GB2149105A - Method of diagnosing infection - Google Patents

Abstract

A method of serological diagnosis, especially diagnosing bovine leukemia virus infection in cattle. The method includes providing a test plate having a plurality of wells. At least some of the wells are coated with an antigen e.g. bovine leukemia virus antigen. An ELISA buffer including a blocking agent to resist nonspecific binding is introduced into the wells. The test sera is introduced into the wells either directly or by premixing with the blocking solution. An enzyme analyte is introduced into the wells and subsequently a substrate-chromagen is introduced into the wells. The reaction is read or stopped. By visual determination based upon the color of the wells, either with the naked eye or with the aid of appropriate apparatus, a determination is made. <IMAGE>

Description

SPECIFICATION Method of diagnosing infection This invention relates to a method of diagnosing infection and, more specifically, it relates to an enzyme-linked immunosorbent assay for effecting serological diagnosis of bovine leukemia virus infection in cattle.

It has been known to employ direct methods of diagnosing bovine leukemia virus infection in cattle. Such methods generally involve isolation and culturing of lymphocytes.

This approach tends to be cumbersome and time consuming and has limited the practical routine use of this approach. In addition, it has generally been found that such direct methods are less sensitive than serological methods.

It has been known that bovine leukemia virus (BLV) infection in cattle results in production of viral antibodies specific for a glycoprotein (gyp51) which is present in the viral surface and for the major core virion protein (either p25 or p24). As these antibodies provide an accurate indicator of bovine leukemia virus infection, their detection by means of serological tests is an effective approach for identifying bovine leukemia virus infected cattle.

One know serological test for this purpose is the agar gel immunodiffusion test (ABID).

In such a test an antigen preparation, which generally is predominantly gp51, is-intro- duced into one well in a thin layer of agar gel.

A serum sample which is to be tested is placed in another well. If bovine leukemia virus antibodies are present in the serum, they will bind with BLV gp antigen and form a visible line in the agar between the wells. This test, however, has been known to be less sensitive than other tests, particularly in the early stages of infection and, on occasion, to provide false negative reactions. Further, weak precipitation reactions result in equivocal results. Also, as the gp antigen used in such tests is a crude preparation, it tends to contain fetal calf serum proteins in high concentrations. These proteins can give precipitin lines with bovine serum containing isoantibodies.

Such antibodies are common among cattle immunized with vaccines containing bovine proteins, among cattle pre-immunized against babesiosis and piroplasmosis by transfusion of bovine blood and among multiparous cows. In principle, the precipitin lines due to bovine protein in the gp51 antigen preparations should give reactions of non-identity or partial identity with the adjacent control precipitin lines. However, under routine testing conditions, such reactions may be very difficult to recognize or may be subject to clerical bias.

This is particularly true in instances where the precipitin lines are weak- or diffused, as is often the case. This results in a subjective and unreliable test result. The only sera which can be accurately assessed using the test is, therefore, sera giving distinct precipitin lines. Yet another disadvantage of this test is that it is not adapted for large scale use as it is not easy to automate and requires a relatively long incubation time, i.e. on the order of forty-eight hours.

It is also known to use a radioimmunoprecipitation assay with purified gp51 labeled with radioactive iodine as a tracer (RlAgp).

This test has been known to be more reliable than the AGID test particularly where the antibody titers are not very high. One of the problems with this test is that special equipment, such as a gamma counter, is needed in order to employ this test. In addition, it is necessary to have special equipment to purify the antigen to a state of homogeneity. Further, this test requires some skill in performing the test, as well as the need to employ suitable safety procedures in handling the radioactive material.

Another known type of test is the enzymelinked immunosorbent assay (ELISA) which offers the advantage of being practical and as inexpensive as the AGID test, while providing results which are acceptable. Also, it does not involve some of the burdens experienced with the RIA gp test. See, for example, Ressang et al. "Enzyme Linked Immunosorbent Assay For The Detection of Antibodies to Bovine Leukosis Virus", Ann. Rech. Vet. 1978,9(4) pp.

