Generation and characterization of new HER2 monoclonal antibodies

Vasconcellos, Flávia Aleixo; Aleixo, Pedro Bandeira; Stone, Simone C.; Conceição, Fabrício Rochedo; Dellagostin, Odir A.; Aleixo, José Antônio Guimarães

doi:10.1016/j.acthis.2012.07.003

Cited by 10 publications

(8 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The purity of the antibody was determined by SDS-PAGE and western blot analysis. The affinity constants of antibodies were identified by SPR (11,12).…”

Section: Biological Activity Of Human S100b Protein In Promotingmentioning

confidence: 99%

“…Antibody titer determination. Antibody titers were measured by indirect ELISA (11,12). The microtiter plates were coated with ~100 µl of recombinant S100B (4 µg/ml) and then incubated at 4˚C overnight.…”

Section: Biological Activity Of Human S100b Protein In Promotingmentioning

confidence: 99%

“…The absorbance was determined at 450 and 630 nm wavelengths, respectively. The specification of the mAbs were examined using competitive ELISA (11,12) (Table II).…”

Section: Biological Activity Of Human S100b Protein In Promotingmentioning

confidence: 99%

See 2 more Smart Citations

Functional expression, characterization, and application of human S100B

Wu¹,

Zhou²,

Xiao³

et al. 2017

Oncology Reports

View full text Add to dashboard Cite

The EF-hand calcium-binding protein S100B presents a wide range of biological activities and functions. This binding protein is involved in various human diseases, including cancer, brain trauma and ischemia, neuro-degenerative disease (Alzheimer's disease), and psychiatric disorders. In this study, we prepared human S100B protein and its monoclonal antibodies. Human S100B protein was expressed in Escherichia coli, successfully purified by diethylaminoethyl cellulose anion-exchange chromatography, and then identified by western blot analysis. Monoclonal antibodies (mAbs) were produced by the standard hybridoma method and validated by enzyme-linked immunosorbent assay and western blot analysis. The prepared human S100B protein and its mAbs demonstrated potential biological activities. The KD of one mAb is approximately 4.72x10-8 mol/l, and its cross reactivity is low with human S100A4, mouse S100A4, and human S100A1. Recombinant Soluble S100B can promote the migration and invasion of HeLa cells. The expression of S100B protein in tumor tissues can be detected effectively by using the prepared monoclonal antibodies. Increasing concentration of the anti-human S100B mAbs showed a reduced expression of the S100B protein. Subsequently, the expression of p53 increased significantly (P<0.05) in A375 cells. A significant increase in apoptosis in A375 cells was observed with increasing S100B mAb concentration. Results showed that our prepared S100B mAbs were suitable for detecting S100B expression in human tissues, furnishing promising tools for further functional investigation and clinical applications.

show abstract

“…The purity of the antibody was determined by SDS-PAGE and western blot analysis. The affinity constants of antibodies were identified by SPR (11,12).…”

Section: Biological Activity Of Human S100b Protein In Promotingmentioning

confidence: 99%

Section: Biological Activity Of Human S100b Protein In Promotingmentioning

confidence: 99%

See 1 more Smart Citation

Functional expression, characterization, and application of human S100B

Wu¹,

Zhou²,

Xiao³

et al. 2017

Oncology Reports

View full text Add to dashboard Cite

show abstract

“…No fluorescence staining was observed in the negative control cells. The MAbs were tested by immunohistochemistry and were found to be effective on binding HER2 protein in formalinfixed, paraffin-embedded tissue sections from human (1) and canine breast cancer. (2) Specific Antigen Identified Human and canine HER2 protein.…”

