Production and purification of anti-rhombomys opimus immunoglobulins

Zoonotic cutaneous leishmaniasis [ZCL] is an increasing public health problem in some endemic regions. Horseradish peroxidase [HRP] conjugated rabbit anti-Rhombomys opimus [R. opimus] Ig is needed for immunoblotting and ELISA tests used to explore the immune response of the rodents against the sand fly saliva. In this study, the production of HRP conjugated rabbit anti-R. opimus Ig was conducted for the first time. Rhombomys opimus Ig was purified from serum by protein G affinity chromatography column and injected into rabbit to produce anti-R. opimus Ig antibody. The titration of antibody against R. opimus Ig in rabbit serum was checked using indirect ELISA. Rabbit anti-R. opimus Ig was purified by Sepharose-4B-R. opimus Ig column. Reactivity of this antibody was assessed by indirect ELISA and was conjugated to HRP by periodate method. Approximately 3.5 mg Ig was purified from 1 ml R. opimus serum using protein G affinity chromatography column. The molecular weight of purified R. opimus Ig was estimated about 150 kDa by SDS-PAGE. Nearly 2.3 mg rabbit anti-R. opimus Ig was purified from 1 ml immunized rabbit serum. The purified antibody was conjugated to HRP and the optimum titer of HRP conjugated rabbit anti-R. opimus Ig was determined as 1:8000 using direct ELISA. HRP conjugated rabbit anti-Gerbil IgG has been produced by a few companies, but to our knowledge HRP conjugated rabbit anti-R. opimus Ig is not commercially available. Production of HRP conjugated rabbit anti-R. opimus Ig is considerably helpful for immunological studies of R. opimus, the main reservoir host of ZCL in Iran as well as some other countries

Production and characterization of anti-Her 2 monoclonal antibodies

Breast cancer is the most common cancer among women in the world. Early diagnosis of this cancer is a key element for its treatment. One of the approaches for diagnosis of breast cancer is detection of its tumour-associated markers. Hence, Her2 has been the main focus of the researches in the field. For diagnosis of Her2 overexpression, monoclonal antibodies [mAb] reacting against Her2 were produced in this study. For this purpose, two peptides from extracellular domain of Her2 were selected and the mAbs reacting against them were produced by hybrodoma technology. Reactivity of these antibodies were then evaluated in different immunological assays including ELISA, Immunoflurescence [IF], western blot [WB] and immunoprecipitation [IP]. Total of 5 clones were produced from two separate fusions, and antibody isotyping revealed that all clones were IgM. These mAbs showed appropriate reactivities in the following assays: ELISA, immunofluresence by staining of breast cancer cell line [SKBR3], WB and IP by detecting the 185 KD band of Her2. In conclusion, it seems that the mAbs are useful diagnostic tools for detection of Her2 expression in patients with breast cancer

[Monoclonal antibody production against human spermatozoal surface antigens]

As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund's adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization

[Extraction and purification of ferritin from liver tissue]

Ferritin with molecular weight of 450 kDa is the most important iron storage protein and is made of 24 subunits consisting of light and heavy chains. Each ferritin molecule is able to store 4500 Fe[3+] molecules. The aim of this study was to determine the preparation of highly pure ferritin for usage in diagnostic and research systems. In this study, ferritin was extracted and purified by homogenizing liver tissue, heating at 75 degrees centigrade, ammonium sulfate fractionation and gel filtration chromatography on sephadex G-200 column. The purify of ferritin was improved by using recycling chromatography. Resulted protein was electrophoresed on polyacrylamide gel in the presence of sodium dodecylsulfate [SDS-PAGE]. Existence of ferritin was confirmed by ELISA test and potassium ferricyanide staining of gel. Silver nitrate staining of gel was used to confirm the purity of ferritin. Electrophoresis of ferritin under reducing conditions in presence of 2- mercapto ethanol was done to show the subunits [19 and 21 kDa] of ferritin. This purification method resulted in very pure ferritin and the yield was 100 microg/gr of wet liver tissue. Electrophoresis of ferritin under reducing conditions in presence of 2- mercapto ethanol showed the both subunits [19 and 21 kDa] of ferritin. Highly pure ferritin resulted by this method is appropriate for diagnostic and research purpose and the yield is reasonable comparing other studies

[Production and characterization of a murine monoclonal antibody recognizing a conformational epitope on hCG]

Human chorionic gonadotropin hormone [hCG] belongs to glycoprotein hormones family. Other members of this family include follicle stimulating hormone [FSH], luteinizing hormone [LH] and thyroid stimulating hormone [TSH]. All these hormones consist of a common alfa and a distinct beta subunit. There is a strong similarity between the members of these hormones. Therefore, detection and quantitative measurment of these hormones require production of monoclonal antibodies specific for non-overlapping epitopes on the beta chain or a conformational epitope specific for each hormone. In this study a murine monoclonal antibody against the hCG dimer molecule was produced by hybridoma technology. The specificity of the antibody was assessed by ELISA and Immunoblotting using a panel of highly purified and recombinant forms of glycoprotein hormones including: native hCG and hLH, recombinant hCG, beta hCG, hCG alpha, beta hCG carboxyl terminal peptide covering amino acid residues 109-145 [beta hCG-CTP], recombinant TSH and native FSH, as well as urine proteins [UP]. It was found that the monoclonal antibody reacted with, dimer recombinant and urine purified hCG and hLH, but not with the reduced form of the hormone, nor with recombinant beta hCG, alpha hCG, TSH, native FSH and UP. Using beta hCG-CTP fragment with different concentrations to monitor inhibition of hormone- monoclonal antibody interactions, no interference was observed. This implies that the epitope recognized by the monoclonal antibody is different from that presented by beta hCG-CTP. These results suggest that the monoclonal antibody recognizes a conformational epitope located at the dimer form of hCG molecule and closely associated with the beta subunit of the hormone