scholarly journals Human Kallikrein 13: Production and Purification of Recombinant Protein and Monoclonal and Polyclonal Antibodies, and Development of a Sensitive and Specific Immunofluorometric Assay

2003 ◽  
Vol 49 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Carl Kapadia ◽  
Albert Chang ◽  
Georgia Sotiropoulou ◽  
George M Yousef ◽  
Linda Grass ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Amniotic Fluid ◽  
Polyclonal Antibodies ◽  
Biological Fluids ◽  
Expression System ◽  
Immunohistochemical Analysis ◽  
Cross Reactivity ◽  
Yeast Expression System ◽  
Tissue Extracts ◽  
Cancer Tissues

Abstract Background: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids. Methods: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies. A sandwich-type immunoassay was developed with these antibodies. The assay was used to measure hK13 in various biological fluids and tissue extracts. Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections. Results: The hK13 immunoassay had a detection limit of 0.05 μg/L and showed no cross-reactivity with homologous kallikreins. The assay was linear at 0–20 μg/L, and within-and between-run CVs were <10% (n = 12). hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate. hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues. hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate. hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues. Recovery of active enzyme added to milk or amniotic fluid was 70–98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors. In fluids and tissue extracts, hK13 was found in its free (∼30 kDa) form. Conclusions: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.

scholarly journals Immunofluorometric Assay of Human Kallikrein 10 and Its Identification in Biological Fluids and Tissues

2001 ◽  
Vol 47 (2) ◽  
pp. 237-246 ◽  
Author(s):  
Liu-Ying Luo ◽  
Linda Grass ◽  
David J C Howarth ◽  
Pierre Thibault ◽  
Huy Ong ◽  
...  
Keyword(s):  
Biological Fluids ◽  
Expression System ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Yeast Expression System ◽  
Disease States ◽  
Pichia Pastoris Yeast ◽  
Sandwich Type ◽  
Kallikrein 2 ◽  
Concentration Changes

Abstract Background: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. Methods: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. Results: The hK10 immunoassay has a detection limit of 0.05 μg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 μg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. Conclusions: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.

scholarly journals Human Kallikrein 8: Immunoassay Development and Identification in Tissue Extracts and Biological Fluids

2003 ◽  
Vol 49 (1) ◽  
pp. 87-96 ◽  
Author(s):  
Tadaaki Kishi ◽  
Linda Grass ◽  
Antoninus Soosaipillai ◽  
Chigusa Shimizu-Okabe ◽  
Eleftherios P Diamandis
Keyword(s):  
Dynamic Range ◽  
Biological Fluids ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Cross Reactivity ◽  
Tissue Extracts ◽  
Highly Sensitive ◽  
Sandwich Type ◽  
Chromatographic Procedure ◽  
First Time

Abstract Background: The serine protease human kallikrein 8 (hK8; neuropsin), a new member of the human kallikrein family, was predicted to be secreted; thus, it is expected to be present in biological fluids. The aim of this study was to develop a sensitive and specific immunoassay for hK8 (hK8-ELISA) and establish the distribution of hK8 in tissue extracts and biological fluids. Methods: Recombinant hK8 was produced in a baculovirus expression system and purified with a three-step chromatographic procedure. Purified hK8 was injected into mice and rabbits for antibody generation. A highly specific and sensitive sandwich-type immunoassay (ELISA) was developed using the rabbit and mouse antisera to hK8. The hK8-ELISA was then used to study the distribution of hK8 in various biological fluids and tissue extracts. Results: The dynamic range of the hK8-ELISA was 0.2 (detection limit) to 20 μg/L, and imprecision (CV) was <10% within this range. This hK8-ELISA was specific for hK8 and had no detectable cross-reactivity with other members of the human kallikrein family. With this assay, hK8 was detected in tissue extracts of esophagus (highest concentrations), skin, testis, tonsil, kidney, breast, and salivary gland and in the biological fluids breast milk (highest concentrations), amniotic fluid, seminal plasma, and serum. Furthermore, in some cancer cell lines, the concentration of hK8 was regulated by steroid hormones. Conclusions: We report for the first time production of recombinant hK8 protein, generation of antibodies, and development of a highly sensitive and specific immunoassay for quantification of hK8 in tissue extracts and biological fluids. This assay can be used to explore the potential of hK8 as a marker of cancer or other conditions.

scholarly journals Development of an Immunofluorometric Assay and Quantification of Human Kallikrein 7 in Tissue Extracts and Biological Fluids

