranes and pads of successively decreasing porosity, the last being a 0.45 micron membrane. Before filtration through the 0.45 micron membrane, sodium azide (NaN3) is added to the liquid in sufficient amount to provide a concentration of 0.1% by weight. As is well known in the art, sodium azide acts as a preservative. The final filtration, through a 0.45 micron or smaller porosity filter, should be done in a laminar flow environment using sterile equipment and techniques.
The sterile solution may be then stored at 2-8C until ready for subdivision into reagent sized containers for use in the immunoassay. In the preferred, 100 test set, 7.5 ml of negative control serum are provided.
The positive control serum is produced from recalcified plasma from blood units which test positively for antigens associated with hepatitis. From each positive unit, a 1%
sample is taken, and these are pooled together to form a trial pool. The trial pool is first heat treated for 10 hours at 60C to inactivate any hepatitis causative agent in the sample.
When the pool has cooled to room temperature, a portion is removed and titrated against a standard antibody using the CEP
technique to check that the antigen activity has been retained.
To the trial pool is then added a sufficient amount of silica to provide a concentration of 20 g per liter of serum. The serum is then stirred using a stirring bar at room temperature for two hours after which it is centrifuged at 9,000 rpm for 30 minutes at 2-8C. The precipitate is discarded.

1~628~L

1 The supernatant is then titrated using CEP against a standard reference antibody. If the titer has remained at satisfactory levels, the total volume of all serum units may be pooled together and subjected to the same process as just ~ -described for the trial pool.
The trial pool and main pool are then combined and diluted with a sufficient amount of negative control serum to obtain optimum results with the positive control serum in the immunoassay of the invention. Preferably, the reading of the positive control serum in the test of the invention, in absorbence units x 1000, should be greater than 2000. This diluted positive control serum is then filtered through suitable media as before described, using a successive range of decreasing porosity. Before the final filtration through a 0.45 micron or smaller membrane, 0.1~ by weight sodium azide is added. As with the negative pool, the final filtration should be done under aseptic conditions in a laminar flow environment.
The weakly positive control serum may be made by diluting the strong positive control with negative serum. The reading of the weakly positive serum as determined by the test of the invention should be between 600 and 1000. In the 100 test set of the preferred embodiment of the invention, 2.5 ml of strong positive control and 2.5 of weak positive control are supplied.

D. Miscellaneous Reagents and Equipment Two hundred or more glass, disposable, flat bottomed vials are supplied which have a diameter slightly greater than .5 inch, i.e., sized to match the 0.50 inch diameter of the discs. One hundred of these vials are used for the initial incubations and washings of the test, the other 100 are employed ;%8~.

1 for the final incubation with the enzyme substrate. Accordingly, the test set enables 100 assays to be conducted simultaneously.
As set forth in more detail below, before one insoluble solid disc is added to each of the 100 vials, a 0.05 ml portion of horse globulin test sample diluent is added to each vial. This step is taken as a precaution to eliminate non-specific reactions in the first incubation of the test samples with the insoluble discs. Although the antibody coated on the disc is purified and highly reactive with hepatitis antigens, there is occasionally present in human serum or plasma a substance capable of reacting with horse globulin per se that can thus form a bridge between the disc and the enzyme labeled conjugate, thus resulting in a false positive reaction. The horse globulin added in the first step binds this substance so it is not free to react with the disc. As disclosed above, the antibody coated on the disc is produced by immunizing a horse with hepatitis associated antigen collected from human blood. In the 100 test set of the invention, a 5.5 ml solution comprising 330 mg of horse globulin dissolved in phosphate buffered saline (PBS) is supplied.

F. Preparation of Substrate The preferred substrate for the enzyme reaction in the test is p-nitrophenylphosphate which is dissolved to a con-centration of 1 milligram per ml in sodium carbonate buffer, the concentration of which is 0.028 molar sodium carbonate and 0.001 molar magnesium (pH 9.8).
At this point, it should be noted that other substrates with suitable pH buffering agents, as appear in Table I below, may be used.

