Agar Gel Precipitation (AGP) assays

The Agar Gel Precipitation (AGP) assay is a very specific immunological technique, frequently used in poultry serology. The technique has been described and used for several poultry diseases, either to detect antibodies using a known, specific antigen, or to detect or identify antigens using monospecific antisera. Although it is increasingly being replaced by ELISAs, the AGP assay is the only routine assay in which the specificity of the observed reaction can be directly compared with a known positive control serum. When used in flock monitoring or diagnosis, (herd) sensitivity of the AGP assay is usually adequate.

Antigens and antisera for AGP

The AGP assay visualises the antigen-antibody complexes formed after antigens and samples have diffused in a semisolid medium. We produce the necessary antigens for use in its own laboratory and for sale. We also offers several antisera which can be used as control sera. These antisera have been carefully selected to yield optimum results with the technique used. All antisera for AGP assays are monospecific, directed at groups of antigens. These sera will detect but not differentiate between serotypes of a single viral pathogen. These antisera can also be used to detect or identify unknown antigens in samples obtained in egg or tissue culture (virus isolation).

 

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA, short for Enzyme-Linked Immunosorbent Assay, is a widely used diagnostic technique for detecting the presence of antibodies or antigens in a sample. It relies on the specific binding between antibodies and antigens. The process involves coating a plate with the sample, incubating, adding a secondary antibody linked to an enzyme, introducing a substrate for the enzyme and measuring the resulting colour change.

To ensure that the quality of results is maintained at a consistently high level, checks are carried out at a minimum of four levels: at the time of test selection, on each batch of test material used, three types of control samples on each ELISA plate, and finally through our Proficiency testing schemes (PTS).

 

Haemagglutination Inhibition (HI) assays

Several avian pathogens haemagglutinate the red blood cells of birds and sometimes other animals. This haemagglutination usually occurs ‘spontaneously’ as a result of the presence of natural receptors (haemagglutinin) on the virus surface. Some examples are: such as Avian Influenza Virus (AI), Newcastle Disease Virus (NDV), Egg Drop Syndrome ’76 Virus (EDS), Mycoplasmas, and Infectious Bronchitis Virus (IBV). In the case of Infectious Bronchitis Virus, most antigens need a special treatment to make them haemagglutinating. The Haemagglutination Inhibition (HI) assay is based on the inhibition of this specific haemagglutination by antivirus antibodies in the test sample, which bind and neutralise the viral haemagglutinin receptor.

Antigens and antisera for HI

The HI assay is usually carried out in U or V shaped microtitre plates (8x12) using 25 μL volumes of antigen containing 4 or 8 haemagglutinating units (HAU) and 25 micro-litre volumes of serially diluted samples. Red blood cells are obtained from birds free of antibodies against the virus concerned and used as a 1% or 2% v/v suspension. The antigen is available at GD. 

Our antisera for HI assays are monospecific, unless otherwise indicated. The antisera are specially selected to yield higher titres with homologous serotypes than with heterologous serotypes, but care should be taken in interpreting the results of IBV HI assays. We offer HI antisera for the most important haemagglutinating avian pathogens including many Avian Influenza types and Mycoplasma. The Mycoplasma antisera can also be used as positive controls in the rapid plate agglutination assays.

 

Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a powerful molecular biology technique used to amplify a specific segment of DNA. It allows researchers to produce millions of copies of a specific DNA sequence from a tiny sample in a short period of time. PCR works by repeatedly heating and cooling the reaction mixture, which contains DNA template, primers, DNA polymerase and nucleotides. Each cycle of heating and cooling doubles the number of DNA molecules, resulting in exponential amplification. PCR has many applications, including genetic research, infectious disease diagnostics, forensic analysis and paternity testing. Its speed, sensitivity and specificity have revolutionised molecular biology and biotechnology.

GD uses the PCR technique to detect and characterise viruses, bacteria and parasites. Many of our antigens can be used as positive controls in PCR assays. With PCR, we only detect the genetic material of the bacteria or virus that we suspect may be causing the disease. Unlike many other tests, with PCR you know what you are looking for before you start the test. PCR cannot be performed directly on the sample material. It requires species-specific sample preparation and DNA extraction. A PCR can be applied to DNA from many different sample materials. An FTA card is a special filter paper with the unique property of rendering all bacteria and viruses harmless. They are no longer infectious. In addition, the DNA is protected because it binds to the paper and can be shipped at room temperature. To release the DNA in an FTA card, a few discs are punched out of the card and washed with a specific liquid, releasing the DNA into the washing liquid. This washing liquid is then used for DNA extraction suitable for PCR studies.

 

Rapid plate aglutination (RPA)

The Rapid Plate Agglutination test is a rapid test that takes only a few minutes and is performed on a white polyacrylate plate that allows light to pass through. It detects antibodies in blood samples, particularly against bacteria such as Mycoplasma gallisepticum and Mycoplasma synoviae, Salmonella pullorum and Salmonella gallinarum. Execution of the test: (1) Serum and purple coloured antigen are mixed on a polyacrylate plate, (2) The plate is gently shaken for two minutes, (3) After two minutes the plate is placed on a light box, (4) Purple clumps indicate antibodies to M.g. and (5) Positive sera are noted and collected for a confirmatory test.

The RPA test requires a high quality system to perform the test at a consistently high level. There are at least four levels of control: when the test is selected, when each batch (the test material) is used, on each test day three types of control samples are tested several times, and finally the Proficiency testing schemes.

 

Virus Neutralisation Test (VNT)

The Virus Neutralisation Test (VNT) measures the amount of antibodies in the blood. It shows whether the animal is protected against the virus by previous infection or vaccination. The VNT measures the amount of antibodies in the blood that can prevent the virus from attaching to the animal's cells. Determination of neutralising antibody eight to twelve serial dilutions of serum (blood sample) are tested. First, a fixed amount of live virus (the antigen) is mixed with the series of dilutions of serum. After an incorporation period, the mixture is added to live cells. Depending on the virus, these may be chicken kidney or liver cells, depending on the virus. If there are enough antibodies in the diluted blood to neutralise all the added virus, the cells will not become infected and remain healthy. If there are no or insufficient antibodies in the dilution, unneutralised virus remains. The cells become infected and die. Damage occurs. The highest dilution of the serum that is still able to protect the cells from infection and protect the cells from infection and damage is called the titer'. The VNT indicates that the chicken has sufficient antibodies in its blood against the virus in its blood (minimum titer), the animal is therefore animal is therefore protected.