We have evaluated the performance of a recently developed immunoassay analyzer, ADVIA Centaur XPT (Siemens, Germany). Precision, linearity, and comparison studies were performed according to the CLSI guidelines. The test items evaluated were ferritin, folate, human epidermal growth factor receptor 2/neu, homocysteine, vitamin B₁₂, B-type natriuretic peptide, creatine kinase–myocardial band, myoglobin, procalcitonin, troponin I. Bio-Rad control materials, linearity materials, and patients' samples were used for the evaluation. For the correlation study, ADVIA Centaur XP (Siemens) were used as comparative methods. The total coefficients of variations (CVs) of the analytes were between 2.5% and 7.0%. The results of linearity evaluation were also acceptable for the range tested. Correlations with comparative methods were good. The overall analytical performance of ADVIA Centaur XPT is acceptable for the immunology analyzer. Therefore, ADVIA Centaur XPT is expected to be widely used.
Allergy and Immunology ; Creatine ; Ferritins ; Folic Acid ; Homocysteine ; Humans ; Immunoassay ; Myoglobin ; Natriuretic Peptide, Brain ; Receptor, Epidermal Growth Factor ; Statistics as Topic ; Troponin I ; Vitamins

OBJECTIVETo investigate the correlation between BRAF V600E mutation and anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs) therapeutic effects in metastatic colorectal cancer.

METHODSStudies were included into meta-analysis to investigate the association between BRAF V600E mutation and clinical outcome in metastatic colorectal cancer patients treated with anti-EGFR MoAbs.

RESULTSA total of 7 studies were included in this meta-analysis. The 7 studies included 1352 patients in total, sample sizes ranged from 67 to 493. Objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were collected from included studies and were used to assess the strength of the relation. In patients with wild-type KRAS, the pooled odds ratio for ORR of mutant BRAF over wild-type BRAF was 0.27 (95% CI=0.10-0.70). BRAF mutation predicted a deterioration in PFS and OS in wild-type KRAS patients treated with anti-EGFR MoAbs (hazard ratio=2.78, 95% CI=1.62-4.76; hazard ratio=2.54, 95% CI=1.93-3.32).

CONCLUSIONBRAF V600E mutation is related to lack of response and worse survival in wild-type KRAS metastatic colorectal cancer patients treated with anti-EGFR MoAbs.

Antibodies, Monoclonal ; immunology ; Colorectal Neoplasms ; immunology ; pathology ; Humans ; Mutation ; Neoplasm Metastasis ; immunology ; Proto-Oncogene Proteins B-raf ; genetics ; Receptor, Epidermal Growth Factor ; immunology

OBJECTIVETo construct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein, ScFv/tP, carrying small interfering (si)RNA directed against the heat shock protein Hsp47, a collagen-binding glycoprotein, in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.

METHODSA single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences. Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein, internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining. The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells, the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Western blotting; in addition, effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8, flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).

RESULTSIndirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells, as well as increased the cells' apoptosis remarkably.

CONCLUSIONThe ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosis via blockade of Hsp47 expression.

Apoptosis ; Cell Proliferation ; HSP47 Heat-Shock Proteins ; genetics ; Hepatic Stellate Cells ; cytology ; Humans ; Protamines ; metabolism ; RNA, Messenger ; RNA, Small Interfering ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; Transfection

OBJECTIVETo evaluate the correlation of clinical effect and prognosis between patients with metastatic colorectal cancer (mCRC) and different K-ras status.

METHODSThe clinical characteristics, chemotherapeutic regimens and survival of 153 mCRC patients with different K-ras status were analyzed retrospectively.

RESULTSThe median overall survival (OS) in patients without K-ras mutation were 31.7 months, significantly longer than 21.3 months in the patients with K-ras mutation (P = 0.037). The median progression-free survival (PFS) and OS in patients who received chemotherapy followed by anti-EGFR antibody treatment were 11.5 and 39.3 months, respectively, significantly longer as compared with the PFS and OS in those received chemotherapy in combination with anti-EGFR antibody concomitantly (5.7, P = 0.02, and 28.7 months, P = 0.034, respectively).

CONCLUSIONSK-ras status is a prognostic biomarker for mCRC patients treated with anti-EGFR antibody. The combination settings of anti-EGFR in combination with chemotherapy may improve survival of mCRC patients with wild-type K-ras status.

Aged ; Antibodies, Monoclonal ; therapeutic use ; Antineoplastic Agents ; therapeutic use ; Antineoplastic Agents, Phytogenic ; therapeutic use ; Camptothecin ; analogs & derivatives ; therapeutic use ; Colorectal Neoplasms ; genetics ; pathology ; surgery ; therapy ; Combined Modality Therapy ; Disease-Free Survival ; Female ; Follow-Up Studies ; Genes, ras ; Humans ; Liver Neoplasms ; secondary ; therapy ; Lung Neoplasms ; secondary ; therapy ; Male ; Middle Aged ; Mutation ; Organoplatinum Compounds ; therapeutic use ; Receptor, Epidermal Growth Factor ; immunology ; Retrospective Studies ; Survival Rate

OBJECTIVETo evaluate the immunohistochemical detection of epidermal growth factor receptor(EGFR) mutations using two EGFR mutation-specific monoclonal antibodies: delE746-A750 and L858R.

