---
title: "ELISA Assay Explained: Principle, 4 Types & Plate Selection"
canonical: "https://www.biomat.it/applications-techniques/elisa-assay/"
description: "How ELISA works: principle, the 4 assay formats, ELISA vs CLIA vs FIA, and how to select the right microplate surface. Technical guide by Biomat."
type: "technical-guide"
author: "Alice Marmieri — Head of Strategy & Scientific Communication, Biomat srl"
manufacturer: "Biomat S.r.l."
updated: "2026-07"
---

# ELISA Assay: Principle, Types and Plate Selection

## What is an ELISA assay?

The Enzyme-Linked Immunosorbent Assay (ELISA), also called Enzyme Immuno Assay (EIA), detects and quantifies specific biomolecules — antigens, antibodies, hormones — in biological fluids such as serum, plasma, saliva and urine.

It uses a biomolecule (typically an antigen or antibody) labelled with an enzyme (e.g. peroxidase or alkaline phosphatase), forming a conjugate with both immunological and enzymatic activity. One component is immobilised on a plate; when a substrate reacts with the enzyme it produces a colour change measurable by spectrophotometer.

ELISA is among the most sensitive and reproducible plate-based technologies, and is rapid and easily automated.

## How the test works

1. **Coating** — a specific target is passively adsorbed onto a 96-well polystyrene plate (the solid phase).
2. **Conjugate binding** — the bound target is complexed with an excess of enzyme-linked specific anti-target (the conjugate).
3. **Incubation and washing** — excess unbound conjugate is washed away.
4. **Detection** — enzyme activity of the bound conjugate is measured using a chromogen-substrate that changes colour; light absorption is converted to numeric values.

## The 4 types of ELISA

| Type | Configuration | Target being evaluated |
| --- | --- | --- |
| **Direct** | Target antigen bound to the well, recognised by an enzyme-labelled primary antibody | The antigen bound to the well |
| **Indirect** | Antigen bound to the well; antigen-specific primary antibody added, then a secondary enzyme-conjugated antibody binds the primary | The primary antibody |
| **Competitive** | Antigen bound to the well; sample (containing the same antigen) plus conjugated anti-antigen antibody added. Sample antigen competes with coated antigen — more antigen means lower signal | The inhibitor antigen |
| **Sandwich** | Two antibodies against two different epitopes: a capture antibody bound to the well, an enzyme-conjugated detection antibody | The antigen |

## ELISA vs CLIA vs FIA

CLIA and FIA share the same reaction principle as ELISA but differ in reagents and microplate type:

| Technique | Detection | Plate colour |
| --- | --- | --- |
| **ELISA** | Enzyme-linked, colorimetric | Clear |
| **CLIA** | Chemiluminescence | White |
| **FIA** | Fluorescence | Black |

They are three methodologies of the same macro-category (immunological tests), not three different products. The base plate is the same; the surface may be the same or different.

## Key applications

- **Infectious diseases:** antibodies against bacterial, viral or fungal infections
- **Tumour markers:** assessing cancer presence
- **Autoimmune diseases:** measuring autoantibodies
- **Allergic diseases:** antibodies against allergens (rhinitis, asthma, dermatitis)
- **Endocrinology:** hormones (LH, FSH, Prolactin, T3, T4, TSH, Testosterone, Cortisol, hCG)
- **Blood transfusion screening:** Hepatitis A/B/C, HIV, Syphilis

## ELISA microplates: design and material

Made of pure low-fluorescence polystyrene. Mould design optimises optical quality to reduce background; radius-edged inner well bottoms improve washing efficiency; the rim protects the bottom from scratches; SBS-compliant for automated processing.

- **Well capacities:** 350 µl, 360 µl, 400 µl
- **Formats:** breakable strip, strip (12×8), solid
- **Colours:** transparent (ELISA), white (luminescence), black (fluorescence)

Polystyrene is naturally hydrophobic (Medium Binding). Surface treatment such as irradiation breaks benzene rings, yielding carboxyl (COOH) and hydroxyl (OH) groups that allow hydrophilic/ionic interactions (High Binding). Passive adsorption forces, in increasing strength: hydrophobic interactions, ionic interactions, van der Waals forces, hydrogen bonding.

