CA1233746A

CA1233746A - Method of diagnosing infection

Info

Publication number: CA1233746A
Application number: CA000466346A
Authority: CA
Inventors: Jorge F. Ferrer; Richard M. Thorn
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 1983-10-26
Filing date: 1984-10-25
Publication date: 1988-03-08
Also published as: GB8426839D0; FR2554127A1; GB2149105A; FR2554127B1; GB2149105B; BE900911A; JPS60173472A

Abstract

ABSTRACT OF THE DISCLOSURE
A method of serologically diagnosing bovine leukemia virus infection in cattle. The method includes pro-viding a test plate having a plurality of wells. At least some of the wells are coated with a 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.
Appropriate control wells may be provided. Intermediate washing and incubation stages are employed. Among the pre-ferred blocking agents are those selected from the group consisting of goat serum, chicken serum, guinea pig and sheep serum.

Description

~33~

Thi~ invention relates to a method of diagnosing infection and, more specifically, it relates to an enzyme-linked i~munosorbent assay for effecting sexological diagnosis of bovine leukemia virus infection in cattle.
~t has been known to employ direct methods of diagnosing b~ine leukemia virus infection in cattle. .Such methods generally involve isolation and ~ulturing of lymph~cytes. This approach tends to be cumbersome and time conguming and has limited the practical routine use of this approach. In addition, ;t has gener~lly been found that su~h ~~~
irect methods are less sensitive than ~erological methods.
It has been known that bovine leukemia virus (BL~3 nfection in cattle results in production of viral antib~dies pe~iic for a glycoprotein (gp51) which is pr~sent in the viral : .15 urface and for the m~jor core virion protein (either p2~ or 2i). As these antibodies provide an accurate indicator of ~vine leukemia ~irus:inEection, their detection bv means of : erological tests is an effective approach for identifying ~ov~ne eukemia virus infected cattle.
One known serological.test for ~his purpose is the agar ~ el i~munodiffusion test IAGID). In such a ~est an antigen . '~" .
. ' _ preparation, which generally is predominantly gp51, is introduced 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 antib~dies 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 ~o be less sensitive than other tests, particularly in the early stages of infection and, on occasion, to provide false neqative reactions. Further, weak precipitation reactions result in equivocal results. Als~, 1~ as the gp antigen used in such tests is a crude preparation, i~
tends to contain ~etal calf serum proteins in high concentrati~ns. These proteins can give precipitin lines with bovine serum c~ntaining isoant;bodies. Such anti~odies are common among cattle immunized with vaccines containing bovine proteins, among cattle pre-immuni zed against babesiosis and piroplasmosis by transf usion of bovine bloc>d and among ~ s~
~ultiparous cows~ In principle, the precipitin lines due to _ b~vine protein in the gp51 antigen preparations should give . reactions of non-identity or partial identity with the adjacent control precipitin lines. ~owever, under routine testing ondi~ions, such reactions may be very d;fficult to recognize or nay be subject to clerical bias. This is particularly true in ns~ances where the precipitin lines are weak or diffused, as is ~ften ~he case. This results in a subjective and unreliable test ~esult. The only sera which can be accurately assessed using the test is, therefore, sera giving distinct precipitln lines. Yet an~ther disadvantage ~f this tést is that it is not adapted for arge scale use as it is n~t easy to automate and requires a

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33 ~ ~ ~
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 (RIAgp). This test has been known to be more reliable than the AGID test particularly where the anti~ody 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 ~s the need to employ suitable safety procedures in handling the radioactive material.
Another known type of test is the enzyme-linked ~15 immunosorbent assay (FJLISA~ which offers the advantage of being practical and as inexpensive as the AGID testr while providing ~ e-r~sults which are acceptable. Also, it does not involve some of the burdens experienced with the ~IA 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 Enzoo~ic Bovine Leukosis~, Fourth Internatlonal Synposion on Bovine Leukosis, pp. 173-184; Todd et al., "Use of Controlled Antigen to Improve the Enzyme: Linked Immunosorbent Assay for Enzoot~ic Bovine Leukosis A~tib~dies, Veterinary Record (198~ 110, pp. 307-308; Poli et al., "Microplate Enzyme-Linked Immunosorbent Assay for Bovine Leukemia ~ 3.
.,,-~ 7~L~