663-666; Behrens et al., "Comparative Studies on the Enzyme Linked Immuno Sorbent Assay and Immunodiffusion Test and the Diagnosis of Enzootic Bovine Leukosis", Fourth International Synposion on Bovine Leukosis, pp. 173-184; Todd et al., "Use of Controlled Antigen to Improve the Enzyme Linked Immunosorbent Assay for Enzootic Bovine Leukosis Antibodies, Veterinary Record (1982) 110, pp. 307-308; Poli et al., "Microplate Enzyme-Linked Immunosorbent Assay for Bovine Leukemia Virus Antibody", Journal of Clinical Microbiology, January 1981, pp. 46-48; Graves et al., "Comparison of Enzyme Linked Immunosorbent Assay with an Polykaryocytosis Inhibition Assay and the Agar-Gel Immunodiffusion Test for the Detection of Antibodies to Bovine Leukemia Virus", Am. J.

Vet, Res. Volume 43, Number 6, pp.

960-966; Biancifiori et al., "Elisa Tests for Detection of Antibodies to Enzootic Bovine Leukosis Virus", Fourth International Symposium on Bovine Leukosis, pp. 167-172 and Todd et al., "An Enzyme-Linked Immunosorbent Assay for Enzootic Bovine Leukosis Virus Antibodies", Vet. Rec. 1980, 107 pp.

124-126.

One of the serious problems with the known ELISA tests is the fact that sera from some cattle not known to be infected with BLV give false-positive reactions. The falsepositive reactions appear to be due to the binding of immunoglobulin to the plastic vessels used for the ELISA test. Depending on the ELISA procedure used, about 1-30% of BLV-free cattle sera can give false-positive reactions, which is a totally unacceptible rate.

To some extent this problem may be minimized by comparing the reactions of a test serum in vessels with and without antigen.

However, if the reaction in the vessel without antigen is too high, one cannot score the sample accurately.

Because of the apparent frequency of falsepositive reactions in these known systems, there remains a very substantial need for an ELISA test which in respect of specificity, sensitivity and practicality will be superior to the previously know ELISA Tests and will also be as effective in these respects as the RIA gp Test.

The present invention has met the abovedescribed need by providing an ELISA test wherein a test plate having a plurality of wells is provided. At least some of the wells are coated with a bovine leukemia virus antigen.

After incubation and washing, blocking of the non-specific binding sites may be accomplished by applying a solution of a suitable animal serum. The test sera may be admixed with the blocking animal serum solution or introduced directly into the wells. After this has been accomplished additional incubation and washing is effected. An enzyme chemically bound to an antibody ("enzyme analyte") which recognizes bovine immunoglobulin is introduced into the wells, followed by further incubation and washing to remove unbound analyte. A substrate containing a chromagen is introduced into the wells and a further incubation step is provided. The reaction is thenread or stopped and the presence or absence of bovine leukemia virus infection may be determined visually either with the naked eye or through use of a suitable instrument, such as a spectrophotometer, for example.Preferred materials and process parameters are employed.

It is an object of the present invention to provide a method of serologically diagnosing bovine leukemia virus which method is highly specific, has desired sensitivity and is practical for routine use with results being available within a few hours.

It is a further object of this invention to provide an ELISA test which effectively reduces the reactions of BLV-antibody free sera to an insignificant level.

It is another object of this invention to provide an ELISA test which does not require the use of specialized equipment or radioactive materials.

It is a further object of the present invention to provide an ELISA test which is inexpensive to- employ.

It is a further object of the invention to provide an ELISA test which does not require the use of specifically trained personnel.

It is another object of the present invention to provide an ELISA test which reduces nonspecific binding of BLV antibody containing sera.

It is a further object of the present invention to provide an ELISA test which is adapted for visual determination of the results by the naked eye.

These and other objects of the invention will be more fully understood from the following description of the invention on reference to the illustration appended hereto.

Figure 1 is a flow diagram of a preferred form of the invention.

Figure 2 is a top plan view of the plate usable in the present invention.

Figure 3 is a top plan view of a well of the plate of Fig. 2.

Figure 4 is a cross-sectional view of the well of Fig. 3 taken through 4-4.

