Production and purification of polyclonal antibody against bovine immunoglobulins in rabbits

Antibodies are important tools in medical researches which have led to many advances in this field. Anti-bovine immunoglobulins and its conjugate with horse radish peroxidase (HRP) is used to diagnosecows’ disease by ELISA or western blotting tests. In this study, the production, purification and horse radish peroxidase (HRP) conjugation of polyclonal IgG against bovine immunoglobulins in rabbits werecarried out. Three 6-month-old New Zealand White rabbits were immunized by bovine immunoglobulins in combination with Freund’s adjuvant. Purified antibody (using ion-exchange chromatography) waslabeled to HRP. Direct enzyme linked immunosorbent assay (ELISA) was used to determine the optimum titer and cross reactivity of HRP conjugated IgG. The purity of various IgG preparations wasabout 98%. The optimum dilution of prepared HRP conjugated IgG was 1:12800. This conjugated IgG has no cross reactivity with sheep and goat immunoglobulins at optimized dilution. This study showedthat ion-exchange chromatography could be an appropriate method for purification of IgG antibodies

Production and purification of polyclonal anti-hamster immunoglobulins in rabbits

Polyclonal antibodies are mixtures of monoclonal antibodies that were produced against different epitops. The goal of this project is to know the production, purification and horseradish peroxidase (HRP) conjugation of polyclonal antibodies against hamster immunoglobulins in rabbits. 300 ìg/300 ìl of ten hamster immunoglobulins was mixed with the same volume (300 ìl) of adjuvant and injected into three 6-month-old white New Zealand rabbits. Anti hamster rich rabbits serums were isolated from whole blood and precipitated with ammonium sulfate in the final concentration of 50%. The precipitate was dialysed against phosphate buffered saline (PBS) (pH: 7.4) and applied to ion exchange chromatography (IEC) on diethylaminoethyl (DEAE)-sepharose 6B with tris-phosphate (pH: 8.1), andtris-phosphate contain 50 mM NaCl buffer. The purity of produced antibody was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. Then purifiedimmunoglobulin G (IgG) was conjugated with HRP. For exact measurement of conjugated IgG titer and evaluating of cross reaction, enzyme linked immunosorbent assay (ELISA) test was designed. Since IEC is a more simple and inexpensive method for the purification of IgG, we obtained a protein with approximate purity of 95%. Produced IgG showed high titer and high specificity in the designed ELISA. Purified antibody and its conjugation with HRP are used in research and diagnosis of hamster disease.Key words: Production, purification, hamster immunoglobulins

Production and purification of polyclonal antibody against attenuated and wild type Leishmania infantum in dogs

Antibodies are still widely used in several programs including early research, imaging, Targeting drug delivery system, Affinity chromatography, flowcytometry technic, diagnosis and treatment. Purification of antibody is a standard approach for detection of infection agent in different species. The reservoir hosts for Leishmania infantum are Dogs and they have active role in the transmission of leishmania to humans by the bite of a sand fly belonging to genus Phlebotomus and Lutzomiya. Consequently, elimination of dogs in endemic areas and vaccination of dogs contributes to reduction of the human and canine VL cases. Serological antibody tests such as IFAT (Indirect Fluorescent Antbody Test), DFAT (Direct Fluorescent Antbody Test), ELISA (Enzyme-Linked Immunosorbent Assay), PCR (Polymerase chain Reaction Assay) have been extensively used to investigate canine infection with L. infantum. In this study we produced and purified polyclonal antibody against attenuated and wild type leishmania infantum in dogs. Anti-leishmania in dog serums precipitated with ammonium sulphate. The IgG recovered from ammonium sulphate precipitation was subject to ion exchange chromatography (IEC) and the purity of IgG was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. The purity of proteins were above 95 and then purified IgG was conjugated with FITC. We determined optimum titer of dog IgG by observation parasites under fluorescent microscope. The optimum dilution of prepared FITC conjugated dog IgG was 1: 400. This polyclonal antibody can be used for other applications in research, diagnosis and clinic. © 2020 - IOS Press and the authors. All rights reserved

Role of Apelin/APJ axis in cancer development and progression

Apelin is an endogenous peptide, which is expressed in a vast board of organs such as the brain, placenta, heart, lungs, kidneys, pancreas, testis, prostate and adipose tissues. The apelin receptor, called angiotensin-like-receptor 1 (APJ), is also expressed in the brain, spleen, placenta, heart, liver, intestine, prostate, thymus, testis, ovary, lungs, kidneys, stomach, and adipose tissue. The apelin/APJ axis is involved in a number of physiological and pathological processes. The apelin expression is increased in various kinds of cancer and the apelin/APJ axis plays a key role in the development of tumors through enhancing angiogenesis, metastasis, cell proliferation and also through the development of cancer stem cells and drug resistance. The apelin also stops the apoptosis of cancer cells. The apelin/APJ axis was considered in this review as an attractive therapeutic target for cancer treatment. © 2020 Medical University of Bialysto

Targeting Cytokines: Production and Characterization of Anti-TNF-α scFvs by Phage Display Technology.

The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications

Potential anticancer effects of cell wall protein fractions from Lactobacillus paracasei on human intestinal Caco-2 cell line

Abstract: Consumption of probiotics has an important role in colorectal cancer prevention. In this study, we aimed to explore that the cell wall protein fractions from Lactobacillus paracasei could induce apoptosis on Caco-2 cell line. The cell wall proteins from L. paracasei were fractionated by gel filtration chromatography (F1, F2 and F3) and characterized by polyacrylamide gel electrophoresis (SDS-PAGE). The anticancer properties were evaluated using MTT assay and Annexin V-FITC/PI staining. Administration of L. paracasei increased a significant concentration- and time-dependent anti-proliferative effect on Caco-2 cell line, determined by cell viability assays. However, a dramatic decrease in cell viability of Caco-2 cells was observed at the concentration of 100 µg ml−1 of F1 L. paracasei for 72 h (58% cell viability, P < 0·05) The results showed that F1 L. paracasei could induce apoptosis in Caco-2 cancer cell line by increased in annexin V and propidium iodide staining for 72 h (up to 90·6%, P < 0·001). These results indicated the importance of the anticancer effects of cell wall protein fractions of L. paracasei in human colon carcinoma Caco-2 cell line. Thus, cell wall protein fractions of L. paracasei can be a potential chemotherapeutic agent against Caco-2 cell lines. Significance and Impact of the Study: Significance and Impact of the Study: Our findings revealed that the newly identified cell wall protein fractions from probiotic Lactobacillus paracasei inhibit the cell growth of human colon carcinoma cell line (Caco-2), and the results indicated that the cell wall proteins from L. paracasei can be a potential chemotherapeutic agent against Caco-2 cell line