AVIAN ENCEPHALOMYELITIS ANTIBODY DETECTION

Abstract: A mammalian anti-avian encephalomyelitis antiserum comprising the immunoglobulin fraction of mammalian blood serum containing antibodis to avian encephalomyelitis which antiserum is substantially free of anti-chicken immunoglobulin. The antiserum is used in a ELISA assay for the detection of avian encephalomyelitis.

Description: This invention relates to a process of immunosorbent analysis for the detection and measurement of specific avian antibodies and to antibody traps for use therein. The measurement of the level of antibody to avian encephalomyelitis (AEV) in the bodies of chickens is a matter of considerable economic importance to the poultry industry. For many years the most reliable measurement of these antibodies has been the virus neutralisation test of Calnek and Jenick. The method is, however, time-consuming and not entirely reliable. The embryo-susceptibility test of Taylor and Schelling offered some improvement in performance but is also unacceptably slow to produce results. The most promising development to date has been the enzyme-linked immunosorbent assay (ELISA), which was shown by Garrett et al to be a specific, sensitive and reproducible test for the detection of antibodies against A.E.V. In a typical Elisa test, a liquid biological test sample is presented to an antibody trap in the form of a solid supporting substrate the surface of which has been treated to render it selectively abosrbent to the specific antibodies to be detected. The adsorbed antibodies are then identified by, for example, coupling them to enzymes or radioactive tracers, which can subsequently be detected by conventional analytical techniques. The adsorptive sites are provided by antigen corresponding to the antibody to be detected and the solid supporting substrate is, for example, a plastic strip or well. Some viruses do not absorb satisfactorily onto such substrates, however and it is difficult to prepare consistently reliable antibody traps by this direct route. The problem has been overcome by first coating the substrate with antibody for the antigen, which is readily absorbed onto the plastic to give a surface of reproducible uniformity. The required antigen is then adsorbed onto the treated substrate to complete the antibody trap. In principle the method is a satisfactory one. However, it has been accepted generally that all of the biological materials used in the analysis must be in a highly purified state in order to avoid masking the test and reducing its sensitivity through competing non-specific side-reactions with impurities. When using the Elisa technique for detecting AEV, for example, the virus is first harvested from the yolk sacs 5 of innoculated eggs. Mammalian antibodies to AEV are then prepared drom the serum of animals immunised with the viral material extracted from the eggs. The complexity of this technique leads to it being very time consuming and expensive in the use of large 0 quantities of reagents. It is accordingly an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art. Accordingly, in a first aspect there is provided a 5 .mammalian anti-avian encephalomyelitis antiserum comprising the immunoglobulin fraction of mammalian blood serum containing antibodies to avian encephalomyelitis which antiserum is substantially free of anti-chicken immunoglobulin. D Preferably, the mammalian anti-avian encephalomyelitis antiserum is a goat antiserum. By immunoglobulin we mean three classes of immunoglobulin, IgG, IgA and IgM having molecular weights of about 155,000, 180,000 (monomeric) or 3000,00 (dimeric) and 5 900,000 respectively, and which have the function of anti-bacterial, anti-viral and anti-toxin antibodies. The mammalian antibodies which we raise against chicken immunoglobulin would react with all three of these immunoglobulin classes. 