The presence of antibodies to Newcastle disease virus in chickens is detected by serological testing. The results of these tests are used for three purposes.
1. To assess the efficacy of Newcastle disease vaccine in laboratory and field trials.
2. To assess the level of Newcastle disease virus antibodies in the field.
3. Serum known to contain antibodies to Newcastle disease virus is used to confirm the presence of Newcastle disease virus in a test sample of allantoic fluid. Such a sample would be obtained during the isolation of virulent Newcastle disease virus. See Section 15.
There are two assays commonly used to carry out serological testing for Newcastle disease virus antibodies.
1. Haemagglutination inhibition (HI) test. The HI test is a convenient and commonly used assay that requires cheap reagents and is read by eye.
2. ELISA (Enzyme linked immunosorbent assay). This is a colourimetric assay and requires the use of a sophisticated instrument to read the optical density of the reactions. ELISA kits for Newcastle disease virus antibody detection are prepared and sold commercially. Detailed instructions are supplied with the kits. They are usually quite expensive.
In this manual a protocol for the HI test based on the test described by Allan and Gough will be used for serological testing. (Allan and Gough, 1974 a.)
Preparation of serum
Serum samples are collected for testing for the presence of antibodies to Newcastle disease virus. Blood is collected as described in Section 7. The blood forms a clot in the syringe in a few minutes. Once the blood clots, the syringes of blood can be kept with the needle upright to prevent serum filling the needle cap. The serum will separate from the clot within a few hours at room temperature or in approximately 2 hours at 37°C. Storage at 37°C will help the serum separate from the clot.
1. Remove the plunger from the syringe. Transfer the serum to a microfuge tube by pouring or using a glass Pasteur pipette.
2. Dispose of needles, syringes, clots and pipettes in appropriate containers.
3. Often the serum will contain red blood cells. Centrifuge for 30 seconds in a microfuge centrifuge or allow to settle under gravity overnight at 4°C to pellet the cells. Do not freeze the samples at this step. Freezing will lyse the red blood cells.
4. Transfer clear serum to a second tube.
5. Remember to label each tube after the transfer of the serum. This ensures the serology results can be applied to individual birds and groups.
Storage of Serum
Store ampoules of serum at -20°C.
Storage at 4°C is acceptable for a short period, up to 2 weeks.
Notes on serum samples
Samples collected in the field may end up at room temperature overnight and often do not separate well.
It has been noted at the John Francis Virology Laboratory that in some samples the serum does not separate from the clot. In these cases, the whole clot is centrifuged. This usually results in a small amount of serum separating.
It is important that the serum samples are clear. Pink samples contain lysed red blood cells. When pink samples are tested observe both test and control samples carefully to determine effect of the colour on the results of the test.
Pink coloured samples can affect results of an ELISA, which is a colourimetric assay.
An overview of the Haemagglutination Inhibition (HI) test
Antibody response to the haemagglutinin protein in the Newcastle disease virus envelope can be measured by the HI test.
When serum containing these antibodies is mixed with Newcastle disease virus, the antibodies bind to the haemagglutinin protein in the envelope of the virus. This blocks the haemagglutinin protein from binding with the receptor site on chicken red blood cells.
Thus the haemagglutination reaction between the virus and the red blood cells is inhibited.
By performing two-fold serial dilutions on the serum prior to testing, the concentration of the serum antibodies can be expressed as an HI titre to the log base 2.
Standardization of the HI test
It is important that there is correlation between the results of tests carried out by different technicians and in different laboratories. For this reason HI tests should be standardized both within a laboratory and between laboratories.
Standardization is achieved by following a standard protocol. This will include:
Using a standard 4 HA units of Newcastle disease virus antigen.
Using standard positive anti-serum and negative serum.
Including a serum control for each test serum to detect the presence of non-specific agglutinins.
Using a standard 1 percent dilution of red blood cells.
The use of control serum in the HI test
Positive and negative control sera are tested to avoid errors in the interpretation of the results of the HI test. Inconsistencies in the results of the HI test may be caused by variations in reagents and procedures.
Examples of possible variations include:
The source and exact percentage by volume of the red blood cells.
The exact amount of antigen used in the HI test.
Accuracy of dilutions.
