GB1571196A - Immunoassay - Google Patents

Description

(54) IMPROVEMENTS IN OR RELATING TO IMMUNOASSAY (71) We, CORDIS CORPORATION, a Corporation organised and existing under the laws of the State of Florida, United States of America, residing at 3901 Biscayne Blvd., Miami, Florida 33137, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention is concerned with improvements in or relating to immunoassay for hepatitis.

The invention provides a process for detecting 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 containing 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 antibodv 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 to produce a detectable reaction product; and, (f) detecting any reaction product present in the solution to detect the presence of antigens associated with hepatitis.

The invention also provides a process for detecting the presence of hepatitis antigens in a liquid sample comprising the steps of (a) incubating the sample with a hepatitis antibody reactive with said antigens and inmobi- lised on an insoluble member, (b) incubating the insoluble member resulting from step (a) with an en2yme-tagged hepatitis antibody reactive with said antigen, (c) separating the insoluble member from any unbonded substances, and (d) correlating the remaining enzymatic activity exhibited by the insoluble member, to the presence of hepatitis antigen to be detected.

The invention also provides a test set adapted for use in detecting the presence of antigens associated with hepatitis in a test sample comprising: (a) an insoluble polymeric solid having a layer of protein reactive groups grafted onto its surface and having hepatitis associated antibody bonded to said reactive groups, the surfaces on said solid being configured to comprise a field of high and low points to provide a solid which, when placed in a fiat-bottomed vial, will be substantially in contact with any solution in the vial while minimizing surfaceto-surface contact, said solid being for immobilizing hepatitis antigens from the sample; (b) an alkaline phesphatase-antibody conjugate reagent for labeling hepatitis antigen immobilized on said solid;; (c) an enzyme substrate capable of being chemically changed under the catalytic in fluence of the alkaline phosphatase; and (d) a fiat-bottomed vial sized to receive said solid.

The invention also provides a test set adapted for use in detecting the presence of antigens associated with hepatitis in a test sample comprising: (a) an insoluble polymeric solid having antibody reactive with antigens associated with hepatitis bonded thereto for immobilizing hepatitis antigens from the sample, the surfaces on said polymeric solid being configured to comprise a field of high and low points to provide a solid which, when placed in a flat bottomed vial, will be substantially in contact with any solution in the vial while minimizing surface-tosurface contact; (b) an alkaline phosphatase-hepatitis antibody conjugate reagent for labeling hepatitis antigens immobilized on said solid; (c) a quantity of p-nitrophenylphosphate for being exposed to the alkaline phosphatase to detect the presence thereof on said solid;; (d) quantities of control sera negative, weakly positive, and strongly positive for antigens associated with hepatitis for comparison with the test sample; (e) an enzyme substrate buffer solution having a pH of the order of 9.8 for optimizing the catalytic effect of the alkaline phosphatase on the p-nitrophenylphosphate; (f) a horse globulin solution for minimizing nonspecific immobilization of enzyme on said insoluble solid; and (g) a flat-bottomed vial sized to receive said solid.

The invention also provides a reagent comprising an enzyme-tagged hepatitis antibody.

The invention also provides a reagent for use in immunoassay of antigens associated with hepatitis comprising immunochemically active hepatitis antibody conjugated with enzymatically active alkaline phosphatase.

The descriptive matter which follows is by way of example of the invention only and not by way of limitation thereof.

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 comprising 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- antibodyantigen- enzyme tagged antibody.

After the second incubation, the member is washed again to remove unreacted enzymeantibody 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 od antigen or antigens, is determined by a spectrophotometer 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 furnished in the form of a kit which has an extended shelf life and greatly simplifies the procedure.

