Production and Characterization of Anti‐recombinant Human Erythropoietin (rhEPO) Monoclonal Antibody

Jin, Yan; Wang, Shan; Mi, Jiebo; Guo, Zhiqiang; Chang, Wen‐Bao

doi:10.1081/ias-120027229

Cited by 8 publications

(6 citation statements)

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“…The SMX-MAb was fully characterized and the results showed that the SMX-MAb was of the subclass IgG 1 and the affinity constant of the SMX-MAb with SMX-HSA was 2.3 )/10 8 l/mol. The affinity constant of MAb normally ranges from 10 5 Á/10 9 L/mol (Yan et al 2004). The results proved that the immunogenicity of SMX-HSA was so high that it induced high immuno-response to produce high affinity antibody.…”

Section: Discussionmentioning

confidence: 79%

“…The antibody's specificity determines the specificity and sensitivity of the immunoassay. The K aff value represents the combined stability of Ag Á/ Ab, which is a crucial parameter that can reflect the quality of the Ab (Yan et al 2004). In order to produce highly specific antibodies to SMX and to select better hybridoma cells to generate monoclonal antibodies only recognizing SMX, SMX was bound to HSA to form an antigen for animal immunization.…”

Section: Discussionmentioning

confidence: 99%

“…The affinity constant (K aff ) of SMX-MAb K aff is an important constant, which can reflect the combinative ability of antigen and antibody. It is a critical factor that directly influences the detection limit of SMX (Kohler & Milstein 1975, Yan et al 2004. According to the revised the method of Beatty et al (1987), the affinity constant (K aff ) can be determined by ELISA using serial dilutions of both coated antigen and anti-SMX McAb (Figure 4).…”

Section: Determination For Subclass Of Smx-mabmentioning

confidence: 99%

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Preparation and characterization of anti-sulphamethoxazole (SMX) monoclonal antibody

Shen

Guo-juan²,

Li³

et al. 2005

Food and Agricultural Immunology

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Anti-sulphamethoxazole (SMX) monoclonal antibody (MAb) has a significant role in monitoring of SMX residue levels in edible animal products. In this study, SMX was diazotizated and coupled to human serum albumin to form an antigen for animal immunization. Anti-SMX hybridomas were prepared by cell fusion using an enzyme-linked immunosorbent assay (ELISA) method to screen for positive hybridomas. A monoclonal antibody (MAb) against SMX was secreted by the hybridomas and its specificity was characterized by ELISA, SDS-PAGE and Western-blot. The results showed that the SMX-MAb was of the IgG1 type and its molecular weight was 162 000 Daltons. The affinity constant of MAb for the conjugated antigen was 2.3)/10 8 l/mol. The detection limit of 50% inhibition rate in the range 10 pg/mL to 10 ng/mL was 0.82 ng/mL and the recoveries of SMX were above 90%. There was no crossreaction between MAb and SMX analogues.

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Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Determination For Subclass Of Smx-mabmentioning

confidence: 99%

See 1 more Smart Citation

Preparation and characterization of anti-sulphamethoxazole (SMX) monoclonal antibody

Shen

Guo-juan²,

Li³

et al. 2005

Food and Agricultural Immunology

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show abstract

“…Only indirect competition-enzyme linked immunosorbent assay can provide the real thermodynamic affinity of the antibody for its antigen. They can be applied to a varity of experimental situations, some of which are reviewed in Goldberg et al [13,27]. The method used here is adapted from friguet.…”

Section: Discussionmentioning

confidence: 99%

Determination of dissociation constants of Anti-ALP monoclonal antibodies by an ELISA – based method

Naseri

Moazzeni

Purfathollah

et al. 2015

HAB

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“…The fusion procedure and positive clone screening method were the same as reported previously. (9) Six cell clones that produced antibody against GP-1D8 were selected and expanded, followed by single-cell cloning for three times using a limiting dilution method. (9) One clone of the hybridoma-secreting antibody against GP-1D8 was characterized.…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of 66‐kDa glycoprotein from human breast carcinoma

et al. 2007

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We extracted a 66-kDa glycoprotein (GP-1D8) from breast invasive ductal carcinoma tissues. The monoclonal antibody (mAb) against GP-1D8 was prepared in our laboratory. Western blotting with the purified protein using the mAb demonstrated a single band of 66 kDa. Immunocytochemical and immunohistochemical analysis revealed strong expression of GP-1D8 protein in the cytoplasm of MCF-7 cells and different types of breast carcinoma tissues, but GP-1D8 is absent in normal breast and benign breast tumor tissues. Glycosylation analysis showed GP-1D8 contained methylated salic acid. GP-1D8 was identified using mass-spectrometric techniques and N-terminal sequencing. These data were used to identify the protein through the SWISSPROT protein sequence database and BLAST homology search. These results showed GP-1D8 had some similarity to human albumin precursor. Co-immunoprecipitation assays of lysate from MCF-7 cells and mass spectrometric analysis revealed the interaction of GP-1D8 with β β β β-tubulin. This is the first time human breast carcinoma tissues and MCF- T he identification of proteins with change levels as biomarkers might be useful for the early detection of cancer, monitoring disease progression, prediction of disease recurrence and evaluation of treatment efficacy. Breast cancer is now the most prevalent cancer in women worldwide.(1) Although some biomarkers, such as CEA, HER2, mammaglobin-A, MUC1 and CA15.3 have been studied as breast cancer-associated antigens, (2-7) the detection sensitivity and specificity of all these markers are low. To date, there has been no specific biomarker for breast cancer.In the present study, the authors extracted a 66-kDa glycoprotein (GP-1D8) from breast invasive ductal carcinoma tissues, purified the GP-1D8 glycoprotein with affinity chromatography, and then identified the GP-1D8 glycoprotein using western blot analysis, N-terminal sequencing and mass-spectrometric techniques. The authors also compared the GP-1D8 expression in normal breast tissues, benign breast tumors (breast fibroadenoma), different types of human breast carcinoma tissues and human breast cancer MCF-7 cells. Co-immunoprecipitation and mass spectrometric experiments were carried out to identify GP-1D8-interacting proteins. Materials and methodsProtein extracts. Fresh samples of eight breast invasive ductal carcinoma tissues were obtained from the School of Oncology, Peking University, Beijing, China. All patients gave written informed consent and the use of these samples was approved by the Hospital Ethical Committee. The pathology diagnosis of the formalin fixed paraffin-embedded tissue sections were confirmed using HE staining. The fresh lumpectomy specimens (3 g for each sample) were excised from the surrounding adipose tissue and washed with PBS (0.137 M NaCL, 2.7 mM KCL, 8.0 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , PBS) three times, the tissues were cut into 1-2-mm 3 size pieces, washed with PBS three times, frozen with liquid nitrogen, and then ground to a fine power using a mortar and pestle. The powder...

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Production and Characterization of Anti‐recombinant Human Erythropoietin (rhEPO) Monoclonal Antibody

Cited by 8 publications

References 8 publications

Preparation and characterization of anti-sulphamethoxazole (SMX) monoclonal antibody

Preparation and characterization of anti-sulphamethoxazole (SMX) monoclonal antibody

Determination of dissociation constants of Anti-ALP monoclonal antibodies by an ELISA – based method

Purification and characterization of 66‐kDa glycoprotein from human breast carcinoma

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