0 In a further aspect of the present invention there is provided a method of preparing an apparatus for use in a method for analysing a sample for avian encephalomyelitis which method includes (a) providing 5 (i) a solid support (ii) a mammalian anti-avian encephalomyelitis antiserum substantially free of anti-άicken immunoglobulin, and (iii) an avian encaphalomyelitis antigen, (b) coating the solid support with the mammalian anti-avian encephalomyelitis antiserum, and (c) coating the coated support so formed with the avian encephalomyelitis antigen. Preferably the mammalian antiserum is a goat antiserum. In a preferred form, the method further includes (b') incubating the solid support for a period sufficient to allow the antibodies in the antiserum to become attached to the solid suppor , and (c 1 ) incubating the coated support for a period sufficient to allow the antigen to be absorbed onto the coated substrate. In a further aspect of the present invention there is provided an apparatus for enzyme-linked immunosorbent analysis including (i) a solid support, (ii) a mammalian anti-avian encephalomyelitis antiserum coated on said support, and (iii)an avian encephalomyelitis antigen bound to said coated support. Preferably the apparatus described above includes (i) a solid plastic support, (ii) a goat anti-avian encephalomyelitis antiserum coated on said support, and (iii)an avian encephalomyelitis antigen bound to said coated support. In a still further aspect of the present invention there is provided a method of analysing a sample for avian encephalomyelitis virus which method includes (i) providing (a) a sample, (b) an enzyme-linked mammalian species immunoglobulin conjugate, and (c) an apparatus referred to above, (ii) adding a portion of the sample to the apparatus, and (iii) dding the conjugate to the apparatus bearing the sample. Preferably the method of analysing a sample further includes (iia) incubating the apparatus for a period sufficient to allow the specific antibody in the sample to become attached to the bound antigen, and (iiia)incubating the apparatus for a period sufficient to allow reaction between the conjugate and captured antibody. The general method of carrying out the analysis embodying the invention, is as follows. The partially purified anti-virus antiserum (mammalian anti-AEV) is coated into a solid plastic support. A standard preparation of AEV antigen is bound onto the antiserum. The test sample of serum is then reacted with the coated support. Any AEV antibody in the test serum will react with the bound AEV antigen. No non-specific binding of chicken antibody will occur because the anti-chicken immunoglobulin has been removed from the anti-virus antiserum. The coated support is now treated with a suitable enzyme linked anti-chicken immunoglobulin which will bind specifically onto the previously bound antibodies from the test serum. The amount of enzyme linked anti-chicken immunoglobulin bound is measured by an enzymic assay. The enzymic assay thus gives a measure of the amount of AEV antibodies in the test serum. In a further aspect of the present invention there is provided a test kit for analysing a sample, which test kit includes (a) a solid support, (b) a mammalian anti-avian encephalomyelitis antiserum which is substantially free of anti-chicken immunoglobulin in a suitable container,and (c) an enzyme-linked mammalian species immunoglobulin conjugate in a suitable container. ^ - Preferably, the test kit further includes (a) quality control mammalian species serum in a suitable container, and (b) a mammalian species immunoglobulin diluent in a suitable container. The invention is further illustrated by the following example which describes the preparation of an analytical kit for the detection of AEV, the preparation of the various consistuent components of the kit and its method of use. It should be understood, however, that this discussion is illustrative only and should not be taken as a restriction on the generality of the description above. Example The preparation of the various reactive components of the kit is as follows: (A) Avian Encephalomyelitis Virus (AEV) : The Van Rockel strain of AEV is innoculated into the yolk sacs of 7 day embryonating specific pathogen-free eggs using approximately 100 mean embryo infective doses per egg. After a further 12 days, the embryos are harvested and the AEV partially purified as described by Matsumoto M. and Murphy M.L. , "Avian Diseases" 1977, 2jL,300). After the extraction with trichloro-tri-fluoro ethane (as described by the above authors) the virus is precipitated in 4% polyethylene glycol 6000, resuspended in 0.1 M Tris-HCl pH8.0, 0.5 M NaCl, using 1 ml of this buffer for every embryo harvested. This virus preparation is then dialysed against this buffer for 3 days at 4°C. (B) Goat Antibody to Avian Encephalomyelitis Virus: Goat antibody to AEV is prepared from the serum of goats immunised