As used herein, reference to visual determination by the "naked eye" shall be deemed to include determination by an individual wearing or using corrective lenses of any type, but shall exclude the use of equipment such as spectrophotometers.

Among the important features contributing to the success of the ELISA test of the present invention is the use of animal serum as an agent for blocking non-specific binding of bovine immunoglobulin without meaningfully interfering with specific reactions. Also of consequence are the unique washing procedures employed after the first and second incubation stages. The substrate-chromagen solution also contributes meaningfully to the successful result as a result of its high sensitivity, low toxicity and stability.

In a preferred method of practicing the present invention the following sequence of operations will be performed.

With reference to Fig. 1, the process will be summarized with details of each stage to be described hereinafter. A suitable plate, having a plurality of wells and being adapted to test a number of samples is provided. (For convenience of reference, numbers have been assigned to the process steps of Fig. 1). About one-half of the wells are coated with BLV antigen 2, after which the wells are incubated 4 and washed 6 to remove unbound antigen.

The plate may then be stored 8, if desired. An ELISA buffer which comprises (a) a blocking serum for blocking the non-specific binding of bovine immunoglobulins to the plastic vessel in (b) high ionic strength buffer is added to the wells 14 and the sera to be tested is also introduced into the wells 14, followed by incubation 16 and, washing 18 to remove unbound antibody. The ELISA buffer and sera may be mixed first and the mixture is introduced into the wells followed by the incubation 1 6 and washing 1 8 steps. An alternate, but less preferred approach would be to intro duce the ELISA buffer into the wells, incubate, wash to remove excess material and then introduce the test sera into the wells in the ELISA buffer without serum and then incubate and wash the wells.The test sera should be added to at least one well with antigen and one well which is without antigen. Enzyme analyte 20 is then added to the wells followed by incubation 22 and washing 24 to remove unbound analyte. Substrate chromagen 26 is then added to the wells followed by incubation 28. The reaction may then be stopped 30 or read.

In the plate 38 shown in Fig. 2, a series of 96 wells which are preferably substantially cylindrical are provided. The plate may be a unitary plastic member with the wells being of substantially equal size and having a capacity of about 100 to 300 microliters (ul). The plate may be composed of any suitable material such as polystyrene or polyvinyl chloride, for example. In the form shown the wells are arranged in a series of columns (1-12) and rows (A-H). In general, it is preferred to alternative rows when antigen coating is effected in order to facilitate scoring of the results. For example, wells in rows A, C, E and G may be antigen coated and wells in rows B, D, F and H may be devoid of antigen coatings. Wells 40, 42 may serve as wells for the weak positive control serum with antigen and wells 44, 46 may serve as the wells for the weak positive control serum with no antigen.

In order to resist undesired flow of material from one well to an adjacent well, as is shown in Figs. 3 and 4, the plate may have wells which are surrounded with an annularly continuous upwardly projecting barrier ring 48.

In addition to the wells which will be treated in the fashion to be described with reference to Fig. 1, it is preferred that certain additional control serum and control wells be provided. The nature of these control wells and function of same will be described hereinafter.

Example 1 A plate, which may be a polystyrene microtiter plate of the above-described type is employed. One-half of the wells are initially coated with bovine leukemia virus antigen.

This may be accomplished by harvesting the supernatant fluid of BLV producing cells, removing large debris by centrifugation and centrifuging the BLV in the clarified supernatant onto a dense layer of sucrose. The BLV cells may be obtained from any suitable source, as will be known to those skilled in the art, but it is preferred to employ a monolayer cell culture that produces BLV continuously and is substantially free of other virus and adventitious agents. An example of such known means employing BLV-infected bat lung cells such as is taught by Graves and Ferrer, in Cancer Research, Vol. 36 pp.

4152-4159, 1 976. Another suitable source is BLV-infected fetal lamb kidney cells as is that taught by Van Der Maaten et al., Bibliotheca Haematologica, Vol. 43, pp. 360-363 (1976). The virus is recovered and adjusted to about 5 to 20 micrograms per millimeter (ug/ml) with phosphate or tris buffered saline (0.15M NaCI, 0.05M PO4 buffer, pH 7.0-7.4) ("PBS"). The antigen solution is added to the wells in quantities t about 200 ul/well in alternate rows. The plate is then subjected to a first incubation at about 3 to 25"C. for about 1 to 1 7 hours. After such incubation, the unbound antigen is removed by a first washing with any suitable isotonic solution, such as phosphate or tris buffered saline solution.

