ELISA Kit for Dihydrotestosterone (DHT)

5α-DHT; 5α-Dihydrotestosterone; Androstanolone; Stanolone

Specificity

This assay has high sensitivity and excellent specificity for detection of Dihydrotestosterone (DHT).
No significant cross-reactivity or interference between Dihydrotestosterone (DHT) and analogues was observed.

Recovery

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

MatrixRecovery range (%)Average(%)
serum(n=5)79-104101
EDTA plasma(n=5)98-105102
heparin plasma(n=5)78-9995

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Dihydrotestosterone (DHT) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Dihydrotestosterone (DHT) 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 Dihydrotestosterone (DHT) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:81:16
serum(n=5)83-101%98-105%95-103%78-101%
EDTA plasma(n=5)82-93%98-105%98-105%93-103%
heparin plasma(n=5)80-88%98-105%80-102%78-99%

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

ReagentsQuantityReagentsQuantity
Pre-coated, ready to use 96-well strip plate1Plate sealer for 96 wells4
Standard2Standard Diluent1×20mL
Detection Reagent A1Assay Diluent A1×12mL
Detection Reagent B1×120µLAssay Diluent B1×12mL
Reagent Diluent1×300µLStop Solution1×6mL
TMB Substrate1×9mLInstruction manual1
Wash Buffer (30 × concentrate)1×20mL

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 50µL standard or sample to each well.
    And then add 50µL prepared Detection Reagent A immediately.
    Shake and mix. Incubate 1 hour at 37°C;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
7. Add 50µL Stop Solution. Read at 450 nm immediately.

Test principle

This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to Dihydrotestosterone (DHT) has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled Dihydrotestosterone (DHT) and unlabeled Dihydrotestosterone (DHT) (Standards or samples) with the pre-coated antibody specific to Dihydrotestosterone (DHT). After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of Dihydrotestosterone (DHT) in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of Dihydrotestosterone (DHT) in the sample.

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