ELISA                                  

Human PSA

Catalogue Number 1P325

For the quantitative determination of Human prostate specific antigen concentrations in serum - plasma - body fluid - celiac fluid - tissue homogenate.

This package ｉnsert must be read in its entirety before using this product.

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.   

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INTENDED USE AND TEST PRINCIPLE

This immunoassay kit allows for the quantitative determination of Human PSA concentrations in serum, plasma, cell culture supernates ａnd other biological fluids. The Stop Solution changes the color from blue to yellow ａnd the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of PSA in the sample, this PSA ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples ａnd allow the operator to produce a standard curve of Optical Density versus PSA concentration. The concentration of PSA in the samples is then determined by comparing the O.D. of the samples to the standard curve.

MATERIALS REQUIRED BUT NOT SUPPLIED

1. 37 ℃ incubator

2. Standard microplate reader capable of measuring absorbance at 450 nm

3. Precision pipettes, disposable pipette tips ａnd Absorbent paper

4. Distilled or deionized water

SAMPLE COLLECTION AND STORAGES

Serum - Use a serum separator tube ａnd allow samples to clot for 30 minutes before centrifugation for 20 minutes at approximately 1000×g. Remove serum ａnd assay immediately or aliquot ａnd store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates ａnd other biological fluids - Remove particulates by centrifugation ａnd assay immediately or aliquot ａnd store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Note:  The samples shoule be centrifugated dequately ａnd no hemolysis or granule was allowed.

REAGENTS PROVIDED

PRECAUTIONS

1.         Do not substitute reagents from one kit lot to another. Standard, conjugate ａnd microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.         Allow kit reagents ａnd materials to reach room temperature （20-25°C） before use. Do not use water baths to thaw samples or reagents.

3.         Do not use kit components beyond their expiration date.

4.         Use only deionized or distilled water to dilute reagents.

5.         Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

6.         Use fresh disposable pipette tips for each transfer to avoid contamination.

7.         Do not mix acid ａnd sodium hypochlorite solutions.

8.         Serum ａnd plasma should be handled as potentially hazardous ａnd capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from Human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious ａnd good laboratory practices should be followed.

9.         All samples should be disposed of in a manner that will inactivate viruses.

10.     Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.

11.     Substrate Solution is easily contaminated. If bluish prior to use, do not use.

12.     Substrate B contains 20% acetone, keep this reagent away from sources of heat or flame.

13.     Remove all kit reagents from refrigerator ａnd allow them to reach room temperature （ 20-25°C）.

ASSAY PROCEDURE

1.         Prepare all Reagents before starting assay procedure . It is recommended that all Standards ａnd Samples be added in duplicate to the Microtiter Plate.

2.         First, secure the desired number of coated wells in the holder, then add 50 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.

3.         Add 50 μL of Enzyme Conjugate to each well. Mix well. Complete mixing in this step is important. Cover ａnd incubate for 1 hour at 37°C.

4.         Wash the Microtiter Plate using one of the specified methods indicated below:

5.          Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with Wash Solution （1X）, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of FIVE washes. After final wash, invert plate, ａnd blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly hen washing the plate to assure that all strips remain securely in frame.

6.         Automated Washing: Aspirate all wells, then wash plates FIVE times using Wash Solution （1X）. Always adjust your washer to aspirate as much liquid as possible ａnd set fill volume at 350μL/well/wash （range: 350-400μL）. After final wash, invert plate, ａnd blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.

7.         Add 50μL Substrate A ａnd 50μL Substrate B to each well. Cover ａnd incubate for 15 minutes at 37°C. Protect from light.

8.         Add 50 μL of Stop Solution to each well. Mix well.

9.         Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.

CALCULATION OF RESULTS

1.         This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. （450 nm） obtained for each of the six standard concentrations on the vertical （Y） axis versus the corresponding concentration on the horizontal （X） axis.

2.         First, calculate the mean O.D. value for each standard ａnd sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.

3.         To determine the amount in each sample, first locate the O.D. value on the Y-axis ａnd extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis ａnd read the corresponding concentration.

4.         Any variation in operator, pipetting ａnd washing technique, incubation time or temperature, ａnd kit age can cause variation in result. Each user should obtain their own standard curve.

5.         The sensitivity by this assay is 0.1 ng/ml.

6.         Standard curve

ASSAY RECORD TEMPLATE

Use this plate layout to record standards ａnd samples assayed.