Time allowed for the antibody/antigen reactions to occur.
Quality of test serum samples (haemolysed serum samples may be responsible for non-specific reactions)
Two standard sera are used.
1. A standard negative control serum known to contain no antibodies to the Newcastle disease virus. It has no HI titre and does not agglutinate chicken red blood cells.
2. A standard positive control serum also known as a standard anti-serum. The HI titre of the anti-serum will have been established by repeated titration.
Variability in HI titres of standard positive serum
It is sometimes observed that the standard HI titre of the standard anti-serum tested on the same day with the same antigen preparation and protocol may vary. A difference in HI titre of one dilution that is one log base 2 (21) can be regarded as due to random errors and an inherent variability in biological responses. However if the titre of the standard positive serum differs by more than one dilution or log base 2, then the test is invalidated. In this case, fresh antigen must be prepared and tested and the HI test repeated.
Three categories of standard sera
1. International standard reference serum. Certain laboratories prepare reference serum to a very high standard. Standard reference Anti-Newcastle disease serum is available for purchase. It is used as a reference serum in the preparation of national and laboratory standard sera.
The National Institute for Biological Standards and Control (NIBSC) in the United Kingdom can supply an international standard reference serum. The potency of the international reference serum has been determined by the supplier and is expressed in International Units (IU) per ampoule of freeze-dried serum.
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2. National standard serum is prepared by a national laboratory and distributed to collaborating laboratories as required. This serum is prepared by mixing sera with known HI titres. A series of HI tests are carried out to establish the HI titre of the national standard. This is compared with the HI titre of the international standard reference serum if available. The national standard serum is then stored in multiple aliquots and distributed to collaborating laboratories within a country.
3. Laboratory standard serum is prepared by some laboratories in order to conserve supplies of national standard serum. The laboratory standard is prepared by mixing serum samples with known HI titres. Comparative HI testing of the laboratory standard and the national standard is used to establish the HI titre of the laboratory standard.
Preparation of national and laboratory standard sera
Each laboratory will require a supply of negative and positive control serum in order to carry out HI tests on serum samples.
Collection of HI negative serum
Collect serum from chickens that have had no exposure to Newcastle disease virus. The serum should show no inhibition of viral haemagglutination activity when tested by the HI test. It is often difficult to find serum without any HI activity. In this case use serum with low HI titres of 21.
Collection of HI positive serum
There may be stored serum that is suitable for this purpose. If not, vaccinate several chickens with I-2 Newcastle disease vaccine. Two weeks after the primary vaccination, give a booster vaccination. Two weeks later, take a serum sample from each chicken and test the HI titre. Collect extra serum from chickens with a titre of 25 or above. The volume of blood collected from each chicken depends on the size of the chicken. Pool all the serum samples with HI titres of 25 or above and test the HI titre of the pooled serum.
Comparative HI testing of international reference and national standard sera
Information supplied by NIBSC in 2001 indicated their international standard reference contained 320 IU per ampoule.
The serum can be reconstituted in PBS. A suitable volume of PBS would be 2 mL, which would give 4 IU in the 25 µL test sample. Refer to instructions provided by the manufacturer.
Use the standard HI test described in this section to determine the titre of the international reference standard and the national standard.
Test both samples in triplicate on the same microwell plate.
Carry out a series of at least three tests on different days.
Once the HI titre of the international standard serum and the national standard serum have been established, prepare a series of two-fold dilutions of the serum that are spread around the endpoint used to establish the HI titres. Titrate the dilutions that range from complete inhibition to no inhibition as an additional check on the HI titres. The results of testing these dilutions will confirm the HI titre of the undiluted serum.
Example of confirming HI titre of standard samples by testing a series of dilutions of the sample.
Repeated testing of the pooled serum established the HI titre at 25.
Prepare 1/8, 1/16, 1/32 and 1/64 dilutions of the pooled serum.
Test each dilution for HI titre.
Confirmatory results: The results tabled confirm that the HI titre of the serum used to prepare the dilutions had a titre of 25.
Table 3: Results confirming HI titre of serum = 25
The test results will give a HI titre for both the International reference serum and the national laboratory serum being tested. The HI titre for 4 IU of the International reference serum can be used to determine the number of IU in the national reference serum.
