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Antigen Titration
Plate Layout-Antigen Detection
ELISA
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Coating of ELISA microtitre plates with coating (trapping) antibody
1. Coat the required number of ELISA plates with an optimal dilution of rabbit antiserum (usually 1/5000), 50 µl to each well.
2. Cover plates and leave overnight in a humidity cabinet. Alternatively, the plates can be coated at 37ºC for one hour.
3. After incubation, empty plates and wash with ELISA wash fluid by filling and emptying wells three times. Empty residual fluid from plates by tapping plate onto several layers of absorbent paper using an abrupt downward hand motion.
4. Plates are now ready for test assay and give best results if used immediately.
Preparation of control sera
Three control sera are required:
Strong positive serum (> 85% inhibition at1/32 dilution)
Weak positive serum (55-85% inhibition at 1/32 dilution)
Negative serum (< 40% inhibition at 1/32)
Control sera will either be provided ready prepared or directions will be given as to how each serum can be generated by mixing positive and negative sera. Control sera should contain preservatives (sodium azide or merthiolate), should be kept frozen in small aliquots, and each aliquot should not be subjected to repeated freeze/thawing cycles.
Antigen titration
As part of the procedure for setting up the liquid phase ELISA for the first time, and each time a new batch of antigen is received or prepared, it is necessary to to perform an antigen titration to determine the optimal working dilution.
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