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 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 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 Analysis of free and protein-bound nitrotyrosine in human plasma by a gas chromatography/mass spectrometry method that avoids nitration artifacts

2000 ◽  
Vol 345 (3) ◽  
pp. 453-458 ◽  
Author(s):  
Matthew T. FROST ◽  
Barry HALLIWELL ◽  
Kevin P. MOORE
Keyword(s):  
Reactive Nitrogen Species ◽  
Plasma Proteins ◽  
Biological Fluids ◽  
Plasma Concentrations ◽  
Normal Subjects ◽  
Gas Chromatography Mass Spectrometry ◽  
Highly Sensitive ◽  
Acidic Conditions ◽  
First Time

Measurement of nitrotyrosine in biological fluids and tissues is increasingly being used to monitor the production of reactive nitrogen species in vivo. The detection of nitrotyrosine in vivo has been reported with the use of a variety of methods including immunoassay, HPLC and GLC/MS. The validity of HPLC and immunoassays have been questioned with regard to their selectivity and sensitivity limits. In principle, the measurement of nitrotyrosine by GLC/MS permits a highly specific, highly sensitive and fully quantitative assay. The nitration of tyrosine under acidic conditions in the presence of nitrite is well documented. Derivatization for the full quantification of nitrotyrosine by using GLC/MS can lead to the artifactual nitration of tyrosine if performed under acidic conditions in the presence of nitrite. We describe a novel alkaline method for the hydrolysis and derivatization of nitrotyrosine and tyrosine, and demonstrate its applicability to the measurement of plasma concentrations of both free and protein-bound nitrotyrosine and tyrosine. A detection limit of 1 pg for nitrotyrosine and 100 pg for tyrosine has been achieved. Our method allows, for the first time, the analysis of free and protein-bound nitrotyrosine and tyrosine in biological samples. The plasma concentrations (means±S.E.M.) of free tyrosine and nitrotyrosine in eight normal subjects were 12±0.6 μg/ml and 14±0.7 ng/ml respectively. Plasma proteins contained tyrosine and nitrotyrosine at 60.7±1.7 μg/mg and 2.7±0.4 ng/mg respectively.

scholarly journals Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits

2000 ◽  
Vol 151 (7) ◽  
pp. 1513-1524 ◽  
Author(s):  
Ruwanthi N. Gunawardane ◽  
Ona C. Martin ◽  
Kan Cao ◽  
Lijun Zhang ◽  
Kimberly Dej ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Sequence Motifs ◽  
Microtubule Nucleation ◽  
Sequence Comparisons ◽  
Baculovirus Expression ◽  
Ring Complex ◽  
And Function ◽  
First Time ◽  
Centrosomal Proteins

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.

scholarly journals Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 130
Author(s):  
Cécile Féraudet Tarisse ◽  
Céline Goulard-Huet ◽  
Yacine Nia ◽  
Karine Devilliers ◽  
Dominique Marcé ◽  
...  
Keyword(s):  
Food Poisoning ◽  
Cross Reactivity ◽  
Threat Detection ◽  
Enzyme Immunoassays ◽  
Staphylococcal Enterotoxins ◽  
Strain Characterization ◽  
Highly Sensitive ◽  
Genes Encoding ◽  
Contaminated Food ◽  
First Time

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly S. aureus. Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of S. aureus, contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI.

scholarly journals Human Kallikrein 4: Quantitative Study in Tissues and Evidence for Its Secretion into Biological Fluids

2005 ◽  
Vol 51 (8) ◽  
pp. 1432-1442 ◽  
Author(s):  
Christina V Obiezu ◽  
Shannon JC Shan ◽  
Antoninus Soosaipillai ◽  
Liu-Ying Luo ◽  
Linda Grass ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Serine Proteases ◽  
Biological Fluids ◽  
Affinity Purification ◽  
Tissue Expression ◽  
Cross Reactivity ◽  
Prostate Tissue ◽  
Wide Range ◽  
Sandwich Type ◽  
Kallikrein 4

