[ANALYSIS OF EFFECTIVENESS OF USINGJIgY FROM CHICKEN IN SANDWICH METHOD OF HBsAg TESTING].

AIM: Study antigen-binding ability of polyclonal antibodies (PCA) of chicken compared with monoclonal -antibodies (MCA) of mice in the model of interaction with HBsAg. MATERIALS AND METHODS: Mice MCA 18C8 and MKA F3/F4 (IgG) were used, effective in enzyme immunoassay sandwich method of HBsAg determination (with a minimal detection dose of 0.017 ng/ml), and affinity purified anti-HBsAg PCA of chicken (IgY), obtained from 2 immunized birds (PCA No. 1 and PCA No. 2). The ability of antibodies to bind HBsAg was evaluated by analytical sensitivity (slope of binding curve) of solid-phase enzyme immunoassay system using mice MCA and chicken PCA. RESULTS: PCA No. 2 has provided a statistically significant 40% increase of analytical sensitivity, compared with <

[Effect of pH of Adsorption Buffers on the Number and Antigen-Binding Activity of Monoclonal Antibodies Immobilized on the Surface of Polystyrene Microplates].

The change in the concentration and antigen-binding activity of 28 monoclonal antibodies was studied after their adsorption on the surface of polystyrene microplates in buffers with different pH values (1.0, 2.8, 7.5, 9.6, and 11.9). We used 16 clones to the HIV p24 protein and 12 clones to the surface antigen of Hepatitis B Virus. The binding efficiency of adsorbed antibodies to the labeled antigen was evaluated by the slope of the linear region of the binding curve to the concentration axis. It was shown that the antigen-binding activity of six antibodies (21.5%) statistically significantly increased after adsorption at pH 2.8 and 11.9 as compared to pH 7.5 and 9.5. The maximum amount of antibodies was found to be adsorbed on the solid surface at pH 7.5. The analysis of the binding of 125I-HBs-antigen to adsorbed antibodies made it possible to evaluate the concentration of active antibodies on the polystyrene surface. It was shown that the increase in the antigen-binding activity was due to an increase in the proportion of antibodies with retained activity after adsorption at pH 2.8 and 11.9. Under these conditions, about 20% of the antibodies retained their antigen-binding activity, and 6% did so after immobilization at pH 7.5.

[Effect of conditions of monoclonal antibody adsorption on antigen-binding activity].

The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013-0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.

[Use of pH-sensitive immobilized monoclonal antibodies for optimization of immunoenzyme sandwich technique of detection of HBsAg of hepatitis B virus].

AIM: To develop highly sensitive sandwich technique for identification of surface hepatitis B virus antigen (HBsAg) in serum and analyse of possible improvement of solid phase for immunoenzyme sandwich technique of HBsAg identification through variation of pH-dependent sorption of monoclonal antibodies on the surface of immune plates. MATERIALS AND METHODS: Calibration curves for identification of HBsAg in sandwich techniques using 36 possible binary combinations of monoclonal antibodies of our panel (including high affinity antibodies to HBsAg produced by 6 hybridomas) were compared. Immobilization of antibodies on solid phase (by passive sorption) was performed at different pH values (2.8, 7.5, and 9.5). RESULTS: Analysis of panel of antibodies to HBsAg produced by 6 hybridomas revealed pH-dependent monoclonal antibodies (18C8), which immobilization at low pH values together with detecting antibodies F4F3 allowed to greatly improve sensitivity of the sandwich technique. Minimal credibly detectable concentration of HBsAg in sera of persons infected with hepatitis B virus was 0.013 - 0.017 ng/ml. Validation of sandwich technique was performed on certified panel of serum samples with various concentrations of HBsAg (different serotypes). CONCLUSION: Highly sensitive sandwich technique for detection of HBsAg was developed. It was shown that analysis of panel of monoclonal antibodies on pH-dependence could be used as simple methodical approach for optimization of immunoenzyme sandwich techniques for detection of different antigens.

Physical and biochemical characteristics of biological fluids in rats with modeled thermal injury.

Lactate dehydrogenase activity, crystallogenic and initiator characteristics of biological substrates were evaluated by enzymological and crystalloscopic analysis of rat serum and urine. Changes in these characteristics of biological media in combined thermal injury were shown. This approach is effective for evaluation of the metabolic status in rats with experimental burn disease.

[Regulatory peptides and psychomotor development in infants].

