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Chemiluminescent Sandwich ELISA - Protocol

The LumiAb Chemiluminescent Sandwich ELISA is our newest ELISA kit. It utilizes the traditional sandwich immunoassay platform that generates rapid light output as a signal for a variety of targeted biological samples such as cell lysates, sera, and supernatants We are happy to introduce LumiAb as a trusted tool to provide a convenient and reliable solution for our customers research needs.

Introduction:

The LumiAb™ Chemiluminescent ELISA Kit contains the components necessary for quantitative determination of natural or recombinant protein concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a “Sandwich” Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a “sandwich” format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator.

The capture antibodies coated to the bottom of each well are specific for a particular epitope on proteins while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase.

After incubation and “sandwiching” of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a sensitive luminescent reaction to ensue upon substrate addition.

When the Peroxide Enhancer solution is added, the reaction catalyzed by peroxidase yields light that is representative of the antigen concentration. After a brief incubation, the microplate can be read with a luminometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Materials:

  • Reagent
  • Microstrips Coated w/ Capture Antibody
  • Protein Standard
  • Biotinylated Dectection Antibody
  • 400x Streptavidin-HRP
  • 15x Wash Buffer
  • Assay Diluent
  • Enhancer Solution
  • Peroxide Solution
  • Adhesive Plate Sealers


  • Quantity
  • 12 x 8-Well Microstrips
  • Varies (Lyophilized)
  • Varies (Lyophilized)
  • 30 μl (400x)
  • 50 ml (15x)
  • 50 ml (1x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 2 Sheets

Assay Protocol:

1. Dilute Protein Standard with Assay Diluent within the range of the Protein Standard in a series of microfuge tubes. Mix each tube thoroughly by inverting several times or by vortexing lightly to ensure proper equilibration. Add 100 μl of each serial dilution step into the wells of a specified row or column of the 96-well microtiter plate in duplicate or triplicate and incubate at room temperature for 2 hours. Unknown Samples of Interest can be serial diluted with Assay Diluent to concentrations within the detection range of this assay kit and added to the plate at 100 μl per well. Blank Control is defined as 100 ul of Assay Diluent per well. Seal the microplate air-tight using parafilm if readily available.

2. Aspirate the protein standard solution out of the microplate wells. If your lab does not have a vacuum-based aspirator, you may dump the solutions from the microplate into a waste container and blot 3-4 times on a stack of paper towels until most or all of the liquid is removed from the wells. Dilute the 15x Wash Buffer to 1x using pure H2O. Add 300-400 μl of 1x Wash Buffer to each well being used and gently shake for 5-7 minutes on an orbital shaker. Perform this wash step 4 times consecutively.

3. After the 4th wash step, dilute the detection antibody solution 100x to 1x with Assay Diluent. Ensure that there is enough detection antibody solution for all wells being used Mix the test tube either by inverting several times or vortexing to ensure proper equilibration. Ensure that there is enough detection antibody solution for all wells being used. Add 100 μl of the diluted detection antibody solution into each well, seal the plate and incubate at room temperature for 2 hours.

4. Remove the detection antibody solution out of the microplate wells by either vacuum-based aspirator or paper towel blotting. Perform 4 consecutive wash steps with gentle shaking between each wash.

5. Dilute the 400x Streptavidin-HRP by 1:400 using Assay Diluent to a 1x Streptavidin-HRP solution.

6. After the 4th wash step, add 100 μl of 1x Streptavidin-HRP solution into each well and incubate at room temperature for 30 minutes.

7. Remove the 1x Streptavidin-HRP solution out of the microplate wells by either vacuum-based aspirator or paper towel blotting. Perform 4 consecutive wash steps with gentle shaking between each wash. Prepare the Peroxide and Enhancer Solution by bringing it to room temperature without exposure to light as these may degrade the substrate. Mix the Peroxide and Enhancer solution in a 1:1 ratio andstore in room temperature.

8. After the 4th wash step, add 100 μl of the Peroxide/Enhancer solutions into each well and incubate at room temperature for light development for 5 minutes. The microplate should be kept out of direct light by either covering with an opaque object or putting it into a dark room. The microplate is now ready to be read by the luminometer.