Abstract Background: Human kallikrein 4 (hK4) is a proteolytic enzyme belonging to the tissue kallikrein family of serine proteases. Previous tissue expression studies have demonstrated highest KLK4 mRNA expression in prostatic tissue, but there has been only limited evidence for the presence of hK4 protein in prostate and other tissues and in corresponding biological secretions. Methods: To investigate the concentrations of hK4 in tissues and biological fluids, we developed a new hK4-specific sandwich-type immunoassay using a monoclonal antibody as the capture reagent. Results: The assay has a detection limit of 0.02 μg/L and <0.1% cross-reactivity toward any of the other 14 human kallikreins. Twelve of 40 tissue extracts prepared from various human tissues contained detectable hK4 concentrations (0.68–7143 ng/g of total protein), with healthy prostate tissue containing the highest amount of hK4. Examination of 16 malignant and 18 benign prostate tissues revealed no significant differences in hK4 protein content, and the tissues contained a wide range of values (benign, <0.02 to 801 ng/g; malignant, <0.02 to 824 ng/g). Among the biological fluids tested, seminal plasma and urine contained widely varying amounts of hK4; concentrations in 54 urine samples were <0.02 to 2.6 μg/L, whereas concentrations in 58 seminal plasma samples were 0.2–202 μg/L. Affinity purification of hK4 from seminal plasma and subsequent mass spectrometry demonstrated the secreted nature of hK4 in seminal plasma. Conclusions: hK4 is found primarily in prostate tissue and is secreted in seminal plasma. Its value as a novel prostatic biomarker needs to be defined further.

scholarly journals Distribution of 15 Human Kallikreins in Tissues and Biological Fluids

2007 ◽  
Vol 53 (8) ◽  
pp. 1423-1432 ◽  
Author(s):  
Julie LV Shaw ◽  
Eleftherios P Diamandis
Keyword(s):  
Serine Proteases ◽  
Biological Fluids ◽  
Expression Data ◽  
Breast Cyst Fluid ◽  
Breast Cyst ◽  
Reverse Transcription Pcr ◽  
Tissue Extracts ◽  
Abundance Patterns ◽  
Sandwich Type ◽  
Cervicovaginal Fluid

Abstract Background: Kallikreins (KLKs) are a group of 15 secreted serine proteases. Some KLKs are established or candidate cancer biomarkers, but for most the physiological function is unknown. We characterized the protein and mRNA abundance patterns of all 15 KLKs in multiple panels of human tissues and biological fluids. Methods: We used sensitive and specific sandwich-type ELISAs for each KLK. Reverse transcription PCR was used for transcript amplification. Multiple panels of human tissue extracts (adult and fetal) were tested, along with various biological fluids. Results: Quantitative protein expression data on 7 sets of adult and 3 sets of fetal tissues were collected for all 15 KLKs. KLKs were also quantified in the following biological fluids: seminal plasma, breast milk, follicular fluid, breast cyst fluid, breast cancer cytosol, amniotic fluid, ovarian cancer ascites, cerebrospinal fluid, cervicovaginal fluid, and urine. The data were used to generate heat maps of KLK concentrations in tissues and fluids and categorize KLK abundance as highly restricted (KLK2 and KLK3 in prostate), restricted (KLK5 in skin, salivary gland, breast, and esophagus; KLK6 in brain and central nervous system; KLK7 in esophagus, heart, liver, and skin; KLK8 in breast, esophagus, skin, and tonsil; KLK13 in esophagus and tonsil), or wide (KLKs 1, 4, 9, 10, 11, 12, 14, and 15). Conclusions: Quantitative KLK concentrations in tissues and fluids aid in the elucidation of KLK function, and coexpression patterns provide clues for KLK participation in proteolytic cascades.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

Sign in / Sign up

Export Citation Format

Share Document