Validation Report #029855

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Human β2-microglobulin (B2M) ELISA Kit (ABIN365026) 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: #029855
Validated on: 04/01/16

Summary

Antigen

Human β2-microglobulin (B2M)

Catalog number
Supplier

Cusabio

Supplier catalog number
Lot number

C738151804

Method validated
Laboratory
Validation number

29855

Positive Control

Human pooled serum (Biochemed, Lot#BC033016HSPMG)

Negative Control

Chicken serum (Biochemed, Lot#BC03316CSPMG)

Notes

β2-microglobulin was easily detected in diluted and undiluted positive samples (up to 8-fold dilutions). Very low signal was seen in the negative control. Spike showed very good recovery (~82%).

Independent Results

Full Methods

ELISA kit

  • Antigen: Human β2-microglobulin (B2M)
  • Catalog number: ABIN365026
  • Supplier: Cusabio
  • Supplier catalog number: CSB-E04883h
  • Lot number: C738151804

Controls

  • Positive control: Human pooled serum (Biochemed, Lot#BC033016HSPMG)
  • Negative control: Chicken serum (Biochemed, Lot#BC03316CSPMG)
  • Standard curve: 0, 0.025, 0.1, 0.5, 2.5, 10 μg/mL β2-microglobulin provided in the ELISA kit
  • Spike control: 2.5 μg/mL standard premixed with chicken serum in a 1:1 ratio

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 OD value for each standard on the x-axis against the concentration on the Y-axis using CurveExpert 1.4 (CUSABIO). An exponential equation was used for the best fit through the points on the graph.
  8. The CurveExpert Analyze feature was used to calculate human β2-microglobulin concentrations of the samples based on their Average Absorbance values.

Experimental Notes

  • The concentration of human β2-microglobulin in human and chicken sera was measured according to the manufacturer’s directions.

Figures

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Figure 1: Graph of corrected-average absorbance (OD 450 nm) readings plotted for standard curve samples.
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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 an exponential equation fit (Concentration = ae^(bx), a =5.1E+001, b =-1.3E+001)
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