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.

Quantitative analysis of the products of IgG chain recombination in hybrid hybridomas based on affinity chromatography and radioimmunoassay.

On the model of a hybrid hybridoma (quadroma) to alpha-endorphin (END) and horseradish peroxidase (HRP), we have elaborated a general approach to analyse H and L chain interactions in hybrid hybridomas and to evaluate their efficiency as producers of bispecific antibodies (bAbs). This strategy is based on quantitative analysis of quadroma produced Abs by affinity chromatography and radioimmunoassay. First, Abs produced by quadroma cells in culture media (IgG pools from three quadroma clones) were fractioned with respect to specificity. Second, Ab concentrations in each fraction (bispecific, anti-END, anti-HRP and inactive) were measured by specific radioimmunoassays, using rabbit antiserum against mouse IgG and 125I-labelled affinity purified quadroma Abs. Then the experimentally obtained Ab distributions were compared with the predicted Ab distributions for different models of IgG chain recombination in quadroma cells (random H/L pairing, preferential homologous H/L association). As follows from these models, in a random H/L recombination the yield of bAbs in quadroma produced IgG cannot exceed 12.5%, and the ratio of bAbs and inactive Abs cannot exceed 0.5. In the analysed clones the yield of bAbs amounted to about 30% of total IgG, and the ratio of bAbs and inactive Abs was about 5-8, giving strong evidence for preferential homologous H/L association in these cells. The ratio of anti-HRP and anti-END Abs was about 10:1, suggesting unequal production of parental IgG chains in quadroma cells. The result of quantitative analysis of quadroma IgG was further supported by two-dimensional gel analysis of affinity-purified fractions of quadroma IgG and of two parental mAbs.

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.

[Bispecific monoclonal antibodies: the isolation and study of their antigen-binding properties]. / Monoklonal'nye bispetsificheskie antitela: poluchenie i izuchenie antigensviazyvaiushchikh svoistv.

Bifunctional antibodies (bABs) having a double specificity to alpha-endorphin (alpha-END) and horseradish peroxidase (HRP) were produced by hybridoma technology. The antibodies constituted about 28-29% of all immunologically active IgG secreted by hybrid hybridoma (quadroma). The quadroma was isolated by fusion of two murine hybridomas (anti-HRP and anti-alpha-END) with distinct phenotypes: double mutant AMD(R)/NAT(S) and its wild type. To produce the double mutant phenotype, an actinomycin D-resistant (AMD(R)) mouse myeloma was used to initiate one of the parental hybridomas. bABs were purified from quadroma culture medium and ascitic fluids by sequential HRP-sepharose and alpha-END-sepharose affinity chromatography. With radioimmunoassay, the affinity of the individual anti-alpha-END combining sites of bABs was shown to be identical to that of parental monoclonal antibodies. Binding to the second antigen (HRP) did not affect the binding of bABs to alpha-END. bABs proved to be efficient for the determination of endorphins and their precursor proopiomelanocortin in immunohistology and immunoblotting.

[The interaction of bifunctional monoclonal antibodies with antigens studied by a radioimmunological method]. / Izuchenie vzaimodeistviia bifunktsional'nykh monoklonal'nykh antitel s antigenami radioimmunologicheskim metodom.

The quadroma produced bifunctional antibodies (bAbs) with double specificity to alpha-endorphin (alpha-END) and horseradish peroxidase (HRP) were compared with the parental anti-alpha-END monoclonal antibodies (mAbs) in respect to their binding to alpha-END. bAbs were purified from quadroma culture medium by sequential HRP-sepharose and alpha-END-sepharose affinity chromatography. Using radioimmunological method the affinity of the individual anti-alpha-END combining sites of bAbs was shown to be identical to that of parental mAbs. Binding to the second antigen (HRP) didn't affect binding of bAbs to alpha-END.

Construction of a quadroma to alpha-endorphin/horseradish peroxidase using an actinomycin D-resistant mouse myeloma cell line.

A hybrid hybridoma (quadroma), secreting antibodies with double specificity to alpha-endorphin (alpha-EP) and horseradish peroxidase (HRP), has been produced. The bispecific antibodies constituted about 28-29% of all immunologically active IgG, produced by quadroma. The quadroma was isolated by fusion of two mouse hybridomas (anti-HRP and anti-alpha-EP) with distinct phenotypes: double mutant AMDR/HAT(S), and wild type (AMDS/HATR). A novel strategy for the construction of a double-mutant was applied, based on the use of an actinomycin D-resistant (AMDR) mouse myeloma for initiation of one of the parental hybridomas.

[A new approach to the design of hybrid hybridomas based on the use of an actinomycin D-resistant line of murine myeloma]. / Novyi podkhod k konstruirovaniiu gibridnykh gibridom, osnovannyi na ispol'zovanii aktinomitsin-D-rezistentnoi linii mielomy myshi.