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 varities of viral hepatitis, one having 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 contracted 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 closely resembling serum hepatitis, no matter how contracted, 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 specification 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 include Micro-Ouchterlony, immunodiffusion, complement fixation, immunoelectro-osmophoresis, haemagglutination and haemagglutination inhibition, electron microscopy, and solid phase radioimmunoassay. See British Medical Bulletin, 1972, Vol. 28, No. 2 (Viral Hepatitis) pages 138141 for a brief description od 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 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 manfacturer), 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 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 pre viouslv 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 simulated 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 antibody 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 with other closely related antigens which may have quite dissimilar or insignificant consequences. Thus, it is apparent that both the source and the manner of preparation of the antibody is quite critical in any immuno..ssay.

One manner of detecting hepatitis associated antigen, as earlier mentioned, involves solid phase radio-immunoassay. Such a pro cedure 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 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 1-123.

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 polytetrofluorethylane onto which is grafted a substituted polystyrene, e.g., isothiocyanatow styrene might be useful in performing radioimmunoassay. This polystyrene is an insoluble material having specifically designed surface of protein-reactive groups which may be used to covalently bond proteins to provide a reagent 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 hepatitis. Since decisions on whether to use particular blood units available from a blood center must often be made in a relatively short time period, a sensitive, rapid, easy to perform screening test for 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 ore 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, be cause 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 maintained at hand, they have not satisfied the demands of the ideal test.

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 convelantly bonded to an insoluble member.

The enzyme is exposed to a chemical sub strate which will undergo a chemical change to produce a reaction product in the presence of the enzyme catalyst. The presence of hepatitis is determined by determining the pre sence of the reaction product 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 conjugate is an alkaline phosphatase and the enzyme substrate is pwnitrophenylphosphate. 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 bluffer designed to main tain 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 0.028 M in Na2CO3 and 0.001 M in Mg.

An immunologically active purified hepatitis antibody conjugated, i.e., chemically linked, with a functional enzyme such as alkaline phosphatase is provided for use in the immunoassay.

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 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 are distorted to an irregular configuration to reduce the disc surface-vial bottom contact. In one preferred embodiment, the opposed surfaces are rendered waffle-like by passing the disc through a press prior to attachment of the antibody.

Accordingly, it is one 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 fiuids.

Another object of the invention is to provide a unitized test set which is adapted for the performance of a sensitive, reproducible immunoassay of antigens 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.

The test set may contain 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 immunoassays of antigens associated with hepatitis.

An insoluble matrix is provided useful as described in the above-mentioned test which has a high concentration of purified, hepatitis associated antibody distributed uniformly over its entre surface, and such that the antibody will not be dislodged by mechanical or chemical forces to which it may be subjected during use. In this regard, the attach ment is effected such that the antibody remains functional, i.e., capable of participating in its immunological reaction and immunochemically unaltered by its attachment.

The insoluble antibody coated solid has an improved shape which is easy to handle during inimunoassay and which is designed to expose a constant and a greater portion of its coated surface to the solutions used during incubations.

The above 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.

Fig. 1 is a flow art chart summarizing the steps of a process embodying the present invention; Fig. 2 is a perspective view of the preferred insoluble member used in the process and a test set embodying the present invention; Figs. 3, 4, and 5 depict three incubation stages of immunoassay; and Fig. 6 is a schematic representation showing the ;'sandwich" structure developed during positive immunoassay of a test sample.

The method of detecting hepatitis embodyins the present invention is carried out in four distinct stages as is illustrated in Fig. 1.

The first stage involves reacting the immobilized antibody on the insoluble member 10 (see Fig. 2) with an antigen present in the test serum as is shown in Fig. 3. This reaction 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 color. The color change acts as an indication of the presence of antigens associated with hepatitis. The last stage involves reading the degree of color and comparing the value obtained with a standard of control.

The hepatitis detection test set embodying the invention which contains all materials needed in the test outlined 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 hereinafter. The kit is distributed, in, e.g., a box, containing reagents and controls to be stored at 2--8"C until used. In addition, vials sufficient for conducting 100 tests are provided.

One hundred to one hundred and five poly- meric 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 antibodyalkaline 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 form a 1 mg/ml solution, becomes relatively unstable. Consequently, the solution must be prepared just prior to use.

