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ELISA Kit for Defensin Beta 103A (DEFb103A)

DEFB103; DEFB3; HBD-3; HBD3; HBP-3; HBP3; Beta-defensin 3; Defensin-like protein

Specificity

This assay has high sensitivity and excellent specificity for detection of Defensin Beta 103A (DEFb103A).
No significant cross-reactivity or interference between Defensin Beta 103A (DEFb103A) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Defensin Beta 103A (DEFb103A) and the recovery rates were calculated by comparing the measured value to the expected amount of Defensin Beta 103A (DEFb103A) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 91-98 95
EDTA plasma(n=5) 89-102 93
heparin plasma(n=5) 89-104 96

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Defensin Beta 103A (DEFb103A) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Defensin Beta 103A (DEFb103A) 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 Defensin Beta 103A (DEFb103A) 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) 82-89% 85-97% 95-104% 79-103%
EDTA plasma(n=5) 80-95% 99-105% 82-91% 92-99%
heparin plasma(n=5) 89-97% 88-95% 79-101% 99-105%

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
TMB Substrate 1×9mL Stop Solution 1×6mL
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 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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Magazine Citations
Plos Pathogens HIF-1α Is Essential for Effective PMN Bacterial Killing, Antimicrobial Peptide Production and Apoptosis in?Pseudomonas aeruginosa?Keratitis PubMed: PMC3715414
Scientific Reports Measurements of AMPs in stratum corneum of atopic dermatitis and healthy skin–tape stripping technique Pubmed:29374283
PeerJ Antibacterial activity of human defensins against Staphylococcus aureus and Escherichia coli 33304659
Animals Fat Encapsulation Reduces Diarrhea in Piglets Partially by Repairing the Intestinal Barrier and Improving Fatty Acid Transport 33375218
Sci Rep Human beta defensin levels and vaginal microbiome composition in post-menopausal women diagnosed with lichen sclerosus 34362937
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