The standard HI test of 25 µL of the international reference serum containing 4 IU and has a HI titre of 8 (23). The national serum tested HI titre of 64 (26).
How many IU does the national serum contain?
4 IU in a sample with a HI titre of 8.
How many (?) IU in a sample with a HI titre of 64.
Not all laboratories use the same protocol for testing HI titre and IU are independent of the test system used.
Serological results of samples expressed in IU should give equivalent results when tested by different systems.
By comparing the HI titre of the standard national serum with the HI titre of the international reference serum enables you to express the results of HI tests in IU. However it is not very often that you will be required to express the results of HI testing in IU.
Most publications of Newcastle disease serology results describe the assay used and express HI titres to log base 2.
Preparing a laboratory standard serum
Each laboratory should receive a national standard serum from a central laboratory. The HI titre and activity in International units of the serum will have been determined by rigorous testing as described above. To conserve the supply of national standard serum some laboratories will decide to prepare a secondary laboratory standard serum.
Collect positive serum as described above. Do comparative testing of the laboratory and national standards. Test each sample in triplicate on the same microwell plate. Repeat several times and analyze HI titres for both samples. Record the HI titre for the laboratory standard.
Store 1 mL aliquots of the laboratory standard at -20°C. This standard is used every time the HI test is carried out and should always show the same HI titre when tested with the 4 HA units of antigen.
Preparing a national standard serum without an international reference serum
Many laboratories will not be able to obtain an international reference serum with which to compare their national standard serum. In this case, more rigorous HI testing of the pooled HI positive serum samples will be required to establish the HI titre of the standard serum. It is suggested that the serum is tested up to ten times. Use freshly prepared 4 HA units of antigen for each test and carry out the test on different days if possible. The results will be a range of HI titres. The most frequently occurring titre can be considered the HI titre of the standard serum. All future HI tests carried out on the standard serum should give this titre.
Storage of standard serum
Once the positive and negative standard sera have been tested, aliquots can be prepared, labeled and stored frozen. Storage at -70°C is optimal (but -20°C is adequate). Do not thaw and refreeze the samples frequently. Representative samples should be thawed and tested to confirm that there is no loss of titre in the storage process.
Preparation of Newcastle disease virus antigen for use in HI tests
Antigen is prepared by inoculating embryonated eggs with a sample of Newcastle disease virus and harvesting the allantoic fluid four days later. Part of the first batch of I-2 Newcastle disease vaccine prepared from the I-2 working seed can be set aside for storage as antigen. A volume of 50 mL to 100 mL is adequate for a large number of tests. Centrifuge the sample at 1 200 g to clarify and remove any contaminating red blood cells. Store the antigen in one mL aliquots at -20°C.
See Sections 6 and 9.
Preparation of 4HA units of Newcastle disease virus antigen
The standard amount of Newcastle disease virus used in the haemagglutination inhibition (HI) test is 4HA units. It is necessary to prepare and test a suspension of Newcastle disease virus containing 4HA units in order to carry out the HI test. This involves a series of following steps.
1. Titrate the stored suspension of virus to be used as the antigen in the HI test. See Section 10 Calculate the HA titre.
2. Calculate the dilution factor required to produce 4 HA units. A simple way is to divide the HA titre by 4.
3. Apply the dilution factor and dilute the original suspension of antigen in PBS to produce an adequate volume of 4HA antigen to carry out the HI test. Allow 2.5 mL for each microwell plate.
4. Titrate the diluted (4HA) suspension of virus. This is a back titration to check the diluted antigen contains 4 HA units.
5. Read HA titre. It should equal 4HA units. If not adjust the dilution and titrate again.
6. Use the 4HA unit dilution of antigen in an HI test to test the standard positive and negative serum. The HI titre of the laboratory standard positive serum should equal the predetermined titre.
The results of the back titration of the diluted antigen and the HI titre of the standard positive are both used to confirm the antigen has been diluted to a concentration equivalent to the standard 4 HA units.
If the HI titre of the positive control serum is less than the standard titre, the antigen is too concentrated. Prepare a new dilution and test again.
Conversely if the HI titre of the positive control serum is too high the antigen is too dilute. Prepare a new dilution and test again.
