This kit is developed to measure the levels of human Kallikrein 3/Prostate Specific Antigen (KLK3/PSA) concentrations in cell culture supernates, serum, plasma or biological fluids.

• These kits were tested to compare the assay range with other competitors and measure the concentration of PSA in serum and plasma

  The detection linear range is 1 to 640 pg/ml

• Sensitivity is 1 pg/ml
• Cell culture supernates or other biological fluids have not been validated for use in this assay

Prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3), P-30 antigen, is a glycoprotein enzyme encoded in humans by the KLK3 gene. PSA is a member of the kallikrein-related peptidase family and is secreted by the epithelial cells of the prostate gland in men and the paraurethral glands as an inactive enzyme (proPSA), a 28 kDa protein with 244 amino acides.The concentration ranges from 0.5 to 2.0 mg/ml in seminal plasma. It becomes activated with chymotrypsin-like enzymatic activity after seven amino acids from the N-terminus removed. Then the major function of this active PSA is to cleave the gel-forming proteins in semen, seminogelin I, and seminogelin II, leading to liquefaction of semen clogs after ejaculation. Besides, PSA has also been found in other tissues or biological fluids, such as breast, urine and saliva with lower concentration.
There is about 70 to 90% of PSA circulating in serum complexes with others, such as ACT/serpin A1 or A3. The remaining 10 to 30% exists as unbound inactive enzyme (free PSA). When measured with available immunoassays, only free PSA and PSA-ACT are detectable and, traditionally, their sum is termed total PSA. If prostatic tissue damage occurs, such as in prostate cancer and benign hyperplasia, excess amounts of PSA will leak into the circulation, resulting in increased serum total PSA levels. Ratios between total and free PSA are frequently found to be lower in prostate cancer than benign hyperplasia. Due to the expression of PSA regulated by androgen, the determination of PSA levels in various biological fluids and cell culture supernates has thus been used to indicate the integrity of the androgen receptor signaling pathway.

This kit was designed for a 3.5 hour solid phase immunoassay to measure the levels of human Kallikrein 3/Prostate Specific Antigen (KLK3/PSA) concentrations in cell culture supernates, serum, plasma or other biological fluids. The recombinant human KLK3/PSA (lyophilized) serves as the standard, antibodies against PSA used to detect, and HRP labeled secondary antibody to amplify the signal, resulting in lower minimum detectable dose (ultra-sensitive), compared with other competitors. Natural human KLK3/PSA showed dose-response curves that were parallel to the standard curves obtained using the kit standard, indicating that this kit can be used to determine relative levels of natural human KLK3/PSA.

Assay Principles:

This kit is developed for a sandwich ELISA format, providing a rapid measurement (3.5 h) of KLK3/PSA. The kit consists of a detachable 96-well pre-coated plate. Standards and samples are pipetted into the wells and any human PSA present is bound onto the plate. After removing any unbound substances, a detector antibody specific for human PSA is added to the wells. After washing to remove any unbound antibody reagent, HRP labeled secondary antibody is added to target the bound antibody to amplify the signal, facilitating a lower minimum detectable dose measurement. After adding HRP Substrate (TMB), TMB turns blue under the catalysis of HRP, and turns yellow after adding stop solution. Measure the OD value with a microplate reader at 450 nm wavelength. Then the concentration of KLK3/PSA is proportional to the OD450 nm value.

Brief protocol (the detailed steps are provided with the product):

1. Detach wells from plate based on your samples and keep at room temperature. Dilute your samples or reconstituted standards in sample dilution buffer and add them to wells (0.1 ml/well). Incubate at 37 °C for 1 hour or room temperature for 1.5 hour. Wash wells four times by wash solution (0.3 ml/well)

2. Add detector antibody to wells (0.1 ml/well). Incubate at 37 °C for 1 hour or room temperature for 1.5 hour. Wash wells as mentioned.

3. Dilute the HRP secondary antibody in wash solution and add to each well (0.1 ml/well). Incubate at room temperature for 0.5 to 1 hour. Avoid light by foil. Wash wells as mentioned.

4. Add TMB to each well (0.1 ml/well) and incubate at room temperature for 10 to 30 minutes. Protect from light. 

5. Stop the substrate reaction by adding stop solution (0.1 ml/well). Absorbance (OD) is calculated as the absorbance at 450 nm.

For research use only. Cell culture supernates or other biological fluids have not been validated for use in this assay.