Antibody coating approach involving gamma globulins from non-immunized animal and second antibody antiserum.
An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with gamma-globulins from non-immunized animal of the same species as that from which the antigen-specific antibody has been developed. Then, an excess of second antibody is added to the tubes and the anti-species specific antibodies present in the antiserum are immunoadsorbed on the immobilized gamma-globulins. Finally, the antigen specific antibody is immunoadsorbed on the immobilized second antibody. We found that the coating protocol developed allows the use of antigen-specific and second antibody antisera dilutions, thus avoiding the need for affinity purification of antibodies. Additionally, it provides solid-phase second antibody with increased binding capacity compared to the directly adsorbed onto the solid second antibody. The advantages of the proposed coating protocol were demonstrated through the development of a solid-phase radioimmunoassay for the determination of total triiodothyronine in human serum samples.
Petrou
P S
PS
Immunoassay Quality Control Lab., I/R.-R.P, N.C.S.R., Demokritos, Aghia Paraskevi, Athens, Greece.
Kakabakos
S E
SE
Koupparis
M A
MA
Christofidis
I
I
D000818:Animals; D000906:Antibodies / Q000097:blood / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007114:Immunization; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology; D007163:Immunosorbent Techniques; D009994:Osmolar Concentration; D011137:Polystyrenes; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D014284:Triiodothyronine / Q000097:blood / Q000276:immunology
D000906:Antibodies; D015415:Biomarkers; D007074:Immunoglobulin G; D011137:Polystyrenes; D014284:Triiodothyronine