scholarly journals Production of monoclonal antibody to quantify serum CA125 concentration in breast and ovarian cancer patients

2020 ◽  
Vol 42 (4) ◽  
Author(s):  
Nguyen Thi Xuan ◽  
Nguyen Trong Ha
Keyword(s):  
Flow Cytometry ◽  
Cancer Cells ◽  
Sandwich Elisa ◽  
Flow Cytometry Analysis ◽  
Brca1 And Brca2 ◽  
Elisa System ◽  
Serum Ca125 ◽  
Capture Antibody ◽  
Hybridoma Cell Lines ◽  
Tumor Growth And Metastasis

Ovarian carcinoma (OC) and breast cancer (BC) are mainly caused by alterations in genes such as BRCA1 and BRCA2, which are involved in differentiation and survival of cancer cells. The protein CA125 (MUC16) is released by cancer cells in most OC and a few BC patients. The cut-off point of CA125 level for tumor growth and metastasis is 35 U/mL. Production of CA125 monoclonal antidody (mAb) to determine expression level of this antigen by ELISA has been well known. In this study, we aimed to generate CA125-specific mAb for developing a new in-house ELISA kit. To this end, BALB/c mice were immunized with CA125 protein and splenocytes of immunized mice were fused with myeloma Sp2/0 cells. Efficiency of mAbs secreted from the hybridoma clones was examined by ELISA and flow cytometry analysis. As a result, among 3 stable hybridoma cell lines identified, A1 clone attained about 90% positive for anti-CA125 mAb, whereas H1 and H3 clones were about 40% and 50% positive for anti-CA125 mAb, respectively. By flow cytometry analysis, anti-CA125 mAb from A1 clone was more specific to CA125 antigen present in OVCAR -3 cells than those from H1 or H3 clone. In addition, the isotype of the obtained mAb was specific IgG1 and Kappa light chain. In conclusion, the mouse anti-human CA125 mAb generated in our lab was specifically binding to CA125 antigen and used as the capture antibody in sandwich ELISA system for early diagnosis as well as monitoring therapeutic response in OC patients.  

scholarly journals Cisplatin Increased the Expression of PD-1 and PD-L1 by YAP1 in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Liyuan Hao ◽  
Yinglin Guo ◽  
Qing Peng ◽  
Zhiqin Zhang ◽  
Shenghao Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
Cancer Cells ◽  
Flow Cytometry Analysis ◽  
Chemotherapy Drugs ◽  
The World ◽  
Hcc Cells

Abstract Hepatocellular carcinoma (HCC) was one of the most malignant cancers in the world. Cisplatin (DDP) was one of the main chemotherapy drugs for HCC, but the mechanism of DDP treatment for HCC remains unclear. In this presentation, we found that DDP inhibited the growth of HCC cells and promoted the expression of PD-1 and its ligand PD-L1 in cancer cells. Meanwhile, flow cytometry analysis revealed that DDP enhanced PD-1-CD8+ T cells expression and decreased PD-1+CD8+ T cells expression. ELISA analysis suggested that DDP decreased TGF-β expression in vivo. Therefore, the study indicated that DDP enhanced PD-1 and PD-L1 expression and inhibited the growth of HCC.

Effect of heterogeneity of HER2 expression on brain metastases of breast cancer.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 635-635
Author(s):  
Mari Hosonaga ◽  
Yoshimi Arima ◽  
Eiji Sugihara ◽  
Norio Kohno ◽  
Hideyuki Saya
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Brain Metastases ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Her2 Expression ◽  
Flow Cytometry Analysis ◽  
Her2 Positive ◽  
The Brain