Regulatory peptides (RP) are an important homeostatic factor. The maternal organism and placenta are substantial sources of RP for fetus during the prenatal period. Not only endogenous, but also exogenous RP play an important role during early postnatal period. In this study, the concentration of exogenous RP (casomorphins-7) and the activity of peptidases (enkephalinases) in the serum of breastfed and bottle-fed infants were estimated. Possible interrelation between these two parameters and the psychomotor development (PMD) of infants were evaluated. Using specially developed RIA, the investigators estimated the presence of human and bovine casomorphins immunoreactivity (CMir) in the serum of breastfed and bottle-fed infants. A distinct correlation of CMir with PMD was demonstrated. The activity of RP-degrading serum enzymes also correlated with PMD level. The role of endo- and exogenous peptides in normal PMD process and in the pathogenesis of early child autism is discussed in the article.

[Platelet serotonergic parameters and clinical symptoms of psychosis in patients with episodic progressive schizophrenia].

A sample included 59 patients, 38 males and 21 females, mean age (M+/-SD) 33,4+/-10,2 years, age-at-onset 26+/-9,5 years, illness duration 7,8+/-6,1 years, with episodic progressive schizophrenia (ICD-10: schizophrenia, paranoid type, F20.0) with continuous course at the stage of exacerbation. Clinical symptoms were assessed using the PANSS. Platelet 5-HT content, 3H-serotonin uptake (Vmax), 3H-imipramine receptor density (Bmax), high- and low molecular weight human serotonin platelet transporter protein immunoreactivity (HMW-SERT and LMW-SERT values) were measured. The most frequent psychotic symptoms were delusions, conceptual disorganization and hallucinations, with the majority of patients experiencing from one to three symptoms. A significant increase of platelet 5-HT content and 3H-imipramine receptor density (Bmax) was found in male patients. In the male group, delusions, conceptual disorganization and hallucinations as well as PANSS psychotic cluster scores were correlated positively with 5-HT content and negatively with HMW-SERT and LMW-SERT values. Possible reasons of the differences in correlations of platelet 5-HT serotonin and serotonin transporter values with psychotic symptoms are discussed. The results are additional evidence for the involvement of serotonergic dysfunction in the pathogenesis of schizophrenic psychoses. They confirm the usefulness of testing of platelet 5-HT content and SERT immunoreactivity as biological markers of schizophrenic psychoses, in particular for male patents.

Analysis of serotonin transporter in human platelets by immunoblotting using site-specific antibodies.

We have produced a panel of site-specific antibodies recognizing different regions of the human serotonin transporter (SERT). This panel included: 1) monoclonal antibodies 23C5 (mAbs 23C5) to the C-terminal region (amino acid residues 597-630); 2) polyclonal antibodies (pAbs) to the N-terminal region (amino acid residues 69-83); 3) pAbs to the region (amino acid residues 86-100) in the beginning of the first transmembrane domain (TMD). The antibodies were produced using recombinant proteins and synthetic peptides (containing certain sequences of SERT) as antigens. These antibodies were purified by affinity chromatography, conjugated to horseradish peroxidase (HRP), and used for immunoblotting analysis of SERT in extracts of human platelets. Sodium dodecyl sulfate extracts were prepared under conditions preventing non-specific proteolytic degradation of the proteins. In platelet extracts, all antibodies were able to detect the 67 kD protein, apparently corresponding to full-length SERT molecule (its theoretical mass is about 70 kD). These antibodies also detected several polypeptides of smaller size (56, 37, 35, 32, 22, and 14 kD), apparently corresponding to N-terminal, C-terminal, and non-terminal SERT fragments. Specificity of immunostaining was confirmed by preincubation of HRP-labeled anti-SERT antibodies with excess of corresponding antigen, which resulted in disappearance of protein band staining. It is suggested that SERT undergoes a programmed proteolytic cleavage (processing) resulting in formation of several SERT-derived polypeptides of smaller size. It is possible that one of the cleaved SERT species is required for serotonin transport activity. Possible sites for specific proteolysis may be located in the region near TMD1 and in the intracellular loop between TMD4 and TMD5.

Analysis of bispecific monoclonal antibody binding to immobilized antigens using an optical biosensor.

The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K(ass)) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K(ass) of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k(diss)) for anti-HRP shoulder of Babs was 21 times higher than k(diss) for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody-enzyme conjugates in the case of binding of bivalent Mabs.

[The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens]. / Sviazyvanie bispetsificheskikh monoklonal'nykh antitel s antigenami, adsorbirovannymi na tverdoi faze.

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.

[Levels and molecular heterogeneity of serotonin transporter protein in platelets of patients with different mental diseases: a comparative analysis with the use of monoclonal and polyclonal antibodies]. / Soderzhanie i molekuliarnaia geterogennost' belka--perenoschika serotonina v trombotsitakh bol'nykh s razlichnymi psikhicheskimi zabolevaniiami: sravnitel'nyi analiz s ispol'zovaniem monoklonal'nykh i poliklonal'nykh antitel.

