ELISA/assay

Human soluble TREM-2 ELISA Kit

DEIA-SY005

96T

Human soluble TREM-2 ELISA Kit

TREM2

sELISA

TREM2

Human

Quantitative

Serum and Plasma

96T

Unopened Kit: Store at 2-8°C for up to 4 month. For longer storage up to 10 months, unopened Standard, Positive Control, Dilution Buffer and Antibody Diluent Solution should be stored at -20°C. Detection Antibody-HRP Conjugate and TMB Substrate Solution should be stored only at 2-8°C. Do not use kit past expiration date.

Human Soluble TREM-2

TREM2

Human

TREM2; triggering receptor expressed on myeloid cells 2; TREM-2; Trem2a; Trem2b; Trem2c

For the quantitative measurement of recombinant and/or natural human soluble TREM-2 from serum and plasma in a sandwich ELISA format.

This Human Soluble TREM-2 ELISA Kit contains the necessary components required for the quantitative measurement of recombinant and/or natural human soluble TREM-2 from serum and plasma in a sandwich ELISA format. This immunoassay contains recombinant human soluble TREM-2 and antibodies raised against this protein. Results from this immunoassay have shown to accurately quantify recombinant and natural soluble TREM-2 samples.

This assay employs the quantitative sandwich ELISA format. The plate is pre-coated with an antibody specific for human soluble TREM-2. The capture antibody can bind to the human soluble TREM-2 in the standard and samples. After washing the plate of any unbound substances, an antibody-HRP conjugate against human soluble TREM-2 is added to the wells. After the last wash to remove any unbound enzyme, a substrate solution is added to the wells and color develops in direct proportion to the amount of human soluble TREM-2 bound in the standard solutions or samples. A standard curve can be established and sample values can be read off the standard curve.

1. sTREM-2 Microplate: 96 wells polystyrene microplate (12 strips of 8 wells) coated with an antibody against human sTREM-2. 2. Human sTREM-2 Standard: 8000 pg/vial of recombinant human sTREM-2 in a buffered protein base with preservative; lyonhilized. 3. Detection Antibody Concentrate: 1.05 mL/vial, 10-fold concentrate of blotlnylated antibody against human TREM-2 with preservative; lyophilized. 4. Positive Control : one vial of recombinant TREM-2; lyophilized. 5. Streptavidin-HRP Conjugate: 120 μL/vial, 100-fold concentrated solution of Streptavidin conjugate to HRP. 6. Dilution Buffer: 45 ml of buffered protein based solution with preservative. 7. Antibody Diluent Solution: 12 ml of buffered protein based solution with preservative. 8. HRP Diluent Solution: 12 mL of buffered protein based solution with preservative. 9. Wash Buffer: 25 ml of 20-fold concentmed buffered surfactant, with preservative. 10. TMB Substrate Solution: 11 ml of TMB substrate solution. 11. Stop Solution: 11 ml of 0.25M HCl solution. 12. Plate Sealer 13. Plastic Pouch

1. Microplate reader capable of absorbance measurement at 450 nm. 2. Microplate shaker (350-400 rpm). 3. Microplate washer or manifold dispenser. 4. 100 ml and 500 ml araduated cylinders. 5. Multi-channel Pipette, Pipettes and pipette tips. 6. Deionized or distilled water.

1. Serum: Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. 2. Plasma: Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge at 1000 x g for 15 minutes and collect plasma. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. 3. Optional: Use Aprotinin (enzyme inhibitor) for ALL sample collection to prevent sample degradation. 0.5 TIU per mL of sample solution. 4. Sample preparation Human Serum and plasma samples may need to be diluted by 4-16 fold. Optimal dilution should be determined by each laboratory for each application with a sample pretest. Use polypropylene test tubes.

Bring all reagents to room temperature before use. 1. Wash Buffer: If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 25 mL of Wash Buffer Concentrate (20-fold) into deionized or distilled water (450 mL) to prepare 500 mL of 1× Wash Buffer. 2. TREM-2 Standard: Reconstitute the TREM-2 standard with 1.0 mL of Dilution Buffer. This reconstitution produces a stock solution of 8000 pg/mL. Allow the standard to sit for at least 15 minutes with gentle agitation until completely dissolved prior to making standard dilutions. Pipette 250 μl Dilution Buffer into tubes 1-8. Use the high standard (8000 pg/ml) to produce a dilution series (see below). Mix each tube thoroughly before the next transfer.The 4000 pg/mL standard serves as the high standard. The Dilution Buffer serves as the zero standard (0 pg/mL). Store the stock solution at-20°C to -70°C for a few days. 3. Positive Control: Reconstitute the Positive Control with 2.0 ml of Dilution Buffer to prepare the 1×working solution. Discard the 1× positive control after use. It Is for one time use only. 4. Detection Antibody Conjugate: Reconstitute the Detection Antibody Concentrate with 1.05 ml of Dilution Buffer to produce a 10-fold concentrated stock solution. For 96 wells test, freshly pipette 9.45 ml of Dilution Buffer into a 15 ml centrifuge tube and transfer 1.05 ml of 10-fold concentrated stock solution to prepare worldna solution. For partial strip test, freshly prepare 900 μl per 8-well strip of working solution. Store the stock of 10-fold concentrated solution at -20°C to 70°C for a few days. Streptavidin-HRP Conjugate: For 96 wells test freshly pipette 10.89 ml of HRP Diluent Solution into a 15 ml centrifuge tube and transfer 110 μl of 100-fold concentrated stock solution to prepare working solution (protect from light). That 1×working solution should be used within 20-30 min. For partial strip test, freshly prepare 900 μl per 8-well strip of working solution. Store the stock of 100-fold concentrated solution at 2°C-8°C for 8 months.

