Validation Report #029475

Share results:

Angiopoietin-like 2 (ABIN578045) 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: #029475
Validated on: 12/03/13

Summary

Antigen

Angiopoietin-like 2 (ANGPTL2)

Catalog number
Supplier

Cusabio

Supplier catalog number
Lot number

T30092379

Method validated
Laboratory
Validation number
Positive Control

Human sera

Negative Control

Goat Sera

Notes

Signal was detected in positive control samples but not in negative control samples.

Independent Results

Full Methods

Primary Antibody

  • Antigen: Angiopoietin-Like 2 (ANGPTL2)
  • Catalog Number: ABIN578045
  • Supplier: Cusabio
  • Supplier Number: csb-e13881h
  • Lot Number: T30092379

Controls

  • Positive control: Serum from normal adult human (specimen known to contain the target
    protein).
  • Negative control: Serum from normal goat (specimen known to not contain the target protein).
  • Standard curve: Serial two-fold dilutions from 100 ng/ml [100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0]
    were generated from the standard provided in the kit using standard/sample diluent buffer.
  • Spike control: Standard diluted in standard/PBS diluent buffer [50 and 0].

Protocol

  • All reagents in the ELISA kit were brought up to room temperature (RT) before use.
  • 100 µL of standard or sample were added to wells in ELISA plate pre-coated with capture
    antibody. All samples and standards were assayed in triplicate.
  • The plate was covered with sealer (provided in kit) and incubated for 2 h at 37°C.
    Unbound material was aspirated and 100 µL of Biotin-Antibody (diluted 1:100 in “Diluent for
    Biotinylated Detection Ab”) was added to each well. Plate was sealed and incubated for 1 h
    at 37°C. Unbound Biotin-Antibody was removed from each well and plate was washed three times with 350 µL of wash buffer (provided in the kit). After the last wash the plate was inverted
    against clean absorbent paper to remove any remaining liquid.
  • 100 µL of HRP-Conjugate (1X) was added to each well. Plate was sealed and incubated for 1
    h at 37°C.
  • After 1 h incubation at 37°C, unbound HRP-Avidin was removed by washing five times
    with 350 µL of wash buffer (provided in the kit). After the last wash the plate was inverted
    against clean absorbent paper to remove any remaining liquid.
  • 90 µL of TMB substrate was added to wells and the plate was covered with a new plate sealer.
    The plate was tapped to ensure mixing and incubated for 25 min at 37°C in the dark.
  • After 25 min, 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 microplate reader set to 450 nm.
  • The triplicate readings for each sample were averaged and the average zero standard optical
    density subtracted to yield ‘corrected absorbance at 450 nm’. 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 Excel. Standard curve was generated by regression analysis with
    four-parameter logistic.
  • An equation (y = 11.973x4 - 32.453x3 + 42.158x2 + 32.504x + 0.073) was derived from the
    standard curve and used to calculate Angiopoietin-Like 2 (ANGPTL2) concentrations in
    samples based on their Average Absorbance values.

Experimental Notes

Nothing to note.

Figures

1394136245360 bbde4908f2f751db85143bc66802cd77.jpg?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 1: Graph of Corrected OD450 nm plotted for standard curve samples.
1394136245366 11ed046a313a7395028c09d523132e2e.jpg?fit=fill&mark=%2freproducibility%2ftsen logo
Table 1: ELISA. ANGPTL2 is present in Human serum and undetectable in goat serum. Spike controls indicate no interference in absorbance readings from the diluent used to prepare standards and sera samples. Absorbance readings (OD450 nm) are shown for standard curve, spike controls and unknown samples. Value for avg reading is derived from the average reading of three samples. Avg reading for 0 ng/ml Standard was subtracted from all avg readings to yield “Corrected OD450 nm “values for Standards, spike controls and unknown samples. Standard deviation is included for all samples. Standard curve was generated by regression analysis with four-parameter logistic. An equation (y = 11.973x4 - 32.453x3 + 42.158x2 + 32.504x + 0.073 ) was derived from the standard curve and used to calculate ANGPTL2 concentrations shown in the Table 1.
Validation badge

Learn more about our validation initiative.