Single-Step Sandwich Immunoassay of Myoglobin with Bifunctional Monoclonal Antibodies

M. B. Smirnova¹, V. A. Nikulina², O. L. Segal¹, E. A. Kizim¹, Y. S. Massino¹, N. N. Ryazanskaya², G. I. Kolyaskina², and A. D. Dmitriev^1*

¹Institute of High Nervous Activity and Neurophysiology, Russian Academy of Sciences, ul. Butlerova 5a, Moscow, 117865 Russia; fax: (095) 338-8500; E-mail: dmitr@rcmh.msk.ru

²Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe Shosse 2/2, Moscow, 113152 Russia; fax: (095) 952-8940

^* To whom correspondence should be addressed.

Received July 8, 1998

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Two protocols for sandwich antigen-capture ELISA of human myoglobin were compared. In the first (routine) variant, 14D6 monoclonal antibodies conjugated to horseradish peroxidase were used as the secondary antibodies. Bifunctional antibodies specific for myoglobin/peroxidase were used as the secondary antibodies in the second variant. The myoglobin-binding site of the bifunctional antibodies was similar to that of the 14D6 antibodies, and the second antigen-binding site of the bifunctional antibodies was bound to horseradish peroxidase. When comparing standard calibration curves, the effective concentration of the bifunctional antibodies and that of antibodies conjugated to horseradish peroxidase were made equal. It is shown that the use of bispecific antibodies as the secondary antibodies does not improve the quality of the parameters tested, i.e., the sensitivity of the assay does not increase and the slope of the calibration curve remains constant.

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KEY WORDS: myoglobin, bispecific antibodies, sandwich method, tetradoma, capture ELISA

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