Rat HMGB-1 (High Mobility Group Protein B1) CLIA Kit (RTES00299)

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat HMGB-1. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat HMGB-1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat HMGB-1, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat HMGB-1. The concentration of Rat HMGB-1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat HMGB-1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat HMGB-1 were tested on 3 different plates, 20 replicates in each plate.

Recovery

The recovery of Rat HMGB-1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Linearity

Samples were spiked with high concentrations of Rat HMGB-1 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
2. Aliquot 100 µL of standard solutions into the standard wells.
3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
6. Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37 °C.
7. Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
10. Add 100 µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37 °C. Protect the plate from light.
11. Determine the RLU value of each well immediately.