The blocking or competitive ELISA test shall be carried out according to the following protocol:

The competitive ELISA using monoclonal antibody 3-17-A3 is capable of identifying antibodies to all known serotypes of bluetongue virus (BTV).

The principle of the test is the interruption of the reaction between BTV antigen and a group-specific monoclonal antibody (3-17-A3) by the addition of test serum. Antibodies to BTV present in the test serum block the reactivity of the monoclonal antibody (Mab) and result in a reduction in the expected colour development after the addition of enzyme labelled anti-mouse antibody, and chromogen/ substrate. Sera can be tested at a single dilution of 1:5 (spot test – Appendix 1) or may be titrated (serum titration – Appendix 2) to give dilution end-point. Inhibition values higher than 50 % may be regarded as positive.

1. 1.

   Appropriate ELISA microtitre plates.

2. 2.

   Antigen: supplied as a cell extracted concentrate, prepared as described below, and stored at either – 20 °C or – 70 °C.

3. 3.

   Blocking buffer: phosphate buffered saline (PBS) containing 0,3 % BTV negative adult bovine serum, 0,1 % (v/v) Tween-20 (supplied as polyoxyethylene sorbiton monolaurate syrup) in PBS.

4. 4.

   Monoclonal antibody: 3-17-A3 (supplied as hybridoma tissue-culture supernatant) directed against the group-specific polypeptide VP7, stored at - 20 °C or freeze-dried and diluted 1/100 with blocking buffer before use.

5. 5.

   Conjugate: rabbit anti-mouse globulin (adsorbed and eluted) conjugated to horseradish peroxidase and kept in the dark at 4 °C.

6. 6.

   Chromogen and substrate: Orthophenylene diamine (OPD-chromogen) at a final concentration of 0,4 mg/ml in sterile distilled water. Hydrogen peroxide (30 %w/v-substrate) 0,05 % v/v added immediately before use (5μl H₂ O₂ per 10 ml OPD). (Handle OPD with care - wear rubber gloves - suspected mutagen).

7. 7.

   1 Molar sulphuric acid: 26,6 ml of acid added to 473,4 ml of distilled water. (Remember Acid must be added to water, never water to acid.)

8. 8.

   Orbital shaker.

9. 9.

   ELISA plate reader (the test may be read visually).

Cc: conjugate control (no serum/ no monoclonal antibody); C++: strong positive serum control; C+: weak positive serum control; C-: negative serum control; Cm: monoclonal antibody control (no serum).

Conjugate control (Cc)

Wells 1A and 1B are a blank control consisting of BTV antigen and conjugate. This may be used to blank the ELISA reader.

Mab control (Cm)

Columns 1 and 2, rows G and H are the monoclonal antibody control and contain BTV antigen, monoclonal antibody and conjugate. These wells represent maximum colour. The mean of the optical density readings from this control represents the 0 % inhibition value.

Positive control (C++, C+)

Columns 1 and 2, rows C-D-E-F. These wells contain BTV antigen, BTV strong and weak positive antiserum respectively, Mab and conjugate.

Negative control (C-)

Wells 2A and 2B are the negative controls, which contain BTV antigen, BTV negative antiserum, Mab and conjugate.

Test sera

For large-scale serological surveys and rapid screening, sera may be tested at a single dilution of 1:5 (Appendix 1). Alternatively, 10 sera may be tested over a dilution range from 1:5 to 1:640 (Appendix 2). This will give some indication of the titre of antibody in the test sera.

1. 1.

   Dilute BTV antigen to pre-titrated concentration in PBS, sonicate briefly to disperse aggregated virus (if sonicator is not available, pipette vigorously) and add 50 μl to all wells of the ELISA plate. Tap sides of plate to disperse antigen.

2. 2.

   Incubate at 37 °C for 60 minutes on an orbital shaker. Wash plates three times by flooding and emptying the wells with non-sterile PBS and blot dry on absorbent paper.

3. 3.