The other material used during the pro cedure 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 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 blood 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 Specification No. 1,387,625.

The preparation of hepatitis antibody de pends first of all on obtaining blood which is known to be positive for antigens associated with hepatitis. Consequently, blood units obtanied from various sources must first be evaluated to determine their suitability for preparing immunospecific purified antibody.

A blood bag segment, containing blood which is believed to be positive for hepatitis antigens, is held at 2--8"C 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 of counterelectrophoresis (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.

Portions of antigens isolated from blood units are used for either stitnulating antibody production in a host animal or purifying the antibody produced by that animal. The anti gens 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 SM solution of CaCl2 on the basis of 0.75 ml CaCl. per 200 ml of plasma. This solution is then incubated at 370C in a water bath for one hour or until a clot forms. After a firm clot forms, the plasma is frozen at 20CC and allowed to thaw at 2-80C 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 immuno absorbent columns.

(1) Preparation of Antigen Pellet for Immunization Hepatitis positive sera which have been subjected to the above process are centrifuged at 10,000 rpm for 30 minutes at 4"C. The supernatant of this centrifugation is distri- buted into ultra centrifuge tubes and centrifuged in, for example, a Beckman L2-65B ultra-cenrrifuge, at 40,000 to 50,000 rpm for 4-20 hours at 4"C. The supernatant in each tube is removed and discarded; the pellet, which contains antigens, is given a preliminary rinse with normal (0.15M) 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 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 40C, - as mentioned above.

The procedure in the preceding 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 solution may be divided into 3 ml aliquots and frozen at -200C 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 plasniaphoresis according to conventional techniques. Alternatively, or in addition, prepara tions 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 immunoabsorptlon purfication step.

Broadly, this preliminary purification is accomplished in three steps. First, plasma 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 antigen-antibody complex formation. 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 be frozen until used.

(3) Preparation od Charcoal Immunoabsorbent Column The preparation of the purified antibody which is used to produce the present reagents is accomplished by subjecting the hepatitis antibody produced as disclosed above to an nnmunospecific extraction process. For a general discussion of this procedure, reference should be made to British Patent Specification No. 1,387,625, the disclosure of which is incorporated herein by preference.

In general, this purification process as utilized herein, takes advantage of the ability of antibodies 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 plastics 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 collected in 500 ml aliquots, each of which are checked for protein content. The column is considered saturated with the antigen when the effluent has a protein 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 runnning 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--8"C 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 colmun 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 have passed through the column and have been separated from the antibody.

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 elute is collected in fractions of appropriate 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, it is subjected to a dual filtration; first, through a 0.45a membrane, and second, through a 0.22o membrane. The filtrate is diluted 1: 3 using distilled water at 2--8"C, 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 compared to against PBS in the first dialysis. After completion of this final dialysis, the 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 Antibody-Enzyme Conjugate Calf intestinal alkaline phosphate is mixed with a solution of the reconstituted antibody in a ratio of 3:1, enzyme to antibody, to a final concentration greater than 10 mg of total protein per ml of solution in PBS (pH 7.4).

The solution is dialyzed thoroughly to remove NH4+ ions.

The dialyzed antibody enzyme mixture is then centrifuged to remove any insoluble material. The protein 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 glutaraldehyde solution per 10 ml antibodyenzyme 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 0.001M Mgx to remove 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), 0.02% NaN3, and 0.001M in MgCl2.

