HMGB1 ELISA 试剂盒

This assay has high sensitivity and excellent specificity for detection of Instant High Mobility Group Protein 1 (HMG1).
No significant cross-reactivity or interference between Instant High Mobility Group Protein 1 (HMG1) and analogues was observed.

• Pre-coated, ready to use 96-well strip plate, flat buttom
• Plate sealer for 96 wells
• Reference Standard
• Standard Diluent
• Detection Reagent A
• Detection Reagent B
• Assay Diluent A
• Assay Diluent B
• Reagent Diluent (if Detection Reagent is lyophilized)
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

• Microplate reader with 450 nm filter.
• Precision single or multi-channel pipettes and disposable tips.
• Eppendorf Tubes for diluting samples.
• Deionized or distilled water.
• Absorbent paper for blotting the microtiter plate.
• Container for Wash Solution

1. Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
2. Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 2,000pg/mL. Firstly dilute the stock solution to 1,000 pg/mL and the diluted standard serves as the highest standard (1,000 pg/mL). Then prepare 7 tubes containing 0.5 mL Standard Diluent and produce a double dilution series by transferring 500 µL each. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 1,000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.2pg/mL, 15.6pg/mL, and the last microcentrifuge tube with Standard Diluent is the blank as 0pg/mL.
3. Detection Reagent A and Detection Reagent B - If lyophilized reconstitute the Detection Reagent A with 150μL of Reagent Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). Briefly spin or centrifuge the stockDetection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively.
4. Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:

1. Making serial dilution in the wells directly is not permitted.
2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting.
4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. Prepare Substrate working Solution within 15 minutes before assay.
6. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
7. Contaminated water or container for reagent preparation will influence the detection result.

1. Prepare all reagents, samples and standards,
   
2. Add 100μL standard or sample to each well. Incubate 2 hours at 37 °C,
   
3. Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37 °C,
   
4. Aspirate and wash 3 times,
   
5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
   
6. Aspirate and wash 5 times,
   
7. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
   
8. Add 50μL Stop Solution. Read at 450nm immediately.

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Instant High Mobility Group Protein 1 (HMG1) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Instant High Mobility Group Protein 1 (HMG1) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

• For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C.
• For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
  Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
• For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.