Production and characterization of monoclonal antibody against human serum albumin.
Hybridomas secreting monoclonal antibodies (MAbs) producing stable, specific and high affinity against human serum albumin (HSA) have been established. The aim of the present study was the production of MAbs that will be potentially used in designing immunoassay methods especially immunochromatography assay kit for screening of microalbuminuria (MAU) in the early detection of diabetic and nondiabetic nephropathy. The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma cell line (SP2). After limiting dilutions three clones producing antibodies were designed as EMRC1-3, which displayed different pattern of fine specificity for HSA and low cross reaction with other proteins as elucidated by inhibition enzyme-linked immunosorbent assay (ELISA). These clones were found to be of immunoglobulin G (IgG) class with k light chain. Subclass determination showed that all three MAbs secreted IgG1 type of antibody. The results of affinity purification for the two selected clones (EMRC1 and EMRC3) displayed high affinity with no cross reactivity with any of the related protein molecules. The stable hybridomas secreting anti-HSA were expanded in 50-mL flasks for large-scale production of the required antibodies. The standard curves were constructed with a sensitivity of 10 pg per well covering up to 100 ng per well. The high binding activity to HSA antigen and having no cross reactivity with other related molecules illustrated the potential application of these antibodies as an immunodiagnostic reagent in designing an immunochromatography assay kit for screening of MAU in diabetic and nondiabetic patients.
Omidfar
K
K
Endocrinology and Metabolism Research Center, Medical Sciences/University of Tehran, Tehran, I.R. Iran. omidfar@tums.ac.ir
Kashanian
S
S
Paknejad
M
M
Kashanian
S
S
Larijani
B
B
Roshanfekr
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D000918:Antibody Specificity; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D012709:Serum Albumin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D012709:Serum Albumin