Direct ELISA Experimental Protocol

Overview

Introduction of Direct ELISA Protocol

The enzyme-linked immunosorbent assay (ELISA) is a technique based on an enzyme-labeled antibody capable of detecting an antigen immobilized to a solid surface, most commonly 96-well or 384-well polystyrene plates. This high-throughput assay relies on unbound materials to be washed off between steps, allowing detection of the enzyme-labeled antibody attached to the antigen. The most commonly used enzymes are alkaline phosphatase (AP) and horseradish peroxidase (HRP). HRP is generally preferred for its lower detection limit. As the technique advances, ELISA has been adopted to other platforms for multiplexing, and the term ELISA has evolved to loosely refer to assays involving antibody detection of analyte. As with any immunoassay, the key to ELISA is the antibody. Antibody affinity and avidity determine the sensitivity and specificity of the assay. Antibody can be employed in several ways in an ELISA assay. It can be directly conjugated to an enzyme to detect plate-immobilized antigen, a form known as direct ELISA. Alternately, a secondary antibody can be used to amplify the signal of the primary antibody bound to the antigen, also known as indirect ELISA. A third method immobilizes antibody onto a plate to capture an antigen in solution followed by detecting with another enzyme-conjugated antibody recognizing a different epitope of the same antigen. This popular method, also known as sandwich ELISA, gives the greatest sensitivity for detecting a specific antigen in a complex sample. A fourth format, also known as competitive ELISA, utilizes a mixture of antibody- antigen and free antibody in liquid phase to interact with plate- immobilized antigens.

Figure 1. Schematic diagram to illustrate the principle of direct ELISA.

This chapter provides the methodology for performing direct ELISA and optimizing performance by chessboard titration. In the direct ELISA, the antigen of interest in fluid phase is immobilized to a microtiter plate through passive absorption. A blocking buffer is added to saturate all unbound sites followed by the binding of enzyme-labeled antibody. Antibody in direct ELISA can be monoclonal or polyclonal. A substrate is added to generate a color change or emit light indicating the presence of antigen. Direct ELISA is useful for qualitative or quantitative antigen detection in a sample, antibody screening, and epitope mapping. Since only one antibody is involved, there is no crossreactivity with secondary antibody, and the assay can be performed in less amount of time compared to other ELISA methods.

Materials of Direct ELISA Protocol

Buffers.

(a) Binding solution: 0.2 M carbonate-bicarbonate, pH 9.4.

(b) Wash solution: Tris-buffered saline (pH 7.4) with 0.1 % (v/v) Tween 20 (TBST).

(d) Dilution buffer: 1 % nonfat dry milk in TBST.

Antigen and antibodies.

(a) Antigen of interest partially purified or purified.

(b) Primary antibody, monoclonal or polyclonal, conjugated to horseradish peroxidase (HRP). Antibody can be purchased in the HRP-conjugated form or self-conjugate with Lightning- Link™ HRP Conjugation Kit.

Supplies and instruments.

(a) White 96-well plates (see Note 3).

(b) 96-well plate cover or adhesive seal.

(d) ELISA plate washer (optional).

(e) Titer plate shaker.

Methods of Direct ELISA Protocol

Antigen Immobilization

Dilute antigen in binding buffer. Antigen should be diluted into a final concentration ranging from 1–100 μg/ml.
Add 100 μl of diluted antigen into each well. Seal the plate with cover or adhesive and incubate for 2 h at room temperature or overnight at 4 °C (see Note 4).
Aspirate off the antigen and wash four times with 200μl wash solution either by hand or ELISA plate washer. This step washes off any unbound antigen.
Optional step: short chemical denaturation (see Note 5).

(a) Prepare 1 M solution of guanidine-HCl.

(b) Add 100μl to each well and incubate for 1 h.

Blocking

Add 200 μl of blocking solution into each well and incubate at room temperature for 30 min to overnight (see Note 6).
Aspirate off the blocking solution. It is not necessary to wash the plate at this point.

