Synergistic effects in antigen capture ELISA using three monoclonal antibodies directed at different epitopes of the same antigen.

Using a panel of monoclonal antibodies (mAb) against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture enzyme-linked immunosorbent assay (ELISA) may be significantly increased by the simultaneous immobilization on a solid phase of two co-operating capture mAbs. This method ("a three-site ELISA") uses three mAbs at different epitopes of the same antigen (two capture/one tracer), unlike the traditional two-site assay, using one capture and one tracer mAbs. We established two-site and three-site ELISA assays for Mb, by varying capture and tracer mAbs. Three-site assays showed 4-6 fold increase in sensitivity, if compared with two-site assays. The model for the effect has been suggested, according to which in three-site ELISA the high-affinity cyclic configurations may be formed by an antigen, two-capture mAbs and the surface of solid phase.

Triple-site antigen capture ELISA for human myoglobin can be more effective than double-site assay.

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.

Single-step sandwich immunoassay of myoglobin with bifunctional monoclonal antibodies.

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.

Study of antigen-binding properties of bispecific antibodies.

Antigen-binding properties of bispecfic antibodies (bAbs) produced by mouse hybrid hybridomas were studied. One of the bAbs held binding sites for two different antigens with relatively high molecular mass: human IgG (M(r) approximately 160,000) and horseradish peroxidase (HRP, M(r) approximately 40,000). Another bAbs showed specificity to antigens differing in molecular mass by more than an order of magnitude: peptide alpha-endorphin (END, M(r) approximately 1600) and HRP (M(r) approximately 40,000). The studied antibodies also contained different immunoglobulin chains. Both heavy chains of the anti-IgG/HRP bAbs molecule were of mouse subclass IgG1. Anti-END/HRP bAbs was formed by a combination of heavy chains which belong to two subclass of IgG: IgG2a and IgG1. bAbs were purified from ascitic fluid by a two-step affinity chromatography on columns with Sepharose-4B conjugated with the corresponding antigen. Radioimmune and immunoenzyme assays were used to analyze antigen-antibody binding and equilibrium constants of association (Ka) for each parental antibody and bAbs were determined by the Scatchard method. No significant changes in the affinity of bAbs antigen-binding sites were observed as compared to the corresponding parental antibodies. It was also shown that bAbs interaction with an excess of one of the antigens did not affect binding of the other antigen to the second bAbs site. Two-dimensional gel electrophoresis was used to analyze the composition of bAbs light and heavy chains specific to END/HRP. This analysis corroborated that bAbs molecules contained light and heavy chains from both parental hybridomas. Hence, it was demonstrated that the hybridoma fusion method can provide bispecific IgG molecules fully preserving antigen binding properties of the parental antibodies.

[Decrease and asymmetry in taste perception in patients with nerve trunk damage]. / Snizhenie i asimmetriia vkusnogo vospriiatiia u bol'nykh s povrezhdeniem nervnykh stvolov.

The purpose of this article was the gustatory perception state study (NaCl solution perception) in patients with one-sided damages of branchial plexus as well as of radial, facial and ulnar nervous trunks. The decrease of gustatory perception was observed in 78 (70.3%) cases, while the lack of such alterations in 33 (29.7%) patients. The gustatory perception disturbances (increase of the absolute threshold on the ipsilateral tongue's half) took place in 82.2% and 69.6% at right and left limbs damages respectively. The results presented testified the strengthening of normally existing asymmetry in gustatory perception especially in patients with left-sided denervation. The degree of gustatory perception restoration may serve as a prognostic test at surgery of such patients.

[Asymmetry of muscular electric excitability in injuries of the brachial plexus]. / Assimetriia élektrovozbudimosti myshts pri povrezhdeniiakh plechevogo spleteniia.

The data are presented on the asymmetry of electric excitability of right and left arm muscles in 22 patients with lesions of the right brachial plexus and 24 patients with left brachial plexus lesioned. Asymmetry included accelerated loss of excitability to short impulse currents in patients with left brachial plexus lesioned, and in more pronounced increase in the volume of threshold current decreasing in length of impulse for the muscles of intact left hand as related to those of intact right hand.