~1~62~1 _ ~-glycerol phosphate (serum), 7.4 ~-glycerol phosphate barbital, 8.6 phenyl phosphate carbonate-bicarbonate, 9-10 ~-naphthyl phosphate barbital, 9.1 p-nitrophenyl phosphate 2A2MlP, 10.25 phenolphthalein phosphate 2A2MlP, 9.90 p-nitrophenyl phosphate 2A2MlP, 10.17 thymolphthalein phosphate carbonate-bicarbonate, 10.0 p-nitrophenyl phosphate diethanolamine, 9.8 4-methylumbelliferyl phosphate carbonate-bicarbonate, 9. 2 The substrates indicated in the Table are all organic phosphate esters. It should be apparent that other organic phosphate esters could be used as substrates for the preferred enzyme, alkaline phosphatase. Furthermore, it should be noted that th~e skilled in the art will have little difficulty selecting a suitable substrate if an enzyme other than alkaline phosphatase is used in the antibody-enzyme conjugate.

G. Procedure To perform the assay according to the invention, 100 vials are set out in racks and each is identified to correspond to a test sample. To each vial is added 0.05 ml of the horse globulin solution, then 0.5 ml of sample is added to 95 of the vials. At the same time, three 0.5 ml samples of negative control serum are placed in each of three vials, a 0.5 ml sample of strong positive control serum is placed in one vial, and a 0.5 ml sample of weakly positive control serum is placed in another vial. To each vial containing the horse globulin and sample, including the control vials, is then added 1 antibody coated disc. The vials, with contents, are incubated for 0.5 hours at 43C in, e.g., a shaking water bath. During this 11~62~31 1 incubation, hepatitis antigens present in the test sample or con-trols will combine with the antibody on the disc.
Prior to the addition of the enzyme-tagged antibody reagent to the vials containing the insoluble members, the super-natant - from the first incubation must be removed and the insoluble members must be washed to remove any unbonded antigen.
The wash solution is preferably a 0.85~ solution of sodium chloride, pH 6.5 - 7.5. After two 2.5 ml washes using this solution, 0.3 ml of the antibody-enzyme conjugate is added to each vial, and the vials are again incubated at 43C for 1 hour with shaking, during which time the enzyme tagged antibody will react with the hepatitis antigens that were fixed to the antibody coated disc during the first incubation.
After addition of the enzyme tagged antibody reagent and a second incubation, the supernatants are aspirated off and the discs in each vial are washed three times with 2.5 ml aliquots of wash solution. This removes unreacted enzyme-antibody conjugate. Each insoluble member is then transferred to a clean vial and 2.5 ml of p-nitrophenylphosphate enzyme substrate-buffer solution is added to each vial ~1 mg pNPP per ml). Since the optimum operational pH of the alkaline phosphatase-p nitrophenyl-phosphate system is 9.8, the enzyme substrate is dissolved in carbonate-Mg++ buffer ~pH 9.8 + .1). This buffer as used comprises an aqueous solution 0.028M in Na2C03 and O.OOlM in Mg++. Forty ml of concentrate may be supplied with the set which, when diluted to 400 ml with distilled water, may be added directly to the 400 mg of pNPPo After addition of the buffered substrate, the vials are subjected to a third incubation for one hour at 43C with shaking. Different substrates must be employed if a different enzyme antibody conjugate is used.