At this point, if desired, the plates may be dried and stored dry at about 4"C. or less for extended periods (which may be in excess of 10 months if desired) prior to use in performing the test.

In the preferred test, as is illustrated in Fig.

1, about 100 to 200 ul (microliters) of the ELISA buffer is added to the wells into which about 10 to 20 ul of the test bovine sera is then introduced. (It is preferred that the ratio of test sera to ELISA buffer be about 1:5 to 1:10.) The ELISA buffer preferably contains about 2 to 20 percent of the animal blocking serum (serum for blocking the non-specific binding of bovine immunoglobulins to the plastic vessel). It is preferred that the animal serum be selected from the group consisting of goat serum, chicken serum, guinea pig serum and sheep serum, with goat serum being preferred. It is also desirable to add to the solution about 0.25 to 2.0 M NaCI and preferably about 0.5 to 1.0 M NaCI and 0.01 to 0.2 M phosphate or tris buffer pH 6 to 8.

The wells are then incubated at about 4-25"C. for about 1-16 hours. An optional washing step 1 2 removes any excess.

An equally preferred alternative is to mix the ELISA buffer with the test sera, and introduce the mix into wells. A less preferred method is to introduce the ELISA buffer into the wells and then incubate at about 4-25"C.

for about 1 to 1 6 hours. A washing step to remove excess material may then be employed. Plates so treated may be stored dry at 4"C. for in excess of three months. If the ELISA buffer is not removed prior to storage, the test serum may be introduced directly into the wells. If the ELISA buffer has been removed prior to storage, the test serum may be diluted and added to the wells or added to wells containing a suitable diluent such as ELISA buffer or ELISA buffer without blocking serum.

As a precaution, it is generally preferred to assay the test sera in duplicate. The plate is then incubated at a temperature of about 3 to 37"C. for about 1 to 16 hours. The wells are subsequently washed in a solution of distilled water and about 0.01 to 0.2 percent of a suitable surfactant or detergent. A suitable surfactant, polyoxyethylene sorbitan monolaurate, is that sold under the trade designation Tween 20 with a concentration with about 0.05% being suitable for this use. Five or more rapid washes can be used, but we prefer four one-minute incubations in the wash solution.

The enzyme-analyte is then diluted in the ELISA buffer (without serum) to about 50 to 70 ng/ml, and preferably bout 50 ng/ml. It is added to the wells in quantities of about 200 ul/weil. The preferred material for this purpose is an antiserum prepared in goats or rabbits which recognize bovine immunoglobulin and which is chemically coupled to horseradish peroxidase. It is preferred that the antiserum be purified such that it contains only antibodies which recognize bovine immunoglobulin, otherwise known as "affinity puri fied". After incubation for a period of about 1 5 to 60 minutes and preferably about 30 minutes at about 20"C. to 37"C., the wells are washed with distilled water in order to remove unbound bovine enzyme analyte.Several rapid washes may be used, but we prefer four one-minute washings in water.

Subsequently, a substrate-chromagen is introduced into the wells in quantities of about 200 ul/well. This solution may consist of H202 in a suitable buffered solution and a suitable chromagen. The chromagen is a substance which is colorless until it donates electrons in the peroxidase reaction, after which it develops intense color. A preferred solution consists of about 0.2 ul 30% H202 per ml of a solution (0.11 mg/ml of pH 4.0 0.05 M citrate buffer) of 2, 2'-azino-di-(3-ethylbenzothiazoline sulfonic acid. This material is available from a number of sources including Sigma Chemical Company. The substrate chromagen containing wells are then subjected to incubation at about 1 5 to 39"C. for about 5 to 60 minutes and preferably about 20 to 30 minutes.The reaction is then read or stopped by a suitable acid addition to the wells. A suitable material for use in stopping the reaction is about 50 to 100 ul of hydrofluoric acid, at concentrations of about 0.1 to 0.5N and preferably of about 0.2N. The pH of the acid is preferred to be about 3.0 to 3.5.