V;rus Antibody", Journal of Clinical Microbioloqy, January 1981, pp. ~6-48; Graves et al., "Comp~r;son of Enzyme Linked Immunosorbent Assay with an Polykaryocytosis Inhibition Assay and the Agar-Gel Immunodiffusion Test for the Detection of Antib~dies to Bovine Leukemia Vir~s", 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 ~ntibodies", 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 ~nown to be infected with BL~ give false-positive reactions. The false-positive reactions appear to be due to the bindir.y 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~
. ~ive false-positive reactions, which is a totally unacceptible rateO To some extent this problem may be minimized by comparing the reactions of a test serum in vessels with and withQut 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 false-positive reactions in these known systems, there remains a very substantial need for an ELISA test which in respect of specif icity, sensitivity ~nd practicality will~be superior to the previously known ELISA Tests and will also ~e as effective in these respects as the RIA ~p Test.
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. ._ ~ 7~

The present invention has met the abt~ve-described need by providing an ELISA test wherein a test plate having a plurality ~f wells is provided. At least some of the wells are l coated with a bvvine leukemia virus antigen. After incubation and washing, blocking of the non-specif ic binding si'ces 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 antib~dy (nenzyme analyte") which recognizes ~ovine immunoglobulin is introduced into the wells, followed by further incu~ation and washing to remove un~ound analyte. A substrate containing a chromagen is introduced into the wells and a further incubation step is provided. The reaction is then read or stopped and the presen^e or absence of bovine leukemia virus infection may ~e determine~ .
visually either with the naked eye or throu~h use of a suitable instrument, such as a spectrophotometer, for example. Preferred materials and process parameters are employed.
It is an objec~ of the present invention to provide a nethod of serologically diagnosing bovine leukemia Yirus which ~ethod is highly specific, has desired sensitivity and is practical for routine use with results being available within a ~ ~2W 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 insignif icant level.

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¦ It is another object o~ this invention to provide an ~ISA test which does not require the use of specialized ~quipment or radioactive materials.
¦ It is a further object of the present invention to provide an ELISA test which i5 inexpensive to empl~y.
It is a further object of the invention to provide an ELISA test which does not require the use of specif ically trained ~ersonnel.
It is another object of thè present invention to provide an ELISA test which reduces non-specif ic binding of BLV
ntibody contain;ng sera.
It is a further object of the present inven'cion to . ?rovide an ELISA test which is adapted for visual determination ~f the results by the naked eye.
These and other objects of the invention will be ~ore : ully understood from the following description of the invent : ~ reference to the illustration appended hereto~

~ ~ Figure 1 is a flow diagram of a preferr~d form of the nvention.
Figure 2 is a top plan view of a plate usable in the ?resent invention~
Figure 3 is a top plan view of a well of the plate of igure 2.
~ ~igure 4:is a cross-sectional view of the well of igure 3 taken through 4-4.
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As used herein, rleference to visual determination by ¦the "naked eye" shall be deemed to include de~ermination ~y an lindividual 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 blockin~ non-specific hinding of bDvine immunoglobulin without meaningfully interfering with ~pecific reactions. Also of conseq~ence are the unique washing procedures employed after the first ~nd second incubation stages~
rhe substrate~chromagen solution also contributes meaningfully to ~he successful result as a result of its high sensitivity, low . oxicity and stability :15 In a preferred method of practicing the present : nvention the following sequence of operations will be perform ~ _ - With reference to Figure l, the process will be ummar.ized with details of each stage to be described : ereinafter. A suitable plate, having ~ plurality of wells and ~ein~ adapted to test a number of samples is provided. (For : : onvenience of reference, numbers have been assigned to the .
' -~.
rocess steps of Figure 1). About one-half of the wells are oated with BLV antigen 2, after which the wells are in~ubated 4 : nd washed 6 to remove unbound antigen. The plate may then be tored 8, if desired. An ELISA buffer which comprises (a~ a ~locking serum for blocking the~ non-specific binding of bovine mmuno~lobulins to the plast;c vessel in (b~ high ionic strength uffer is added to the wells 14 and the sera to be testèd is also ~3~
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 . ollowed by the incubation 16 and washing 18 steps~ An alternate, but less preferred approach would be to introduce 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. En~yme 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 Figure 2, a series of 96 wells which are preferably substantially cylindrical are proYided. The plate may be a unitary plastic member with the wells being of ~ .~.A
. substantially equal 5i ze and having a capacity of about lOa to : 300 microliters (ul). The plate may be composed of any su;table nater;al 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 lternate rows when antigen coating is effected in order to acilitate scoring of the results. For example, wells in rows A, ~, E ana G may be antigen coated and wells in rows B, D, F and H
. nay be devoid of antigen coatings. Wells 40, 42 may serve as ~ells for the weak positive control serum with antigen and wells ..

~3'`~.

