Human Autoimmune Regulator (AIRE) ELISA Kit

 50 MATDAALRRL LRLHRTEIAV AVDSAFPLLH ALADHDVVPE DKFQETLHLK        
100 EKEGCPQAFH ALLSWLLTQD STAILDFWRV LFKDYNLERY GRLQPILDSF      
150 PKDVDLSQPR KGRKPPAVPK ALVPPPRLPT KRKASEEARA AAPAALTPRG        
200 TASPGSQLKA KPPKKPESSA EQQRLPLGNG IQTMSASVQR AVAMSSGDVP     
250 GARGAVEGIL IQQVFESGGS KKCIQVGGEF YTPSKFEDSG SGKNKARSSS     
300 GPKPLVRAKG AQGAAPGGGE ARLGQQGSVP APLALPSDPQ LHQKNEDECA      
350 VCRDGGELIC CDGCPRAFHL ACLSPPLREI PSGTWRCSSC LQATVQEVQP      
400 RAEEPRPQEP PVETPLPPGL RSAGEEVRGP PGEPLAGMDT TLVYKHLPAP     
450 PSAAPLPGLD SSALHPLLCV GPEGQQNLAP GARCGVCGDG TDVLRCTHCA      
500 AAFHWRCHFP AGTSRPGTGL RCRSCSGDVT PAPVEGVLAP SPARLAPGPA      
540 KDDTASHEPA LHRDDLESLL SEHTFDGILQ WAIQSMARPA APFPS  

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 AIRE 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.