Polyclonal (PAb) and monoclonal (MAb) antibodies to CT2-epitope of the C-terminal fragment of serotonin transporter (SERT) protein were used to study the levels and molecular heterogeneity of platelet SERT in healthy donors and patients with affective (AD) and somatoform (SD) disorders, schizoaffective disorder (SAD) and schizophrenia. SERT was found to exist as high molecular wight (HMW) and low molecular weight (LMW) forms separated after electrophoresis. The levels of HMW and LMW forms of SERT were significantly, decreased in mentally ill patients as compared to healthy individuals. Unlike PAb, horse radish peroxidase (HRP)-conjugated MAbs were more sensitive and specific to SERT and could detect the LMW form of SERT as a duplet protein form with MW about 40 and 43 kDa. The MAb to CT2 C-terminal fragment of SERT conjugated with HRP is considered to be a new valuable tool for further investigation of SERT expression, properties, and posttranslation modification in the controls and in patients with different psychopathology.

The comparison of the ability of monoclonal antibodies directed to different proteins (human IgG, human myoglobin and HRP) and bispecific antibodies derived thereof to bind antigens immobilized on a surface of a solid phase.

BACKGROUND: Bindings of mouse monoclonal antibodies (mAbs) and affinity purified bispecific antibodies (bAbs), derived thereof, to antigens adsorbed on immunoplates have been compared, using ELISA and RIA methods. METHODS: The analysed panel of antibodies included mAbs specific to human myoglobin (Mb), human IgG (hIgG) and horseradish peroxidase (HRP) and biologically produced bAbs with double specificity to Mb and HRP, and to hIgG and HRP. RESULTS: The degree of difference between different mAbs and corresponding bAbs varied markedly from antibody to antibody, depending on whether the parental mAbs could bind immobilized antigens bivalently. The observed equilibrium binding constant (K(obs)) for anti-HRP mAbs was 21-38 times higher that of anti-HRP site of bAbs (anti-hIgG/HRP or anti-Mb/HRP, respectively), due to bivalent binding of mAbs. Anti-Mb mAbs also bound bivalently with immobilized Mb. On the contrary, anti-hIgG mAbs bound monovalently with immobilized hIgG in the same conditions. The avidity of anti-Mb/HRP bAbs increased, if both antigens were simultaneously adsorbed on a solid phase. CONCLUSIONS: The obtained data indicate that the use of bAbs in heterogeneous immunoassays instead of traditional mAb-enzyme conjugates hardly can provide the significant gain in assay performance if parental mAbs bind bivalently.

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.

[Brain isoforms of creatine kinase in health and mental diseases: Alzheimer's disease and schizophrenia]. / Mozgovaia izoforma kreatinfosfokinazy v norme i pri psikhicheskikh zabolevaniiakh (bolezn' Al'tsgemera, shizofreniia).

The paper analyzes the authors' own findings and the data available in the literature on the intensity, site, and possible causes of impairment of the creatine-creatine phosphate system of brain energy metabolism in mental diseases, such as Alzheimer's disease (AD) and schizophrenia. Examining the level of cytosolic BB creatine kinase in postmortem AD and schizophrenic's brain structures showed a significant decrease in BB creatine kinase as compared with the similar control brain structures. There was the maximum decline in AD cases. It was considerable as compared with both the control and schizophrenic groups (p < 0.01). The decrement was revealed by various techniques, including the determination of activity, immunological responsiveness and the analysis of two-dimensional protein maps. Immunocytochemical investigation indicated a decrease in responses to BB creatine kinase, mainly in astrocytes. The reduction in cytosolic BB creatine kinase levels is not a result of age, postmortem delay, or psychotic therapy. The causes of lower BB creatine kinase levels in the cell cytosol of the postmortem brain in mental pathology are discussed. The decrement in cytosolic BB creatine kinase in AD and schizophrenia occurs not only in the brain, but also in the peripheral tissues which contain BB creatine kinase. In all cases, it is greater in AD than in schizophrenia. Using immunosorbents with monoclonal antibodies to M-creatine kinase and to B-creatine kinase subunits makes it possible detect BB-creatine kinase in the extracts of human peripheral lymphocytes and platelets. A study of whether there is a relationship between the clinical data of mental patients and the level of BB creatine kinase in their blood elements is assumed to be useful in evaluating BB creatine kinase as a prognostic/diagnostic marker of mental diseases.

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.