Human Thrombomodulin (TM) ELISA Kit

       10         20         30         40         50
 MLGVLVLGAL ALAGLGFPAP AEPQPGGSQC VEHDCFALYP GPATFLNASQ
         60         70         80         90        100
 ICDGLRGHLM TVRSSVAADV ISLLLNGDGG VGRRRLWIGL QLPPGCGDPK
        110        120        130        140        150
 RLGPLRGFQW VTGDNNTSYS RWARLDLNGA PLCGPLCVAV SAAEATVPSE
        160        170        180        190        200
 PIWEEQQCEV KADGFLCEFH FPATCRPLAV EPGAAAAAVS ITYGTPFAAR
        210        220        230        240        250
 GADFQALPVG SSAAVAPLGL QLMCTAPPGA VQGHWAREAP GAWDCSVENG
        260        270        280        290        300
 GCEHACNAIP GAPRCQCPAG AALQADGRSC TASATQSCND LCEHFCVPNP
        310        320        330        340        350
 DQPGSYSCMC ETGYRLAADQ HRCEDVDDCI LEPSPCPQRC VNTQGGFECH
        360        370        380        390        400
 CYPNYDLVDG ECVEPVDPCF RANCEYQCQP LNQTSYLCVC AEGFAPIPHE
        410        420        430        440        450
 PHRCQMFCNQ TACPADCDPN TQASCECPEG YILDDGFICT DIDECENGGF
        460        470        480        490        500
 CSGVCHNLPG TFECICGPDS ALARHIGTDC DSGKVDGGDS GSGEPPPSPT
        510        520        530        540        550
 PGSTLTPPAV GLVHSGLLIG ISIASLCLVV ALLALLCHLR KKQGAARAKM
        560        570
 EYKCAAPSKE VVLQHVRTER TPQRL                                    

Introduction

Test principle

The microtiter plate provided in this kit has been pre-coated with an antibody specific to the index. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to the index. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain the index, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of the index in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Recovery

Matrices listed below were spiked with certain level of recombinant TM and the recovery rates were calculated by comparing the measured value to the expected amount of the index in samples.

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of the index and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Precision

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

Stability

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage conditions.
Note:
To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 2 hours at 37℃;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37℃;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 1 hour at 37℃;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
8. Add 50µL Stop Solution. Read at 450nm immediately.