with the partially purified AEV preparation. The immunisation protocol involves a primary immunisation with AEV virus emulsified in Fruend's complete Adjuvant, given intramuscularly. Subsequent injections comprise 2ml of AEV preparation, given subcutaneously at no less than monthly intervals. Blood samples are taken one week after the immunisation. The blood is taken into plain tubes and the serum separated from the clot. Goat IgG is prepared by caprylic acid fractionation of serum using a modification of a procedure described by Steinbuch M. and Andran R. ("Arch Biochem Biophys", 1969, 134, 279). In this procedure the pH of the serum is adjusted to pH 4.8 + 0.05 with IM acetic acid. Caprylic acid is added, dropwise with stirring,m using 6.8 gm of caprylic acid for every 100 ml serum. The stirring is continued for a further 30 minutes, making sure that the temperature of the suspension does not fall below approximately 15 C. The suspension is then centrifuged at 3000 g for 30 minutes, the - supernatent removed, filtered to remove fat, and neutralised with IM naOH. This solution is then dialysed against phosphate buffered saline (PBS) for 3 days, maintaining the temperature above 15°C. (C) Affinity purified Goat anti-chicken IgG horse radish peroxidase conjugate Chicken IgG is prepared using the caprylic acid method described above. Coat antibody to chicken IgG is prepared from the serum of goats immunised with this chicken IgG preparation. The immunisation protocol is identical to that described above, except that 1-2 mg of chicken IgG is used for each immunisation. Goat IgG is prepared as described above. Affinity purified goat anti-chicken IgG is prepared from the goat IgG anti-chicken IgG using a column of sepharose 4B coupled to chicken IgG and the method described by Cripps et al ("J Immunol Methods", 1983, _57, 197) for affinity purifying human IgA and IgM. A horse radish conjugate is prepared with affinity-purified Goat anti-chicken IgG antibody according to Wilson and Nakane. ("Immunofluorescence and Related Staining Procedures",) Elsevier/North Holland Biomedical Press) . Goat IgG is added to the conjugate to lOmg/ml and the conjugate stored frozen in aliquots. (D) Normal Goat IgG diluent Goat serum is prepared from goat blood collected from abbatoirs. Goat IgG is prepared from this serum using the caprylic acid fractionation method described above. (E) Reference chicken serum The reference chicken serum is a pool of chicken serum prepared from adult chickens which have been 5 exposed to AEV. The amount of antibody against AEV in this pool is quantitated against a standard reference serum which is defined as containing 2 x 10 units of antibody activity per millilitre. (F) Quality control chicken serum 10- The quality control chicken serum is a dilution of field chicken serum in specific pathogen-free serum. This preparation contains sufficient antibody activity to bind approximately 50% of the conjugate bound by the highest concentration of reference 15 chicken serum used. AEV ELISA KIT - PROCEDURE A. CONTENTS (STORE FROZEN) Avian encephalomyelitis virus antigen ELISA ANTIGEN 20. Anti-avian encephalomyelitis virus antibody ELISA ANTIBODY Affinity-purified, horse radish peroxidase conjugated anti- ELISA CONJUGATE chicken IgG 25 Reference serum (100 AbU/500ul) REFERENCE SERUM Quality Control Serum (15 AbU/50ul) QUALITY CONTROL SERUM Diluent Protein Additive DILUENT ADDITIVE B. Reagent Preparation 1. Antibody coated plates : Add ELISA ANTIBODY to 5 ml of 30 0.1 M sodium carbonate buffer pH 9.5. Place 50ul in each well of solumns 2-12 (i.e. leave column 1 empty) .Incubate overnight at room temperature, and then store at 4°C. 2. Elisa buffer : 6.05 gm Iris-hydroxymethylaminomethane 14.4 gm NaCl 35 0.37 gm EDTA 146 ml 0.2 M HC1 0.25 ml Tween 20 2 00..55 mmll 55 xx 1100 2 MM T.himerosal Make to 500 ml in distilled water and adjust to pH 8.0. Add the DILUENT ADDITIVE to 60 ml ELISA BUFFER 3. Washing Solution : Dulbecco's "A" phosphate buffered saline with 0.1% tween 20. 