Detection of total free light chains (FLC) of immunoglobulins and their ratio (kappa/lambda quotient) are used in diagnostics and monitoring of multiple myeloma and other gammapathies, primary amyloidosis and multiple sclerosis. Previously described immunoassays with monoclonal antibodies (Mabs) against cryptic and constantly exposed epitopes of FLC failed to recognize rare variants of lambda Bence-Jones proteins and a significant proportion of lambda chains excreted with urine. Aiming to improve this approach, a novel murine Mab (IgG2b coded as 1C8) was employed, which specifically binds free lambda chains but doesn’t interact with native IgA, IgG, and IgM. The novel Mab recognized an epitope exposed at free lambda chains in peripheral blood of healthy donors and patients with multiple myeloma. It is not destroyed or masked upon renal filtration.The aim of this study was to determine basic features of improved assay system, and to estimate its potential in diagnostics of monoclonal gammapathies. The mixtures of three Bence-Jones proteins of either kappa- or lambda- types purified from the urine of multiple myeloma patients were used as calibrator samples.Improved immunometric assay is able to detect free kappa and lambda chains in serum and urine at a scale of 1 to 100 ng/ml, thus being three orders more sensitive than, e.g., detection levels of Freelite method based on polyclonal antibodies.A novel assay allows to detect free kappa and lambda chains at comparable levels in serum or urine, and to deduce kappa/lambda ratio. The proposed assay is able to detect FLC in 10,000-fold excess of whole IgG molecules. The calibrating plots for both antigens are linear on log-log scales, with very similar slopes. Detection thresholds for kappa or lambda chains proved to be 5 and 3 ng/ml, respectively. Mean concentrations of free kappa chains in sera of healthy donors were 6.7±2.1, in urine, 4.2±3.8 mcg/ml. Mean concentrations of free lambda chains were 4.7±1.96, and 1.6±1.0 mcg/ml, respectively. This method, if applied to serum and urine samples from multiple myeloma patients, revealed free light chains were similar to the paraproteins detected by means of electrophoresis/immunofixation. The values of kappa/lambda ratios corresponded to the types of gammapathies revealed.

Endoglin (CD105) is the marker of endothelial and mesenchymal stem cells and the component of TGF-β, BMP-9 and BMP-10-binding receptor complexes. Its expression is significantly increased on blood vessels endothelium of ischemic tissues and growing tumors. Measurement of concentration of the soluble endoglin in the serum or urine is used as a method for diagnosing cancer and pregnancy disorders. The aim of this work was to create a novel family of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. Murine myeloma cells' derived recombinant protein representing the whole extracellular part of endoglin was used as an antigen. F1(SJL/JxBALB/c) mice were the donors of immune splenocytes. Hybridoma screening procedures were performed using E. coli-produced copies of the antigen, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight distinct epitopes were produced. Eight antibodies bind membrane form of endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by western blot. Sandwich ELISA system was designed in order to measure soluble endoglin in cell culture medium.

AIM:To develop and characterize by immunochemical methods the panel of monoclonal antibodies (MAbs) recognizing different antigenic determinants ofhuman secretory component (SC) molecule.MATERIALS AND METHODS:sIgA and SC were obtained from colostrum by combination of ion-exchange chromatography and gel filtration. Recombinant SC was expressed in Escherichia coli cells transformed by construction that contained fragment of gene coding extracellular domain of receptor for polymeric Ig. MAbs were produced and studied using hybridoma technologies and different methods of immunoenzyme analysis respectively.RESULTS:Panel comprising 10 MAbs against human SC of which 4 types of antibodies recognize cryptic epitopes of free SC and other 6 types recognize epitopes exposed both on SC and sIgA. MAbs panel contains antibodies interacting with conformational and linear epitopes of antigen. Three from obtained MAbs bind to SC epitopes which structure is determined by presence of carbohydrate residues in the molecule of antigen. Immunometric systems were developed which allow to differentially detect free SC and sIgA.CONCLUSION:Developed and characterized MAbs panel recognizing different epitopes of SC molecule opens new opportunities for laboratory and basic research of human secretory immunity.

AIM:To obtain and study of immunochemical characteristics of monoclonal antibodies (MAbs) to CagA cytotoxin of Helicobacter pylori employing recombinant fragments of CagA protein.MATERIALS AND METHODS:Standard methods of construction and selection of hybridomas, different variants of immunoenzyme analysis and immunoblotting were used. Molecular genotyping of H. pylori cultures by amplification of cagA gene fragments was performed.RESULTS:Panel of MAbs recognizing 4 different linear epitopes on the CagA molecule, three of which are localized in conservative parts of cytotoxin and one--in variable region of CagA, was developed. On the basis of two obtained antibodies, system of two-center immunoenzyme assay for quantitative detection of CagA protein which is characterized by high sensitivity and specificity, was developed. Obtained MAbs allow to differentiate CagA-positive and CagA-negative strains of H. pylori by immunochemical methods.CONCLUSION:Employing pure recombinant fragments of CagA protein, first panel of MAbs to CagA cytotoxin of H. pylori was developed and characterized. Obtained MAbs open perspectives for study of the H. pylori cytotoxin molecule and construction of immunodiagnostic assays aimed on detection of CagA antigen.

The serum and liquor of 70 patients with multiple sclerosis (MS), 54 patients with other neurological diseases and 20 controls were examined for free light chains (FLC) of immunoglobulins type chi and lambda. The findings were compared to blood-brain barrier competence, liquor pleocytosis, IgG intrathecal synthesis 86% MS patients exhibited high levels of liquor chi-type FLC which appeared early in the disease course irrespectively of MS phase and pattern. Simultaneous high content of lambda-type FLC was reported in 52% of the cases. Patients in need of differential diagnosis were characterized by normal or reduced (in spinocerebellar ataxia) concentrations of FLC except for the conditions with impaired blood-brain barrier.