CLIA Kit for High Temperature Requirement Factor A4 (HTRA4)

HtrA Serine Peptidase 4

Specificity

This assay has high sensitivity and excellent specificity for detection of High Temperature Requirement Factor A4 (HTRA4).
No significant cross-reactivity or interference between High Temperature Requirement Factor A4 (HTRA4) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant High Temperature Requirement Factor A4 (HTRA4) and the recovery rates were calculated by comparing the measured value to the expected amount of High Temperature Requirement Factor A4 (HTRA4) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 80-98 90
EDTA plasma(n=5) 79-103 99
heparin plasma(n=5) 82-93 88

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level High Temperature Requirement Factor A4 (HTRA4) 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 A4 (HTRA4) 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 A4 (HTRA4) 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) 90-105% 81-88% 89-103% 90-105%
EDTA plasma(n=5) 88-95% 94-105% 78-91% 80-91%
heparin plasma(n=5) 79-95% 88-95% 80-91% 78-90%

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 Upregulation of HtrA4 in the placentas of patients with severe pre-eclampsia PubMed: 22964307
J Clin Endocrinol Metab Human HtrA4 Expression Is Restricted to the Placenta, Is Significantly Up-Regulated in Early-Onset Preeclampsia, and High Levels of HtrA4 Cause Endothelial Dysfunction - See more at: http://press.endocrine.org/doi/abs/10.1210/jc.2014-3969#sthash.XcUOSx2Z.dpuf PubMed: 25946029
Journal of Biomolecular Screening HtrA3 Isoform-Specific ELISAs for Early Detection of Preeclampsia. pubmed:27932697
Catalog No. Related products for research use of Homo sapiens (Human) Organism species Applications (RESEARCH USE ONLY!)
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