Human Resistin ELISA Development Kit

(Catalog #K4559-1000; 10 x 96 well plates; Store kit at –20^oC)

I. Description:

Human Resistin ELISA Development Kit contains the key components required for the quantitative measurement of natural and/or recombinant human Resistin in a sandwich ELISA format within the range of 16-2000 pg/ml. Using the ELISA protocol described below, the recommended microplates, reagents and solutions, the components supplied in this kit are sufficient to assay hResistin in approximately 1000 ELISA plate wells.

 

1. 
   

   Kit Components:

   
   

Capture Antibody: 200 μg of affinity purified anti-hResistin. Reconstitute in 1-ml sterile water for a concentration of 200 μg/ml.

Detection Antibody: 25 μg of biotinylated affinity purified anti-hResistin. Reconstitute in 0.25 ml sterile water for a concentration of 100 μg/ml.

Human Resistin Standard: 1 μg of recombinant hResistin. Reconstitute in 1 ml sterile water for a concentration of 1 μg/ml.

Avidin Peroxidase Conjugate: 60 μl vial.

 

2. 
   

   Storage Conditions:

   
   

The reconstituted Capture Antibody and Detection Antibody can be stored at 4^oC for up to 2 months or aliquoted and stored frozen at < –20^oC for up to 6 months.

Reconstituted hResistin Standard can be kept at 4^oC for 1 week, or aliquoted and stored frozen at < –20^oC for up to 2 months.

Upon receipt, Avidin Peroxidase Conjugate should be aliquoted into ten 6 μl vials and stored at < –20^oC for up to 2 years.

 

3. 
   

   Recommended Materials:

   
   

ELISA microplates (NUNC Maxisorp, Cat.# 4420404);

Tween-20 (BioVision, Cat.# 2109-100);

BSA (BioVision, Cat.# 2119-10);

ABTS Liquid Substrate Solution (Sigma, Cat.# A3219);

PBS [10X] (BioVision, Cat.# 2113-500). 

Recommended Solutions:

PBS: Dilute 10X PBS to 1PBS in ddH₂O

Wash Buffer: 0.05% Tween-20 in PBS

Block Buffer: 1% BSA in PBS

Diluent: 0.05% Tween-20, 0.1% BSA in PBS

 

4. 
   

   Plate Preparation:

   
   

0. 
   

   Dilute Capture antibody with PBS to a concentration of 2 μg/ml. Immediately, add 100 μl to each ELISA plate well. Seal the plate and incubate overnight at room temperature.

   
   


1. 
   

   Aspirate the wells to remove liquid and wash plates 4 times using 300 μl of wash buffer per well. After the last wash invert plate to remove residual buffer and blot on paper towel.

   
   


2. 
   

   Add 300 μl block buffer to each well. Incubate for at least 1 hour at room temperature.

   
   


3. 
   

   Aspirate and wash plate 4 times.

   
   

 

 

 

 

5. 
   

   ELISA Protocol:

   
   

Standard/Sample: Serial dilute standard from 2 ng/ml to zero in diluent. Immediately add 100 μl of standard or sample to each well in triplicate. Incubate at room temperature for at least 2 hours.

Detection: Aspirate and wash plate 4 times. Dilute detection antibody in diluent to a concentration of 0.125 μg/ml. Add 100 μl per well. Incubate at room temperature for 2 hours.

Avidin Peroxidase: Aspirate and wash plate 4 times. Dilute one 6 μl aliquot of Avidin Peroxidase 1:2000 in diluent for total volume of 12 ml. Add 100 μl per well. Incubate 30 minutes at room temperature.

ABTS Liquid Substrate (ABTS Substrate should be at ambient temperature prior to use): Aspirate and wash plate 4 times. Add 100-μl substrate solution to each well. Incubate at room temperature for color development. Monitor color development with an ELISA plate reader at 405 nm with wavelength correction set at 650 nm.

Note: Reliable standard curve are obtained when either O.D. readings do not exceed 0.2 units for the zero standard concentrations, or 1.2 units for the highest standard concentration. The plate should be monitored at 5 minute intervals for approximately 30 minutes.