Validation Report #029584

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Defensin, alpha 3, Neutrophil-Specific (DEFa3) ELISA Kit (ABIN419782) has been independently validated for ELISA by the Science Exchange network of experts.

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

Validation number: #029584
Validated on: 01/22/14

Summary

Antigen

Human Defensin, alpha 3, Neutrophil-Specific (DEFa3)

Catalog number
Supplier

USCNK

Supplier catalog number
Lot number

L131225503

Method validated
Laboratory
Validation number
Positive Control

Human serum

Negative Control

Mouse serum

Notes

Minimal levels of signal detected in negative control. Percent recovery of the spiked samples shows that there is matrix interference. Dilution of >50 fold is required for accurate measurement of the analyte in human serum samples.

Independent Results

Full Methods

Primary Antibody

  • Antigen: Defensin, alpha 3, Neutrophil-Specific (DEFa3)
  • Catalog number: ABIN419782
  • Supplier: USCNK
  • Supplier catalog number: SEE135Hu
  • Lot number: L131225503

Controls

  • Positive control: normal human serum
  • Negative control: mouse serum
  • Standard curve: serial two-fold dilutions from 30 ng/ml [30, 15, 7.5, 3.75, 1.875, 0.9375, 0] were generated from the standard provided in the kit using standard diluent buffer.
  • Spike control: standard diluted in human or mouse serum [6 ng/mL].
  • Control lyophilized powder was dissolved in 150uL standard diluent buffer (7.39 ng/mL)

Protocol

  • All reagents in the ELISA kit were brought up to room temperature (RT) before use.
  • 100 µl of each sample was added per well to the micro ELISA plate well. All samples and standards were assayed in triplicate.
  • The plate was covered with sealer (provided in kit) and incubated for 120 mins at 37°C.
  • Liquid was removed from each well by pipette.
  • Detection Reagent A was diluted 100 fold in Assay Diluent A. 100 µl of diluted detection reagent A was added to each well and the plate was sealed. The plate was tapped to ensure mixing and incubated for 60 mins at 37°C.
  • Wells were washed with 300 µl wash buffer three times. Each wash involved fully aspirating the liquid from each well by pipette. After the last wash the plate was inverted against clean absorbent paper to remove any remaining liquid.
  • Detection Reagent B was diluted 100 fold in Assay Diluent B. 100 µl of diluted detection reagent B was added to each well and the plate was sealed. The plate was tapped to ensure mixing and incubated for 30 mins at 37°C.
  • Wells were washed with 300 µl wash buffer five times. Each wash involved fully aspirating the liquid from each well by pipette. After the last wash the plate was inverted against clean absorbent paper to remove any remaining liquid.
  • 90 µl of Substrate Solution was added to each well and the plate was covered with a new plate sealer. The plate was tapped to ensure mixing and incubated at room temperature in the dark.
  • After about 10 mins, when an apparent gradient appeared in the standard wells, the reaction was terminated by adding 50 µl of Stop Solution to each well.
  • The optical density (OD value) of each well was read using a micro-plate reader set to 450 nm.
  • The triplicate readings for each sample were averaged and the average zero standard optical density subtracted. A standard curve was generated by plotting the mean OD value for each standard on the y-axis against the concentration on the x-axis using Softmax Pro softare.
  • The equation y = (A-D)/(1 + (x/C)^B) + D was used to calculate IL-6 concentrations of the samples based on their average OD values.

Experimental Notes

  • Percent recovery of the spiked samples shows that there is matrix interference. Dilution of >50 fold is required for accurate measurement of the analyte in human serum samples.

Isotype Control Antibody

Secondary Antibody

Additional Information

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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Table 1: ELISA. DEFa3 could be detected in human serum (positive control) after 10 fold dilution. Spike controls indicate that there is interference from the human serum matrix and a dilution of >50 fold is required. Mouse serum was used as negative control, there were residual levels of DEFa3. Control sample concentration is expected to be 7.39 ng/mL
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Table 2: Value for Average Reading is derived from the average of three readings (OD 450nm). The Average Reading for BLANK (0 ng/ml) was subtracted from all Average Readings to yield Average Absorbance values for Standards. Standard deviation is included for all samples. An equation y = (A-D)/(1 + (x/C)^B) + D was generated from the standard curve and used to calculate DEFa3 concentrations shown in the Figure 1.
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