In addition to alkaline phosphatase, other enzymes may be used. 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 2. glycerol dehydrogenase 3. glyoxylate reductase 4. L-lactate dehydrogenase 5. malate dehydrogenase 6. glucose 6-phosphate dehydrogenase 7. mannitol l-phosphate dehydrogenase 8. glucose oxidase 9. galactose oxidase 10. L-amino acid oxidase 11.D-amino acid oxidase 12. polyphenol oxidase 13. ascorbate oxidase 14. catalase 15. peroxidase 16. cholinesterase 17. phospholipase C 18. er-amylase 19. Iysozyme 20. ,B-galactosidase 21. amyloglucosidase 22. ,B-glucuronidase 23. carboxypeptidase A 24. urease 25. inorganic pyrophosphatase 26. aldolase 27. carbonic anhydrase 28. histidase The enzyme that is used for tagging the antibody is selected with several considerations in mind.These considerations 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 availa utility of the enzyme and the cost of the enzyme.

C. Preperation of Antibody Coated Insolube Member The next step in the preparation of the reagents is to 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 Gr4t Copolymets, to G. W. Tregear et al., discloses an insoluble continuous polymeric substance comprising a polymeric backbone onto which side chains of another polymer or copolymer are grafted. By suitable choice of the grafted polymer, it is possible to chemically link biological substances to the insoluble matrix. A product which is disclosed in the above patent; is commercially available in a disc form under the traderume PROTAPOL DI/1 from Imperial Chanical Industries of Australia and New Zealand (ICIANZ).

The PROTAPOL DI/1 comprises a poly tetreiuoroetbylene backbone having ishio- cyanatopolystyrene groups grafted unifottly over its surface and is designed for use in radioimmunoassay. The discs, as presently available, are approximately 0.01 inches chick and 0.5 inch in diameter.

In accordance with one important embodiment of the matrix, as shown in Fig. 2, each disc 10 is provided with a waffle-like pair of surfaces 11 comprising a first series of liner ridges 12 and a second series of linear ridges 14 which form grids. Ridges 12 and 14 are preferably perpendicular to each other and define a plurality of square depressions 16. The sides of each ridge 12 and 14 taper upwardly from adjacent pairs of depfes6ions 16.to form a line defining the top of the ridge.

It should be noted that in order to facilitate description, 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 mre to disfigure the polymeric material the disc without actually puncalring the dise This is important because the disc has a Xe layer on its surface. Thus, penetra ion of the disc would expose interior portiorts which no antibody can be bonded. Exposure of the polytetrafluoroethylene layer would actu ally result in a disc which would have a lower bonding capacity.

The main consideration is to provide a disc matrix with surfaces which, when placed in a flat bottomed vial 15, will be substtially 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 and it will be realized, that 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 0.1M NaHCO3 (pH 9.6) for each 5.0 mg of antibody. In general, the procedure for attachment involves contacdng the waffled discs with the dilute solution at 2-80C for 8 to 16 hours, with agitation.

Afterwards, the antibody solution is discarded and the discs are washed twice with successive volumes of 0.1M NaHCO,, pH 9.6, phos phate buffered saline, and cold (28oC) 0.3% bovine serum albumin in phosphate buffered saline with 0.5% TWEEN (Registered Trade Mark) 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 2-80C until ready for use.

Example.

The following procedure was used to prepare 8,000 discs, each of which were first treated with the press to produce the desred 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 solution is adjusted to 0.05 mg/ml in a final volume of 800 ml in 0.1M NaIlCO, (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 conitaining tne 8,000 discs, and the bottle is rotated for 16 hours, e.g., overnight, at 2-80C 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 (2-8 C) 0.1M 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 (0.01M sodium phosphate, 0.15M NaCI, 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; (0.3%).

The discs are finally washed with two suc cessive 1 liter volumes of a solution of cold crystalline bovine serum albumin (pH 8) at a concentration of 2 mg/ml. This step is per formed to provide a protein environment for the protein on the disc. The discs, after 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 complee, a sheet of filter paper is used to cover them. Buffer 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.

(1) Preparation of Control Serum In order to obtain meaningful data from the immunoassay, 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 iadioimmunoassay technique. To each unit that is clearly negative for hepatitis antigen, 5M CaCI2 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 37"C in a water bath until a clot forms. The clotted plasma units are then frozen at --20"C and stored for at least 12 hours. The plasma units are then allowed to thaw at 2-80C 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--8"C.

