Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies

Lynette M. Boscato*, Gregory Egan, Margaret C. Stuart

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


A one-step two-site immunoradiometric assay for the measurement of free β subunit of human chorionic gonadotrophin (β-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free β subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free β subunit. Although the level of hCG "cross-reaction" was low when studied using conventional techniques, the apparent β-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of 125I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.

Original languageEnglish
Pages (from-to)393-401
Number of pages9
JournalAnalytical Biochemistry
Issue number2
Publication statusPublished - 1 May 1985


  • binding assays
  • clinical chemistry
  • computer methods
  • immunochemical methods
  • monoclonal antibodies
  • protein hormones


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