ABSTRACT
Improvement in the sensitivity and specificity of UMELISA techniques as a result of the introduction of monoclonal antibodies superseding the use of polyclonal antibodies was studied. For this purpose, we performed a comparison of the results in the functioning of the screening programs before and after the introduction of monoclonal antibodies. We analyzed some parameters such as sensitivity, specificity and detection limit in the UMELISA HBsAg PLUS and UMELISA TSH techniques. The sensitivity and specificity parameters were evaluated by means of comparison with the commercial assays. The detection limit was calculated as the fluorescence of the calibrator 0 + 2 standard deviations. Since the introduction of MAb obtained for us an increase in the sensitivity, specificity, and positive predictive value was evidenced. On the other hand, the use of the MAb guarantees better stability in the diagnostic kit production process.
Subject(s)
Allergy and Immunology/instrumentation , Antibodies, Monoclonal/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Blood Donors , Calibration , Hepatitis B Surface Antigens/analysis , Hepatitis B Surface Antigens/blood , Humans , Hypothyroidism/diagnosis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Spleen cells from BALB/c mice immunized with recombinant TmpA were fused with mouse myeloma cells (P3/X63-Ag8), and five hybridomas secreting monoclonal antibodies were obtained. These hybridomas specifically recognize TmpA and do not cross-react with other molecules such as recombinant HBsAg of HBV and synthetic HCV core peptides. The monoclonal antibodies were IgG1 subclass and ascitic fluid from these hybridomas was purified by affinity chromatography on Protein A-Sepharose CL-4B column to isolate the IgG1 active fraction. The affinity constant of these monoclonal antibodies ranged from 6.4 x 10(8) and 1.73 x 10(10) M(-1).
Subject(s)
Antibodies, Monoclonal/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Immunoassay/methods , Membrane Proteins/chemistry , Membrane Proteins/immunology , Recombinant Proteins/chemistry , Spleen/metabolism , Animals , Antibody Specificity , Chromatography, Affinity , Epitopes , Hybridomas/metabolism , Immunoglobulin G/chemistry , Kinetics , Mice , Mice, Inbred BALB C , Peptides/chemistry , Protein Binding , Recombinant Proteins/immunology , Spleen/cytology , Syphilis/diagnosis , Treponema pallidum/metabolismABSTRACT
Spleen cells from BALB/c mice immunized with free native human chorionic gonadotropin hormone beta-subunit (beta hCG) were fused with mouse myeloma cells (P3/X63-Ag8) and one hybridoma secreting monoclonal antibodies (MAbs), was obtained. This hybridoma specifically recognizes beta hCG and does not cross-react with other human glycoprotein hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (hCG). The MAb was of the IgG(1) subclass and ascitic fluid from this hybridoma was purified by affinity chromatography on Protein A-Sepharose CL-4B column to isolate the IgG(1) active fraction. The affinity constant of this MAb was 1.5 x 10(10)M(-1).
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Chorionic Gonadotropin/chemistry , Chorionic Gonadotropin/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Chromatography, Agarose , Dose-Response Relationship, Drug , Down Syndrome/diagnosis , Epitopes , Follicle Stimulating Hormone/chemistry , Humans , Hybridomas , Immunoglobulin G/metabolism , Kinetics , Luteinizing Hormone/chemistry , Mice , Mice, Inbred BALB C , Spleen/cytology , Spleen/metabolism , Thyrotropin/chemistryABSTRACT
A monoclonal antibody (MAb) directed against human immunoaffinity purified trypsinogen has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro-enzyme-linked immunosorbent assay (UMELISA). The MAb was purified by affinity chromatography on protein A-sepharose, and MAb had a high affinity for trypsinogen with the affinity constant equal 2.06 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 but did not react with the same protein after reduction. The antibody seem to be directed against conformational epitope. The MAb obtained was characterized immunologically and used to develop UMELISA for detection Trypsin. This monoclonal assay enabled the detection of 2.8 ng/mL.
