General instruction sheet for all freeze dried viral antigens.
1. Intended Use
As a specific antigen for antibody detection in immunoassay procedures. This freeze dried antigen has been prepared and standardised for the enzyme-linked immunosorbent assay (ELISA) plate test.
This product is presented as two glass vials closed with a rubber bung and plastic screw cap. One vial contains positive antigen as a freeze-dried pellet and is labelled accordingly. The second vial contains negative material as a freeze-dried pellet and is appropriately labelled. In both vials the pellets are under vacuum.
The positive vial contains virus antigen derived from cell or egg culture along with extracts from that cell culture and medium. The negative vial is prepared in an identical fashion to the positive vial with the exception that the cell culture was not seeded with virus.
4. Method of Use
a) Reconstitution: It is good procedure to only reconstitute the product immediately before use. Reconstitution may be with a suitable aqueous diluent. However, standardisation has been based on the use of a bicarbonate buffer(pH 9.6). This antigen has been prepared for reconstitution to 5.0 ml which provides a sufficient volume to coat one 96-well microtitre plate at 50 microlitres per well. If buffer is injected through the bung, take care that pressure changes do not lift the bung with subsequent loss of antigen. Similarly, if the bung is removed before releasing vacuum, antigen powder may escape from the vial.
b) Coating plates or strips: Place 50 microlitres of reconstituted antigen in each well and stand overnight at +4oC. Coated plates or strips not required for immediate use may be stored without drying at -70oC (or below) before removal of antigen, subsequent washing and application of any blocking procedures. Using high binding polystyrene ELISA plates in this way will generally yield optical densities of between 1.0 and 3.0 using positive sera at dilutions between 1:50 and 1:200 with OPD as a substrate.
5. Precautions and Warnings
a) This product is not guaranteed sterile. If reconstituted material is to be stored it should be kept at +4oC for short periods (24 hours) only.
b) Non-specific binding of globulins in test sera to the polystyrene plate can be effectively reduced by making serum dilutions in Phosphate Buffer containing 0.1% tween 20 and 3% w/v dried skim milk.
c) Traces of bovine serum proteins (BSP) may be present in this product. Animals yielding antiserum for testing may have been vaccinated with products containing bovine serum proteins (or otherwise made contact with BSP); a result of this is significant binding to negative antigen, which masks the specific reaction. This problem may be reduced or eliminated by adsorbing the test sera with foetal bovine serum prior to testing. An effective way of doing this is to mix equal volumes of undiluted test sera and undiluted foetal bovine serum and stand overnight at +4oC.
Prior to reconstitution the product may be stored at +4oC for at least 12 months without loss of activity. Refreezing the product after reconstitution may result in some loss of activity. Storage after reconstitution should be at -70oC or lower. Coated plates or strips may be stored without drying at -70oC or at +4oC after drying.