Regenerating Islet-Derived Family, Member 4 (REG4) ELISA Kit

Antigen: Regenerating Islet-Derived Family, Member 4 (REG4)

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN365758

Additional Information

Method type: Sandwich ELISA

Detection Method: Colorimetric

Alternative name: regenerating gene IV (REG-4)

HGNC: 22977

UniProt: Q9BYZ8

Cross-Reactivity (Details): No significant cross-reactivity or interference between Human REG4 and analogues was observed.

Sample Type: Serum, Plasma

Sample Volume: 50 - 100 µL

Assay Time: 1 - 4.5 h

Plate: Pre-coated 8 x 12 strip plate

Components: 1. Assay plate (12 x 8 coated Microwells). Quantity: 1(96 wells)
2. Standard (Freeze dried). Quantity: 2
3. Biotin-antibody (100 x concentrate) Quantity: 1 x 120 µl
3.HRP-avidin (100 x concentrate). Quantity: 1 x 120 µl
4. Biotin-antibody Diluent. Quantity: 1 x 10 ml
5. HRP-avidin Diluent. Quantity: 1 x 10 ml
6. Sample Diluent. Quantity: 1 x 20 ml
7. Wash Buffer (25 x concentrate). Quantity: 1 x 20 ml
8. TMB Substrate. Quantity: 1 x 10 ml
9. Stop Solution. Quantity: 1 x 10 ml
10. Adhesive Strip (For 96 wells). Quantity: 4
11. Instruction manual

Description: Synonyms: GISP, REG-IV, RELP, OTTHUMP00000014031|gastrointestinal secretory protein|regenerating gene type IV

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human REG-4 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Specificity: This assay has high sensitivity and excellent specificity for detection of Human REG4.

Sensitivity: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined the mean O.D value of 20 replicates of the zero standard added by their three standard deviations.

Minimum Detection Limit: 0.078 ng/mL

Detection Range: 0.312 ng/mL - 20 ng/mL

Assay Precision:

• Intra-assay Precision (Precision within an assay): CV%<8% Three samples of known concentration were tested twenty times on one plate to assess.
• Inter-assay Precision (Precision between assays): CV%<10% Three samples of known concentration were tested in twenty assays to assess.

Application Details

Principle: This assay employs the quantitative sandwich enzyme immunoassay technique.

Protocol: Antibody specific for REG4 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any REG4 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for REG4 is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of REG4 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Sample Collection:

• Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 ×g. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
• Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 ×g at 2-8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.

Assay Procedure: Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. It is recommended that all samples and standards be assayed in duplicate.

• 1. Prepare all reagents, working standards, and samples as directed in the previous sections.
• 2. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.
• 3. Add 100 µL of standard and sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37°C. A plate layout is provided to record standards and samples assayed.
• 4. Remove the liquid of each well, don't wash.
• 5. Add 100 µL of Biotin-antibody (1x) to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37°C. (Biotin-antibody (1x) may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.)
• 6. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (200 µL) using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 2 minutes, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash Buffer by aspirating ordecanting. Invert the plate and blot it against clean paper towels.
• 7. Add 100 µL of HRP-avidin (1x) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
• 8. Repeat the aspiration/wash process for five times as in step 6.
• 9. Add 90 µL of TMB Substrate to each well. Incubate for 15-30 minutes at 37°C. Protect from light.
• 10. Add 50 µL of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
• 11. Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. 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.

Calculation of Results: Using the professional soft 'Curve Expert 1.3' to make a standard curve is recommended, which can be downloaded from our web. Average the duplicate readings for each standard and sample and subtract the average 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, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the REG4 concentrations 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 have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Application Notes: Detection wavelength: 450 nm

Material not included: 1. Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
2. An incubator which can provide stable incubation conditions up to 37° C +/- 0.5° C.
3. Squirt bottle, manifold dispenser, or automated microplate washer.
4. Absorbent paper for blotting the microtiter plate.
5. 100 mL and 500 mL graduated cylinders.
6. Deionized or distilled water.
7. Pipettes and pipette tips.
8. Test tubes for dilution.

Storage: 2-8°C

Storage Comment: Unopened: Store at 2 - 8° C. Do not use past kit expiration date.
Opened: May be stored for up to 1 month at 2 - 8° C. Try to keep coated assay plate in a sealed aluminium foil bag, and avoid the damp.

Restrictions: For Research Use only

Publications

Product: Zheng, Xu, Yu et al.: "The role of Reg IV gene and its encoding product in gastric carcinogenesis." in: Human pathology, Vol. 41, Issue 1, pp. 59-69, 2009 (PubMed).

Tao, He, Ma et al.: "Evaluation of REG4 for early diagnosis and prognosis of gastric cancer." in: Human pathology, 2011 (PubMed). Method employed by authors: ELISA (Sample species: Human).