## High binding vs medium binding

| | High Binding | Medium Binding |
| --- | --- | --- |
| Surface | Hydrophilic (chemically modified) | Hydrophobic polystyrene |
| Binding capacity | 400–500 ng IgG/cm² | 100–200 ng IgG/cm² |
| Best for | Proteins of varying hydrophilicity; maximum sensitivity | Proteins with hydrophobic regions, e.g. antibodies > 10–20 kDa |
| Uniformity | CV < 5% | CV < 5% |

High Binding is highly selective and shows high affinity even at very low amounts (efficient coating from ~1 µg/ml; coupling with an inert protein such as BSA helps stabilise at low concentrations).

Uniformity data measured with HIgG coating, anti-HIgG-HRP detector, TMB substrate.

## Which surface for which target

- Hydrophobic biomolecule > 10 kDa → [Medium Binding](https://www.biomat.it/product/microplates/96-well-plates/medium-binding-96-well-plates/)
- Hydrophilic biomolecule > 10 kDa → [High Binding](https://www.biomat.it/product/microplates/96-well-plates/high-binding-96-well-plates/)
- Biotinylated biomolecule → [Streptavidin](https://www.biomat.it/product/microplates/96-well-plates/streptavidin-plates/)
- Mammalian IgG → Protein A / Protein G plates (Fc binding)
- Peptide < 5 kDa with NH2 group → [Carboxylated](https://www.biomat.it/product/microplates/96-well-plates/carboxylated-plates/) (covalent via EDC)
- Peptide < 5 kDa with COOH group → [Aminated](https://www.biomat.it/product/microplates/96-well-plates/amine-activated-96-well-plates/) (covalent via EDC)
- Human immunoglobulin quantification → [Anti-Human IgG/IgM/IgA](https://www.biomat.it/surfaces/anti-human-igg-igm-iga/)

## Quality

Medical-grade raw materials; plates moulded in clean conditions under strict quality control; all lots tested. Validation against a competitor on 232 sera (94 positive, 128 negative) confirmed 100% concordance of sensitivity and specificity; regression analysis met R ≥ 0.95.

## ELISA test example

A typical indirect ELISA calibration curve (anti-Tetanus Toxoid antibodies, plate coated with Tetanus Toxoid antigen) shows optical density increasing proportionally with target concentration. Using an HRP conjugate with TMB + H₂O₂ as chromogen/substrate, a yellow colour indicates the target protein is present — the more intense the colour, the higher the concentration.

## Frequently asked questions

**What coatings are available for your 96-well ELISA plates?**
High-binding (optimal protein/antibody adsorption), medium-binding (general biomolecule applications), low-binding (minimises non-specific interactions), plus customised coatings on request. Over 24 coated surfaces are available.

**What is the difference between Medium Binding and High Binding microplates?**
Medium Binding plates are purely hydrophobic polystyrene interacting mainly through hydrophobic interactions. High Binding plates have a mixed hydrophobic/hydrophilic surface from chemical modification, enabling stronger adsorption through ionic bonds and Van der Waals forces — suited to different biomolecules by polarity.

**How is consistency and reliability ensured?**
Each batch undergoes strict QC including coating uniformity, well-depth accuracy and optical clarity, for high reproducibility across ELISA formats. A Certificate of Quality is released for every lot.

**Can custom formats or modifications be ordered?**
Yes — different well geometries, colours, surface chemistries, optional barcoding and bespoke packaging. See [Custom Product Development](https://www.biomat.it/services/custom-microplates/).

**What is the shelf life and how should plates be stored?**
Up to 24 months when stored correctly: sealed in original packaging, protected from sunlight, at 2–8°C.

**Is technical support provided for protocol optimisation?**
Yes — assistance with coating recommendations, sample/reagent handling, plate setup and troubleshooting, plus technical documentation and expert consultation.

## Related

- [Choosing the Right Microplate](https://www.biomat.it/applications-techniques/choosing-microplate-for-assay/)
- [Medium and High Binding Well Plates: adsorption test data](https://www.biomat.it/applications-techniques/medium-and-high-binding-well-plates/)
- [All 96-well plates](https://www.biomat.it/our-products/microplates/96-well-plates/) · [All surfaces](https://www.biomat.it/surfaces/)
- [ELISA reagents](https://www.biomat.it/our-products/reagents-for-elisa/)

For Research Use Only.

## Manufacturer

Biomat S.r.l., Via Trento 124, 38061 Ala (TN), Italy. ISO 9001:2015 certified. Founded 1992. Contact: info@biomat.it | +39 0464 357951 | [Request a quote](https://www.biomat.it/request-quote/)