Twenty grams of silica, e.g., AEROSIL (Registered Trade Mark)-3 80, 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, the silica can be removed by filtration through appropriate filter media.

The supernatant is then further processed by filtration through, for example, MILLI PORE (Regstered Trade Mark) 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 (NaN2) 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--8"C 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 recalcihed 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 600C 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--8"C. The precipitate is discarded.

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 pre ferred 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 0.5 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 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 glo bulin test sample diluent is added to each vial. This step is taken as a precaution to eliminate nonspecific 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 concentration 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.

The figures to the right in Table I are pH values.

TABLE I ss-glycerol phosphate (serum), 7.4 ss-glycerol phosphate barbital, 8.6 phenyl phosphate carbonate-bicarbonate, 9-10 ss-naphthyl phosphate barbital, 9.1 p-nitrophenyl phosphate 2A2M'1P, 10.25 (2-amino-2 methyl-l propanol) phenolphthalein phosphate 2A2M1P, 9.90 p-nitrophenyl phosphate 2A2M1P, 10.17 thymolphthalein phosphate carbonate-bicarbonate, 10.0 p-nitrophenyl phosphate diethanolamine, 9.8 4-methylumbelliferyl phosphate carbonate-bicarbonate, 9.2 The substrate 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 those 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, 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 borse 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 43"C in, e.g., a shaking water bath. During this incubation, hepatitis antigens present in the test sample or controls 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 supernatant 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 antibodyenzyme conjugate is added to each vial, and the vials are again incubated at 43 0C for 1 hours 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-nitrophenyl- phosphate enzyme subtsrate-buffer solution is added to each vial (1 mg pNPP per ml).

Since the optimum operational pH of the alkaline phosphatase-p nitrophenylphosphate system is 9.8, the enzyme substrate is dissolved in carbonate-Mgs buffer (pH 9.8 +0.1).

This buffer as used comprises an aqueous solu tion 0.028M in Na2CO3 and 0.001M in Mug". Forty ml of concentrate may be sup plied with the set which, when diluted to 400 ml with distilled water, may be added directly to the 400 mg of pNPP. After addi tion of the buffered substrate, the vials are subjected to a third incubation for one hour at 43"C with shaking. Different substrates must be employed if a different enzyme anti body conjugate is used.

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 fjr this purpose.

The enzyme substrate solution disclosed abcve changes from a colorless liquid to one having a yellow color in the event enzyme is present on the disc, i.e., in those vials containing samples positive for antigens associ ated 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 spectre; photometer against a distilled water blank.

When the negative controls read less than 600 (absorbance units X 1000), they are con sidered as proper standards against which to compare 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 concentration of hepatitis antigens in the sample may be obtained.

Examples.

Five controls should be assayed with each group of unknowns-three negative controls, one strong positive control, 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 disposable tip for each transfer to avoid cross-contamination.

1. Preset water bath at 430C.

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. Piper 0.05 ml (one drop) of horse glo bulin 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 globulin 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 iden tification; 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 43"C in a water bath with shaking attachment set for mild agitation for 30 minutes.

7. After incubating the sample with the antibody disc, completely aspirate all supernatants from each vial. Wash the 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 1210C).

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 43 0C 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-nitrophenyl phosphate substrate solution prepared in Step 10.

14. Incubate vials for one hour at 43"C 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. 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 cuvette should be rinsed with distilled water before reading the absorbance of the next sample. When nondisposable cuvettes are used, the cuvettes 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 absorbence 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 classifying a reactive serum as positive for antigens associated with hepatitis, Caation n 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, con firmed by neutralization with horse antiserum, must be considered positive for hepatitis B antigen. As is noted above, confirmation test ing 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 test ing in accordance with the present invention is accomplished as follows: The positive test sample is tested in dupli cate. 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 exposde to normal horse serum.

After one half hour of incubation, the sub sequent 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 denstities will tend to give quantitative information concerning the concentration of hepatitis antigens present in the sample.

Limitations to the Procedure 1. Nonrepeatable reactives: If repeat testing on a reactive sample shows the value is less than the 100 cutoff value, the test is presumed to be a nonrepeatable reactive and is considered negative for antigens associated with hepatitis. The original result may be due to errors in technique, such an inadquate 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 in tended 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 2"C and 50 C.

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 2"C and 50 C.

It should also be noted that the process embodying 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 semi, urine, saliva, and cerebrospinal fluid.

Results A representative showing of the results of tests performed in accordance with the present invention appear below. The results shown in Table A are the results of tests 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 embodying the present invention.

TABLE A TABLE B TABLE C CEP RIA CEP Neg Test (O.D. < 1000) Test (O.D. x 1000) Test (O.D. x 1000) Sample OD405 Sample OD405 Sample OD405 72 2807 65 2745 88 0 73 2802 68 2675 89 0 79 2815 69 266 90 0 82 2822 75 1354 91 23 121 2831 77 265 92 42 201 2768 81 953 93 0 202 2780 87 1383 94 0 205 2749 123 1688 97 38 98 0 206 2758 124 2739 99 0 208 2795 125 1741 99 100 0 209 2800 204 285 101 0 210 2764 243 2466 102 0 213 2776 261 2636 214 2804 103 0 2795 104 0 217 2795 203 0 218 2800 207 2746 207 0 219 2746 220 2718 211 8 226 2695 212 0 215 0 228 2782 216 0 232 2760 234 2774 224 0 235 2764 225 0 238 2760 22? 0 238 2760 239 2755 229 0 244 2730 230 0 TABLE t (Contlnued) TABLE A (Continued) Neg CEP + ~~~~~~~~~~~~~~~~~~~~~~ Test (O.D x 1000) Test (O.D. x 1000) Sample OD405 Sample OD405 231 0 250 707 233 45 251 2761 240 0 252 2778 241 0 253 2783 242 0 254 2742 255 2743 256 2784 Attention is directed to our divisional patent applications Nos. [11699/79] (Serial No.

260 2775 1,571,197) and [20659/79] (Serial No.

1,571,198).

WHAT WE CLAIM IS:- 1. A process for detecting 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 containing 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; (e) exposing the insoluble member to a substrate solution which the enzyme of the tagged antibody reacts upon to enable a chem ical change in the substrate to take place to produce a detectable reaction product. and, (f) detecting any reaction product present in the solution to detect the presence of antigens associated with hepatitis.

2. A process according to claim 1, wherein the reaction product is detected by measuring the optical absorption of the substrate solution.

3. A process according to claim 1 or claim 2, wherein the enzyme is alkaline phosphatase.

4. A process according to claim 1 or claim 2, wherein the enzyme is selected from alcohol dehydrogenase, glycerol dehydrogenase, glyoxylate reductase, L-lactate dehydrogenase, malate dehydrogenase, glucose 6-phosphate dehydrogenase, mannitol l-phosphate dehydrogenase, glucose oxidase, galactose oxidase, L-amino add oxidase, D-amino acid oxidase, polyphenol oxidase, ascorbate oxidase, catalase, peroxidase, cholinesterase, phospholipase C, a-amylase, lysozyme, ss-galactosidase, amyloglucosidase, XB-glucuronidase, carboxypepti- dase A, urease, inorganic pyrophosphatase, aldolase, carbonic anhydrase, and histidase.

5. A process according to claim 3, wherein the substrate is p-nitrophenyl phosphate.

6. A process according to claim 3, wherein the substrate is selected from ,B-glycerol phosphate, phenyl phosphate, B-naphthyl phosphate, phenolphthalein phosphate, thymol phthalein phosphate, and 4-methylumbelliferyl phosphate.

7. A process according to any one of the preceding claims, wherein the insoluble member is a polymeric member having an antibody reactive with an antigen associated with hepatitis covalently bonded to its surface.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (28)

**WARNING** start of CLMS field may overlap end of DESC **. TABLE t (Contlnued) TABLE A (Continued) Neg CEP + ~~~~~~~~~~~~~~~~~~~~~~ Test (O.D x 1000) Test (O.D. x 1000) Sample OD405 Sample OD405 231 0 250 707 233 45 251 2761 240 0 252 2778 241 0 253 2783 242 0 254 2742 255 2743 256 2784 Attention is directed to our divisional patent applications Nos. [11699/79] (Serial No. 260 2775 1,571,197) and [20659/79] (Serial No.

1,571,198).

WHAT WE CLAIM IS:- 1. A process for detecting 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 containing 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; (e) exposing the insoluble member to a substrate solution which the enzyme of the tagged antibody reacts upon to enable a chem ical change in the substrate to take place to produce a detectable reaction product. and, (f) detecting any reaction product present in the solution to detect the presence of antigens associated with hepatitis.

2. A process according to claim 1, wherein the reaction product is detected by measuring the optical absorption of the substrate solution.

3. A process according to claim 1 or claim 2, wherein the enzyme is alkaline phosphatase.

4. A process according to claim 1 or claim 2, wherein the enzyme is selected from alcohol dehydrogenase, glycerol dehydrogenase, glyoxylate reductase, L-lactate dehydrogenase, malate dehydrogenase, glucose 6-phosphate dehydrogenase, mannitol l-phosphate dehydrogenase, glucose oxidase, galactose oxidase, L-amino add oxidase, D-amino acid oxidase, polyphenol oxidase, ascorbate oxidase, catalase, peroxidase, cholinesterase, phospholipase C, a-amylase, lysozyme, ss-galactosidase, amyloglucosidase, XB-glucuronidase, carboxypepti- dase A, urease, inorganic pyrophosphatase, aldolase, carbonic anhydrase, and histidase.

5. A process according to claim 3, wherein the substrate is p-nitrophenyl phosphate.

6. A process according to claim 3, wherein the substrate is selected from ,B-glycerol phosphate, phenyl phosphate, B-naphthyl phosphate, phenolphthalein phosphate, thymol phthalein phosphate, and 4-methylumbelliferyl phosphate.

7. A process according to any one of the preceding claims, wherein the insoluble member is a polymeric member having an antibody reactive with an antigen associated with hepatitis covalently bonded to its surface.

8. A process according to any one of the

claims 1 to 6, wherein the insoluble member comprises a water insoluble polymeric member having (i) a layer of reactive groups grafted onto its surface said reactive groups being capable of covalently bonding to a hepatitis antibody, and (ii) surfaces configured to provide the member with surfaces which, when placed in a flat-bottomed vial, will be substance tially in contact with any solution in the vial while minimising the surface-to-surface contact between the member and the bottom of the vial.

9. A process according to any one of the preceding claims, 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.

10. A process according to any one of the preceding claims, wherein the sample incubated in step (a) is a body fluid.

11. A process according to claim 10, wherein the body fluid is selected from serum, plasma, components of plasma, components of serum, urine, saliva, and cerebrospinai fluid.

12. A process according to any one of the preceding claims wherein the antigen that is detected is the antigen associated with hepatitis B.

13. A process for detecting the presence of hepatitis antigens in a liquid sample comprising the steps of (a) incubating the sample with a hepatitis antibody reactive with said antigens and immobilised on an insoluble member, (b) incubating the insoluble member resulting from step (a) with an enzyme-tagged hepatitis antibody reactive with said antigen, (c) separating the insoluble member from any unbonded substances, and (d) correlating the remaining enzymatic activity exhibited by the insoluble member, to the presence of hepatitis antigen to be detected.