44, 46 may serve as the wells for the weak positive control serum with no antigen.
In order to resist undesired ~low of material from one well to an adjacent well, as s shown in Figures 3 and 4, the plate may have wells which are surrounded with an annularly continuous upwardly projecting barrier r;ng 48.
In addition to the wells which will be treated in $he Eashion to be described with reference to Figure l, it is preferred that certain additional control serum and control wells be provided. The nature of these control wells and function of ;ame will be described hereinafter.
Example l A plate, which may be a polystyrene microtiter plate of the ab~ve-described type is employed. One-half of the wells are nitially coated with bovine leukemia virus antigenO This may be ~ccomplished by harvesting the supernatant fluid of BLV proauc ~ ~-ells, removing large debris by centrifuqation and centrifuging :he BLV in the clarified supernatant onto a dense layer of iucrose. The BLV cells may be obtained from any suitable source, s will be known to those skilled in the art, but it is preferred :o employ a monolayer cell culture that produces BLV continuously nd is substantially free of other virus and adventitious gents. An example of such known means employing BLV-infected ~at lung cells such as is taught by Graves and Ferrer ~ in Cancer esearch, Vol. 36, pp. 4152-4159, 1976. Another suitable source s BLV-infec~ed fetal lamb kidney cells as is that ~ught by Yan er Maaten et al., Bibliotheca Haematologica, Vol. 43, pp. 360-62 (1976). The virus _s recovered and adjusted to about 5 to 20 ' ~.
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~23~37~i micrograms per millimeter (ug/ml) with phosphate or tris buffered saline ~0.15M NaCl, 0.05M PO4 buffer, pH 7.0 - 7. 4~ tllPBsn) ~ The antigen solution is added to the wells in quantities to about 200 ul/well in alternate rows~ The plate is then subjected to a ~irst incubation at about 3 to 25C for ahout 1 to 17 hours.
~fter such incubation~ the unbound antigen is removed by a ~first ~ashing with any suitable isotonic solution, such as phosphate or ris buffered saline solution.
At this point, if desired, the plates may be dried and tored dry at about 4C or less for extended periods ~which may e in excess of 10 months if desired) prior to use in performing he test.
In the preferred test, as is illustrated in Figure 1, out 100 to 200 ul (microliters~ of the ELISA buffer iS added to he wells into which about 10 to 20 ul of the test bovine sera i5 hen introduced. tIt iS preferred that the ratio of test sera ~ .
. ~ISA buffer be about 1:5 to 1:10.) The ELISA buffer preferably ntains about 2 to 20 percent of the animal blocking serum serum for blocking the non-specific binding of bovine ¦ mmunoglobulins to the plastic vessel). It is preferred that the nimal serum be selected from the group consisting of goat serum, hicken serum, guinea pig serum and sheep serum, with goat serum eing preferred. It is also desirable to add to the solution bout 0.25 to 2.0 M NaCl and preferably about 0. 5 to 1.0 M NaCl nd 0.01 to 0.2 M phosphate or tris buffer p~ 6 to 8~ The wells re then incubated at about 4-~2;5~C for about 1-16 hours9 An : ptional washing step 12 removes any excess.

lZ~;37ql6 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 4-25C for abou~ 1 to 16 hours. A washing step to remove excess material may then be employed. Plates so treated may be stored dry at 4C for in excess of three months. If the E~ISA 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 qenerally preferred to assay the test sera in duplicate. The plate is then incubated at a temperature of about 3 to 37C for about 1 to 16 hours. The wells are subsequently washed in a solution of distilled wate ~ ~:
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 ~0 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 about SG
ng/ml. It is added to the wells in ~uantities of about 200 ul/well. The preferred materi`al for this purpose is an antiserum prepared in goats or rabbits which recognize bovine immunoglobulin and whioh is chemically coupled to horseradish 11.
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peroxidase. It is preferred that the antiserum be purified such that it contains only antibodies which recognize b~vine immunoglobulin, otherwise known as "affinity puriied". After incubation for a period of about 15 to 60 minutes and preferably about 30 minutes at about 20C to 37C, 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 suhstrate-chromagen is introduced into the wells in quantities of about 200 ul/well. This solution may consist of H2O2 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% H2O2 per ml of a solution (O.ll mg/ml of pH 4.0 0. n5 M
citrate buffer3 of 2, 2'-azino-di-(3-ethylbenzothiazoline ~ulfonic acid This material is available from a number of sources including Sigma Chemical Company. The substrate chromagen containin~ wells are then subjected to incubation at about 15 to 39C for about 5 to 60 minutes and preferably about 20 to 30 minutes. ~he reaction is then read or stopped by a suitablP acid addition to the wells. A suitable material for use in stopping the reaction is about 50 to lO0 ul of hydrofluoric acid, at concentrations of about O.l to 0.5N and preferably of about 0.2N. The p~ of the acid is preferred to be about 3.0 to