., : :

llG6Z81 1 Two drops (0.1 ml) of 3M NaOH solution are then added to each vial to terminate the reaction. Each test set is supplied with 15 ml of 3M sodium hydroxide for this purpose.
The enzyme substrate solution disclosed above changes from a colorless liquid to one having a yellow colour in the event enzyme is present on the disc, i.e., in those vials containing samples positive fcr antigens associated with hepatitis.
The supernatant from the negative control are pooled together in a suitable vial and their absorption is read at 405 nm in a spectrophotometer against a distilled water blank. When the negative controls read less than 600 (absorbance units x 1000), they are considered as proper standards against which to com-pare the test results. Using the pooled negative control samples as a blank, test sample and positive control values are read, and the results are recorded as absorbance units x 1000. With some spectrophotometers, it is possible to insert a negative control in the instrument, adjust the reading to zero, and read the value of the test samples directly. An unknown test sample whose optical density times 1,000 is greater than 100, using the pooled negative controls as a blank, is considered positive for antigens associated with hepatitis. This value has been selected to limit nonrepeatable positives which, if present, generally result from errors in laboratory technique.
The readings of the test samples may also be compared with the weak positive and strong positive control samples.
Thus, not only the presence, but an indication of the concen-tration of hepatitis antigens in the sample may be obtained.

Examples ~ive controls should be assayed with each group of unknowns--three negative controls, one strong positive control, 11~6;2~1 1 and one weak positive control. These should be subjected to the same process and incubation times as the test samples.
Caution: Use a clean pipet or disposale tip for each transfer to avoid cross-contamination.
1. Preset water bath at 43C.
2. Number two sets of vials to correspond to test sample identification and controls, and place vials in vial holders.
The first set of these vials will be used for the incubation of test specimens and controls with the antibody coated disc and with the antibody-enzyme tagged reagent. The second set of vials will be used in Step 12 for the substrate reaction.
3. Pipet 0.05 ml (one drop) of horse globulin reagent into the bottom of the first set of vials. Nonspecific false positives resulting from antibodies present in certain human sera which react with horse globulin, advantageously are essentially eliminated by using horse globuline in the test sample diluent.
4. Into this first set of vials pipet 0.5 ml of each test sample into the bottom of the vial having the corresponding sample identification; pipet 0.5 ml of the positive and negative controls into the bottom of their respective vials.
5. Transfer an antibody-coated disc to each vial of the first set. Keep the surface of the discs clean. They should be transferred with clean forceps or a suction-tipped cannula.
They should not be handled with the fingers.
6. Incubate the vials at 43C in a water bath with shaking attachment set for mild agitation for 30 minutes.
7. After incubating the sample with the antibody disc, com-pletely aspirate all supernatants from each vial. Wash the 1~36Z~31 1 discs by adding 2.5 ml of isotonic saline solution to all the vials. Repeat this procedure so that each disc is washed two times. To aid in removing all fluid, tilt the vial holder while aspirating. Shake the vial holder after each addition of wash solution. The liquid waste collected in the container attached to the aspirator should be autoclaved before disposal (minimum of one hour at 121C).
8. After final wash and aspiration, add 0.3 ml of the enzyme-labeled antibody solution to each vial.
9. Incubate the vials for one hour at 43C in the water bath with the shaking attachment set for mild agitation.
- 10. Prepare p-nitrophenyl phosphate substrate by rinsing the contents of one vial of p-NPP (100 mg) into 100 ml of diluted substrate buffer. (The latter is prepared by adding 10 ml of concentrated sodium bicarbonate buffer to 90 ml of distilled water.) Rotate gently to mix; solution should occur immediately.
Note: This substrate solution must be prepared on the day it is to be used. When not being used it should be refrigerated. Any solution remaining after 24 hours should be discarded.
11. Aspirate the supernatant and wash three times as in Step 7.

12. Transfer the discs to the second set of identically numbered clean vials prepared in Step 2.

13. To each vial containing a disc, add 2.5 ml of the p-nitro-phenyl phosphate substrate solution prepared in Step 10.

14. Incubate vials for one hour at 43C in a water bath with the shaking attachment set for mild agitation.

15. Add two drops of 3M sodium hydroxide solution (approximately 0.1 ml total) to all the vials to terminate the reactions.

1~6281 1 Shake vials in holder to mix reagents well. The absorption readings must be made within four hours after terminating the reactions.

