Single Radial Immunodiffusion (SRID) Testing Service

Single radial immunodiffusion (SRID) is an agar gel precipitation test used to determine the quantity or concentration of an antigen in a sample. Antibody of known specificity is distributed evenly in an agar gel, and a sample containing the antigen of interest is placed in a well within the gel. Antigen then diffuses radially from the well, and a precipitin ring forms at the point of antibody-antigen equilibrium. Antigen concentrations are determined by measuring the diameter of precipitin rings and extrapolating using standard curves.

WHO's guidelines specify that the vaccine producers must determine the potency at the time of release and throughout the approved shelf life of the product. Single radial immunodiffusion (SRID) is currently the gold standard for influenza vaccine potency. In each case, the assays are based on the diffusion of viral antigen into an agarose gel containing specific antibodies to the antigen being measured. The interaction between antigen and antibody produces a precipitation zone whose size is directly proportional to the amount of antigen applied. A potency value for unknowns is obtained by comparing the sizes of zones produced by unknown preparations to the sizes of zones obtained with a calibrated reference of known antigen content.

Once the specific reference antigens and antibodies are prepared and the test standardized, it is a remarkably simple technique which, unlike agglutination assays, is very reproducible, relatively unaffected by minor variations in test conditions, and is far less time consuming and cumbersome than in vivo assays for potency. Similar SRID assays have been used experimentally to determine the potency of inactivated polio and rabies vaccines. For influenza and rabies, disruption of the virions with a detergent is necessary to permit the diffusion of the appropriate antigens, whereas, with polio, intact virions are allowed to diffuse.

Applications

• Used in immunology to determine the quantity or concentration of an antigen in a sample.
• Estimation of the immunoglobulin classes in serum.
• Estimation of IgG, IgM antibodies in serum.
• To determine the relative purity of an antigen preparation.
• Serological surveys.

Our Procedures

• Wells are introduced into a slab of agarose containing the antibody at a concentration that will give zones of an appropriate size.
• Serial dilutions of the antigen to be assayed are then placed in the wells and the antigen diffuses until it reaches the zone of equivalence with the antibody dilution, where a precipitin ring forms.
• The agarose is washed free of excess antibody, dried and stained with coomassie blue or some other appropriate agent to visualise the rings, whose size is measured.
• The greater the antigen content the larger the ring area. The reference is run in the same way and the ring sizes compared to measure antigen potency expressed relative to the reference.

Our Features

• As SRID has been used for decades there is a great deal of historic data and experience.
• It measures the antigen thought to be most relevant to protection, specifically that in the native conformation.
• It can be used to assay the potency of the final finished product which is almost always multivalent.

Our Capabilities

• Influenza virus (IFV)
• Poliovirus
• Rabies virus

(Other viruses or microorganisms may be available on request)

We combine infection and analytical expertise to provide our clients with the most powerful portfolio of antiviral and antimicrobial in vitro testing services. Facing an increasing demand for new antiviral and antimicrobial compounds for the treatment of infectious diseases, Creative Diagnostics can test these compounds in vitro to determine their potential efficacy in vivo models.

Reference:

1. Philip D. Minor. Assaying the Potency of Influenza Vaccines. Vaccines (Basel). (2014) 3(1): 90-104.
2. Laura R. Kuck, et al. VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines. Vaccine. (2018) 36: 2937-2945.