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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. Dmitriev2*

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