4. Reference serum dilutions : Ref. 2: 0.5 ml Reference serum + 0.5 ml ELISA BUFFER (50 AbU/50ul) Ref. 3: 0.2 ml Reference serum + 0.8 ml ELISA BUFFER (20 AbU/50ul) Ref. 4: 0.1 ml Reference serum + 0.9 ml ELISA BUFFER (10 AbU/50ul) Ref. 4: 0.1 ml Ref. 2 + 0.9 ml ELISA BUFFER (2 AbU/50ul) 5. Add ELISA ANTIGEN to 5 ml ELISA BUFFER 6. Add ELISA CONJUGATE to 5 ml ELISA BUFFER 7. Substrate solution : the substrate recommended for use in this ELISA is repurified 5-aminosal-icylic acid (5-amino-2-hydroxybenzoic acid, Merck). This is prepared by mixing 9 g of 5-aminosalicylic acid, and 9 g of sodium eta bisulphite in 1 litre of distilled water at 80 C. To this suspension add 4 g of activated charcoal, stir for 5 minutes and then filter into a conical flask sitting in ice. After cooling the filtrate, the precipitate is collected on filter paper in a Buchner funnel and washed twice with 50 ml distilled water at 4°C. This precipitate is collected, dried and stored in the dark at room temperature. The substrate solution is prepared by dissolving 1 g of repurified 5-aminosalicylic acid in 1 litre 0.01 M sodium phosphate buffer pH 6.8 with 0.34 g of EDTA. The final pH of this solution varies between 5.9 and 6.0. This solution can be frozen in suitable sized aliquots for storage. For use the substrate solution is thawed, and 20 ul of 100 volume hydrogen peroxide added per 100 ml of subsrrate solution. 8. Test Sera : the test sera are all diluted 1:100 in ELISA Buffer (generally lOul test serum to 990ul ELISA Buffer). C. ASSAY PROCEDURE 1. Wash coated plate 3 x with washing solution. 2. Add 50ul of the ELISA ANTIGEN solution to each coated well, and incubate for 1 hour at room temperature. 3. Wash 3 x in washing solution 4. Add 50ul of reference serum dilutions, quality control serum, and test serum, in duplicate, to the wells of the plate (Refer to item F for recommended plate layout). 5. Wash 3 x in washing solution. 6. Add 50ul of ELISA CONJUGATE solution to each well, incubate for 1 hour at room temperature. 7. Wash 3 x in washing solution. 8. Add lOOul of the substrate solution, incubate for 1 hour at room temperature. 9. Read the absorbance values at 492 nm. D. CALCULATION OF RESULTS 1. Express the mean absorbance value for each duplicate reference, quality control or test serum as a percentage of the absorbance units for Ref. 1 (i.e. % bound). 2. Put % bound for each reference sample against the logarithm of the AbU content. 3. Calculate the AbU content of each test serum sample by interpolation of the % bound using the reference curve. 4. The quality control sample should record a value of approximately 15 AbU per 50ul. E. INTERPRETATION OF RESULTS Serum samples can be interpreted as follows. Interpretation Score <5 AbU negative 0 5-20 AbU low positive 1 20-100 AbU positive 2 >100 AbU strong positive 3 Each serum sample can be assessed individually as above or an overall flock index can be assessed as follows. The number of serum samples in each division is multiplied by the score for that group, and the total for the flock determined. This is expressed as a percentage of the potential maximum which is 3 x the number of serum samples. The ELISA index can be interpreted as >50 flock immune 30 - 50 immunity status doubtful <30 flock susceptible Note: It is not essential to use a plate reader to determine the absorbance value of each sample and determine the AbU as above. The ELISA Index can be determined by classifying each serum as falling within each group by visually comparing the colour of each sample with those of the reference samples. These sera can be assigned a score and the ELISA index determined as above. The wells of flat bottom microtitre trays were each coated with 50 ul of Goat IgG anti-AEV in 0.1 M sodium carbonate buffer, pH 9.5. The antibody was adsorbed to the plastic surfaces for 18 hours at room temperature, after which the plates were stored at 4°C. The wells were washed thrice with phosphate-buffered saline pH 7.2 with 0.1% Tween 20 (PBS-tween), and 50 ul of ELISA antigen diluted in ELISA buffer (0.1 M Tris-HCl pH 8.0, 0.5M NaCl, 0.002M EDTA, 0.05% tween 20, 5 x 10 M thimerosal) was added to each well and incubated for 1 hour at room temperature. The relative concentrations of Goat anti-AEV and ELISA antigen were predetermined by chequerboard titration. The wells were again washed thrice with PBS-tween and 50 ul serum samples, diluted 1:100 in ELISA buffer containing 1 mg/ml normal Goat IgG (ELISA buffer-G) were added, in duplicate, to the wells. A series of 6 duplicate samples of dilutions of a reference positive serum was also added to each plate. After 1 hour of inbucation at room temperature, the wells were again washed thrice in PBS-tween and 50 ul of the affinity purified Goat anti-chicken