16. Pool the three negative controls and read the absorption at 405 nm in a photometer against a blank of distilled water. Record the result as absorbance units X 1000. If the negative control reads greater than 600, the assay is unsatisfactory and must be repeated. When a flow-through photometer is used and strong positive samples are encountered, the curvette should be rinsed with distilled water before reading the absorbance of the next sample.
When nondisposable curvettes are used, the curvette must be rinsed with distilled water following any positive reading.

17. With the pooled negative control, adjust the instrument to 0 absorbance. Determine the absorbance of each reaction mixture, recording the results as absorbance units X 1000. With some photometers, the instrument cannot be adjusted to 0 with the pooled negative control samples.

In this case, the reading of the negative controls must be subtracted from the reading of each sample.

Evaluation of Results Unknown test samples whose absorbance units X 1000 values are greater than 100 are considered to be reactive, using the pooled negative controls as a blank. It may be desired to repeat the test on samples considered reactive. Before classi-fying a reactive serum as positive for antigens associated with hepatitis, confirmation must be obtained by testing with the Cordis Confirmatory Test Set (Cat. No. 783-950). This assay must be performed on all reactive samples. A reactive serum, 1~6~281 1 confirmed by neutralization with horse antiserum, must be considered positive for hepatitis B antigen. As is noted above, confirmation testing is required in order to evaluate the results. Confirmation testing is required by the Food and Drug Administration as well as by the laws of many states.
Briefly, confirmation testing in accordance with the present invention is accomplished as follows:
The positive test sample is tested in duplicate. After the first step in which the sample is exposed to the disc in each vial, following washing, one disc is exposed to antibodies specific for the hepatitis antigens; and, the other disc is exposed to normal horse serum. After one half hour of incubation, the subsequent procedures are identical to those carried out in the routine screening assay. If a sample is positive for hepatitis, the specimen which was exposed to the horse antibody will have a very low value; whereas, the disc exposed to the normal horse serum will have a high value equivalent to that found in the routine screening procedure.
Samples which have low concentrations of the hepatitis antigen, tend to have low final read out values. Those which have a high concentration of hepatitis antigens will have maximum values. Within a narrow range of concentration differences, the resulting read out optical densities will tend to give quantitative information concerning the concentration of hepatitis antigens present in the sample.

Limitations to the Procedure l. Nonrepeatable reactives: If repeat testing on a reactive sample shows the value is less than the lO0 cutoff value, the test is presumed to be a nonrepeatable reactive and is considered negative for antigens associated with hepatitis.

6Z~

1 The original result may be due to errors in technique, such an inadequate washing.
2. Nonspecific positives: The nonspecific false positives resulting from antibodies present in certain human sera that react with horse globulin are essentially eliminated by using horse globulin in the test sample diluent.
3. Plasma from blood collected in EDTA should not be used.
It should be noted that the incubation periods for various steps and the temperature at which incubation is performed can be accomplished over a wide range of times and temperatures. Thus, this invention is not intended to be limited in any way to the time and temperature of incubation.
For example, the time of the incubation of the insoluble member with the sample may range between 10 minutes and 24 hours, with the temperature of incubation ranging between 2C and 50C. On the other hand, the time of incubation of the insoluble member with the enzyme tagged binding partner ranges between 30 minutes to 24 hours, with the temperature of incubation of the insoluble member with the enzyme tagged binding partner ranging between 2C and 50C.
It should also be noted that the process of the present invention can be used to determine the presence of antigens associated with hepatitis in any body fluid where the antigens are present. Thus the process can be used to determine the presence of these antigens in serum, plasma, components of plasma, components of serum, urine, saliva, and cerebrospinal fluid.
Results A representative showing of the results of tests performed in accordance with the present invention appear below.

~6281 1 The results shown in Table A are the results of tests in accordance with the present invention for samples which test positive for hepatitis by the CEP method. The values given in Table B are the values for test samples which test positively for hepatitis by the radioimmunoassay method yet negative for hepatitis by the CEP method and the values given in Table C test negative for both the RIA and the CEP method.
The numerical values given in Tables A, B and C for the various - test samples are the values obtained by testing with the procedure of the present invention.

