人胰蛋白酶原***肽(TAP)ELISA Kit

 

This immunoassay kit allows for the specific measurement of human trypsinogen activation peptide,TAP concentrati*** in cell culture supernates, serum, and pla***a.

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal

antibody specific for TAP has been pre-coated onto a microplate. Standa*** and samples are pipetted into the wells and any TAP present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for TAP is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TAP bound in the initial step. The color development is stopped and the intensity of the color is measured.

 

Assay plate                                             1

Standard                                               2

Sample Diluent                                          1 x 20ml

Assay Diluent A                                          1 x 10ml

Assay Diluent B                                          1 x 10ml

Detection Reagent A                                      1×120ul

Detection Reagent B                                      1×120ul

Wash Buffer                                           1 x 30ml

(25 x concentrate)

Substrate                                              1 x 10ml

Stop Solution                                           1 x 10ml

Cell culture supernates- Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20° C. ***oid repeated freeze-thaw cycles.

Serum- Use a serum separator tube (SST) and allow samples to clot for 30 minutes before

centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C.

Pla***a- Collect pla***a using EDTA or heparin as an anticoagulant. Centrifuge samples for

15 minutes at 1000 x g at 2 - 8° C within 30 minutes of collection. Store samples at ≤ -20° C. ***oid repeated freeze-thaw cycles.

Note: Citrate pla***a has not been validated for use in this assay.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

1. The kit should not be used beyond the expiration date on the kit label.

2. Do not mix or substitute reagents with those from other lots or sources.

3. If samples generate values higher than the highest standard, further dilute the samples

with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.

4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors h***e been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded.

Wash Buffer - If crystals h***e formed in the concentrate, warm to room temperature and mix gently until the crystals h***e completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer.

Standard - Rec***titute the Standard with 1.0 mL of Sample Diluent. This rec***titution produces a stock solution of 50 nmol/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial diluti***. The undiluted standard serves as the high standard (50 nmol/mL). The Sample Diluent serves as the zero standard (0 nmol/mL).

Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100),respectively.

 

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standa*** and samples as directed in the previous secti***.

2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37° C.

3. Remove the liquid of each well, don’t wash.

4. Add 100 uL ofDetection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.

5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive cubate for 1 hours at 37° C.

7. Repeat the aspiration/wash as in step 5.

8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.

9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

10. Determine the optical density of each well within 30 minutes, using a microplate reader set

to 450 nm.

 

Specificity
This assay recognizes recombinant and natural human TAP. No significant cross-reactivity or interference was observed.

Important Note:

1.       The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

2.       It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standa***, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is ***ailable.

3.       Duplication of all standa*** and specimens, although not required, is recommended.

4.       When mixing or rec***tituting protein soluti***, always ***oid foaming.

5.       To ***oid cross-contamination, change pipette tips between additi*** of each standard level, between sample additi***, and between reagent additi***. Also, use separate reservoirs for each reagent.

6.       To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

***erage the duplicate readings for each standard, control, and sample and subtract the ***erage zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, c***truct a standard curve by plotting the mean absorbance for each standard on the y-axis against

the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the TAP concentrati*** versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples h***e been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

1.       Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Refer to the package label for the expiration date.

2.       Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 

3.       A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm w***elength is acceptable for use in absorbance measurement.

The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.