Visual inspection, in a manner to be described hereinafter, will permit determination as to whether bovine leukemia virus infection is present in the serum being tested.

In order to provide for a more efficient test, certain controls are preferably employed in the present invention.

In the preferred practice of the present invention, each plate will be provided with at least four wells for each serum to be tested and four control wells to ensure that the assay has been performed under suitable conditions and to facilitate evaluation of the results.

Each test serum, diluted as described above in the ELISA buffer will be incubated in two uncoated wells and in two wells previously coated with BLV antigen. (While the test may be performed with individual as distinguished from double wells, i.e., two of each type, this provides an extra safeguard and eases visual scoring.) The no antigen control determines the degree to which the particular test serum binds non-specifically to the well. The sensitivity and specificity of the test depends on the difference in binding between the wells coated with the antigen and the wells not so coated.

Ideally, with a BLV antibody containing serum, there is strong binding to the antigen coated wells and no appreciable binding to the uncoated wells. The degree of binding translates into color intensity with strong binding resulting in a definite color such as a dark green and no binding appearing colorless.

To determine if the assay has optimum sensitivity, two uncoated and two BLV antigen coated wells are incubated with a weak positive serum. The titer of this weak positive serum is adjusted to give the minimum color reaction which will be considered positive.

With a 96 well plate the use of four wells for the weak positive control serum, leaves 92 wells for testing. This permits duplicate testing of 23 test sera.

One of the advantages of the process of the present invention is the fact that an accurate test determination may be made visually by comparing the color which develops in the bovine leukemia virus coated wells with the color in the control wells. This may be accomplished with the naked eye or by the use of suitable equipment such as a spectrophotometer, for example.

With respect to visual determination with the aid of an instrument such as a spectrophotometer, the absorbance at about 405 to 41 5 mm of each well may be determined by means of any suitable commerical ELISA spectrophotometer. The OD values of BLV antibody free sera would generally be below 0.4 units and the differences in OD values given by the same sera in the wells with bovine leukemia virus antigen and in wells with control antigen would generally be less than 0.05 units.

In order to provide additional guidance regarding a specifically preferred embodiment of the invention, a further example will be considered.

Example 2 Referring once again to Fig. 2 for general guidance, alternate rows of a polystyrene microtiter plate having 96 generally cylindrical wells are provided with a 200 ul of sucrose cushion purified BLV antigen at about 10 ug/ml in phosphate buffer solution. The con trol (untreated) rows are not treated with anything. The test plate is then incubated at 4"C.

for 1 6 hours. After the incubation the un bound antigen is removed by washing with phosphate buffer solution. Two hundred ul ELISA buffer is added to all wells and then 20 ul of the weak positive serum or test sera is added to two wells coated with BLV antigen and to two adjacent wells not coated with BLV antigen. All of the sera are tested against both BLV antigen containing wells and control anti gen containing wells. As is preferred, all test sera are assayed in duplicate. After incubation for 2 hours at room temperature, the wells are washed four times with 0.05% of a suitable detergent, such as Tween 20 (a polyoxyethy lene sorbitan monolaurate). Each wash is a one-minute incubation in the wash solution.

Subsequently, the affinity purified rabbit antibovine immunoglobulin chemically coup led to horseradish peroxidase-conjugated is diluted to about 60 ng/ml in ELISA buffer and is added to the wells (200 ul/well) which have been incubated with test sera and con trol bovine sera. After incubation for about 30 minutes at about 25"C., the wells are washed with distilled water four times in order to remove unbound enzyme analyte. This wash ing contributes to further reduction of non specific reactivity. About 200 ul of a sub strate-chromagen solution having a concentra tion of 0.11 mg 2,2'-azino-di-(3-ethylbenzothi- azoline) sulfonic acid and 0.2 ul 30% H202 per ml of pH 4.0 citrate buffer is then added.

If it is desired to read the results spectropho tometrically or to preserve the results, the reaction is stopped after about 25 minutes by adding 50 ul of 0.2 N hydrofluoric acid, of pH 3.3. The test results are then determined visually either with the naked eye or by the use of a suitable instrument such as a spectro photometer.