TABLE A TABLE B TABLE C
CEP+ RIA+ CEP- Neg .
Test (O.D. x 1000) Test (O.D. x 1000) Test (O.D. x 1000) Sample oD405 SampleoD405 Sample oD405 217 2795 104 o 218 2800 203 o 219 2746 207 o 226 2695 212 o 228 2782 215 o 30232 2760 216 o 235 2764 225 o 1 TABLE A (contd.) TABLE C (contd.) .
CEP+ Ne~
. _ _ _ _ _ Test (O.D. x 1000) Test (O.D. x 1000) Sam~le OD405 Sam~le OD405 . . . _ . .

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiment is therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

~., - -

Claims (23)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for determining the presence of antigens associated with hepatitis in a sample comprising the following steps:

(a) incubating the sample with an antibody immobilized on an insoluble member, said insoluble member being a water insoluble polymeric matrix having a layer of reactive groups grafted onto its surface, said reactive groups being capable of covalently bonding to an antibody or antigen, said antibody being co-valently bonded to said reactive groups, the antibody being reactive with the antigens associated with hepatitis, the incubation enabling a bond to be formed between the antibody and an antigen present in the sample to produce an insoluble member having an antigen bonded thereto in the event antigens associated with hepatitis are present in the sample;
(b) separating the insoluble member from any unbonded substances;
(c) incubating the insoluble member with a solution con-taining tagged antibody reactive with an antigen associated with hepatitis, the tagged antibody being tagged with an enzyme capable of effecting a reaction of a substrate to produce a detectable reaction product, the incubation being conducted to enable the tagged antibody to bond to any antigen bonded in step (a) to the antibody on the insoluble member;
(d) separating the insoluble member from the enzyme tagged antibody solution to remove any unbonded tagged antibody therefrom;
1. Claim 1 continued .....
   