IgG conjugate (diluted in ELISA buffer-G) was added to each well, and incubated for 1 hour at room temperature. The wells were again washed thrice in PBS-tween, and 100 ul of the substrate was added to each well. After 1 hour, the absorbance of 492 nm was measured for each well using a Titertek Multiskan. The absorbance of the lowest dilution of the reference serum was specified as 100% bound B n^ an< ^ t ^ ιe absorbance of each sample was expressed as a percentage of B n . A reference curve was constructed by plotting the percentage bound in each reference sample against the logarithm of its antibody activity content, with the B π sample for the studies reported herein being defined as containing 1000 antibody units per 50 ul of a 1:100 dilution. Sample antibody activities were read off this curve and reported in this paper as antibody activity in 50 ul of the diluted serum. 5 The dose-response curve for the reference serum: The reference serum used in these studies was a pool of high titre field sera from a variety of sources (neutralisation index = ) . This pool was defind as having 2 x 10 antibody units per ml, which, when diluted for use in 10 the ELISA contained 1000 antibody units per 50 ul. A composite does-response curve for 10 assay runs using this serum is presented in Figure 1. Relationship between end-point title and antibody units: Sera from a variety of sources, and exhibiting a 15 wide range of antibody levels were assayed both by end-point titration, and by antibody unit content. The end-point titre was defined as that dilution of serum at which the absorbance was 0.2. This titre was estimated by interpolation on logarithmic-linear graph paper. The results of this analysis, 20. presented in Figure 2, demonstrate that there is a close relationship betwen these parameters. The results in the rest of this paper are reported as antibody units per 50 ul of a 1:100 dilution of the test serum. Assessment of assay characteristics. 25 Specificity: The relationship between the virus neutralization test and the ELISA was used to assess the ELISA for measurement of antibodies against AEV. A comparison of these two assays in the rank-orders given to a number of chicken 30 sera is presented in Table 1. This analysis demonstrated significant correlation between these tests in their ranking of sera by antibody activity against AEV. the relationship between these two assays was further investigated by testing their agreement on the classification of the above sera as 35 either positive or negative for antibody activity (Table 2). The specificity of the ELISA was also assessed by analysing sera from specific-pathogen-free chickens which had been immunised with other viruses. These results (Table 3) domonstrate that antibody activity was only detectable in the serum of birds inoculated with AEV. Inoculation with Newcastle disease virus, infectious bronchitis virus, heomagglutinating avian adenovirus, avian reoviruses, and reticuloendot-heliosi-s virus failed to induce a detectable antibody response. Sensitivity: The relative abilities of the ELISA and the virus neutralisation test to detect an antibody response to primary vaccination of specific-pathogen-free chickens with AEV was used to assess the relative sensitivities of the two assay systems (Table 4). These results indicate that a primary antibody response to vaccination is detectable within 9 days in the majority of individuals, with strong responses being detected within 15 days post inoculation. Comparison of the end-point titres by ELISA with neutralisation indices for a number of sera is presented in Table 1. These results further indicate that the two assay systems are of comparable sensitivity. Reproducivility: Three pools of sera were prepared containing high, mid and low levels of antibody units as detected by ELISA. These samples (in duplicate), were included in each ELISA run, and the within- and between-assay variance for each measurement range was calculated as described by Rodbard (1974). These results are presented in Table 5. The specificity of this ELISA has been demonstrated by comparison with virus neutralization, and by testing sera from specific-pathogen-free chickens which had been immunised with a variety of other viruses. Both the ELISA and virus neutralization tended to rank a number of sera in the same order in terms of antibody activity. Many of these sera were