Bring all reagents and samples to room temperature before the start of the assay. Blank, standard dilutions, positive control and samples should be assayed in duplicate. ELISA Protocol may need further optimization. 1. Prepare all reagents and working standards as directed in the previous sections. 2. Add 100 μL of Dilution Buffer to Blank wells. 3. Add 100 μl of Standard dilutions In reverse order of serial dilution #8-1, samples, or positive control per well. Cover with plate sealer. Incubate for 2 hours on microplate shaker at room temperature. 4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with 1x Wash Buffer (300 μL) using a squirt bottle, 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. 5. Add 100 μL of 1× Detection Antibody working solution to each well. Cover with plate sealer. Incubate for 2 hours on microplate shaker at room temperature. Protect from light. 6. Repeat the aspiration/wash as in step 4. 7. Add 100 μL of Substrate Solution to each well. Incubate for 60 minutes on microplate shaker at room temperature. Protect from light. 8. Repeat the aspiration/wash as in step 4. 9. Add 100 μL of Substrate Solution to each well. Incubate for 20-25 minutes on mlcroplate shaker at room temperature. Protect from light. 10. Add 100 μl of Stop Solutlon to each well. 11. Determine the optical density of each well using a microplate reader set to 450 nm within 5 minutes.

Average the duplicate readings for each standard, positive control and sample, and subtract the average zero standard optical density. Create a standard curve by reducing the data uslrc computer software capable of generating a log-log or 4-parameter curve fit. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Intra-assay Precision 4-6% Inter-assay Precision 4-9%

31.25-4000 pg/mL

15 pg/mL

1. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. 2. This ELISA kit should not be used beyond the expiration date on the kit label. 3. Do not mix reagents with those from other lots or sources. 4. It is important that the Dilution Buffer selected for the standard curve be consistent with the samples being assayed. 5. Each laboratory must determine the optimal dilution factors for the samples being assayed with a pretest. 6. Any modifications in buffers, pipetting technique, washing technique, incubation time or temperature, as well as kit age can cause a change in signal. 7. Not all interfering factors have been tested in the immunoassay, therefore the possibility of interference cannot be excluded. 8. This kit should be handled by those persons who have been trained in and can follow the principles of good laboratory practice. Wear protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken while handling solutions in this kit to avoid contact with skin or eyes, especially with the stop solution because it contains diluted hydrochloric acid. Wash immediately with water in case of contact on skin or eyes.

For the quantitative measurement of recombinant and/or natural human soluble TREM-2 from serum and plasma in a sandwich ELISA format.

1. sTREM-2 Microplate: 96 wells polystyrene microplate (12 strips of 8 wells) coated with an antibody against human sTREM-2. 2. Human sTREM-2 Standard: 8000 pg/vial of recombinant human sTREM-2 in a buffered protein base with preservative; lyonhilized. 3. Detection Antibody Concentrate: 1.05 mL/vial, 10-fold concentrate of blotlnylated antibody against human TREM-2 with preservative; lyophilized. 4. Positive Control : one vial of recombinant TREM-2; lyophilized. 5. Streptavidin-HRP Conjugate: 120 μL/vial, 100-fold concentrated solution of Streptavidin conjugate to HRP. 6. Dilution Buffer: 45 ml of buffered protein based solution with preservative. 7. Antibody Diluent Solution: 12 ml of buffered protein based solution with preservative. 8. HRP Diluent Solution: 12 mL of buffered protein based solution with preservative. 9. Wash Buffer: 25 ml of 20-fold concentmed buffered surfactant, with preservative. 10. TMB Substrate Solution: 11 ml of TMB substrate solution. 11. Stop Solution: 11 ml of 0.25M HCl solution. 12. Plate Sealer 13. Plastic Pouch

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet