DE2643829A1 - PROCEDURE FOR DETERMINING THE PRESENCE OF ANTIGENT ASSOCIATED WITH HEPATITIS - Google Patents

Description

^D * · ^ΙΝ »DIPL.-ΙΝβ. M. SC. DIPI PHY ·. DR. HÖGER - LEGAL RIGHTS - GRIESSBACH - HAECKER PATENT LAWYERS IN STUTTGART

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Cordis Corporation Miami, Florida, 33137 U.S.A.

Method for determining the presence of an antigen attributable to hepatitis

The invention relates to a method for determining the Presence of an antigen assigned to hepatitis in a sample.

Hepatitis, ie inflammation of the liver _B , occurs mostly as a result of an infection or a blockage of the biliary ducts _o Man

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suggests that there are two different types of viral hepatitis, one of which has a longer incubation period than that other. If a patient contracted hepatitis and was known to have contracted it parenterally, then one called this type of hepatitis in the past "serum hepatitis". However, if the patient has no known hepatitis parenterally, but rather orally, this type of hepatitis was called "infectious". It is reported, however That, in addition to having overlapping incubation times, is called "infectious hepatitis" too parenterally, and so-called "serum hepatitis" can also be contracted orally. Therefore, the terms "serum hepatitis" and "infectious hepatitis" cannot be used to distinguish these two forms of hepatitis, although one may assume It is possible that there are two different forms of hepatitis caused by at least two different active substances. For this reason it has been proposed to use the terms "hepatitis A" for the form similar to "infectious hepatitis" and "Hepatitis B" to be used for the form similar to "serum hepatitis".

The present invention is preferably directed to the detection of the antigen or antigens that are related to hepatitis of type B are to be assigned. Patients who develop this form of hepatitis, which is very similar to serum hepatitis these antigens are often in their blood regardless of how they got the hepatitis. At this point It should be noted that there is no reliable evidence for the presence of any of the hepatitis A or any other hypothetical hepatitis C antigen.

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The disease represents "serum hepatitis" or hepatitis B. poses a serious and not negligible clinical problem. Because of the importance of this problem, there are many of detection methods for hepatitis have been developed. To this include "micro-Ouchterlony", immunodiffusion, complement fixation (complement fixation), immunoelectro-osmophoresis, hemagglutination and hemagglutination inhibition, electron microscopy, and radioimmuno detection in the solid phase. A short description each of these methods can be found in British Medical Bulletin, 1972, Vol. 28, No. 2 (Viral Hepatitis), p. 138 to 141.

Immuno-electro-osmophoresis or counter-electrophoresis (counterelectrophoresis CEP) provides a quick and easy method for the detection of the hepatitis antigen and its Antibody. However, this technique is somewhat less sensitive than, for example, complement fixation. you The main advantage is that the verification can be completed in two hours. Because of the low sensitivity However, the CEP method is no longer used by the Food and Drug Administration agreed.

The possibility of using the radio-immuno-detection (radioimmunoassay RIA) for routine diagnostic examinations is considered somewhat limited, and not just because of the but also relatively complicated, specialized and expensive equipment that is necessary for performing the test because of the extreme care that must be taken when handling radioactive isotopes. The mark

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with radioactive isotopes poses a serious health risk, requires constant monitoring and a permit the atomic energy authorities (for users and manufacturers) and finally raises problems with the disposal of the waste. Even so, this technique for immunodetection is fairly widespread at the moment.

All immunological detection methods naturally depend on a basic characteristic of all antibodies and antigens, namely, their ability to react with a specific complementary antigen or antibody. When an antibody is added to a serum containing its antigen, the antibody and the antigen form a compound or a compound Complex and can fall out of solution. In most of the testing procedures mentioned above, the presence of the Antigen detected in human sera using this simple process.

You already have labeled antibodies to identify different ones Used antigens. When an antibody which reacts specifically with a certain antigen in a known manner from the globulin portion of the serum or plasma of a host animal is isolated which has been stimulated to form this antibody, the antibody can be labeled in a known manner will. By combining the antibody with a marker, e.g. - as mentioned above - with a physical one detectable substance such as a radioisotope or with fluorescent chemicals, the presence of the antibody can be detected will. In the diagnostic application it will be

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the labeled antibody attach itself to the antigen to be detected, provided that this counter-antigen is in a prepared one Sample is present. The presence of the antigen in the sample can then be confirmed by the detection of the labeled antibody in the Sample to be confirmed.

For diagnostic purposes, a labeled antibody should be used be specific enough for a particular antigen that it only reacts with the antigens that it is desired to detect will. Cross-reactions with other, similarly structured antigens, but with very different and insignificant consequences must be avoided. This is why It is clear that both the origin and the preparation of the antibody are quite critical in any immunodetection.

As already stated, one of the methods for detecting the antigen to be assigned to hepatitis comprises radio-immunodetection in the solid phase. One such method is described in U.S. Patent No. 3,867,517. After that, the Implementation of this proof initially a unilaterally closed Tube or an insert to be used together with this made of cast polystyrene covered with an antibody layer. This is achieved by using the part to be covered an antibody solution or incubated. Afterward the unknown sample is brought together with the tube or insert coated in this way, so that the antibodies react with the antigens present in the sample. The tube is then washed and exposed to antibodies, marked with the radioactive isotope J-125. It is then washed again to remove all unbound,

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remove labeled antibody. If there are antigens in the sample, the polystyrene tube becomes a sandwich (or insert), the antibody, the antigen and the antibody labeled with J-125. The one with the J-125 Radiation emitted by labeled antibodies is then counted and compared with a reference sample.

It has also been reported that a polytetrafluoroethylene disc was used when performing radioimmunodetection on which a substituted polystyrene, e.g., isothiocyanostyrene, is layered or "grafted" may be useful. This polystyrene is an insoluble substance with specifically formed surfaces of protein-reactive groups that can be used to covalently bind proteins as reagents in the bio-detection method.

Recently, an important alternative is to label antibodies with radioisotopes or fluorescent chemicals has been developed. This consists of labeling antibodies with an enzyme. One such method is in US Pat U.S. Patents 3,654,090 and 3,791,932. An enzyme-linked immunodetection for alphafetoprotein after competitive or the sandwich method is described in Clinica Chemica Acta, Volume 48 (1973), pages 15-18.

In comparison with the radio-immunodetection, the marking offers with enzymes have a number of important advantages. For example, every enzyme-labeled molecule in the final mixture takes part in the detection or participate in the measurement. With radioisotope labeling, on the other hand, only a small fraction of the labeled molecules take up

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participate in the actual measurement, since only a very small part of the radioisotopes in the final mixture disintegrates during the measurement time. When labeling with enzymes, each labeled molecule takes part repeatedly in the detection or measurement, since the Enzyme affects many substrate molecules, which then form detectable end products. Such reactions can take up to 100 times per minute, which increases the sensitivity considerably. A radioisotope, on the other hand, decays only once during the measurement, afterwards it no longer takes part in the verification. An enzyme-labeled reagent also has long life, while the isotope-labeled reagents continually disintegrate, causing serious storage problems raise. Enzyme-labeled reagents also pose minimal health risks while handling Considerable health risks arise with radioisotopes and molecules labeled with them. After all, you can with one Immunodetection with enzyme-labeled molecules a simple one and use relatively cheap equipment. In contrast, the success of radioimmuno-detection depends on its efficiency the decay detection and thus on the quality of a very expensive detection apparatus.

Although finding an antigen related to hepatitis in a person's blood is not synonymous with a history of the disease hepatitis, studies have shown that a high incidence of hepatitis occurs when a patient has received blood in which tests for the presence of hepatitis-related antigens has been established. Because the decision whether to use certain blood reserves available at a blood center

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can, often has to be taken in a relatively short time a sensitive, quick and easy-to-perform overview test for hepatitis that does not require expensive equipment, of the utmost importance. Although the different ones used in the past for the detection of hepatitis antigens Tests have been satisfactory to a certain extent, but they all have one or more drawbacks afflicted.

Every year thousands die from hepatitis or have to be hospitalized. It has long been believed that in order to get this disease under control, the starting point would have to be to develop a technique for examining the blood that could be made available worldwide and easily and routinely performed. Although a number of such detection methods are now available, they have not yet been able to meet the requirements of this ideal test, as they are relatively insensitive _/ involve long and detailed procedures, the use of which is more complicated and not readily available in most hematological laboratories Devices and equipment require or can only be carried out with reagents that are highly unstable and therefore cannot be stored for long periods of time or handled without risk to health.

It is the object of the invention to propose a method for the detection of antigens to be assigned to hepatitis, which quickly, easily, accurately and routinely determines the presence of antigens in body fluids. This task

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is achieved according to the invention by a method, which the includes the following steps:

(a) Bringing (incubating) the sample with a

; held immovably on an insoluble body, antibodies reactive with the hepatitis antigen, which when brought together a bond between Antibodies and antigen present in the sample forms and an insoluble body with bound to it Antigen is produced if there are hepatitis antigens in the sample,

(b) separating the insoluble body from any unbound substance,

(c) Bringing together (incubating) the insoluble body with a labeled solution containing antibodies reactive with the hepatitis antigens, the labeled antibodies are labeled by means of an enzyme which can act on a substrate in such a way that that a detectable reaction product is formed, and wherein the bringing together is carried out such that the labeled antibodies with each in step (a) with an antibody can bind to the insoluble body bound antigen,

Cd) Separating the insoluble body from that of the enzyme-labeled Antibody-containing solution to remove any unbound, labeled antibody therefrom,

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(e) Bringing (incubating) the insoluble body into contact with a substrate solution to which the enzyme of the labeled Antibody acts and thereby brings about a chemical change in the substrate, in which a detectable reaction product is formed,

(f) detecting any reaction product present in the solution, to detect the presence of hepatitis antigens.

Further preferred embodiments of the method according to the invention are the subject of the subclaims.

The invention thus represents direct immunodetection for antigens to be assigned to hepatitis, using the "sandwich" principle is applied. When performing the detection, the antigen to be detected is between those reacting with it Antibody layers embedded. An antibody layer is labeled with an enzyme. The other antibody layer is covalently bound to an insoluble body. The enzyme is brought into contact with a chemical substrate contained in The presence of the enzyme catalyst undergoes a chemical change and thereby forms a reaction product. The presence of the hepatitis antigen is detected by the presence of the reaction product.

According to the invention, a detection unit is also provided or to create a detection kit for hepatitis antigens. The three most important reaction partners of the test set are the insoluble, polymeric solid bodies to which hepatitis antigens are attached

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reactive antibodies are bound to the enzyme-labeled hepatitis antibodies and an enzyme substrate which is chemically changed under the catalytic influence of the enzyme and thereby forms a verifiable end product. In a preferred embodiment of the invention, the enzyme with which the antibody that binds to the antigen is labeled, an alkali phosphatase and the enzyme substrate p-nitrophenyl phosphate. The test set can also contain control sera, e.g. negative for the presence of hepatitis antigens, weak positive and strongly positive sera, a solution of horse globulin, which is added to the test sample to reduce the frequency of non-specific reactions between the test sample and

₍ A buffer to keep the pH of the enzyme substrate solution in the most favorable range for the reaction, and a large number of vials, the size of which is selected so that contact between the insoluble body and the When alkali phosphatase and p-nitrophenyl phosphate are used as the enzyme-enzyme substrate system, the buffer is an aqueous 0.028 molar Na ₂ CO ₃ and 0.001 molar Mg ⁺⁺ solution.

In the context of the invention, an immunologically active, purified 'Suggested hepatitis antibodies for use in immunodetection using a functional enzyme such as Alkali phosphatase, conjugated, i.e. chemically bound.

In the context of the invention, a disk-shaped, insoluble body or matrix for use in

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Immunodetections suggested. The matrix has a multitude of groups which react with proteins and which are evenly attached or "grafted" onto their surface. Purified hepatitis antibodies are covalently bound to these reactive groups and thus form an outer layer of hepatitis antibodies.

In the immunodetection, the insoluble body is placed in a flat-bottomed vial and filled with a liquid one Sample just covered. In order for the test to be reproducible, it is important that the antigens in the sample are compatible with the whole Come into contact with the surface of the antibody-coated disc. The inventor has found that the use a flat or flat disk on the surface has led to a loss of sensitivity. Therefore are the two opposite surfaces of the disc according to the invention deformed, in particular irregularly deformed, to the contact of the disc surface with the bottom of the vial or to keep the glass small. In a preferred embodiment of the invention, the surfaces of the Wafer-like disc in which the disc is pressed before the antibodies are deposited.

The exemplary embodiments described are aimed at detecting the antigen that can be assigned to hepatitis B. However, there is no reason why the method of the present invention cannot also be used for the detection of antigens could be assigned to the other types of hepatitis, once the antigens of these types of hepatitis are identified Has.

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If one, as proposed according to the invention, a test set with the necessary reagents, the reaction vessels and the like and additionally with standard control samples, namely with regard to the hepatitis antigens -negatives, weak and strong positive samples, one can thus advantageously largely rule out errors when carrying out the test. It also increases the accuracy and sensitivity of the test. It is particularly advantageous if the test set contains laboratory equipment with which the individual steps the process can be standardized »

It is particularly advantageous if a hepatitis-antibody-enzyme complex is used in the method is used in which both the antibody and the enzyme have their chemical of interest Retain properties. The complexes proposed according to the invention can be abolished for a long time without them lose their immunological or catalytic properties; in addition, they are compared to those associated with hepatitis Antigens highly reactive.

Preferably, enzymes are bound to the hepatitis antibodies, which catalyze a reaction with a high reaction rate so that the concentration of the end product is precisely determined with the help of a photometric detection device can be.

According to the invention, the insoluble matrix has a high concentration of purified hepatitis antibodies evenly distributed over the entire surface. These are to the Matrix bound so that they are neither mechanical nor by

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chemical forces to which they are subjected during the procedure may be, can be moved from the place. The connection to the matrix is chosen so that the function of the Antibody is retained, i.e. that it can continue to participate in the immunological reaction and through the attachment to the matrix remains immunochemically unchanged.

The following description of preferred embodiments of the invention is used in conjunction with the drawing for more detailed information Explanation. Show it:

1 shows a flow chart of the individual steps of the method according to the invention;

Fig. 2 is a perspective view of an insoluble body according to the invention;

3 shows a view of a reaction vessel during the first phase of the method according to the invention;

4 shows a view of a reaction vessel during the second phase of the method according to the invention;

5 shows a view of a reaction vessel during the third phase of the method according to the invention ; and

6 shows a schematic representation of the sandwich structure, which is built up during the process according to the invention.

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The invention is first described in general below and then detailed below. The method according to the invention for the detection of antigens to be assigned to hepatitis is carried out in four successive steps, such as in Fig. 1 is shown schematically.

In the first step of the inventive method one leaves the antibodies arranged immovably on an insoluble body 10 (FIG. 2) with the antigen in the test serum react as shown in FIG. 3. This reaction makes the antigens immobile on the insoluble ones Body bound, so that when reacting with an enzyme-labeled antibody in the next process step, as in 4, the labeled antibody is also immovably bound. As shown in Fig. 5, the enzyme is with brought together a suitable substrate, which reacts under the influence of the enzyme and undergoes a color change in the process. This change in color serves as evidence of the presence of the hepatitis-related antigens in the sample. Of the The final step is to determine the degree of discoloration and this value with a standard sample to compare.

The detection set according to the invention contains all of the above required materials and is intended for 100 tests. Larger and smaller sets can of course be made can be produced by proportionally increasing or decreasing the amount of reactants. The set will usually sold in a container containing the reactants and control substances until use

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should be kept at temperatures of 2 to 8 C. In addition, jars or bottles are provided in a quantity which is sufficient for 100 tests.

100 to 105 polymer discs with waffle-like surfaces, as shown in Fig. 2 are attached. They carry a layer of antibodies that react with hepatitis antigens can; the antibody layer is bound and lyophilized on the surface of the discs. The exact nature of this insoluble solid disks and how they are made are discussed below.

The second reactant in the kit comprises a sample of a compound of hepatitis antibodies with alkaline phosphatase. The exact nature of this reactant, method of preparation, and other alternatively useful reagents are discussed described below. This reactant is supplied as a ready-to-use solution.

A third one contained in the test set according to the invention Reaction partner consists of 400 mg of a p-nitrophenyl phosphate enzyme substrate. This substance is stable in powder form, but becomes relatively unstable when dissolved in a buffer is to form a solution with a concentration of 1 mg / ml. Therefore, the solution must always be immediately before the Use to be made.

The others used during the process of the invention Materials include a specific amount of equine globulin, sample dilution, various buffer solutions, and various Washing solutions. The exact nature and manufacture

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of these reagents and materials are described below.

A. Preparation of the antibody

In order to be able to carry out the method according to the invention, must you have antibodies that react with the antigen or the aritigens that can be assigned to hepatitis. It will care pointed out that such an antibody exists and the present invention therefore does not refer to the use of one specific antibody should be restricted.

A reactive antibody can be prepared by purifying blood from a host animal that a known Antigen sample has been injected .. One with a hepatitis antigen reactive antibody can be prepared essentially by the method described in British Patent 1,387,625 will.

The preparation of hepatitis antibodies depends first of all from receiving blood which is known to be positive antigens to be assigned to hepatitis. Therefore must Blood samples from different sources on hers first Suitability to be examined to allow the preparation of immunospecific, purified antibodies according to the invention.

A segment of blood vessels with blood believed to relate to Hepatitis antigen positive is kept in an upright position at a temperature of 2 to 8 ° C so the blood cells settle in the lower half. The plasma

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is separated from the cells and the titer of the undiluted plasma and a sample diluted 1:16 in, normal saline solution is ser ~~ Counterelectrophoresis (CEP) determined against a standard antibody. If both the undiluted plasma sample and the If the sample diluted 1:16 are positive, the blood is considered usable for the preparation of purified antibodies viewed. This preparation is described below.

Part of the antigens isolated from the blood is either used to stimulate antibody production in the host animals or to purify the antibodies produced by the animal. Before using the antigens for any of the specified purposes they must be subjected to a prior isolation process.

The plasma is placed in a sterile vacuum container and clumped out by adding a 5 molar CaCl ^ solution, with 0.75 ml CaCl are added to 200 ml plasma. This solution is then placed in a water bath for about an hour at about Maintained 37 ° C until a lump is formed. After a solid lump has formed, the plasma is frozen at -20 ° C and thawed again at 2 to 8 ° C to allow the lump to shrink or retract. The serum will separated from the lump and, if necessary, filtered and is then ready to prepare a hepatitis antigen clot or pellets for immunization or for use in immuno-absorbent columns.

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(1) Prepare the antigen pellet or antigen glob for immunization

Hepatitis positive sera following the process outlined above are centrifuged at 10,000 rpm for 30 minutes at 4 ° C. The part floating on top or the protruding part is distributed in ultracentrifuge tubes after centrifugation and, for example, in a Beckman L2-65B Ultra-. centrifuge at 40,000 to 50,000 rpm for 4-20 hours at 4 ° C. The supernatant in each tube is poured off and thrown away; the lumpy residue or pellet containing the antigens is treated in advance with a normal Washed out (0.15 molar) saline solution.

A small amount of normal saline is then added to each centrifuge tube and the contents of a sonication subject to break up the lump. The suspensions in the sonicator tubes are then combined and evenly distributed in clean tubes filled with normal saline solution. This solution is again in the Beckman L2-65B centrifuge centrifuged at 40,000 to 50,000 rpm for 4-20 hours at 4 ° C as described above.

The process described in the previous paragraph can To be repeated 5 times or more.

The lump or pellet material is placed in a possible after removing the supernatant from each centrifuge tube a small amount of normal saline solution. A sample is mixed with a standard hepatitis antibody sample

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resembled. If counterelectrophoresis (CEP) shows a pellet titer of 1:25 or higher, the pellet may or Lumps are used for immunization. The combined Antigen clumps can be divided into 3 ml equal amounts and frozen at -20 ° C for future use will.

(2) Production and pre-purification of the hepatitis antibodies

A sample of the antigen clot or pellet prepared as described above is taken on the morning of immunization to an equal amount of a complete FREUND adjuvant is added and an emulsion is prepared according to methods known to those skilled in the art. The antigen will then a host animal, e.g. a horse, in the manner known per se, in order to stimulate the production of hepatitis antibodies. The immunized horses are blood drawn or they are subjected to plasmapheresis, known Techniques are applied. Alternatively or in addition, preparations without adjuvant can be used with the addition of the immunizing agents in a different manner be used.

The collected blood must be used for isolation of the hepatitis antibodies prior to the final immuno-absorption purification step be treated. This preliminary cleaning is carried out in three steps. First, the plasma is recalcified from the host animal. Then the serum is mixed with a sufficient amount of normal human plasma (NHP) to prevent others than to precipitate antibodies associated with hepatitis by the fact that the formation of insoluble antigen-antibody complexes

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is directed. The absorbed antiserum is with the help of the CEP tested for hepatitis antibodies. Eventually be the antibodies reacting with the hepatitis antigens are precipitated with ammonium sulfate. This material can be used up to be frozen.

(3) Production of Activated Carbon Immunoabsorbent Columns

For the preparation of the purified, for the production of the inventive Reaction partner or reagent used antibody is the hepatitis antibody obtained in the manner described above subjected to an immunospecific extraction process. For a general discussion of this procedure, see British Patent 1,387,625 (Bradish et al) dated March 19, 1975 referenced, which is entitled "Immunospecific Separation of Antigens and Antibodies". By mentioning this patent specification their disclosure is incorporated into the present application.

In general, the purification process used in the present invention utilizes the ability of those with hepatitis antigens reacting antibodies to form complexes with these antibodies. This complex formation leads to the exclusion of others foreign antibodies and proteins that are inevitably present in the antibody samples obtained from the blood drawn.

A column is created by pre-washed, sorbent charcoal is packed into a glass or plastic tube using known techniques. An association of antigens becomes prepared from at least six different serum samples

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get a mixture of different hepatitis antigens. This association is then applied to a protein concentration set between 1 and 2 mg protein per ml of solvent, this being determined with the aid of UV absorption.

in order to bind the antigen to the charcoal, it is diluted Solution introduced at a flow rate of about 300 to 1000 ml per hour. Around 75 mg of protein should be per gram of activated charcoal (Charcoal) are deposited in the column. The outflow from the columns is collected in equal parts of 500 ml, each of which is checked for protein content. The column can be viewed as saturated with the antigen when the discharge has a protein content approximately equal to that of the starting material. The bed is through one the column flowing stream of phosphate buffered saline (PBS) washed until the Outflow at a wavelength of 280 nm no longer shows any significant detectable absorption.

In order to wash out any loosely attached proteins, the coal bed is filled with freshly prepared 5 molar sodium iodide solution rinsed, which contains 200 mg sodium thiosulfate per liter. Afterward the sodium iodide solution is flushed out of the column, by passing a sufficient amount of PBS through them. After a final wash of the bed with PBS, which Contains 1 mg per ml of sodium azide (protective agent), the column can be stored at a temperature of 2 to 8 ° C until use will.

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Preparation of the antibody for use in an enzyme compound and together with an insoluble solid

The antibodies purified in the manner described above are freed from ammonium sulfate and diluted with PBS, whereby for one part of antibody, two parts of buffer solution are added. The column is drawn up and arranged so that the political groups can be collected. The antibody solution is fed continuously with a flow rate of about 200 ml per column Hour added. The discharge is collected and checked for protein and hepatitis antibodies to determine when the column is saturated with antibodies. As soon as saturation is reached, the column bed is washed out with PBS, to remove loosely absorbed proteins.

At this stage, the immobile antigens attached to the coal column have none with the antibodies that react with them Bond formed. Other foreign proteins and antibodies not specific to the absorbed antigens react have passed through the column and been separated from antibodies.

In order to break this antibody-antigen bond and to rinse out the purified antibodies _/ a 5 molar sodium iodide solution prepared immediately before use is introduced into the column. The amount of sodium iodide should be sufficient to remove all antibodies bound in the column. The flow through the column is set to at least 200 ml per hour, and the

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washed liquid is collected in fractions of an appropriate volume. The total amount collected should be at least as large as that of the sodium iodide solution supplied.

When each fraction of the purified antibodies is collected, it is subjected to a double filtering, first through a 0.45, u membrane and then through a 0.22, u membrane. That The filtrate is diluted in a ratio of 1: 3 with distilled water from 2 to 8 ° C, e.g. 200 ml of the filtrate become 400 ml added distilled water. These diluted antibody fractions are then for example in an AMICON concentrator given with an XM-50 membrane and concentrated.

In a final purification step, the concentrated, purified antibodies are dialyzed. After dialysis, the antibodies are removed and centrifuged. The supernatant is dialyzed at least 24 hours against a ₁ O 01-molar sodium phosphate solution, while the first dialysis against PBS is performed. After the end of this last dialysis, the antibody concentration is measured »

The antibodies are then examined for their activity in comparison with a standard antigen with the aid of the CEP technique in order to determine the antibody content. If this is acceptable, “ they are brought into a liophyllous state and kept until they are used.

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B. Preparation of the antibody-enzyme complex

Alkaline phosphatase from the intestines of calves (intestinal alkaline phosphatase from calves) is reacted with a solution of the reabsorbed antibody in a ratio of enzyme mixed to antibody like 3: 1 until a final concentration is reached at which more than 10 mg total protein per ml of solution of PBS (pH 7.4) is reached. The solution is fully dialyzed to NH. -Ions to remove.

The dialyzed antibody-enzyme mixture is then centrifuged to remove any insoluble matter. The protein content of the supernatant is adjusted to 10 mg / ml by adding PBS-Mg solution. 8% glutaraldehyde is added to this solution in such a way that 1 ml of glutaraldehyde solution is added to 10 ml of antibody-enzyme solution Stirred slowly for 3.5 to 20 minutes " whereby the antibodies and the enzymes form a chemical bond with the help of glutaraldehyde. The solution of the complex is then dialyzed against PBS, which contains ^{0.00r MMg ++ to remove the glutaraldehyde.}

The dialyzed material is then centrifuged and the supernatant is diluted by adding an aqueous solution that is 0.05 µmolar in terms of tris (hydroxymethyl) aminometane (pH8) buffer, 1% normal human albumin (crystalline), 0 , 02% NaN ₃ and 0.001 molar in MgCl ₂ .

In the context of the present invention, other enzymes can be used in addition to or in addition to alkali metal phosphatase. Indeed there

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there is an almost endless list of enzymes that are! covalent with connect to the antibody. From the number of different enzymes, the following breakdown indicates particularly interesting enzymes.

1. Alcohol dehydrogenase

2. Glycerol dehydrogenase

3. Glyoxylate reductase

4. L-lactate dehydrogenase

5. Malate dehydrogenase

6. Glucose-6-phosphate dehydrogenase

7. Mannitol-1-phosphate dehydrogenase

8. Glucose Oxyase

9ο galactose oxidase 10 »L-amino acid oxidase 11. D-amino acid oxidase 12o polyphenol oxidase 13o ascorbate oxidase

14. Catalase

15. Peroxidase

16. Cholinesterase

17. Phospholipase C

18. «C - amylase

19. Lysozymes

20. β-galactosidase

21. Amyloglucosidase

22. β- Glucuronidase

23. Carboxypeptxdase A

24. urease

25. Inorganic pyrophosphatase

26. Aldolase

27. Carbonic anhydrase

28. Histidase.

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The enzyme used to label the antibody is under Chosen taking into account a number of considerations. These include the stability of the enzyme and its easy detection of the enzyme, the ability of the enzyme to withstand the conditions of covalent bond with the antibody that Availability of the enzyme and its cost.

C. Preparation of an Antibody Coated Insoluble Body

The next step in the preparation of the reactants is the covalent attachment of a portion of the purified antibodies to an insoluble body. For this purpose, the insoluble body must have reactive groups or attachment sites that react with the specific antibody used in the bio-detection.

U.S. Patent 3,700,609 (G.W.Tregear et al) entitled "Grafted Copolymers" which was disclosed in this way is made the subject of the present application, an insoluble, continuous polymer substance is described, which has a polymeric "backbone" backbone) or framework,

.to which;

'' Side chains of other polymers or copolymers are attached, With a suitable choice of the attached or "grafted" polymers, it is possible to chemically attach biological substances to the to bind insoluble matrix. One of the products mentioned in the aforementioned US patent is commercially available in the form of a Disk under the trade name PROTAPOL DI / 1 from Imperial Chemical Industries of Australia and New Zealand (ICIANZ) available.

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The PROTAPOL D1 / 1 includes a polytetrafluoroethylene backbone or skeleton, on the surface of which isothiocyanopolystyrene groups are evenly attached or grafted on. It is intended for use in radio immunodetection. As the discs currently available, they are approximately 0.25 mm thick and approximately 12.5 mm in diameter.

According to a preferred embodiment of the matrix according to the invention, each disc 10 has two waffle-like shapes Surfaces 11 on which a first group of linear extending ribs 12 and a second group of linearly extending ribs 14, the ribs 12 and 14 lattice form (Fig. 2). The ribs 12 and 14 are preferably arranged perpendicular to one another and therefore form a plurality of square depressions 16. The side faces of each Ribs 12 and 14 run from adjacent pairs from recesses 16 roof-shaped upwards and form a linear ridge at the tip of the rib. It is on top pointed out that the ribs 12 and 14 are shown greatly exaggerated for better clarity in the drawing.

This desired shape of the discs is achieved in that the disks pass between rollers which have protrusions on their surface for embossing the desired configuration, have in the slices. The rollers are designed in such a way that they exert enough pressure to move the polymer material to deform in the disc without perforating the disc. This is important, as the disc has a reaction layer on its surface. Because of this, would a perforation of the disc inner parts of the same exposed

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place to which no antibodies can be bound. Exposing the polytetrafluoroethylene layer would therefore result in a disc that has a lower binding capacity.

The main idea in the formation of the matrix is that the disk-shaped matrix should have surfaces that are at the Place the disc on the flat bottom of a vial or jar essentially completely in contact with the the sample to be examined, i.e. the contact surfaces between the matrix and the bottom of the vial or glass

1>
chens (should be kept as small as possible. In another preferred embodiment of the disk according to the invention, the surfaces of the disk are designed in such a way that they have areas with high and low-lying points.

The antibodies prepared in the manner described above and present in the lyophilized form are taken up again by adding 100 ml of a 1 molar NaHCO '(pH 9.6) solution per 5.0 mg of antibody. For attachment, the weapon-shaped surfaces are brought together with the diluted solution for about 8 to 16 hours at a temperature of 2 to 8 ^° C. with movement. The antibody solution is then poured off, and the discs are washed twice with successive amounts of 0.1M NaHCO ₃ , pH 9.6, and phosphate-buffered saline and cold {2-8 ° Cj 0.3% bovine serum albumin Washed in phosphate buffered saline with 0.5% TWEEN 20. After another wash with crystalline bovine serum albumin and freezing over dry ice, lyophilization is carried out and the disks are kept at one temperature

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stored at 2 to 8 C until use.

Although the description refers to the preparation of disks to which hepatitis antibodies are bound, it is It is clear to the person skilled in the art that the panes according to the invention can also be used for the immovable attachment of an almost endless Number of proteins can be used with benefit. Such is the increased contact between the test sample and the disc suitable for use in tests that require binding of the following proteins; Antibodies for drugs, such as Digoxin, opiates and steroids; Antibodies from natural products such as insulin or other hormones / and specific metabolic enzymes found in the blood and others Body fluids are found.

example

The following procedure was used to prepare 8,000 discs, each of which was first press treated to obtain the desired shape described above. For a lot of 8000 slices you need 40 mg of hepatitis antibody, ie 5 mg per 1000 slices. The protein content of the resumed hepatitis antibodies is adjusted to 0.05 mg / ml in a final volume of 800 ml in 0.1M NaHCO ₃ (pH 9.6). The entire 800 ml amount of the buffered antibodies are then poured into a 1000 ml screw-top bottle containing a tight container containing 8000 discs, and the bottle is rotated for 16 hours, e.g., overnight, and at one temperature Maintained from 2 to 8 ° C so that the slices at each

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Slowly tumble rotation »Then the liquid poured out of the bottle and removed, and the slices are placed in a 2-liter bottle with a large opening given.

The disks are washed twice with successive 1 liter quantities of cold (2-8 ° C) 0.1M NaHCO ₃ , pH 9.6, after which the buffer is removed. The disks are then washed one more time, this time with two successive 1 liter quantities of cold buffer (0.01M sodium phosphate, 0.15M NaCl, pH 7.4). When the remaining buffer is removed, the disks are washed a third time, this time with two consecutive 1 liter quantities of cold bovine serum albumin solution (0.3%).

The discs are finally filled with two consecutive 1 liter quantities of a solution of cold, crystalline bovine serum albumin (pH 8) at a concentration of 2 mg / ml. This step is done in order for the proteins to create a protein environment on the disc. After removing the remaining washing liquid, the discs are placed on bowls (about 23 χ 23 cm), each with a bow Filter paper laid out and filled with 200 ml of a solution "of crystalline Bovine serum albumin are filled. After the slices have been transferred, they are covered with a sheet of filter paper. The buffer is then completely removed and the slices are quick frozen on dry ice for 30 minutes.

The contents of the dishes are then lyophilized and the dry ones Slices removed and stored in a sealed container. .

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(1) Preparation of the control serum

In order to obtain meaningful results with the immunodetection, it is necessary to provide negative and positive control sera, with which a given test sample can be compared. The preparation of these control sera is described below in individually described.

The negative control serum is produced from human plasma which has been examined, for example, using the radio-immuno-detection technique and which has been found to be negative with regard to antigens that can be assigned to hepatitis. _{A 5M CaCl 2} solution is added to each such amount, which is clearly negative with regard to hepatitis antigens, in order to achieve clumping. About 0.75 ml of CaCl ₂ solution are used per 200 ml of plasma. This plasma is then kept in a water bath at 37 ° C until a lump forms. The clumped plasma parts are then frozen at -20 ° C and stored for at least 12 hours. These plasma pieces are then thawed at 2 to 8 ° C and the serum is collected. If the serum appears excessively cloudy, it may be desirable to clarify it by centrifugation, for example at 9000 revolutions for 30 minutes and at a temperature of 2 to 8 ° C.

20 grams of silica, for example AEROSIL-380, are per Liter of serum was added and mixed with this for 2 hours at room temperature to remove lipoproteins and the serum to stabilize. The mixture is then centrifuged and the precipitate removed. If desired, the pebble

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acid can be removed by filtration using suitable filter media.

The supernatant is then processed further by filtering it is, for example, by MILLIPORE or HORM membranes and -Cushion with successively decreasing porosity, being the last one is a 0.45 / U membrane. Before filtration through the 0.45, u membrane becomes the liquid sodium azide (NaN.,) in sufficient Amount added to achieve a concentration of 0.1 percent by weight. As is known, sodium azide acts as a Protective agent. The final filtration through the 0.45, u filter or a filter with even smaller pores should be in a laminar Flow environment take place; the equipment and the entire duct should be sterile.

The sterile solution can then be stored at temperatures of 2 to 8 ° C should be retained until broken into smaller containers such as those used for immunodetection. By doing The preferred 100-test kit provides 7.5 ml of the negative control serum.

The positive control serum is obtained from recalcified plasma from amounts of blood which are related to the hepatitis associated antigens are positive. A 1% sample is diverted from each positive unit and these samples become poured together to give a test sample. This test sample is first heat-treated for 10 hours at 60 ° C, to inactivate any hepatitis-causing agent in the sample. When the test sample is at room temperature has cooled, a portion is removed and its titer with

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Using the CEP technique against a standard antibody determined to check whether the antigen activity has been preserved is. A sufficient amount of silica is then added to the test sample to achieve a concentration of 20 g per liter To obtain serum. The serum is then stirred with the aid of a stirrer at room temperature for 2 hours; after that is at Centrifuged 9000 rpm for 30 minutes at a temperature of 2 to 8 ° C. The precipitate is removed.

The titer of the supernatant is then determined using the CEP technique against a standard reference antibody. If the If the titer has remained at a sufficient level, the total amount of all serum units can be added together and in the in the same way as just described for the test sample has been treated.

The test sample and the main sample are then combined and mixed with a sufficient amount of a negative control serum diluted in order to achieve optimal results with the positive control serum in the immunodetection according to the invention achieve. The display of the positive control serum in the test according to the invention should preferably be greater than 2000 measured in absorption units χ 1000. This diluted positive control serum is then filtered through suitable media as previously described, taking into account the size of the pores gradually decreases. Before the final filtration, be through a .45, u membrane or a membrane with smaller pores 0.1 weight percent sodium azide added. As with the negative Control sample, the last filtration should be carried out under aseptic conditions and in a laminar flow environment will.

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The weakly positive control serum can be prepared by diluting the strongly positive control serum with negative serum will. The one in the implementation of the erfxndungsgema.ssen The value of the weakly positive serum resulting from the detection method should be between 600 and 1000. In that for 100 tests The preferred test set designed is 2.5 ml of the strongly positive control serum and 2.5 ml of the weakly positive control serum attached.

D. Different reactants and equipment

200 or more glass vials or jars for disposal with a flat bottom are included, which is something have a diameter of more than 12.5 mm, i.e. that are so large that those with a diameter of 12.5 mm Fit slices in. 100 of these bottles or jars are used for the first bringing together of the substances (incubation) and the washout used the other 100 for the final contact (incubation) with the enzyme substrate. The test set allows 100 verifications to be carried out at the same time.

Before the insoluble, solid disc is inserted into each of the 100 vials or jars, each vial 0.05 ml of a horse globulin sample dilution was added, as detailed below. That is used as a precautionary measure carried out to the first contact (incubation) of the sample to be examined with the insoluble disc avoid non-specific reactions. Although the antibody layer on the disc is purified and high grade

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is reactive with hepatitis antigens, a substance is occasionally found in human serum or plasma which can react with horse globulin itself and thus create a bridge between the disc and the enzyme-labeled complex forms, resulting in a false positive reaction. The horse globulin added in the first step binds these Substance so that it is no longer free and can no longer react with the disc. As stated above, the Antibodies bound on the disc produced by having a horse associated with the hepatitis, from the human Blood collected antigens is immunized. In the test set intended for 100 tests, 5.5 ml of a 330 mg Phosphate-buffered saline (PBS) containing horse globulin is included.

F. Preparation of the substrate

The preferred substrate for the enzyme reaction in the test is p-nitrophenyl phosphate, which has a concentration of 1 mg / ml is dissolved in a sodium carbonate buffer, the concentration of which is 0.028 molar with respect to sodium carbonate and 0.001 molar in terms of magnesium (pH 9.8).

At this point it should be noted that other substrates with suitable pH buffer substances are also used as listed in Table I below.

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Board ϊ

ß-glycerol phosphate (^ -glycerol phosphate Pheny! Phosphate β-NaphthyIpho sphat p-nitropheny! phosphate

Phenolphthalein phosphate p-nitrophenyl phosphate Thymolphthalein phosphate p-nitrophenyl phosphate 4-methylumbelliferyl phosphate

(Serum) 7j4 Barbital 8 _? 6 carbonate-bicarbonate 9- "barbital 9.1 2A2M1P, 10.25

2A2M1P, 9 ο 90 2Ä2M1P "10.17 Carbonate bicarbonate 10.0 Diethanolamine , carbonate bicarbonate,

Di © indicated in the panel substrates are all organic Phosphatesterο It is clear _f that other organic Phosphatester as a substrate for the preferred enzyme alkaline phosphate ase can be used "Even if other enzymes are used as the alkali-phosphatase" are relatively easy to suitable Find substrates »

procedure

In order to carry out the detection according to the invention, 100 vials or jars are placed in racks and each of them is marked as belonging to a sample. 0.05 ml of the horse globulin solution are added to each vial, then 5 ml of sample liquid _{are added to 95 of the vials for 0 s.} At the same time, 0.5 ml of the negative control serum is added to each of three vials, 0.5 ml of the strongly positive control serum into one vial and 0.5 ml of the weakly positive control serum into another vial.

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Into each vial containing the Pferdeglobulin and the sample F also in the control vial, an antibody-coated disk is then inserted. The vials and their contents are kept (incubated) at 43 ° C. for half an hour, for example in a moving water bath. During this incubation, any hepatitis antigens present in the test sample combine with the antibodies on the discs.

Before the reagent with the enzyme-labeled antibodies is added to the vials "in which the insoluble body is located, the supernatant from the first incubation must be removed" The insoluble bodies must also be washed to remove any unbound antigen. The washing solution is preferably an O _ir 85% solution of sodium chloride, pH 6.5 to 7,5c After two washes of 2.5 ml of this solution v / ground 0.3 ml of Äntikörper-enzyme complex in each glass filled, -and the jars are again kept at 43 ° C for one hour with agitation (incubated). During this time, the enzyme-labeled antibodies react with the antigens attached to the antibody-coated disk during the first incubation.

After adding the enzyme-labeled antibodies and a second incubation, the supernatant is sucked off; the slices in each vial are washed three times with 2.5 ml of the washing solution each time. This will remove the enzyme-antibody complexes that did not react. Each insoluble body (wheel) is then placed into a clean vial and 2.5 ml of a _i p-nitrophenyl phosphate enzyme substrate buffer solution is added to each vial (1 mg p-nitrophenyl phosphate per ml).

