Solid phase radioimmunoassay for cyclic AMP using staphylococcal protein A-antibody adsorbent.
A radioimmunoassay for cyclic AMP has been developed using protein A containing staphylococci as an immunoabsorbent. Protein A containing heat-killed staphylococci (Cowan I) are coated with rabbit antiserum raised against the 2'-O-succinyl derivative of cyclic AMP coupled to human serum albumin. After washing with a Tween 20 containing buffer, antibody coated staphylococci are diluted with heat-killed staphylococci devoid of protein A (staphylococcus epidermidis) and mixed with [125I]-2'-O-succinyl cyclic AMP tyrosine methyl ester, standards or unknowns. At the end of the incubation, separation of bound and free labelled antigen is achieved by bound and free labelled antigen is achieved by centrifugation. The results are comparable to those obtained with a precipitation assay using polyethylenglycol 6000. Acetylation prior to radioimmunoassay increases sensitivity about 80-fold. 50% depression of zero dose binding occurs at 15--16 femtomoles acetylated cyclic AMP. The crossreactivity with cyclic GMP, ATP, ADP, 5'-AMP and adenosine is extremely low. The present technique is an attractive alternative to the second antibody method or polyethylenglycol precipitation.
D000107:Acetylation; D000818:Animals; D000907:Antibodies, Bacterial / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins / Q000276:immunology*; D003429:Cross Reactions; D000242:Cyclic AMP / Q000032:analysis*; D006168:Guinea Pigs; D007164:Immunosorbents; D066298:In Vitro Techniques; D008168:Lung / Q000032:analysis; D011233:Precipitin Tests; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods; D013210:Staphylococcus / Q000276:immunology*; D013211:Staphylococcus aureus / Q000276:immunology
D000907:Antibodies, Bacterial; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins; D007164:Immunosorbents; D000242:Cyclic AMP