obtained from chickens shortly after a primary immunisation with AEV. Some of the differences between the two tests in terms of rank order of these sera may relate to the differential sensitivities of the two assay systems to different antibody populations, as it is known that the ELISA is particularly affected by the avidity of the antibody papulation (Butler et al, 1981). An important practical aspect of the specificity of the ELISA is its discrimination between positive and negative sera, in respect of which there was very close agreement with virus neutralisation. There was no detectable antibody when a range of sera from specific-pathogen-free chickens, immunised with a variety of viruses, was tested by ELISA. Some of these viruses can cause problems in egg production, as can AEV. These observations indicate that the ELISA described in this paper measures antibody specific for AEV, but does not detect antibody against a variety of other viruses. Both the ELISA and virus neutralisation detected a response to primary immunisation within 9 days. This observation indicated that both tests have equivalent sensitivity in a practical sense. Observations on outbreaks of avian e cephalomyelitis in the field support this finding that the ELISA can detect a primary response to AEV within two weeks of infection. Substantial rises in titre can be readily demonstrated in sequential serum samples taken on week apart. The evaluation of antibody activity by reference to a calibration curve has enabled an evaluation of the reproducibility of this ELISA. Estimation of the mid-range value was highly reproducible both within- and between-assay, and compares favourably with analogous results published for competitive-inhibition enzyme immunoassays. As expected, the extreme range samples exhibited greater variance, although the values obtained indicate that this ELISA is reproducible over a wide range. These figures also compare favourably with an evaluation of reproducibility for antibody measurements undertaken by end-point titration. Table 1: Relationship between Neutralisation Index and Antibody units (by ELISA) for the measurement of antibodies to avian encephalomyelitis . Serum No. Neutralisation Index Antibody Units 1-26 . 1.0 <5.0 27 2.2 18 28 <1.0 6.4 29 1.1 <5.0 30 1.7 6.0 31 1.3 <5.0 32 2.4 5.5 33 3.0 84 34 3.0 38 35 2.4 15 36 1.2 26 37 1.2 18 38 3.7 28 39 1.2 i 7 40 2.3 39 a. The rank correlation coefficient (R < -) for the relationship between Neutralisation Index and Antibody unit measurement on each serum was 0.56 (p<D.05). Table 2: Classification of sera by ELISA and virus Neutralisation. a. A positive serum uas defined as one having greater than 10 antibody units per 50 μl of a 1:100 dilution of serum. b. A positive serum uas defined as one having a neutralisation index of greater than 1.0. Table 3: Measurement of antibodies to Avian encephalomyelitis virus, infectious bronchitis virus (IBV), Newcastle disease virus (NDU), and hae agglutinating avian adenovirus (HAV-) in the sera of specific-pathogen-free chickens - immunised with a variety of viruses. Antibody measurement Virus Inoculum AEV b IBV NDV HAV AEV 1 70 -ve -ve -ve 2 40 3 24 . 36 c IBV 9 4 1 -ve 2 2 5 3 2 6 \DV 1 11 -ve 2 ' 2 II II 2 8 HAV II 11 -ve 2 8 Reticuloendo- theliosis virus II 11 II -ve Foul Pox virus II It II II Reovirus II II II II Adenovirus II II II II a. Antibody measurements for IBV, NDV and HAV were performed by haemagglutinat inhibition. b. AEV antibody measured as antibody units per 50 μl of a 1:100 dilution of se c. Negative values correspond to less than 10 antibody units per 50 ul of a 1: dilution of serum. Table 4: Detection of a primary antibody response to vaccination with AEV by both ELISA and virus neutralisation. Days post inoculation . 15 Virus Neutralisation all -ve 5/6 +ve 6/6 -t-ve FLISA all -ve 4/6 ÷ve 6/6 +ve a. See Table 2 for the definitions of positive in each of the tests. Table 5: Within- and between-assay co-efficient of variation for three serum pools representing high-, mid-range, and low levels of antibody activity. % Co-efficient of variation Mean antibody Serum Pool Within Assay unit concentration' Between Assay Quality Control 1 (HIGH) 397 12.24 16.19 Quality Control 2 (MID) 53 5.57 9.77 Quality control 3 (LOW) 15 8.89 11.36 a. Expressed as antibody units per 50 ul of a 1:100 dilution of serum. b. Calculated as described by Rodbard (1974).