Example 3 Referring to Figure 2, once again, a further example will be considered assuming that a specific sample of test sera has been intro duced into wells A3, A4, B3, B4 and that wells Al, A2, B1, B2 function as weak posi tive control wells a direct visual reading may be made. Row A has wells provided with antigen and row B has wells without antigen.

After the test procedure has been employed, wells Al, A2 will be of a darkened color (green) representing the minimum color inten sity to be regarded as a positive test result and negative antigen control wells B1, B2 will be clear. Negative antigen control wells B3, B4 will be clear. A dark color (green) in wells A3, A4 at the level of the color in wells Al, A2 or at a more intense level will be regarded as a positive test result. If wells A3, A4 are clear or of lesser color than wells Al, A2 the test result is negative. It will be appreciated that the test could be performed with Al, B1 serving as the weak positive control and wells A3, B3 as the test wells. It is preferred to employ two wells for each function, however, as an extra confirmation of accuracy.

The method of the present invention contributes to improved specificity by reducing the background color to a negligible level in the antigen control wells. The specificity of the test is such that even weak-positive sera can be identified accurately as a result of the virtual absence of undesired background in control wells. The test of the present invention is also practical in that personnel without specialized training and without requiring special equipment can process a large number of samples in a relatively short period of time.

Further, the test is economical to employ and the test plates and associated materials have an acceptable shelf life. An accurate visual determination can be made either with the naked eye or by employing suitable equipment.

While for clarity of description herein an example employing a microtiter plate with 96 wells has been illustrated and described, it will be appreciated that other types of plates, different numbers of wells and other means of providing a group of receptacles for single or multiple tests such as a group of separate tubes or other receptacles may be employed.

For convenience of reference herein the term "wells" shall be deemed to encompass such alternate approaches.

Whereas particular embodiments of the invention have been described above for purpose of illustration, it will be appreciated that by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

Claims (22)

1. A method of serologically diagnosing bovine leukemia virus infection in cattle including the steps of providing a test plate having a plurality of wells, coating at least some of said wells with a bovine leukemia virus antigen, effecting a first incubation of said plate, effecting a first washing of said plate to remove unbound antigen, blocking non-specific binding sites by applying a solution of blocking animal serum to said wells, adding test sera to at least some of said wells, effecting a second incubation of said plate, effecting a second washing to remove unbound components, introducing an enzyme analyte into said wells, effecting a third incubation of said plate, effecting a third washing to remove unbound bovine serum, introducing a substrate chromagen into said wells, effecting a fourth incubation of said plate, and visually determining by color whether antibodies to bovine leukemia virus exist in said test serum.

2. A method as claimed in claim 1, including introducing said test sera into said blocking solution prior to introduction of said blocking solution into said wells.

3. A method as claimed in claim 1, wherein after introducing said blocking animal serum solution into said wells and before said second incubation said test sera is introduced into said wells and mixed with said blocking animal serum.

4. A method as claimed in claim 3, wherein said blocking animal serum solution is a high ionic strength buffered animal serum solution and said buffered animal serum solution contains about 2 to 20 percent animal serum.

5. A method as claimed in claim 1, including the steps of providing at least one negative antigen control well for each sample of test serum, introducing no bovine leukemia virus antigens into said negative antigen control wells, and introducing said blocking animal serum solution and said test sera into said negative antigen control wells.

6. A method as claimed in claim 5, wherein the reaction is stopped after said fourth incubation.

7. A method as claimed in claim 5, including the steps of providing at least one minimum positive serum control well, and introducing into said wells the minimum sera concentrations of antibodies deemed to be positive, whereby the test sera results may be compared with said minimum positive serum control well, and visully determining whether said test serum is positive by comparing the difference of its color reaction in said antigen coated and the negative antigen test wells with the differences of the color reactions of the said minimum positive control serum.

8. A method as claimed in claim 1, wherein said blocking animal serum solution contains a material selected from the group consisting of goat serum, chicken serum, guinea pig serum and sheep serum.