14. A test set adapated for use in detecting the presence of antigens associated with hepatitis in a test sample comprising: (a) an insoluble polymeric solid having a layer of protein reactive groups grafted onto its surface and having hepatitis associated antibody bonded to said reactive groups, the surfaces on said solid being configured to comprise a field of high and low points to provide a solid which, when placed in a flatbottomed vial, will be substantially in contact with any solution in the vial while minimizing surface-to-surface contact, said solid being for immobilizing hepatitis antigens from the sample; (b) an alkaline phosphatase-antibody conjugate reagent for labeling hepatitis antigen immobilized on said solid;; (c) an enzyme substrate capable of being chemically changed under the catalytic in fluency of the alkaline phosphatase; and (d) a flat-bottomed vial sized to receive said solid

15. A test set according to claim 14, further including sera known to contain different con centrations of antigens associated with hepatitis.

16. A test set according to claim 15, wherein said sera include sera negative, weakly posi tive, and strongly positive for antigens associ ated with hepatitis.

17. A test set according to any one of claims 14, 15 and 16, further including a globulin solution for minimizing nonspecific immobilization of enzyme on said insoluble solid.

18. A test set according to any one of the preceding claims, wherein the alkaline phosphatase enzyme substrate is pnitro- phenylphosphate.

19. A test set according to any one of rhe preceding claims, further including an alkaline phosphatase substrate buffer for op timizing the catalytic influence of the alkaline phosphatase on said substrate.

20. A test set according to claim 18, further including an enzyme substrate buffer having a pH of the order of 9.8 for optimizing the catalytic influence ob the alkaline phosphatase on the pWnitrophenylphosphate.

21. A test set according to claim 20, wherein said buffer comprises an aqueous solution 0.028M in Na2CO3 and .001M in Mg.

22. A test set adapted for use in detecting the presence of antigens associated with hepatitis in a test sample comprising: (a) an insoluble polymeric solid having antibody reactive with antigens associated with hepatitis bonded thereto for immobilizing hepatitis antigens from the sample, the surfaces on said polymeric solid being configured to comprise a field of high and low points to provide a solid which, when placed in a flat-bottomed vial, will be substantially in contact with any solution in the vial while minimizing surface-to-surface contact; (b) an alkaline phosphatase-hepatitis antibody conjugate reagent for labeling hepatitis antigens immobilized on said solid; (c) a quantity of pnitrophenylphosphate for being exposed to the alkaline phosphatase to detect the presence thereof on said solid; ; (d) quantities of control sera negative, weakly positive, and strongly positive for antigens associated with hepatitis for comparison with the test sample; (e) an enzyme substrate buffer solution having a pH of the order of 9.8 for optimizing the catalytic effect of the alkaline phosphatase on the p-nitrophenylphosphate; (f) a horse globulin solution for minimizing nonspecific immobilization of enzyme on said insoluble solid; and (g) a flat-bottomed vial sized to receive said solid.

23. A reagent comprising an enzyme-tagged hepatitis antibody.

24. A reagent for use in immunoassay of antigens associated with hepatitis comprising immunochemically active hepatitis antibody conjugated with enzymatically active alkaline phosphatase.

25. A reagent according to claim 23, wherein said enzyme is selected from alcohol dehydrogenase, glycerol dehydrogenase, glyoxylate reductase, L-lactate dehydrogenase, malate dehydrogenase, glucose 6-phosphate dehydrogenase, mannitol l-phosphate dehydrogenase, glucose oxidase, galactose oxidase, L-amino acid oxidase, D-amino acid oxidase, polyphenol oxidase, ascorbate oxidase, catalase, peroxidase, cholinesterase, phospholipase C, amylase, lysozyme, P-galactosidase, amyloglucosidase, il3-glucuronidase, carboxypeptidase A, urease, aldolase, carbonic anhydrase, inorganic pyrophosphatase, and histidase.

26. A process of determining the presence of hepatitis antigens substantially as hereinbefore described with reference to Figure 1 of the accompanying drawings.

27. A test set substantially as hereinbefore described with reference to the accompanying drawings.

28. A reagent comprising an enzyme-tagged hepatitis antibody substantially as hereinbefore described.