CLIA Kit for High Temperature Requirement Factor A1 (HTRA1)

HtrA; L56; ORF480; PRSS11; HtrA Serine Peptidase 1; Protease,Serine,11; IGFBP5-Protease; High-temperature requirement A serine peptidase 1

Specificity

This assay has high sensitivity and excellent specificity for detection of High Temperature Requirement Factor A1 (HTRA1).
No significant cross-reactivity or interference between High Temperature Requirement Factor A1 (HTRA1) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant High Temperature Requirement Factor A1 (HTRA1) and the recovery rates were calculated by comparing the measured value to the expected amount of High Temperature Requirement Factor A1 (HTRA1) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 82-103 90
EDTA plasma(n=5) 78-101 96
heparin plasma(n=5) 98-105 101

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level High Temperature Requirement Factor A1 (HTRA1) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level High Temperature Requirement Factor A1 (HTRA1) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of High Temperature Requirement Factor A1 (HTRA1) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 78-95% 96-104% 90-98% 85-103%
EDTA plasma(n=5) 98-105% 79-101% 82-94% 78-102%
heparin plasma(n=5) 83-91% 92-99% 82-91% 83-97%

Stability

The stability of 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 condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
Substrate A 1×10mL Substrate B 1×2mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 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 100µL Substrate Solution. Incubate 10 minutes at 37°C;
8. Read RLU value immediately.

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Magazine Citations
Placenta Alterations of maternal plasma HTRA1 level in preeclampsia complicated by IUGR PubMed: 23083999
Placenta Upregulation of HtrA4 in the placentas of patients with severe pre-eclampsia PubMed: 22964307
Exp Gerontol Association of frailty with the serine protease HtrA1 in older adults Pubmed:27058767
International Journal of Clinical and Experimental Pathology HtrA1 up-regulates expression of MMPs via Erk1/2/Rock-dependent pathways files:ijcep0069706.pdf
Pregnancy Hypertension Association between first trimester plasma htra1 level and subsequent preeclampsia: a possible early marker?
American journal of translational research HtrA1 upregulates the expression of ADAMTS-5 in HNPCs via the ERK/NF-κB/JNK signaling pathway Pubmed: 31497227
J Matern Fetal Neonatal Med First trimester HtrA1 maternal plasma level and spontaneous preterm birth Pubmed: 32102578
BMC Geriatr Cross-sectional associations among P3NP, HtrA, Hsp70, Apelin and sarcopenia in Taiwanese population 33743591
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