   (e) exposing the insoluble member to a substrate solution which the enzyme of the tagged antibody reacts upon to enable a chemical change in the substrate to take place and to produce a detectable reaction product; and (f) detecting any reaction product present in the solution to determine the presence of antigens associated with hepatitis.
2. 2. A process as claimed in claim 1 wherein the reaction product is detected by measuring the optical absorption of the substrate solution.
3. 3. A process as claimed in claim 1 wherein said tagged antibody comprises immunochemically active hepatitis antibody conjugated with enzymatically active alkaline phosphatase.
4. 4. A process as claimed in claim 3 wherein the substrate solution comprises p-nitrophenyl phosphate.
5. 5. A process as claimed in claim 1 wherein said reactive groups are capable of covalently bonding to a hepatitis antibody and having surfaces which have been distorted to provide the matrix with surfaces which, when placed in a flat-bottomed vial, will be substantially in contact with any solution in the vial while minimizing the surface-to-surface contact between the matrix and the bottom of the vial.
6. 6. A process as claimed in claim 1 wherein the value obtained in detecting any reaction product is compared with the value obtained from a sample known to contain antigens associated with hepatitis.
7. 7. A process as claimed in claim 1 wherein the sample incubated in step (a) is a body fluid.
8. 8. A process as claimed in claim 7 wherein the body fluid is selected from the group consisting of serum, plasma, components of plasma, components of serum, urine, saliva, and cerebrospinal fluid.
9. 9. A process as claimed in claim 1 wherein the antigen that is determined is the antigen associated with hepatitis B.
10. 10. A method for the detection of a hepatitis antigen in a liquid sample containing said antigen, comprising the steps of:
    (a) providing a quantity of the antibody associated with said antigen, said antibody being bound to a water-insoluble, water-insuspensible, solid carrier, said solid carrier being a polymeric matrix having a layer of reactive groups grafted onto its surface, said reactive groups being capable of covalently bonding to an antibody or an antigen, said antibody being covalently bonded to said reactive groups;
    (b) contacting and incubating said liquid sample with said solid carrier of step (a) to form a reaction mixture and eliminating unbonded substances from said solid carrier;
    (c) providing a solution of a second quantity of said antibody, said second quantity of said antibody being co-valently linked to an enzyme;
    (d) contacting and incubating the solid carrier resulting from (b) with said solution of enzyme-linked antibody of (c), separating the solid carrier from the solution of enzyme-linked antibody, and eliminating unbonded substances from said solid carrier; and (e) exposing said solid carrier containing said enzyme-labelled antibody of step (d) to a chemical substrate which will undergo a chemical change to produce a reaction product in the presence of an enzyme catalyst on said antibody to enable the presence of hepatitis antigen to be detected.
11. 11. A process for determining the presence of antigens associated with hepatitis in a sample comprising the following steps:
    (a) incubating the sample with an antibody immobilized on an insoluble member, said insoluble member being a water in-soluble polymeric matrix, the antibody being reactive with the antigens associated with hepatitis, the incubation enabling a bond to be formed between the antibody and an antigen present in the sample to produce an insoluble member having an antigen bonded thereto in the event antigens associated with hepatitis are present in the sample;
    (b) separating the insoluble member from any unbonded substances;
    (c) incubating the insoluble member with a solution con-taining tagged antibody reactive with an antigen associated with hepatitis, the tagged antibody being tagged with an enzyme capable of effecting a reaction of a substrate to produce a detectable reaction product, the incubation being conducted to enable the tagged antibody to bond to any antigen bonded in step (a) to the antibody on the insoluble member;
    (d) separating the insoluble member from the enzyme tagged antibody solution to remove any unbonded tagged anti-body therefrom;
    (e) exposing the insoluble member to a substrate solution which the enzyme of the tagged antibody reacts upon to enable a chemical change in the substrate to take place and to produce a detectable reaction product; and (f) detecting any reaction product present in the solution to determine the presence of antigens associated with hepatitis.
12. 12. A process as claimed in claim 11 wherein the reaction product is detected by measuring the optical absorption of the substrate solution.
13. 13. A process as claimed in claim 11 wherein said tagged antibody comprises immunochemically active hepatitis antibody conjugated with enzymatically active alkaline phosphatase.
14. 14. A process as claimed in claim 13 wherein the substrate solution comprises p-nitrophenyl phosphate.
15. 15. A process as claimed in claim 11 wherein the value obtained in detecting any reaction product is compared with the value obtained from a sample known to contain antigens associated with hepatitis.
16. 16. A process as claimed in claim 11 wherein the sample incubated in step (a) is a body fluid.
17. 17. A process as claimed in claim 16 wherein the body fluid is selected from the group consisting of serum, plasma, components of plasma, components of serum, urine, saliva, and cerebrospinal fluid.
18. 18. A process as claimed in claim 11 wherein the antigen that is determined is the antigen associated with hepatitis B.
    
    19. A method for the detection of a hepatitis antigen in a liquid sample containing said antiqen, comprising the steps of:
    (a) providing a quantity of the antibody associated with said antigen, said antibody being bound to a water-insoluble, water-insuspensible, solid carrier, said solid carrier being a polymeric matrix;
    (b) contacting and incubating said liquid sample with said solid carrier of step (a) to form a reaction mixture and eliminating unbonded substances from said solid carrier;
    (c) providing a solution of a second quantity of said antibody, said second quantity of said antibody being co-valently linked to an enzyme;
19. Claim 19 continued .....
    
    (d) contacting and incubating the solid carrier result-ing from (b) with said solution of enzyme-linked antibody of (c), separating the solid carrier from the solution of enzyme-linked antibody, and eliminating unbonded substances from said solid carrier; and (e) exposing said solid carrier containing said enzyme-labelled antibody of step (d) to a chemical substrate which will undergo a chemical change to produce a reaction product in the presence of an enzyme catalyst on said antibody to enable the presence of hepatitis antigen to be detected.
    
