ELISA LAB

core principle is the highly specific antigen-antibody interaction, with detection via a colorimetric change produced by a reporter enzyme.

What is the principle behind ELISA?

ELISA

plate-based assay designed to detect and quantify soluble substances such as peptides, proteins, antibodies, and hormones

Direct ELISA

Antigen is immobilized, and an enzyme-linked primary antibody binds directly.

Indirect ELISA

A primary antibody binds the antigen, followed by an enzyme-linked secondary antibody for detection.

Indirect ELISA

increases sensitivity but involves more steps.

Serial dilution

stepwise dilution where each step has the same dilution factor, used to determine the approximate concentration of antibodies in a sample.

ensures sample concentration falls within the assay's detectable range.

what is the purpose of serial dilution in ELISA

Start with tube 1: 5 µL ATS + 240 µL buffer (approx. 1:48).
Add 120 µL buffer to tubes 2–7.
Transfer 120 µL from one tube to the next, mixing each time.
Tube 8 serves as a negative control.

Describe the two-fold dilution procedure used in the experiment.

Coating with antigen (tetanus toxoid)
Blocking nonspecific sites with skim milk
Sample application (anti-tetanus antibody)
Secondary antibody (PAPO) application
Substrate (TMB) addition and stopping with H₂SO₄

What are the five main steps in the ELISA procedure?

TMB (Tetramethylbenzidine)

reacts with horseradish peroxidase to produce a blue color

yellow, presences of anti-tetanus antibodies

If the reaction of TMB (Tetramethylbenzidine) with horseradish peroxidase is stopped with acid, what does it indicate? what color?

By reading absorbance at 450 nm using a microplate reader and comparing values against a standard curve to determine anti-tetanus antibody concentration.

How is ELISA quantification achieved?

coating buffer (carbonate-bicarbonate, pH 9.4)

facilitates the adsorption of the antigen (tetanus toxoid) onto the polystyrene surface of the ELISA plate.

PAPO

horseradish peroxidase-conjugated secondary antibody that binds to human IgG, enabling colorimetric detection upon substrate (TMB) addition.

Protein A and Protein G

These are microbial proteins (from S. aureus and Streptococcus, respectively) that bind to the Fc region of antibodies.

Protein A and Protein G

Used in “poor man’s ELISA” as a less specific but cost-effective alternative to anti-human goat antibodies.

washing

removes unbound substances

false positives

Insufficient washing can cause

sensitivity

excessive washing can lower

Use forward pipetting.
Mix by pipetting up and down.
Avoid touching tube walls.
Maintain 90° angle.
Ideally use fresh tips per component to prevent contamination.

What are pipetting best practices during ELISA?

Coating step: 1–2 hours at RT or overnight at 4°C
Sample and PAPO steps: 1–2 hours ideally, shortened to 15–30 minutes due to time constraints.

What is the optimal incubation time and temperature for ELISA?