   Control wells: Add 100 μl of blocking buffer to Cc wells. Add 50 ul of positive and negative control sera, at a dilution of 1:5 (10 μ l sera + 40 μl blocking buffer), to respective wells C-, C+ and C++. Add 50μl blocking buffer to Mab control wells.

   Spot titration method: Add a 1:5 dilution of each test serum in blocking buffer to duplicate wells of columns 3 to 12 (10 μl sera + 40 μl blocking buffer),

   or

   Serum titration method: Prepare a two-fold dilution series of each test sample (1:5 to 1:640) in blocking buffer across eight wells of single columns 3 to 12.

4. 4.

   Immediately after the addition of the test sera, dilute Mab 1:100 in blocking buffer and add 50 μl to all wells of the plate except for the blank control.

5. 5.

   Incubate at 37 °C for 60 minutes on an orbital shaker. Wash three times with PBS and blot dry.

6. 6.

   Dilute rabbit anti-mouse concentrate to 1/5 000 in blocking buffer and add 50 μl to all wells of the plate.

7. 7.

   Incubate at 37 °C for 60 minutes on an orbital shaker. Wash three times with PBS and blot dry.

8. 8.

   Thaw the O-Phenylenediamine dihydrochloride (OPD) and immediately before use add 5 μl of 30 % hydrogen peroxide to each 10 ml of OPD. Add 50 μl to all wells of the plate. Allow colour to develop for approximately 10 minutes and stop the reaction with 1 Molar sulphuric acid (50 μl per well). Colour should develop in the Mab control wells and in those wells containing sera with no antibody to BTV.

9. 9.

   Examine and record the plates either visually or using a spectrophotometric reader.

Using the software package print out the optical density (OD) values, and the percentage inhibition (PI) for test and control sera based on the mean value recorded in the antigen control wells. The date expressed as OD and PI values are used to determine whether the test has performed within acceptable limits. The upper control limits (UCL) and lower control limits (LCL) for the Mab control (antigen plus Mab in the absence of test sera) are between OD values 0,4 and 1.4. Any plate that fails to conform to the above criteria must be rejected.

If a computer software package is not available print out the OD values using the ELISA printer. Calculate the mean OD value for the antigen control wells, which is equivalent to the 100 % value. Determine the 50 % OD value and manually calculate the positivity and negativity of each sample.

Percentage inhibition (PI) value = 100 – (OD of each test control/Mean OD of Cm) × 100.

The duplicate negative control serum wells and the duplicate blank wells must record PI values between + 25 % and – 25 %, and between + 95 % and + 105 %, respectively. Failure to be within these limits does not invalidate the plate but does suggest that background colour is developing. The strong and weak positive control sera must record PI values between + 81 % and + 100 %, and between + 51 % and + 80 %, respectively.

The diagnostic threshold for test sera is 50 % (PI 50 % or OD 50 %). Samples recording PI values >50 % are recorded negative. Samples that record PI values above and below the threshold for the duplicate wells are considered doubtful; such samples may be re-tested in the spot test and/or titration. Positive samples may also be titrated to provide an indication of the degree of positivity.

Visual reading: Positive and negative samples are easily discernible by eye; weakly positive or strong negative samples may be more difficult to interpret by eye.

1. 1.

   Wash 40-60 roux of confluent BHK-21 cells three times with serum-free Eagle's medium and infect with bluetongue virus serotype 1 in serum-free Eagle's medium.

2. 2.

   Incubate at 37 °C and examine daily for cytopathic effect (CPE).

3. 3.

   When CPE are complete in 90 % to 100 % of the cell sheet of each roux, harvest the virus by shaking any still-attached cells from the glass.

4. 4.

   Centrifuge at 2 000 to 3 000 rpm to pellet the cells.

5. 5.