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Since the optimal pH value of the alkali phosphatase p-nitrophenyl phosphate system is 9.8, the enzyme substrate is dissolved in a carbonate Mg buffer (pH 9.8 + O ₄ ,!). This buffer comprises an aqueous solution, which with respect to Na-CO-. O, O28M and with respect to Mg O, QO1M. 40 ml of concentrate are added to the set according to the invention, which can be added directly to the 400 mg of p-nitrophenylphosphate after dilution to 400 ml using distilled water. After the addition of the buffered substrate, the vials are subjected to a third incubation at 43 ° C. with agitation for one hour. If a different enzyme-antibody complex is used, different substrates must be used.

Two drops (0.1 ml) of a 3M NaOH solution are then poured into given each vial to stop the reaction. For this purpose each test set contains 15 ml of a 3M sodium hydroxide solution added.

The enzyme-substrate solution described above changes from a colorless liquid to a yellow liquid when exposed enzyme is present on the disc, i.e. in the vials that contain samples positive for hepatitis antigens.

The above-described part of the negative control sera is poured together in a suitable glass and its absorption at 405 nm with a spectrophotometer against the blind or comparison "?
'value of ~~ distilled water measured. If the negative control shows less than 600 (absorption units χ 1000), they can be regarded as suitable standard samples against which the test results can be compared. With the cumulative negative control samples

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equal value
The values of the test samples and the positive control samples are now read as blank or f, the results being recorded in absorption units χ 1000. With some spectrophotometers you can insert a negative control substance into the instrument, adjust the reading to zero and read the value of the test sample directly. An unknown test sample, the optical density of which is 1000 times greater than 100, if the combined negative control substances are regarded as the zero value, is regarded as positive with regard to the hepatitis antigens. This value has been chosen to eliminate non-reproducible positive results which, if they occur, are generally the result of an error in operation.

The measured values of the test samples can also be compared with the weakly positive and strongly positive control samples. In this way, not only the presence, but also an indication of the concentration of the hepatitis antigens in of the sample.

Examples

Five control measurements should be carried out with each group of unknown substances, three with a negative control serum, one with a strongly positive control serum and one with a weakly positive control serum. These control samples should be subjected to the same procedure and incubation times as the test samples. Caution: To avoid cross-contamination, a clean pipette or disposable tip should be used for each transfer.
The following steps must be carried out: _ 41 _

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1, heating the water bath to 43 ° C "

2 “Numbering two sets of jars to make them correspond to the labeling of the test samples and control samples and place the vials in a vial holder. The first set of these vials is used for bringing together (incubating) the test samples and Control samples with the discs coated with antibodies and with the enzyme-labeled antibodies used. The second set of vials for the substrate reaction described under item 12.

3. Pipette 0.05 ml (1 drop) of the equine globulin reagent on the bottom of the first set of jars. Non-specific, false positive results from in Antibodies that react with horse globulin present in some human sera are raised by the Advantageously largely eliminated the use of horse globulin as a test sample diluent.

4. Pipette 0.5 ml of each test sample onto the bottom of the vial of the first set, taking into account the appropriate sample identification must be observed; Pipette 0.5 ml of the positive and negative Control sera on the bottom of the appropriate tubes.

5. Insert an antibody coated disc in every glass of the first sentence. The surfaces of the panes must be kept clean. she should be done using clean tweezers or by means of

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be transported with a pointed suction cannula, but not touched with the fingers.

6. Store (incubate) the vials at 43 ° C in a water bath for 30 minutes, using the Bewecrungsvorrichtung is switched to slight Qewecrung.

7. After the sample has been brought together (incubated) with the antibody disk, the supernatant is completely aspirated from every glass. Wash the slices by pouring 2.5 ml of an isotonic saline solution into each vial will. This process must be repeated so that each disc is washed twice. To the To aspirate all of the liquid, the vial holder should be tilted during aspiration. After adding the Washing solution should be shaken in the vial holder. The one in the container assigned to the suction device Collected unusable liquid substances should be stored for at least one hour before throwing them away Temperature of 121 ° C are kept in an autoclave.

8. After the final wash and aspiration, add 0.3 ml of the enzyme-labeled antibody solution to each Glass.

9. Store (incubate) the vials for one hour in a water bath at 43 ° C, with the is switched to slight movement.

a water bath at 43 ° C, with the agitation device

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10. Prepare the p-nitropheny! Phosphate substrate Rinse the contents of one vial of p-nitrophenyl phosphate (100 mg) in 100 ml of diluted substrate buffer. (The latter is made by adding 10 ml of concentrated sodium bicarbonate buffer to 90 ml prepared in distilled water). Swirl gently in a circle to mix; the solution should be immediate. Note: This substrate solution must be prepared on the day it is to be used. Otherwise it has to be cooled down. Any solution that is older than 24 hours should be discarded.

11. Aspirate the supernatant and wash three times as in Step 7.

12. Transferring the slices to the second set of Identically labeled, clean jars prepared according to step.

13. Add 2.5 ml of the p-nitrophenyl phosphate substrate solution, as it was prepared in step 10, in each glass containing a disc,

14. Store (incubate) the vials in a water bath at 43 ° C, which is switched to slight agitation

a water bath at 43 ° C, with the shaker

15. Add two drops of 3M sodium hydroxide solution (approximately 0.1 ml total) to each vial in order to to end the reactions. For good mixing of the

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Reactant shaking the holder. Within four hours of the end of the reaction, the Absorption measurements are carried out.

16. Put the three negative controls together and measure the absorbance at 405 nm in a photometer against a blank value of distilled water. The result is given in absorption units χ 1000. If the negative control gives a value that is greater than 600, the evidence is unsatisfactory and must be repeated. If a flow photometer is used / ended and strong positive samples occur, should be removed from the cuvette with distilled water before determining the absorbance of the next sample be rinsed out. If cuvettes are used that cannot be thrown away, the cuvette must be returned rinsed with distilled water for each positive measurement result.

17. Adjust the instrument to absorbance zero with the combined negative control substance. Determine the absorption of each reaction mixture and write down the results in absorption units χ 1000. With some photometers, the instrument cannot work with the negative control substance that has been poured together can be adjusted to zero. In this case, the value of the negative control substances must be different from the value of each sample subtracted from.

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evaluation of results

Unknown test samples / their measured values, measured in absorption units χ 1000, greater than 100, if you use the cumulative negative control substances as a blank value, are to be regarded as reactive. It can be desirable be able to repeat the test on samples that are considered reactive. Before using a reactive serum as classify positive for antigens associated with hepatitis the positive reaction must be proven with the Cordis Confirmatory Test Set (Cordis Confirmatory Test Set, Catalog No. 783-950). This proof must for all reactive samples to be carried out «A reactive serum, which is produced in this way by neutralization with equine antiserum must be considered positive for hepatitis B antigens. As mentioned above, the attestation must be taken must be carried out to evaluate the results. Proof of confirmation is also provided by by the Food and Drug Administration and by the laws of many "states. Briefly summarized, the verification of confirmation according to the invention is carried out as follows:

The positive test sample is tested in duplicate. After the first Process step in which the sample is brought into contact with the disc in each vial, and after washing becomes a disc with antigens specific for hepatitis Antibodies brought together? the other slice is matched with normal horse serum. This bringing together (Incubation) lasts half an hour, followed by the same procedural steps as described above

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Routine evidence. If the sample is positive for hepatitis antigens then the sample that has been brought into contact with the horse antibodies will have a very low value, while the slice that has been combined with normal horse serum will show a high value that corresponds to that corresponds to the value found in the routine verification procedure.