Claims: 1. A mammalian anti-avian encephalomyelitis antiserum comprising the immunoglobulin fraction of mammalian blood serum containing antibodies to avian encephalomyelitis which antiserum is substantially free of anti-chicken immunoglobulin. 2. A mammalian anti-avian encephalomyelitis antiserum according to claim 1 wherein the antiserum is a goat antiserum. 3. A method of preparing an apparatus for use in a method for analysing a sample for avian encephalomyelitis which method includes (a) providing (i) a solid support (ii) a mamalian anti-avian encephalomyelitis antiserum substantially free of anti-chicken immunoglobulin, and (iii) an avian encaphalomyelitis antigen, (b) coating the solid support with the mammalian anti-avian encephalomyelitis antiserum, and (c) coating the coated support so formed with the avian encephalomyelitis antigen. 4. A method according to claim 3 wherein the antiserum is a goat antiserum. 5. A method according to claim 5 further including (b') incubating the solid support for a period sufficient to allow the antibodies in the antiserum to become attached to the solid substrate, and (c*) incubating the coated support for a period sufficient to allow the antigen to be absorbed onto the coated substrate. 6. An apparatus for enzyme-linked immunosorbent analysis including (i) a solid support, (ii) a mammalian anti-avian encephalomyelitis antiserum coated on said support, and (iii)an avian encephalomyelitis antigen bound to said coated support. 7. An apparatus according to claim 6 including (i) a solid plastic support, (ii) a goat anti-avian encephalomyelitis antiserum coated on said support, and (iii)an avian encephalomyelitis antigen bound to said coated support. 8. A method of analysing a sample for avian encephalomyelitis virus which method includes (i) providing (a) a sample, (b) an enzyme-linked mammalian species immunoglobulin conjugate, and (c) an apparatus according to claim 6, (ii) adding a portion of the sample to the apparatus, and (iii)adding the conjugate to the apparatus bearing the sample. 9. A method according " to claim 8 further including (iia) incubating the apparatus for a period sufficient to allow the specific antibody in the sample to become attached to the bound antigen, and (iiia)incubating the apparatus for a period sufficient to allow reaction between the conjugate and captured antibody. 10. A test kit for analysing a sample, which test kit includes (a) a solid support, (b) a mammalian anti-avian encephalomyelitis antiserum which is substantially free of anti-chicken immunoglobulin in a suitable container,and (c) an enzyme-linked mammalian species immunoglobulin conjugate in a suitable container. 11. A test kit according to claim 10 further including (a) quality control mammalian species serum in a suitable container, and (b) a mammalian species immunoglobulin diluent in a suitable container.

Inventors: SMART IAN JAMES, SMART, IAN, JAMES
Applicants: VICTORIA STATE, THE STATE OF VICTORIA
Application year: 1986
Main classification: C07K15
IPC codes: G01N33