AbstractThe aim of the project was to develop a solid phase enzyme-linked immunosorbent assay (ELISA) for the quantitation of red cell antibodies of any specificity. Binding of negatively charged reagent red blood cells (RBC) was achieved in microplate precoated with poly-L lysine (PLL). The optimum concentrations of PLL and RBC were between 10 and 20 mg/ml and 0.5% volume for volume, respectively.
Experiments using serial dilutions of monoclonal anti-A, anti-B and anti-D incubated in microplate wells coated with the corresponding antigen positive or negative red cells showed that the RBC remain bound throughout the test and the assay can be shown to be specific. The absorbance values obtained were proportional to the concentration of antibody.
When human polyclonal antibodies (anti-A, anti-B and anti-D) and AB serum (which contained no RBC antibodies detectable by conventional techniques) were used, high background reactions were obtained and the discrimination between positive and negative reactions was very poor. Experiments have shown that a significant proportion of the background reactions in the solid phase tests were due to nonspecific binding of antibodies in the test serum to PLL and to the plastic of the microplate wells.
Attempts were made to reduce non-specific binding with varying concentrations of alpha 1 acid glycoprotein, bovine serum albumin, bovine and goat casein, foetal calf serum (PCS), rabbit serum and Tween 20. Of these, 20% PCS gave the best reduction in background noise but the assay was still not discriminating enough for routine testing. Neither the replacement of PLL with lectins nor manipulation of the pH for blocking and incubation reactions did not reduce the non-specific reactions significantly.
In conclusion, the principle of solid phase ELISA can be shown to work for monoclonal antibodies to red cell antigens. With polyclonal antibodies there were high non-specific background reactions which could not be reduced enough to allow a useable dose response relationship for antibody quantitation. Hence, the study of the correlation of the concentration of red cell antibodies by this technique with haemolytic disease of the newborn (HDN) could not be performed.
|Date of Award||Oct 1999|