The hybrid hybridomas (tetradomas) were produced from the fusion of the double mutant actinomycin Dr (ADr)/HATs hybridoma to horseradish peroxidase (HRP) and wild type hybridoma to alpha-endorphin (EP). The double mutant phenotype was constructed using the new strategy, based on the fusion of immune mouse splenocytes with mouse myeloma (X63.Ag8, 653) cell variants, made resistant to 30 ng/ml of AD by stepwise selection. This allowed the direct introduction of the dominant selective marker (ADr) into the hybrid cells. Tetradomas secreted the bispecific monoclonal antibodies (bi Mabs), simultaneously binding to EP and HRP in double antigen ELISA, the ELISA plates covered with EP-bovine serum albumin conjugate. Using rat pituitary the bi Mabs were shown to be effective for immunostaining of EP-producing cells. EP-producing cells.

[The endorphins of the epithelial and subepithelial structures of the rat small intestine and the evolutionary hypotheses of the formation of the mechanisms of the negative regulation of their synthesis]. / Endorfiny épitelial'nykh i subépitelial'nykh struktur tonkogo kishechnika krys i nekotorye évoliutsionnye gipotezy formirovaniia mekhanizmov negativnoi reguliatsii ikh sinteza.

The effects of the agonist of the glucocorticoid hormones dexamethasone and dopamine antagonist--haloperidol on the concentration of immunoreactive alpha-, beta- and gamma-endorphins in duodenum, ileum, and jejunum of rats were studied. Besides the extracts of the intestines, the immunoreactive endorphins were measured in the extracts of their mucosa-submucosa and muscle-serous layers, that allowed to separate the endorphin-producing cells of the nervous system (muscle-serous layer) from endorphin producing cells of endocrine and immune systems (mucosa-submucosa layer). The injection of dexamethasone (0.2 mg per rat, daily for 6 days) caused the reliable decrease in concentrations of all three types of endorphins in mucosa-submucosa and muscle-serous layer of duodenum, ileum, and jejunum. Under the action of haloperidol (0.6 mg per rat, daily for 6 days) the reliable increase of beta-endorphin concentration was noticed only in jejunum. The suggestion is made that two distinct subpopulations of endorphin-producing cells exist in the intestine: in one cells endorphin synthesis is regulated by glucocorticoids, as in the anterior lobe of pituitary, in the other cells the synthesis of endorphins is regulated by dopamine, as in the cells of the intermediate lobe of pituitary. It is suggested that both glucocorticoid and dopamine types of regulation of endorphins synthesis were formed in the intestine or even in the gastric cavity. In process of evolution the cells with glucocorticoid type of regulation gave rise to the anterior lobe of pituitary, the cells with the dopamine type of regulation--to the intermediate lobe.

[Identification of pro-opiomelanocortin and its C-terminal fragments in the mammalian thymus]. / Identifikatsiia proopiomelanokortina i ego C-kontsevykh fragmentov v timuse mlekopitaiushchikh.

Immunoreactive alpha-, beta-, gamma-endorphins and beta-lipotropin were detected in perfused calf thymus extracts at the following concentrations (fmol/mg) tissue, M +/- m): 1.32 +/- 0.08, 1.53 +/- 0.45, 0.0186 +/- 0.0022 and 0.741 +/- 0.157, respectively. It was demonstrated for all ligands tested that the synthetic peptide and increasing amounts of the extract cause a similar displacement of the corresponding 125I-peptide from its complex with specific antiserum. Using the immunoblotting technique with a highly specific antiserum to bovine beta-lipotropin, the extracts of calf thymus, rat thymocytes and bovine hypophysis were found to contain two polypeptides with Mr of 32 and 14 kD, whose mobility corresponds to that of proopiomelanocortin and beta-lipotropin.

[The opioid component of the effect of the lacrimal glands on wound healing]. / Opioidnyi komponent vliianiia sleznykh zhelez na zazhivlenie ran.

Naloxone partially inhibited skin wound contraction and completely blocked acceleration of wound healing in rats with stimulated lacrimal glands. Endorphins were detected in lacrimal glands. Alteration of functional activity of the lacrimal glands produces a considerable effect on pain sensitivity and endorphin blood concentration. Opioid peptides have a considerable effect on the functioning of the lacrimal apparatus and are involved in injury-induced reactions, regulating pain sensitivity, structural homeostasis and producing an anti-stress effect.

[Radioimmunological assay of plasma levels of alpha-gamma-endorphins in healthy donors and patients with endogenous depression]. / Radioimmunologicheskoe opredelenie soderzhaniia alpha-gamma-éndorfinov v plazme krovi zdorovykh donorov i bol'nykh éndogennymi depressiiami.

Radioimmunological procedure enabled to estimate alpha- and gamma-endorphins in human blood plasma without pre-extraction. Basal level of these neuropeptides in peripheric blood plasma of 18 healthy donors constituted: C alpha = 392 +/- 244 pg/ml and C gamma = 20.6 +/- 9.1 pg/ml. Content of these endorphins in blood plasma did not depend on age and sex. Concentration of alpha- and gamma-endorphins were shown to correlate in peripheric blood (r = 0.88); this suggests the overall mechanisms for regulation of their synthesis, secretion and degradation. At the same time, distinct differences in concentrations of alpha- and gamma-endorphins in blood plasma were not found in groups of healthy donors and of patients with endogenous psychoses and pronounced depressive symptoms.