Toxoplasma gondii is an obligate intracellular protozoan that is distributed worldwide. Recently, several tests for avidity of Toxoplasma IgG antibodies have been introduced to help discriminate between recently acquired and distant infections. The study was conducted in Jawaharlal Nehru Medical College and Hospital, India from February 2011 to September 2012. Serum specimens were subjected to Toxoplasma IgM ELISA and IgG avidity ELISA test. Out of 48 patients with abortions, 17 (35.4%) were positive for IgM ELISA, and 8 (16.6%) had low IgG avidity antibodies. Out of 48 patients with other obstetric problems, 23 (47.9%) were positive for IgM ELISA, and 17 (35.4%) had low IgG avidity antibodies. Combining both groups on avidity test, only 25 of 40 (62.5%) IgM-positive women had low-avidity IgG antibodies suggesting a recent T. gondii infection in these women. More importantly, 15 (37.5%) of the IgM-positive women had high-avidity antibodies suggesting that the infection was acquired before gestation The relation of IgM seropositivity with the following risk factors was not found to be statistically significant; contact with cats (0.13), non-vegetarian food habits (0.05), and low socio-economic status (0.49). While, for IgG avidity ELISA, only contact with cats (0.01) was significantly associated with seropositivity. All other risk factors have P-values of >0.05 (not significant). IgG avidity test when used in combination with IgM test was a valuable assay for diagnosis of ongoing or recently acquired T. gondii infection in India.
Abortion, Spontaneous/immunology ; Adolescent ; Adult ; Animals ; Antibodies, Protozoan/*immunology ; *Antibody Affinity ; Cats ; Enzyme-Linked Immunosorbent Assay ; Female ; Food Contamination ; Humans ; Immunoglobulin G/blood/immunology/*physiology ; Immunoglobulin M/blood ; India/epidemiology ; Risk Factors ; Seroepidemiologic Studies ; Toxoplasma/*immunology ; Toxoplasmosis/epidemiology/*immunology ; Young Adult

OBJECTIVETo identify the self-preparation monoclonal antibody which target to clenbuterol, and set up the standard curve to clenbuterol (CL) detection.

METHODSThe affinity constants and activity of the monoclonal antibody which target to CL were determined by ELISA. ELISA was also used to confirm whether the monoclonal antibody had any across-reaction with BSA and CL analogues. The rat ascites which contains the monoclonal antibody target to CL was purified by (NH4)2SO4 salt-out method and further by affinity column. At last, the CL detection standard curve which based on indirect competition ELISA was established.

RESULTSThe ELISA experiment showed that the antibody titer was 10(6) and the monoclonal antibody affinity constants was 2.90 x 10(10) L/mol. The result of the indirect competition ELISA confirmed that the monoclonal antibody had no cross-reaction with BSA and a few kind of CL analogue. CL detection standard curve based on indirect competition ELISA was established, which R2 was 0.9812, and the lowest detectable limit was 1.0 ng/ml.

CONCLUSIONThe standard curve based on indirectly competitioning ELISA was established. The self-preparation monoclonal antibody which target to CL has high affinity and high specific to CL, which had established the foundation to the advanced development of the CL immune test paper and CL ELISA kit.

Animals ; Antibodies, Monoclonal ; chemistry ; isolation & purification ; Antibody Affinity ; Clenbuterol ; immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Limit of Detection ; Rats

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
Amino Acid Sequence ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; Antibody Affinity ; Asthma ; blood ; Base Sequence ; DNA, Complementary ; metabolism ; Escherichia coli ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphocytes ; chemistry ; Peptide Library ; RNA, Messenger ; isolation & purification ; Recombination, Genetic ; genetics ; Ribosomes ; chemistry ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; isolation & purification ; Transformation, Genetic

Abstract: This study is to improve the affinity of scFv-AK404R against VEGFR2. The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain. VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography. The affinity constant of scFvs was measured by competitive ELISA, and the structure of scFvs was analyzed by bioinformatics. The result showed that a library with 6.4x10(5) scFv members was established by electro-transformation. Two mutated clones with high absorbance value were isolated after screening. After purification by affinity chromatography, electrophoretically pure scFv proteins were obtained. The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R, and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity. These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.
Amino Acid Sequence ; Antibody Affinity ; Chromatography, Affinity ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Hydrophobic and Hydrophilic Interactions ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology ; metabolism

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ~17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.
Animals ; Antibodies, Monoclonal, Humanized/*genetics/immunology/therapeutic use ; Antibody Affinity ; Cell Line, Tumor ; Directed Molecular Evolution/*methods ; Epitope Mapping ; Epitopes/genetics/immunology/therapeutic use ; Humans ; *Immunotherapy ; Mice ; Neoplasms/*therapy ; Phosphorylation/drug effects ; Protein Binding ; Receptor, Epidermal Growth Factor/*antagonists & inhibitors/immunology ; Selection, Genetic ; Single-Chain Antibodies/*genetics/immunology/therapeutic use

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Animals ; Antibody Affinity ; Cells, Cultured ; Cytidine Deaminase ; genetics ; metabolism ; HEK293 Cells ; Humans ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Mutation ; Single-Chain Antibodies ; chemistry ; genetics ; immunology ; Somatic Hypermutation, Immunoglobulin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics.

METHODSHybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff).

RESULTSFive hybridoma cell lines, named NT-1, NT-2, NT-3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG(1) isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7 × 10(9) L/mol. The titers and IC(50) values of purified ascite fluids were in the range of (0.64-2.56) × 10(5) and (0.55-1.0) ng/mL, respectively. Of all the cross-reacting steroids, (-NT was the most reactive with the mAbs at 62% with NT-1 mAb and 64% with NT-2 mAb. Negligible cross-reactivity (<0.01%) with other steroids was observed.

CONCLUSIONThe establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.

Animals ; Antibodies, Monoclonal ; Antibody Affinity ; Antibody Specificity ; Cell Line ; Female ; Hybridomas ; Mice ; Mice, Inbred BALB C ; Molecular Structure ; Nandrolone ; chemistry ; immunology ; Plasmacytoma ; Reagent Kits, Diagnostic ; Spleen ; cytology

Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Neoplasm ; biosynthesis ; immunology ; Antibody Affinity ; genetics ; Bacteriophages ; immunology ; Base Sequence ; Cell Line, Tumor ; Cells, Cultured ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Immunohistochemistry ; Liver Neoplasms ; immunology ; therapy ; Plasmids ; genetics ; Single-Chain Antibodies ; chemistry ; immunology

INTRODUCTIONMultiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM.

MATERIALS AND METHODSHere, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86.

RESULTSAnti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A(450)~1.1) showed a 1/3 increase in binding as compared to the fi rst round scFvs (A(450)~0.4) with 100 microg/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A(450)~0.1 to A450~0.15 on 12.5 microg/mL of antigen as compared to low binders (A(450)~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374.

CONCLUSIONSThese studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents.

Antibodies, Monoclonal ; isolation & purification ; Antibody Affinity ; Cell Line ; DNA Helicases ; immunology ; Humans ; Immunoglobulin Idiotypes ; immunology ; isolation & purification ; Immunoglobulin Variable Region ; isolation & purification ; Ku Autoantigen ; Multiple Myeloma ; immunology ; Peptide Library ; Recombinant Proteins