3.5. ~`
Visual inspection, in a manner to ~e described hereinafter, will permit determination as to whether bovine ' 12, ~3~t7~;

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 facili~ate evaluation of the results.
Each test serum, diluted as described above in the ELISA buffer will be incubatea 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 t~ the well. The sensitivity and specificity of the test depends on the difference in binding between the wells coated with thè
antigen and the wells not so coated. Ideally, with a BLV
antibody containing serumr there is strong binding to the antigen coated wells and no appreciable binding to the uncoated wells.
rhe~degree of binding translates into color intensity with strong binding resulting in a definite color such as a daxk 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 ~ite~r of this weak positive serum is adjusted to give the minimum color reaction which will be con-idered positive~ With a 96 well plate the use of four wells ~ 13.
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~ '7~i Eor the weak positive control serum, leaves 9~ 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 nade visually by comparing the color which develops in the bovine Leukemia virus coated wells with the color in the control ~ells. This may be accomplished with the naked eye or by the use ~f 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 ~05 to 415 mm of each well may be determined by means of any ;uitable commerical ELISA spectrophotometer~ The OD values of ~LV antibody free sera would generally be below 0.4 units and the ~ifferences in OD values given by the same sera in the wells with ovine leukemia virus antigen and in wells with control antigen ould generally be less than 0.05 units~ ~ ~ .
~ In order to provide additional guidance regarding a pecifically preferred embodiment of the invention, a further xample will be considered.
.:
~ Example 2 Referring once again to Figure 2 for general guidance, lternate rows of a polystyrene microtiter plate having 96 ~enerally cylindrical wells are provided with a 200 ul of sucrose ~ushion purified BLV antigen at about 10 ug/ml in phosphate ~uffer solu~ion. The control ~untreated) rows are not treated ith anything. The test plate is then incubated at 4C for 16 ours After the incubation the unbound antigen i5 removed by 1 ~ . . ' `.
, 1~.

l~at~
~ashin~ with phosphate buffer solution. Two hundred ul ELISA
buffer is added to all wells and then 20 ul of the weak positive ~erum 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 ~ontrol antigen containing wells. As is preferred, all test sera ~re assayed in duplicate. After incubation for 2 hours at room temperature, the wells are washed four times with 0.05~ of a l uitable detergent, such as Tween 20 (a polyoxyethylene sorbitan nonolaurate). Each wash is a one-minute incubation in the wash olution.
Subsequently, the affinity purified rabbit anti-bovine mmunoglo~ulin chemically coupled to horseradish peroxidase-~onjugate~ is diluted to about 60 ng/ml in ELISA buffer and is 1 Idded to the wells t200 ul/well) which have been incubated with :est sera and control bovine sera. Aft~r incubation for about~
n~nutes at about 25~C, the wells are washed with distilled water our times in order to remove unbound enzyme ana~yte. This ~ashing contributes to further reduction of non-specific ~eactivity. About 200 ul of a substrate-chromagen solution ~aving a concentration-of O.ll mg 2,2'-azino-di-(3-thylbenzothiazoline) sulfonic acid and 0.2 ul 30~ ~2 per ml of ~H 4.0 citrate buffer is then added. If it is desired to read he results spectrophotometrically or to preserve the results, he reaction is stopped after about 25 minutes by adding sn ul of .2 N hydrofluoric acid, of pH 3.3. The test resu7ts are then etermined visually either with the naked eye or by the use of a t ~ ~uitable instrument such as a spectrophotometer.
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. ._ 1233'74ti Example 3 Referring to Figure 2, ~nce again, a further example will be considered assuming that a specific sample of test sera has been introduced into wells A3, A4, B3, B4 and that wells Al, A2, Bl, B2 function as weak positive 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 minimùm color intensity to be regarded as a positive test result and negative antigen control wells Bl, 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 regar~ed as a positive test result. If wells A3, A4 are clear or of lesser color than wells Al, ~2 the test result is negative. It will be appreciated that the test could be performed with Al, Bl 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 speci~icity 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 fraining nd without requiring specia1 equipment can process a 16.

~ '74~; 1 large number of ~amples in a relatively short period of time.
Further, the test is economical to employ and ~he t~st 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 illus~rated and described, it will be appreciatea that other typ~s 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 ~ncompass such alternate approaches.
. Whereas particular embodiments of the invention have been descr;bed above for purpose of illustra ion, it will be .
appreciated that by those skilled in ~he art tha~ numerous -~
~ariations of the detail~ may be ma~e without departing from the invention as described in the appended claims.

Claims

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

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 introducion 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 con-centrations 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.

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 2.0M.
NaCl, 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, in-troducing 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 immunoglobulin 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.

12. 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.

15. 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.

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

17. 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 con-centrations 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. NaCl, 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.