9. A method as claimed in claim 4, wherein said buffered animal serum solution is about 5-10 percent of said animal serum in phosphate buffered saline and said blocking solution has about 0.25M to 2.OM. NaCI, and about 0.01 M to 0.2 M phosphate buffer with a pH of about 6 to 8, and including the steps of admixing test sera with said blocking solution prior to introduction of said sera into said wells, said test sera being diluted by a ratio of about 1:5 to 1::10 by said buffered animal serum solution, introducing test sera into a first series of said wells containing said bovine leukemia virus antigen and a second series of said wells not having said bovine leukemia virus antigens, providing a control serum containing a low titer of bovine leukemia virus antibodies, and introducing said control serum into at least one said well containing said bovine leukemia virus antigen and at least one said well not containing said bovine leukemia virus antigens.

10. A method as claimed in claim 1, including the steps of employing as said enzyme-analyte an affinity purified goat or rabbit anti-bovine immunoglobulin, coupling said affinity purified immunogloblin with horseradish peroxidase, and including as said substrate-chromagen a material for assaying peroxidase.

11. A method of diagnosing infection comprising providing a test plate having a plurality of wells, coating at least some of said wells with an antigen, effecting a first incubation of said plate, effecting a first washing of said plate to remove unbound antigen, blocking non-specific binding sites by applying a solution of blocking serum to said wells, adding test sera to at least some of said wells, effecting a second incubation of said plate, effecting a second washing to remove unbound components, introducing an enzyme analyte into said wells, effecting a third incubation of said plate, effecting a third washing to remove unbound test serum, introducing a substrate chromagen into said wells, effecting a fourth incubation of said plate, and visually determining by color whether antibodies to infection exist in said test serum.

1 2. A method as claimed in claim 11, including introducing said test sera into said blocking solution prior to introduction of said blocking solution into said wells.

13. A method as claimed in claim 1, wherein after introducing said blocking serum solution into said wells and before said second incubation introducing said test sera is introduced into said wells and mixed with said blocking serum.

14. A method as claimed in claim 13, wherein providing said blocking serum solution is a high ionic strength buffered serum solution, and said buffered serum solution contains about 2 to 20 percent serum.

1 5. A method as claimed in claim 11, including the steps of providing at least one negative antigen control well for each sample of test serum, introducing no said antigens into said negative antigen control wells, and introducing said blocking serum solution and said test sera into said negative antigen control wells.

1 6. A method as claimed in claim 15, wherein the reaction is stopped after said fourth incubation.

1 7. A method as claimed in claim 15, including the steps of providing at least one minimum positive serum control well, introducing into said wells the minimum sera concentrations of antibodies deemed to be positive, whereby the test sera results may be compared with said minimum positive serum control well, and visually determining whether said test serum is positive by comparing the difference of its color reaction in said antigen coated and the negative antigen test wells with the differences of the color reactions of the said minimum positive control serum.

18. A method as claimed in claim 11, wherein said blocking serum solution contains a material selected from the group consisting of goat serum, chicken serum, guinea pig serum and sheep serum.

19. A method as claimed in claim 14, wherein said buffered serum solution is about 5-10 percent of said serum in phosphate buffered saline, and said blocking solution has about 0.25M to 2.0M. NaCI, about 0.01 M to 0.2 M phosphate buffer with a pH of about 6 to 8, and including the steps of admixing test sera with said blocking solution prior to introduction of said sera into said wells, said test sera being diluted by a ratio of about 1:5 to 1:10 by said buffered serum solution, introducing test sera into a first series of said wells containing said antigen and a second series of said wells not having said antigens, providing a control serum containing a low titer of said antibodies, and introducing said control serum into at least one said well containing said antigen and at least one said well not containing said antigens.

20. A method as claimed in claim 11, including the steps of employing as said enzyme-analyte an affinity purified goat or rabbit anti-bovine immunoglobulin, coupling said affinity purified immunoglobulin with horseradish peroxidase, and including as said substrate-chromagen a material for assaying peroxidase.

21. A method of serologically diagnosing bovine leukemia virus infection in cattle substantially as hereinbefore described.

22. A method of diagnosing infection substantially as herein before described.