Preparation of 4HA units of antigen for HI test in 10 microwell plates:
The HA titre of the antigen was tested according to the protocol described above. The HA titre = 128
Calculation of dilution factor to prepare 4 HA units: 128/4 = 32
Calculation of volume of 4HA unit dilution of antigen required:
Allow 2.5 mL per plate; total volume required = 10 × 2.5 mL = 25 mL
Apply dilution factor = 25 mL/32 = 0.781 mL = 781 µL
Preparation of the diluted antigen: mix 781 µL of original virus suspension with 24.219 mL of diluent. PBS is a suitable diluent.
Note: In this case it would be easiest to prepare 32 mL of the 4HA antigen. This would use 1 mL of the original suspension diluted in 31 mL of PBS.
1. Fill in recording sheets to record how samples will be dispensed into microwell plates.
2. Calculate the number of plates required and number each plate.
3. Dispense 25 µL of PBS into each well of the plates.
4. Shake each serum sample and dispense 25 µL into the first well and the last (control) well of a row of a microwell plate.
5. Use a multichannel pipette to make two-fold serial dilutions along the row until the second last well from the end. The last well is the serum control. Do not dilute this well. See Appendix 4 for instructions on carrying out two-fold serial dilutions.
6. Add 25 µL of the 4HA dilution of antigen to each well excluding the control wells in the last column. See Section 10 for preparation of 4HA units of antigen
7. Gently tap the sides of the microwell plates to mix the reagents. Cover plates with a lid. Allow to stand for 30 minutes at room temperature.
8. Add 25 µL of 1 percent washed red blood cells to each well including the control wells in the last column.
9. Gently tap the sides of the microwell plates to mix the reagents. Cover the plates with a lid. Allow to stand at room temperature for 45 minutes.
10. Read the settling patterns for each serum sample. Read the control serum well first then read the patterns in the other wells.
11. Record the pattern observed in each well on a microwell plate recording sheet. Determine the endpoint. This is the point where there is complete inhibition of haemagglutination.
12. Record the antibody level for each serum sample. This is expressed as a log base 2. For convenience, the titre is often recorded as just the log index. For example a titre of 26 would be recorded as 6.
Interpretation of results
In the wells where antibodies are present there will be haemagglutination inhibition. The red blood cells will settle as a button.
In the wells where antibodies are absent, the red blood cells will agglutinate.
The end point of the titration is the well that shows complete haemagglutination inhibition. Sometimes it is not easy to determine. Look at the size of the button as an indication of the degree of haemagglutination inhibition. Use the control well as a point of comparison. Be consistent in determining the endpoint.
The neuraminidase enzyme present in the virus particle will eventually break the bond between the virus and red blood cells. This process is called elution.
When elution occurs, the red blood cells are no longer agglutinated. They roll down the side of V-bottom microwell plates to resemble the negative settling pattern, a tight button.
Some Newcastle disease virus strains elute more rapidly and the test must be read before this occurs.
Usually elution takes longer than 45 minutes. A control well with virus and red blood cells is useful to determine elution time.
Figure 22: Titration to determine HI titre
Some sera may contain substances other than antibodies that inhibit viral haemagglutinin. These substances are described as non-specific inhibitors and are rarely observed with chicken serum and Newcastle disease.
Some chicken sera contain substances that will agglutinate chicken red blood cells. The settling pattern of the agglutinated cells is similar to that produced by Newcastle disease virus. These natural agglutinins are present in low concentration. The control serum well will indicate the presence of natural agglutinins.
Adsorption of natural agglutinins
If natural agglutinins are present in a serum sample, they can be removed by adsorption with chicken red blood cells. The serum can then be retested in the HI test.
1. Place 200 mL of the 10 percent washed red blood cells into a microfuge tube.
2. Centrifuge for 15 seconds.
3. Remove the supernatant.
4. Shake the serum sample and remove 500 mL of serum. Some samples may contain less volume. Use a new tip for each sample.
5. Add the serum to the red blood cells in the microfuge tube. Mix gently.
6. Stand for not less than 30 minutes at 4°C.
7. Centrifuge for 15 seconds.
8. Remove the serum (supernatant) immediately and transfer to a clean microfuge tube.
9. Store the adsorbed sera at 4°C overnight or at -20°C for longer periods.