2004 ◽  
Vol 50 (4) ◽  
pp. 709-716 ◽  
Author(s):  
Tadaaki Kishi ◽  
Antoninus Soosaipillai ◽  
Linda Grass ◽  
Sheila P Little ◽  
Edward M Johnstone ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Biological Fluids ◽  
Cross Reactivity ◽  
Protein Quantification ◽  
Kidney Cells ◽  
Human Embryonic Kidney Cells ◽  
Tissue Extracts ◽  
Sandwich Type ◽  
Chromatographic Procedure ◽  
Stratum Corneum Chymotryptic Enzyme

Abstract Background: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids. Methods: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies. Results: The assay had imprecision (CV) <10% through the dynamic range of 0.2–20 μg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine. Conclusions: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.

scholarly journals Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 254
Author(s):  
Shelby S. Szteiter ◽  
Ilse N. Diego ◽  
Jonathan Ortegon ◽  
Eliana Salinas ◽  
Abcde Cirilo ◽  
...  
Keyword(s):  
Snake Venom ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Snake Envenomation ◽  
Disintegrin Domain ◽  
The Common ◽  
Neutralization Ability ◽  
New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

MMP-2 mRNA Expression in Ovarian Cancer Tissues Predicts Patients' Response to Platinum-Taxane Chemotherapy

2019 ◽  
Vol 39 (4) ◽  
pp. 1821-1827 ◽  
Author(s):  
WITOLD JELENIEWICZ ◽  
MAREK CYBULSKI ◽  
ANDRZEJ NOWAKOWSKI ◽  
AGNIESZKA STENZEL-BEMBENEK ◽  
MAŁGORZATA GUZ ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Mrna Expression ◽  
Taxane Chemotherapy ◽  
Cancer Tissues

scholarly journals A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Cécile Beck ◽  
Philippe Desprès ◽  
Sylvie Paulous ◽  
Jessica Vanhomwegen ◽  
Steeve Lowenski ◽  
...  
Keyword(s):  
High Performance ◽  
Neurological Diseases ◽  
Expression System ◽  
Cross Reactivity ◽  
Encephalitis Virus ◽  
Serological Tests ◽  
Multiplex Immunoassay ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Negative Controls

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using theDrosophilaS2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.

scholarly journals Immunoradiometric and immunohistochemical analysis of Cathepsin D in ovarian cancer: lack of association with clinical outcome

1998 ◽  
Vol 78 (12) ◽  
pp. 1645-1652 ◽  
Author(s):  
G Ferrandina ◽  
G Scambia ◽  
A Fagotti ◽  
G D'Agostino ◽  
P Benedetti Panici ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Clinical Outcome ◽  
Cathepsin D ◽  
Immunohistochemical Analysis

scholarly journals miR-214 promotes radioresistance in human ovarian cancer cells by targeting PETN

2017 ◽  
Vol 37 (4) ◽  
Author(s):  
Qin Zhang ◽  
Shuxiang Zhang
Keyword(s):  
Ovarian Cancer ◽  
Ionizing Radiation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ovarian Cancer Cell ◽  
Cancer Cell Lines ◽  
Ovarian Cancer Cells ◽  
Human Ovarian Cancer ◽  
Ovarian Cancer Cell Lines ◽  
Cancer Tissues

Ovarian cancer is one of the leading causes of death among gynecological malignancies. Increasing evidence indicate that dysregulation of microRNAs (miRNAs) plays an important role in tumor radioresistance. The aim of the present study is to investigate whether microRNA-214 (miR-214) was involved in radioresistance of human ovarian cancer. Here, we showed that miR-214 was significantly up-regulated in ovarian cancer tissues and radioresistance ovarian cancer cell lines. Transfection of miR-214 agomir in radiosensitive ovarian cancer cell lines promoted them for resistance to ionizing radiation, whereas transfection of miR-214 antagomir in radioresistance ovarian cancer cell lines sensitized them to ionizing radiation again. Furthermore, we found miR-214 effectively promoted tumor radioresistance in xenograft animal experiment. Western blotting and quantitative real-time PCR demonstrated that miR-214 negatively regulated PTEN in radioresistance ovarian cancer cell lines and ovarian cancer tissues. Taken together, our data conclude that miR-214 contributes to radioresistance of ovarian cancer by directly targeting PTEN.

Sign in / Sign up

Export Citation Format

Share Document