    20. A process for determining the presence of antigens associated with hepatitis in a sample comprising the following steps:
    (a) incubating the sample with an antibody immobilized on an insoluble member, the antibody being reactive with the antigens associated with hepatitis, the incubation enabling a bond to be formed between the antibody and an antigen present in the sample to produce an insoluble member having an antigen bonded thereto in the event antigens associated with hepatitis are present in the sample;
    (b) separating the insoluble member from any unbonded substances;
    (c) incubating the insoluble member with a solution con-taining tagged antibody reactive with an antigen associated with hepatitis, the tagged antibody being tagged with an enzyme capable of effecting a reaction of a substrate to produce a detectable reaction product, the incubation being conducted to enable the tagged antibody to bond to any antigen bonded in step (a) to the antibody on the insoluble member;
20. Claim 20 continued .....
    
    (d) separating the insoluble member from the enzyme tagged antibody solution to remove any unbonded tagged antibody therefrom;
    (e) exposing the insoluble member to a substrate solution which the enzyme of the tagged antibody reacts upon to enable a chemical change in the substrate to take place and to produce a detectable reaction product; and (f) detecting any reaction product present in the solution to determine the presence of antigens associated with hepatitis.
21. 21. A method for detecting a hepatitis antigen in a liquid sample containing said antigen, comprising the steps of:
    (a) providing a quantity of the antibody associated with said antigen, said antibody being bound to a water-insoluble, water-insuspensible, solid carrier;
    (b) contacting and incubating said liquid sample with said solid carrier of step (a) to form a reaction mixture;
    (c) providing a solution of a second quantity of said antibody, said second quantity of said antibody being co-valently linked to an enzyme;
    (d) contacting and incubating the solid carrier result-ing from (b) with said solution of enzyme-linked antibody of (c); separating the solid carrier from the solution of enzyme-linked antibody;
    (e) correlating the enzyme activity of substance bound to the solid phase to the presence of hepatitis antigen to be de-tected.
22. 22. A process for determining the presence of antigens associated with hepatitis in a sample comprising the following steps:
    (a) incubating the sample with an antibody immobilized on an insoluble member, the antibody being reactive with the antigens associated with hepatitis, the incubation enabling a bond to be formed between the antibody and an antigen present in the sample to produce an insoluble member having an antigen bonded thereto in the event antigens associated with hepatitis are present in the sample;
    (b) separating the insoluble member from any unbonded substances;
    (c) incubating the insoluble member with a solution con-taining tagged antibody reactive with an antigen associated with hepatitis, the tagged antibody being tagged with an enzyme capable of effecting a reaction of a substrate to produce a detectable reaction product, the incubation being conducted to enable the tagged antibody to bond to any antigen bonded in step (a) to the antibody on the insoluble member;
    (d) separating the insoluble member from the enzyme tagged antibody solution to remove any unbonded tagged anti-body therefrom;
    (e) exposing the insoluble member to a substrate solution which the enzyme of the tagged antibody reacts upon to enable a chemical change in the substrate to take place and to produce a detectable reaction product; and (f) detecting any reaction product present in the solution to determine the presence of antigens associated with hepatitis.
    
    23. A method for determing the presence of a component of an antigen-antibody reaction in a liquid sample containing the component to be determined comprising the steps of:
23. Claim 23 continued ...
    
    a. providing a given quantity of a first reagent consisting of one component of said reaction selected from the group consisting of an antigen and an antibody bound to the sur-face of water-insoluble, water-insuspensible, solid carrier, and also providing a predetermined amount of the binding partner for the component to be determined when the component in said liquid sample and the component bound to said solid carrier have the same immunochemical properties;
    b. contacting and incubating a given quantity of said liquid sample with said first reagent;
    c. washing said solid carrier;
    d. providing a given quantity of a substance having the same immunological properties as the first reagent previously bound to the solid carrier, said substance being covalently linked to an enzyme;
    e. contacting and incubating the solid phase from step (c) with said enzyme-linked substance;
    f. washing the solid carrier; and g. determining the enzyme activity substance bound to the solid phase which is a measure of the presence and quantity of the component to be determined.