Validation Report #029854

Share results:

Human luteinizing hormone (LH) ELISA Kit (ABIN365639) has been independently validated for Enzyme-linked immunosorbent assay by the Science Exchange network of expert service providers.

The Science Exchange network is used to independently validate research reagents, protocols, and results to ensure accuracy and reproducibility.

Validation number: #029854
Validated on: 04/10/16

Summary

Antigen

Human luteinizing hormone (LH)

Catalog number
Supplier

Cusabio

Supplier catalog number
Lot number

C4621151803

Method validated
Laboratory
Validation number

29854

Positive Control

Human postmenopausal individual serum (Biochemed, Lot#BC033016HSPMG)

Negative Control

Chicken serum (Biochemed, Lot#BC03316CSPMG)

Notes

Human luteinizing hormone (LH) was readily detected in the positive controls (up to 4-fold dilutions). Lower dilutions of the positive control gave lower reading of LH. Spike showed fairly poor recovery (~58%) indicating presence of inhibitory components in serum (chicken) even when it was 4-fold diluted, which is consistent with the lower readings of LH in lower dilutions of human serum.

Independent Results

Full Methods

ELISA kit

  • Antigen: Human luteinizing hormone (LH)
  • Catalog number: ABIN365639
  • Supplier: Cusabio
  • Supplier catalog number: CSB-E12690h
  • Lot number: C4621151803

Controls

  • Positive control: Human postmenopausal female serum (Biochemed, Lot#BC033016HSPMG)
  • Negative control: Chicken serum (Biochemed, Lot#BC03316CSPMG)
  • Standard curve: 0, 2, 5, 20, 40, 75 pIU/mL LH provided in the ELISA kit
  • Spike control: 75 pIU/mL standard premixed with 4-fold diluted chicken serum in a 1:1 ratio. Lower dilutions of chicken serum produced lower readings.

Protocol

  1. 50 μL of standard and samples were added to 96-well strip plates provided in the kit with 50 μL of HRP conjugate. All samples and standards were assayed in duplicate.
  2. The microplate was covered and incubated at 37°C for 1 hr.
  3. Content of the wells was discarded and wells were washed 3 times with 200 μl of washing solution.
  4. 50 μl of Substrate A and Substrate B each was added to each well. The plate was covered and incubated at 37°C for 15 min.
  5. 50 μl of the Stop Solution was added per well.
  6. The optical density (OD value) of each well was read immediately using a microplate reader set to 450 nm.
  7. The duplicate readings for each sample were averaged and the average zero standard optical density subtracted. The corrected average-value was tabulated as Average Absorbance. A standard curve was generated by plotting the mean optical density (OD) value for each standard on the X-axis against the concentration on the Y-axis using CurveExpert 1.4 (CUSABIO). A logistic equation was used for the best fit through the points on the graph.
  8. The CurveExpert Analyze feature was used to calculate human LH concentrations of the samples based on their Average Absorbance values.

Experimental Notes

  • The concentration of human LH in human and chicken sera was measured according to the manufacturer’s directions.

Figures

Mrymivnzsbmhieisecty lh.png?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 1: Graph of corrected-average absorbance (OD 450 nm) readings plotted for standard curve samples.
Yyioz7jvtwuwjcmetnks screen shot 2016 05 16 at 6.06.59 pm.png?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 2: Table of absorbance readings (OD 450 nm) for standard curve, spike controls, negative (chicken serum) and positive (human serum). Value for Average Reading was derived from the average of two readings (OD 450nm). The Average Reading for blank sample (no conjugate added) was subtracted from all Average Readings to yield Average Absorbance values for Standards, spike controls and control samples. Standard deviation was included for all samples. The concentration of samples was calculated using the Analyze feature of the CurveExpert 1.4 software for a logistic equation fit (Logistic Model: y=a/(1+b*exp(-cx)), a =1.2E+2, b =4.1E+1, c =2.5).
Validation badge

Learn more about our validation initiative.