Have specimens with low levels of hepatitis antigens usually low final readings. The samples with a high concentration of the hepatitis antigens, on the other hand, have high measured values. Within a narrow range of concentration differences, the measured value of the optical density is approximately provide a quantitative statement about the concentration of the hepatitis antigens present in the sample.

Limitations of the procedure

1. Non-reproducible reactivity: If repeated detection of a reactive sample shows values that lie below the limit value 100, it can be assumed that the experiment is unreproducibly reactive; it is then assume the sample is negative for hepatitis antigens. The first result can then refer to one Errors in the implementation of the verification can be traced back to, e.g. insufficient washing.

2. Non-specific positive reactions: Non-specific, false positive results from antibodies contained in Some human sera are present and which react with equine globulin are essentially through

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eliminated the use of horse globulin in the test sample solution.

3. Plasma from blood collected in EDTA should not be used.

It should be noted that the residence times (incubation times) for the various process steps and the temperatures at which the bringing together (incubation) is carried out, can be varied within a larger time and temperature range. The invention aims to therefore not to specific times or temperatures of the bringing together (Incubation). For example, the time of bringing together (incubation) of the insoluble Body with the 'sample fluctuate between 10 minutes and 24 hours, the temperature of the incubation between 2 ° C and 50 ° C. On the other hand, the incubation period of the insoluble body with the enzyme-labeled binding partner between 30 minutes and 24 hours, wherein the temperature of the incubation of the insoluble body with the enzyme-labeled binding partner should be between 2 ° C and 50 ° C can.

It should also be pointed out that the method according to the invention can be used to determine the presence of antigens that can be assigned to hepatitis in any body fluid in which antigens are present. Thus, this method can be used to detect the presence of these antigens in serum, plasma, zn determine plasma components, serum components, urine, saliva and cerebrospinal fluid.

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2643823

Results

A representative indication of results which have been achieved when carrying out tests according to the invention are given below. The results in Table A are the results of detection tests according to the invention for samples which are found to be positive for hepatitis when checked using the CEP method. The values in Table B are values for test samples which are positive for hepatitis when examined using the radio-immunodetection method, but negative for hepatitis when examined using the CEP method. The values in Table C are values for test samples which were found to be negative for hepatitis using both the RIA method and the CEP method. The numerical values given in Tables A, B and C for the various test samples are values obtained "in the detection of the with the inventive method. The numerical values are given in absorption units χ 1000 (ODx 1000), the absorption having been measured at a wavelength of 405 nm (OD 405).

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Table A. Table B. (OD χ 1000}
OD4O5 Tabel C. CEP + RIA + CEP- 2745 Neg Test-
sample (ODx 1000)
OD 40 5 Test-
sample 2675 test
sample (OD χ 1000)
OD4O5 72 2807 65 2266 88 0 73 2802 68 1354 89 0 79 2815 69 265 90 0 82 2822 75 953 91 23 121 2831 77 1383 92 42 201 2768 81 1688 93 0 202 2780 87 2739 94 0 205 2749 123 1741 97 '38 206 2758 124 285 98 0 208 2795 125 2466 99 0 209 2800 204 2636 100 0 210 2764 243 101 0 213 2776 261 102 0 214 2804 103 0 217 2795 104 0 218 2800 203 0 219 2746 207 0 220 2718 211 8th 226 2695 212 0 228 2782 215 0 232 2760 216 0 234 2774 224 0 235 2764 225 0 238 2760 227 0 239 2755 229 0 244 2730 230 0 250 2707 231 ' 0 251 2761 233 45

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Table A.
(Continuation) CEP + test
sample (ODx 1000)
OD4O5 252 2778 253 2783 254 2742 255 2743 256 2784 260 2775

Table C (continued)

N eg

Test (O.D.x 1000) sample OD4O5

240 0 241 0 242 0

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Claims (9)

A 41 952 b u - 163 September 27, 19 76 - Claims: 1. Procedure for determining the presence of one of the Hepatitis-associated antigen in a sample, characterized in that it has the following steps includes: (a) Bringing (incubating) the sample with an immobile on an insoluble body held antibodies reactive with the hepatitis antigen, which when brought together a bond between antibodies and forms antigen present in the sample and an insoluble body with bound thereto Antigen is produced if there are hepatitis antigens in the sample, (b) separating the insoluble body from any unbound substance, (c) Bringing (incubating) the insoluble body into contact with a labeled solution containing antibodies which are reactive with the hepatitis antigens, the labeled antibodies being labeled by means of an enzyme which can act on a substrate in such a way that a detectable reaction product is formed, and where the bringing together is conducted in this way _? that the labeled antibodies bind with any antigen bound to the insoluble body in step (a) with an antibody - 52 - 7 0 98-1 5/081 A 41 952 b can enter, (d) separating the insoluble body from the solution containing the enzyme-labeled antibodies, to remove any unbound, labeled antibody therefrom, (e) bringing the insoluble body into contact (incubation) with a substrate solution onto which the The enzyme of the labeled antibody acts and thereby a chemical change in the substrate brings about at which a detectable reaction product is formed, and (f) Detecting any reaction product present in the solution to determine the presence of Detect hepatitis antigens. 2. The method according to claim 1, characterized in that that the reaction product is detected by measuring the optical absorption of the substrate solution. 3. The method according to any one of claims 1 or 2, characterized characterized in that the labeled antibodies comprise immunochemically active hepatitis antibodies which are associated with enzymatically active alkaline phosphatase. 4. The method according to claim 3, characterized in that that the substrate solution contains p-nitrophenyl phosphate. - 53 - ? 098 15/0812 A 41 952 b u - 163 September 27, 1976 264382S 5. The method according to any one of the preceding claims, characterized in that the insoluble body a water-insoluble polymer matrix with a layer more reactive on its surface Includes groups that can form a covalent bond with a hepatitis antibody, and that the Surfaces of the insoluble body are deformed in order to obtain matrix surfaces, which upon insertion the matrix in a flat-bottomed vial is essentially in contact with the solution in the vial come, while the surface-surface contact between the matrix and the bottom of the vial is as small as possible is held. 6. The method according to any one of the preceding claims, characterized in that the detection of each reaction product is compared with a value obtained for a sample known to contain hepatitis antigens. 7. The method according to any one of the preceding claims, characterized in that the in step (a) used Sample is a human liquid. 8. The method according to claim 7, characterized in that the human fluid from the serum, Including plasma, plasma components, serum components, urine, saliva and cerebrospinal fluid Group is selected. - 54 - 7ÖS815 / 08 A 41 952 b u - 163 September 27, 1976 9. The method according to any one of the preceding claims, characterized in that the antigen that can be assigned to hepatitis B is determined. 9815/081