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The importance of serial dilution and colony counting is reflected by the. Serial Dilutions MLAB 1335 Immunology/Serology. Serial Dilutions A serial dilution is any dilution where the concentration decreases by the same quantity in each. Learn more about aseptic technique, dilution. Importance Of Serial Dilution In Serology. Other than that, the diluted solution from this serial dilution can be used to count the concentration of the actual solution. By knowing the dilution factor of certain solution, the calculations of the concentration become easier and systematic. This method is applicable in several fields. Perform a serial dilution, which are a series of dilutions, when the final volume is a large value like 10,000 mL, for example. In this case, make a 1 mL to 100 mL dilution first and from that solution take another 1 mL into another 100 mL. The final solution is a 1 to 10,000 mL (100 mL x 100 mL) dilution.

Introduction

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.

Materials

Syringes with blood samples.
Sterile glass Pasteur pipettes.
Microfuge tubes.
Storage tubes. (1.8 mL Nunc cryotubes are ideal but microfuge tubes are a cheaper alternative.)
Sharps container and discard bag for biological waste.

Method

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.

Temperature.

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

Importance Of Serial Dilution In Serology Lab