Section: Specificitymentioning

confidence: 99%

Monoclonal Antibodies 410G and 33F Against Human and Canine HER2 Protein

2011

Hybridoma

View full text Add to dashboard Cite

A segment of the HER2 gene was amplified by PCR using a cDNA fragment (IMAGE human, Invitrogen, Carlsbad, CA) corresponding to the extracellular portion of the HER2 receptor (residues 76-309). The amplicon was cloned into the pQE30 expression vector. This plasmid was used to transform E. coli BL21(DE3) Star. The purification of recombinant protein was done using the Ä KTAprimeÔ automated liquid chromatography system (GE Healthcare, Piscataway, NJ). The rHER2 protein in the final preparation was analyzed by 15% SDS-PAGE and quantified by the Bradford method. Method of ImmunizationSix-week-old female BALB/c mice were injected intraperitoneally with 70 mg of rHER2 protein in Freund's complete adjuvant. Injections were repeated on days 14, 21, and 28 using Freund's incomplete adjuvant. Mice were boosted intravenously with approximately 10 mg of antigen in saline 3 days before fusion. Parental Cell Line Used for FusionSp2/0-Ag14 myeloma cells (ATCC# CRL-1581). Selection and Cloning ProcedureHybridomas were selected under HAT medium and positive wells were identified using indirect ELISA with rHER2 as the capture antigen. The cells from two wells presenting high antibody activity were cloned twice by the limiting dilution technique, retested, and cultivated for freezing in liquid nitrogen. Heavy and Light Chains of ImmunoglobulinBoth antibodies are IgG1. Light chains were not determined. SpecificityThe MAbs stained MCF-7 cells by immunofluorescence. Specific fluorescence signals dispersed on the cell outer membrane were observed. No fluorescence staining was observed in the negative control cells. The MAbs were tested by immunohistochemistry and were found to be effective on binding HER2 protein in formalinfixed, paraffin-embedded tissue sections from human (1) and canine breast cancer. (2)

show abstract

“…The protumorigenic properties of HER2, such as strong catalytic kinase activity, extracellular accessibility, high expression, and association with poor prognosis, suggest that it is a promising target for antibody-based therapy [4,5]. Several therapeutic methods have been developed to block HER2 activity, thereby suppressing tumor growth, including the use of mAbs such as trastuzumab [6].…”

Section: Introductionmentioning

confidence: 99%

Expression and In Vitro Function of Anti-Breast Cancer Llama-Based Single Domain Antibody VHH Expressed in Tobacco Plants

Park

Lee

Kim

et al. 2020

IJMS

View full text Add to dashboard Cite

Overexpression of human epidermal growth factor receptor type 2 (HER2) is considered as a prognostic factor of breast cancer, which is positively associated with recurrence when cancer metastasizes to the lymph nodes. Here, we expressed the single variable domain on a heavy chain (VHH) form of anti-HER2 camelid single domain antibody in tobacco plants and compared its in vitro anticancer activities with the anti-HER2 full size antibody. The gene expression cassette containing anti-HER2 camelid single domain antibody VHH fused to human IgG Fc region with KDEL endoplasmic reticulum (ER) (VHH-FcK) was transferred into the tobacco plant via the Agrobacterium-mediated transformation. The transformants were screened with polymerase chain reaction and Western blot analyses. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding of the purified anti-HER2 VHH-FcK to the HER2-positive breast cancer cell line, SK-BR-3. Migration assay results confirmed anticancer activity of the plant-derived anticancer camelid single chain antibody. Taken together, we confirmed the possibility of using anti-HER2 VHH-FcK as a therapeutic anticancer agent, which can be expressed and assembled and purified from a plant expression system as an alternative antibody production system.

show abstract

Generation and characterization of new HER2 monoclonal antibodies

Cited by 10 publications

References 23 publications

Functional expression, characterization, and application of human S100B

Functional expression, characterization, and application of human S100B

Monoclonal Antibodies 410G and 33F Against Human and Canine HER2 Protein

Expression and In Vitro Function of Anti-Breast Cancer Llama-Based Single Domain Antibody VHH Expressed in Tobacco Plants

Contact Info

Product

Resources

About