635 Background: HER2-overexpressing or triple-negative [ER(-)/PR(-)/HER2(-)] breast cancers are associated with increased risk of brain metastases. The mechanisms leading to metastasis in each subtype are not well known. Methods: We introduced the wild-type HER2 gene into MDA-MB-231-luc-D3H2LN (231-Luc) cells, which are triple-negative breast cancer cells, and established HER2-expressing (63.2%) cells as 231-Luc-HER2 cells. We investigated the tumor formation following orthotopic inoculation and brain metastasis following intracardiac injection into nude mice. Metastasis was detected by bioluminescence imaging and confirmed in H&E staining and immunohistochemistry of vimentin and HER2 expressions. Flow cytometry analysis was used to detect the proportion of CD44+/CD24- cells, a maker for stem-like breast cancer cells. Results: 231-Luc-HER2 cells formed larger tumors in orthotopic xenograft models compared to 231-Luc cells, however, no significant difference was observed in proliferation in vitro. Neither 231-Luc-HER2 nor 231-Luc metastasized in the brain from the breast after orthotopic inoculation. After intracardiac injections of the 231-Luc-HER2 cells, brain metastasis developed (7/13 mice, 53.8%). Immunohistochemical analysis revealed that most metastasized cells expressed HER2, although we had injected a mixture of HER2-positive and HER2-negative cancer cells. Interestingly, administering Lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, effectively prevented HER2-positive cells to colonize the brain. However, the HER2-negative 231-Luc-HER2 cells developed into brain metastases. In fact, the 231-Luc cells, which are HER2-negative, also metastasized in the brain (10/16 mice, 62.5%). Flow cytometry analysis of the 231-Luc-HER2 cells showed that HER2-positive cells decreased the population of CD44+/CD24- (HER2+/CD44+/CD24-: 86.8% and HER2-/CD44+/CD24-: 96.3%). Conclusions: The mechanism of brain metastases of HER2-positive breast cancer cells is different from that of HER2-negative breast cancer cells. It is therefore important to consider an additional therapeutic approach when dealing with HER2-negative cells in tumors having the heterogeneity of HER2 expression.

scholarly journals Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8055 ◽  
Author(s):  
Thien V. Tran ◽  
Andrei E. Siniavin ◽  
Anh N. Hoang ◽  
My T.T. Le ◽  
Chuong D. Pham ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cancer Cells ◽  
Cell Lines ◽  
Phospholipase A ◽  
Human Lung Cancer ◽  
Flow Cytometry Analysis ◽  
Mcf7 Cells ◽  
Ki 67 ◽  
Cellular Marker

Background Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. Methods The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry. Results MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.

scholarly journals Development of Monoclonal Antibody Based IgG and IgM ELISA for Diagnosis of Severe Fever with Thrombocytopenia Syndrome Virus Infection

2021 ◽  
Author(s):  
Mei Zhang ◽  
Yanhua Du ◽  
Li Yang ◽  
Lin Zhan ◽  
Bin Yang ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Infections ◽  
Serum Samples ◽  
Elisa System ◽  
N Protein ◽  
Capture Elisa ◽  
Igm Elisa ◽  
Hybridoma Cell Lines ◽  
Severe Fever

Abstract Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that possesses a great threat to human health because of the high fatality rate. Method: To develop sensitive and specific sero-diagnosis systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patient serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. Results: The MAb based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA with a sensitivity and specificity of 100%. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while was more sensitive comparing with the indirect IgM ELISA. Conclusions: The MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

scholarly journals A Synergic Potential of Antimicrobial Peptides against Pseudomonas syringae pv. actinidiae

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1461
Author(s):  
Nuno Mariz-Ponte ◽  
Laura Regalado ◽  
Emil Gimranov ◽  
Natália Tassi ◽  
Luísa Moura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antimicrobial Peptides ◽  
Pseudomonas Syringae ◽  
Pathogenic Bacteria ◽  
High Efficiency ◽  
Bacterial Canker ◽  
Membrane Permeation ◽  
Flow Cytometry Analysis ◽  
Pathogenic Agent ◽  
Peptide Mixtures

Pseudomonas syringae pv. actinidiae (Psa) is the pathogenic agent responsible for the bacterial canker of kiwifruit (BCK) leading to major losses in kiwifruit productions. No effective treatments and measures have yet been found to control this disease. Despite antimicrobial peptides (AMPs) having been successfully used for the control of several pathogenic bacteria, few studies have focused on the use of AMPs against Psa. In this study, the potential of six AMPs (BP100, RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) to control Psa was investigated. The minimal inhibitory and bactericidal concentrations (MIC and MBC) were determined and membrane damaging capacity was evaluated by flow cytometry analysis. Among the tested AMPs, the higher inhibitory and bactericidal capacity was observed for BP100 and CA-M with MIC of 3.4 and 3.4–6.2 µM, respectively and MBC 3.4–10 µM for both. Flow cytometry assays suggested a faster membrane permeation for peptide 3.1, in comparison with the other AMPs studied. Peptide mixtures were also tested, disclosing the high efficiency of BP100:3.1 at low concentration to reduce Psa viability. These results highlight the potential interest of AMP mixtures against Psa, and 3.1 as an antimicrobial molecule that can improve other treatments in synergic action.

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