PREPARATION OF CYCLIC-NUCLEOTIDE ANTISERA WITH THYROGLOBULIN-CYCLIC NUCLEOTIDE CONJUGATES

  • 1 January 1980
    • journal article
    • research article
    • Vol. 6  (6) , 461-468
Abstract
Antisera to cAMP and cGMP were obtained by immunizing rabbits with antigens prepared by conjugating the 2''O-succinyl derivative of the cyclic nucleotides to thyroglobulin. The cyclic nucleotide-thyroglobulin conjugates were injected intradermally into multiple sites on the backs of the animals. This immunization procedure resulted in the production of antiserum in 4 of 5 animals capable of binding at a final serum dilution of greater than 1:10,000, 20% of the corresponding iodinated cyclic nucleotide derivative added. The antisera were also highly specific. The antiserum for cAMP had a 2500-fold or greater relative affinity for cAMP than other nucleotides or nucleosides, while that for cGMP had a 5000-fold or greater affinity for 2''O acetylated cGMP than other acetylated nucleotides or nucleosides except for acetylated cIMP. The obstacles to measuring cyclic nucleotides, particularly cGMP, in tissues were also overcome by refining and simplifying the methods for iodination, purification and assay. A disequilibrium incubation was developed as an alternative to the acetylation method to increase the sensitivity of the radioimmunoassay. The levels of both cGMP and cAMP can be determined rapidly and easily in the same tissue sample.

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