METHODSA total of 175 paraffin-embedded lung adenocarcinoma tissue samples previously genotyped by directive DNA sequencing were subject to immunostaining using delE746-A750 and L858R antibodies.

RESULTSThere was no significant difference of mutation detection between DNA sequence analysis and delE746-A750 and/or L858R immunostaining (33.7% vs 30.9%, P > 0.05). The overall sensitivity, specificity, positive predictive value and negative predictive value of immunostaining using these two EGFR mutation-specific antibodies were 83.1%, 95.7%, 90.7% and 90.9%, respectively.

CONCLUSIONWith high sensitivity and good specificity, immunohistochemistry using EGFR mutation-specific monoclonal antibodies is an adequate, easy and cost-effective prescreening method to detect EGFR mutations using paraffin-embedded tissue specimens of lung adenocarcinomas.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Monoclonal ; DNA Mutational Analysis ; Female ; Gene Deletion ; Humans ; Immunohistochemistry ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Paraffin Embedding ; Point Mutation ; Receptor, Epidermal Growth Factor ; genetics ; immunology ; metabolism ; Sensitivity and Specificity

Nanobody is a kind of antibody from camel, which misses light chain. Nanobody has the same antigen binding specificity and affinity as mAb. Moreover, because of its small molecular weight, high stability and easy preparation, nanobody has great value of biomedical applications. In this study, we successfully prepared highly pure antiEGFR nanobody in E.coli using genetic engineering techniques. Cell proliferation assay (CCK-8 assay) and migration experiments (cell scratch test and Transwell assay) indicated that the recombinant antiEGFRnano can significantly inhibit the proliferation and migration of endometrial cancer cells. These results provide a new way of thinking and methods for EGFR-targeted therapy of endometrial cancer.
Adenocarcinoma ; metabolism ; pathology ; Amino Acid Sequence ; Base Sequence ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Endometrial Neoplasms ; metabolism ; pathology ; Escherichia coli ; metabolism ; Estrogens ; metabolism ; Female ; Genetic Vectors ; Humans ; Plasmids ; Receptor, Epidermal Growth Factor ; genetics ; immunology ; Recombinant Proteins ; metabolism ; Single-Domain Antibodies ; genetics ; pharmacology

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.
Antibodies, Bispecific ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Humans ; Plasmids ; Protein Binding ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ~17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.
Animals ; Antibodies, Monoclonal, Humanized/*genetics/immunology/therapeutic use ; Antibody Affinity ; Cell Line, Tumor ; Directed Molecular Evolution/*methods ; Epitope Mapping ; Epitopes/genetics/immunology/therapeutic use ; Humans ; *Immunotherapy ; Mice ; Neoplasms/*therapy ; Phosphorylation/drug effects ; Protein Binding ; Receptor, Epidermal Growth Factor/*antagonists & inhibitors/immunology ; Selection, Genetic ; Single-Chain Antibodies/*genetics/immunology/therapeutic use

Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.
Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Antigens, CD20 ; immunology ; Humans ; Immunoconjugates ; therapeutic use ; Neoplasms ; drug therapy ; immunology ; Protein Engineering ; RANK Ligand ; immunology ; Receptor, Epidermal Growth Factor ; immunology ; Receptor, ErbB-2 ; immunology ; Vascular Endothelial Growth Factor A ; immunology

A recombinant plasmid pET28a-HBcAg-delta n was constructed, in which three mimic B-epitopes of HER family were inserted into the truncated HBc vector. The fusion protein expressed was purified and used to immunize BALB/c mice to induce antibody against the epitopes. Three mimic epitope genes were inserted into the sequences of amino acid residues 78 and 79 of HBcAg by overlap PCR. The PCR product was then cloned into pET28a to construct recombinant expression plasmid which was transformed to E. coli BL21 (DE3) and induced by IPTG. After purification, the fused protein designed HBHE was used to immunize BALB/c mice to detect humoral immunoresponse. The recombinant plasmid was successfully constructed by DNA sequencing analysis. A fusion protein with correct molecular mass was expressed and confirmed by SDS-PAGE. High titre antibody was elicited in the mice immunized with HBHE by indirect ELISA and Western blotting. The HBc particle vector containing three B-epitopes of HER family had been successfully prepared, purified and high titre antibody against HBHE was detected. All these data are helpful in further research of the broad-spectrum anti-tumour effect of combine polypeptide epi-position vaccine of EGFR and HER2.
Animals ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Epitopes ; immunology ; Genetic Vectors ; Hepatitis B Core Antigens ; genetics ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; Random Allocation ; Receptor, Epidermal Growth Factor ; genetics ; immunology ; Receptor, ErbB-2 ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccination ; methods ; Vaccines, Combined ; immunology