   Discard the supernatant and re-suspend the cells in approximately 30 ml of PBS containing 1 % ‘Sarkosyl’ and 2 ml phenylmethylsulphonyl fluoride (lysis buffer). This may cause the cells to form a gel and more lysis buffer may be added to reduce this effect. (NB: phenylmethylsulphonyl fluoride is harmful - handle with extreme caution.)

6. 6.

   Disrupt the cells for 60 seconds using an ultrasonic probe at an amplitude of 30 microns.

7. 7.

   Centrifuge at 10 000 rpm for 10 minutes.

8. 8.

   Store the supernatant at + 4 °C and re-suspend the remaining cell pellet in 10 to 20 ml of lysis buffer.

9. 9.

   Sonicate and clarify, storing the supernatant at each stage, a total of three times.

10. 10.

    Pool the supernatants and centrifuge at 24 000 rpm (100,000 g) for 120 minutes at + 4 °C over a 5 ml cushion of 40 % sucrose (w/v in PBS) using 30 ml Beckmann centrifuge tubes and an SW 28 rotor.

11. 11.

    Discard the supernatant, drain the tubes thoroughly and re-suspend the pellet in PBS by sonication. Store the antigen in aliquots at – 20 °C.

Bluetongue ELISA antigen is titrated by the indirect ELISA. Twofold dilutions of antigen are titrated against a constant dilution (1/100) monoclonal antibody 3-17-A3. The protocol is as follows:

1. 1.

   Titrate a 1:20 dilution of BTV antigen in PBS across the microtitre plate in a twofold dilution series (50 μl/well) using a multichannel pipette.

2. 2.

   Incubate for one hour at 37 °C on an orbital shaker.

3. 3.

   Wash plates three times with PBS.

4. 4.

   Add 50 μl of monoclonal antibody 3-17-A3 (diluted 1/100) to each well of the microtitre plate.

5. 5.

   Incubate for one hour at 37 °C on an orbital shaker.

6. 6.

   Wash plates three times with PBS.

7. 7.

   Add 50 μl of rabbit anti-mouse globulin conjugated to horseradish peroxidase, diluted to a pre-titrated optimal concentration, to each well of the microtitre plate.

8. 8.

   Incubate for one hour at 37 °C on an orbital shaker.

9. 9.

   Add substrate and chromogen as described previously. Stop the reaction after 10 minutes by the addition of 1 Molar sulphuric acid (50 μl/well).

In the competitive assay, the monoclonal antibody must be in excess, therefore a dilution of antigen is chosen which falls on the titration curve (not on the plateau region) which gives approximately 0,8 OD after 10 minutes.

The agar gel immuno-diffusion test shall be carried out according to the following protocol:

Precipitating antigen is prepared in any cell culture system that supports the rapid multiplication of a reference strain of bluetongue virus. BHK or Vero cells are recommended. Antigen is present in the supernatant fluid at the end of virus growth but requires 50 to 100-fold concentration to be effective. This may be achieved by any standard protein concentration procedure; virus in the antigen may be inactivated by the addition of 0,3 % (v/v) beta-propiolactone.

Using the international reference serum and antigen a national standard serum is produced, standardised for optimal proportion against the international reference serum, freeze-dried and used as the known control serum in each test.

Procedure

1 % agarose prepared in borate or sodium barbitol buffer, pH 8,5 to 9,0, is poured into a petri dish to a minimum depth of 3,0 mm. A test pattern of seven moisture-free wells, each 5,0 mm in diameter, is cut in the agar. The pattern consists of one centre well and six wells arranged round it in a circle of radius 3 cm. The central well is filled with the standard antigen. Peripheral wells 2, 4 and 6 are filled with known positive serum, wells 1, 3 and 5 are filled with test sera. The system is incubated for up to 72 hours at room temperature in a closed humid chamber.

Interpretation

A test serum is positive if it forms a specific precipitin line with the antigen and forms a complete line of identity with the control serum. A test serum is negative if it does not form a specific line with the antigen and it does not bend the line of the control serum. Petri dishes must be examined against a dark background and using indirect illumination.