Human Kallikrein 3/PSA DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Kallikrein 3/PSA. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: Kallikrein 3/PSA
The human tissue kallikrein (KLK) gene family contains 15 members that play important roles in cancer. Notably, kallikrein-1, also known as tissue kallikrein, cleaves kininogen to release the vasoactive kinin peptide, bradykinin or lysyl-bradykinin. Kallikrein-3, called prostate specific antigen (PSA), is an established tumor marker that aids in the diagnosis, staging, and follow up of prostate cancer. Kallikrein-4 is specifically expressed in the prostate and over-expressed in prostate cancer. Kallikrein-5 is widely expressed but found at high levels in skin, breast, brain and testis; over-expression is an indicator of poor prognosis in ovarian cancer. Kallikrein-8 is expressed in the brain and is a novel marker of ovarian and cervical cancer.
Human plasma kallikrein, a serine protease, is synthesized in the liver and circulates in the plasma bound to high molecular weight (HMW) kininogen or as a free zymogen. Once activated by its physiological activator, coagulation factor XII, it displays endopeptidase activity towards peptide bonds after arginine (preferred) and lysine. It cleaves HMW kininogen, its major physiological substrate, to release the potent vasodilator peptide bradykinin. It is also able to cleave a number of inactive precursor proteins to generate active products, such as plasminogen and prourokinase.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human Kallikrein 3/PSA DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 6
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Microfluidic-based prostate cancer model for investigating the secretion of prostate-specific antigen and microRNAs in vitro
Authors: Padmyastuti, A;Sarmiento, MG;Dib, M;Ehrhardt, J;Schoon, J;Somova, M;Burchardt, M;Roennau, C;Pinto, PC;
Scientific reports
Species: Human
Sample Types: Cell Culture Supernates
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Starting to have sexual intercourse is associated with increases in cervicovaginal immune mediators in young women: a prospective study and meta-analysis
Authors: SM Hughes, CN Levy, FL Calienes, KA Martinez, S Selke, K Tapia, BH Chohan, L Oluoch, C Kiptinness, A Wald, M Ghosh, L Hardy, K Ngure, NR Mugo, F Hladik, AC Roxby
Elife, 2022-10-25;11(0):.
Species: Human
Sample Types: Cervicovaginal Lavage Fluid
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Changes in concentrations of cervicovaginal immune mediators across the menstrual cycle: a systematic review and meta-analysis of individual patient data
Authors: SM Hughes, CN Levy, R Katz, EM Lokken, MN Anahtar, MB Hall, F Bradley, PE Castle, V Cortez, GF Doncel, R Fichorova, PL Fidel, KR Fowke, SC Francis, M Ghosh, LY Hwang, M Jais, V Jespers, V Joag, R Kaul, J Kyongo, T Lahey, H Li, J Makinde, LR McKinnon, AB Moscicki, RM Novak, MV Patel, I Sriprasert, AR Thurman, S Yegorov, NR Mugo, AC Roxby, E Micks, F Hladik, Consortium
Bmc Medicine, 2022-10-05;20(1):353.
Species: Human
Sample Types: Serum
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Interleukin-13 rs1800925/-1112C/T promoter single nucleotide polymorphism variant linked to anti-schistosomiasis in adult males in Murehwa District, Zimbabwe
Authors: ET Choto, T Mduluza, MJ Chimbari
PLoS ONE, 2021-05-28;16(5):e0252220.
Species: Human
Sample Types: Serum
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Association of schistosomiasis and risk of prostate cancer development in residents of Murehwa rural community, Zimbabwe
Authors: ET Choto, T Mduluza, F Mutapi, MJ Chimbari
Infect Agent Cancer, 2020-10-06;15(0):59.
Species: Human
Sample Types: Plasma
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Two-dimensional differential in-gel electrophoresis proteomic approaches reveal urine candidate biomarkers in pediatric obstructive sleep apnea.
Authors: Gozal D, Jortani S, Snow AB, Kheirandish-Gozal L, Bhattacharjee R, Kim J, Capdevila OS
Am. J. Respir. Crit. Care Med., 2009-09-24;180(12):1253-61.
Species: Human
Sample Types: Urine
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