Immobilized Antigen Detection by Labeled Antibody

Dilute HRP-conjugated antibody in dilution buffer. HRP conjugated antibody should be diluted into a final concentration of 1 μg/ml or follow manufacturer’s instruction.
Add 100 μl of diluted HRP-conjugated antibody into each well. Seal the plate with cover or adhesive and incubate at room temperature for 1 h.
Aspirate off the antibody and wash four times with 200 μl wash solution either by hand or ELISA plate washer.
Prepare ECL substrate according to manufacturer’s instruction.
Add 50–100 μl of substrate into each well.
Place the plate on a titer plate shaker and incubate for 2 min at room temperature.
Read the light signal using PerkinElmer VICTOR ™ X3 Multilabel Plate Reader.

Optimization by Chessboard Titration

A common phenomenon observed in ELISA is the prozone or hook effect. This occurs when either the antigen or the antibody is used at high concentration but gives false low signal. A simple method of optimizing antigen and antibody concentrations is by chessboard titration.

Titration of Antigen

Fill columns 2–11 with 100 μl of binding solution.
Dilute antigen in binding solution into a final concentration of 100 μg/ml for partially purified sample or 10 μg/ml for purified peptides.
Add 200 μl of diluted antigen into A1–H1.
Aspirate off 100 μl of diluted antigen from wells in column 1 and transfer to wells in column 2. Gently pipette up and down three times to mix the content of the well.
Repeat step 4 and dilute antigen from columns 2–11. Leave column 12 with binding buffer only.
Seal the plate with cover or adhesive and incubate for 2 h at room temperature or overnight at 4 °C.
Aspirate off the antigen and wash four times with 200 μl wash solution either by hand or ELISA plate washer. This step washes off any unbound antigen.
Add 200 μl of blocking solution into each well and incubate at room temperature for 30 min to overnight.
Aspirate off the blocking solution. It is not necessary to wash the plate at this point.

Titration of Antibody

Fill rows B–H with 100 μl of dilution buffer.
Dilute HRP-conjugated antibody in dilution buffer into a final concentration of 1 μg/ml.
Add 200 μl of diluted antibody into all 12 wells of row A.
Aspirate off 100 μl of diluted antibody from row A and transfer to row B. Gently pipette up and down three times to mix the content of the well.
Repeat step 4 and dilute antibody from row B to row G. Leave row H with dilution buffer only.
Seal the plate with cover or adhesive and incubate for 1 h at room temperature.
Aspirate off the antigen and wash four times with 200 μl wash solution either by hand or ELISA plate washer. This step washes off any unbound antibody.
Add 50–100 μl of substrate into each well.
Place the ELISA plate on a titer plate shaker and incubate for 2 min at room temperature.
Read the light signal using PerkinElmer VICTOR™ X3 Multilabel Plate Reader.

Notes of Direct ELISA Protocol

HRP-conjugated antibodies should be stored at −20 °C in 50 % glycerol to prevent decrease in signal.
Sodium azide and compounds containing cyanides and sulfides are inhibitors of HRP. Therefore, reagents used in ELISA should be free of these compounds. 0.01 % thimerosal can be used as an alternative antibacterial reagent.
Chemiluminescent HRP-based assays require black or white opaque plates to minimize leaching of light into neighboring wells. White plates are preferred to amplify light signal. ELISA plates are usually flat bottomed and made from polystyrene or polyvinyl chloride. Plates are manufactured to minimize edge effect, but each batch should still be tested to ensure consistency.
Most protein antigens in binding solution can be refrigerated in plate wells for up to a week without much loss in signal. Care should be taken to seal individual wells to prevent evaporation. This may be convenient for screening large quantities of wells. However, the stability of individual antigen on ELISA plates should be tested before proceeding with long-term refrigeration.
Proteins passively absorbed to polystyrene plates result in random orientation, and aggregation may occur affecting the presentation of epitopes to antibody. Adding guanidine-HCl allows partial unfolding of antigen to unmask epitopes covered by aggregation. However, this step also destroys conformational epitopes. In addition, some antibodies may be sensitive to guanidine- HCl. If this is the case, 1 M urea or 1 M SDS can be used as alternatives.
10 % nonfat dry milk in TBST is a typical blocking reagent used in ELISA. However, it can non-specifically react with some antibodies. Alternative blocking agents such as BSA, fish gelatin, or commercial nonprotein-based blocks can be used. A nonprotein- based block has the advantage of not containing IgG which potentially makes nonspecific interaction with antibodies.