Triple-Site Antigen Capture ELISA for Human Myoglobin Can Be More Effective Than Double-Site Assay

V. A. Nikulina¹, E. A. Kizim², Yu. S. Massino², O. L. Segal², M. B. Smirnova², V. V. Avilov³, D. B. Saprygin³, S. P. Smotrov³, G. I. Kolyaskina¹, and A. D. Dmitriev^2*

¹Mental Health Center, Russian Academy of Medical Sciences, Zagorodnoe Shosse 2/2, Moscow, 113152 Russia; fax: (7-095) 952-8940

²Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, ul. Butlerova 5a, Moscow, 117865 Russia; fax: (7-095) 952-8940; E-mail: dmitr@rcmh.msk.ru

³Association of Medical Laboratory Diagnostics, ul. Barrikadnaya 2, Moscow, 117312 Russia

^* To whom correspondence should be addressed.

Received February 4, 1999; Revision received April 8, 1999

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Using a panel of monoclonal antibodies against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture ELISA can be significantly increased by simultaneous immobilization of two cooperating capture monoclonal antibodies on a solid phase. This method ("triple-site ELISA") uses three monoclonal antibodies to different epitopes of the same antigen (two capture/one tracer) unlike the traditional double-site assay using one capture and one tracer monoclonal antibody. We developed double- and triple-site ELISA for Mb by varying the capture and tracer monoclonal antibodies. Triple-site assays showed 4-6-fold increase in sensitivity compared to the double-site assays. A model for this effect is suggested; according to the model, in triple-site ELISA, high-affinity cyclic configurations can be formed by an antigen, two capture monoclonal antibodies, and the surface of the solid phase.

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KEY WORDS: antigen capture ELISA, triple-site ELISA, monoclonal antibodies, synergistic effect, human myoglobin

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