Direct ELISA (Protocol)

Direct ELISA process

ELISA is a plate based assay technique which is used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. It can be of various types such as direct, indirect, sandwich and competitive.

Direct ELISA is a variant of enzyme assay where only one antibody is used which is conjugated directly to the detection enzyme for determining the presence and quantity of an antigen.

In this test, known antigen is coated and immobilized onto wells of microtiter plates. Enzyme conjugated antibody is added to wells and is allowed to react after which washing is done. Suitable substrate is added which on reaction with enzyme gives color reaction.


Direct Enzyme Assay involves following steps:

  • Coating of antigen into plate surface by passive adsorption.
  • Binding of antigen with enzyme conjugated antibody. Washing off unbound antibodies.
  • Addition of substrate for detection of antigen-antibody complex.


Direct ELISA Process


The common procedure for Direct ELISA is outlined as follows:

  1. Coating ELISA plate with testing antigen. Seal plate and incubate overnight at 4°C.


  1. Washing of plate 3 times with Phosphate Buffer Saline-Tween solution (0.05 % Tween-20 in PBS).


  1. Blocking of plate with 0.2% non-fat dry milk in PBS at room temperature for 1 hour at 4°C


  • Note: Milk should be thoroughly dissolved ( 0.5 g milk in 50 ml PBS (1%) for at least 30 minutes at room temperature with rotation or stirring, then dilute to 0.2 %, and keep rotating or stirring for another 10-15 minutes at room temperature).


  • If high background is experienced, 1% milk in PBS could be applied for both blocking and antibody dilution.


Direct ELISA steps


  1. Washing of plate 3 times with Phosphate Buffer Saline-Tween solution.


  1. Incubation with biotinylated, affinity-purified rabbit IgG at room temperature for 1 hour, followed by washing 6 times with Phosphate Buffer Saline-Tween solution.


  1. Incubation with HRP-Streptavidin (1:4000-10,000 dilutions) in 0.2 % milk-Phosphate Buffer Saline (100 μl/well) at room temperature for 1 hour.


  1. Washing of plate for 8 times with Phosphate Buffer Saline-Tween solution.


  1. Development of color using 3,3’,5,5’-tetramethylbenzidine (TMB) as a substrate (100 μl/well) and incubate at room temperature for 15-30 minutes without shaking.


  1. Stop reaction by addition of 2N H2S04 (100 μl/well).


  1. Recording the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.


Download Link: Direct ELISA Protocol.pdf

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