Validation Report #029608

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Cystatin C (CST3) ELISA Kit (ABIN365082) 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: #029608
Validated on: 02/11/14

Summary

Antigen

Human Cystatin C (CST3)

Catalog number
Supplier

Cusabio

Supplier catalog number
Lot number

S29095913

Method validated
Laboratory
Validation number
Positive Control
Negative Control

Rat urine

Notes

Signal was detected in positive control sample and not in negative control sample.

Independent Results

Full Methods

Primary Antibody

  • Antigen: Cystatin C (CST3)
  • Catalog number: ABIN365082
  • Supplier: Cusabio
  • Supplier catalog number: CSB-E08384h
  • Lot number: S29095913

Controls

  • Positive control: Urine from normal adult human (specimen known to contain the target protein).
  • Negative control: Urine from rat (specimens known to not contain the target protein).
  • Standard curve: Serial two-fold dilutions from 500 ng/ml [500, 250, 125, 62.5, 31.2, 15.6, 7.8, 0] were generated from the standard provided in the kit using standard/sample diluent buffer.
  • Spike control: Standard diluted in standard/PBS diluent buffer [62.5 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 hours at 37°C. Unbound material was aspirated but the wells were NOT Washed.
  • 100 µL of Biotin-Antibody (diluted 1:100 in “Biotin-Antibody Diluent”) was added to each well. Plate was covered with sealer (provided in kit) and incubated for 1 hour 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-Avidin Conjugate (diluted 1:100 in “HRP-Avidin Diluent”) was added to each well. Plate was covered with sealer (provided in kit) and incubated for 1 hour 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 and blotted 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 gently tapped to ensure mixing and incubated for 30 min at 37°C in the dark.
  • After 30 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 = -1106.2x4 + 2631.5x3 - 1044.9x2 + 234.72x - 1.4717) was derived from the standard curve and used to calculate CST3 concentrations in samples based on their Average Absorbance values.

Experimental Notes

None

Figures

1392253181194 de4ad1a243080dd25d2c38fc8550e0f0.png?fit=fill&mark=%2freproducibility%2ftsen logo
Figure 1: Graph of corrected OD 450 nm plotted for standard curve samples.
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Table 1: ELISA. Cystatin C (CST3) is present in human urine and undetectable in rat urine. Spike controls indicate no interference in absorbance readings from the diluent used to prepare standards and sera samples. Absorbance readings (OD 450 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” amount of 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 = -1106.2x4 + 2631.5x3 - 1044.9x2 + 234.72x - 1.4717) was derived from the standard curve and used to calculate CST3 concentrations shown in Table 1.
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