ELISA Plate Reader (Microplate Reader) - Principle, Parts, Uses

What is ELISA?

ELISA (Enzyme-Linked Immunosorbent Assay) is a laboratory technique used to detect and quantify antigen or antibody in a given sample.

It is an immunological test in which specific antigen-antibody reaction is used for detection. It is commonly used for detecting toxins, viruses, hormones, proteins and other foreign substances in blood, cell lysate, food sample and other biological samples.

In this method, antibody is attached to the bottom of a microplate well. The sample is then added, and the target antigen binds with the specific antibody. This forms antigen-antibody complex.

After this, an enzyme linked antibody or enzyme linked molecule is added. The enzyme acts on a colourless substrate and converts it into a coloured product. The development of colour indicates the presence of antigen or antibody in the sample.

The intensity of colour is directly proportional to the amount of antigen present in the sample. More colour indicates more antigen and less colour indicates less antigen. The colour intensity is measured by using a microplate reader.

There are mainly four types of ELISA, which are as follows-

Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

ELISA is widely used in diagnosis of infectious diseases, detection of antibodies, antigen detection, food testing, vaccine study and other laboratory research works.

What is ELISA reader?

ELISA reader is a laboratory instrument used to read and measure the result of ELISA test.

It is also known as absorbance microplate reader. It is used to measure the absorbance of liquid samples present in microplate wells. Most commonly, 96 well microplate is used, which helps to test many samples at the same time.

In ELISA reader, a specific wavelength of light is passed through each well of the microplate. The wavelength is selected by using optical filter or monochromator. The detector present on other side measures how much light is absorbed by the sample.

This absorbed light is recorded as optical density (OD). In colorimetric ELISA, the enzyme reacts with substrate and produces colour change. The intensity of this colour is measured by ELISA reader.

The colour intensity is directly proportional to the amount of antigen or biological molecule present in the sample. More colour gives more OD value and less colour gives less OD value.

ELISA reader is simple to use and gives quick quantitative result. It is commonly provided with software for calculation and analysis of result. Most ELISA readers measure absorbance, but some advanced readers can also detect fluorescence and luminescence.

Purpose of an ELISA reader

The following are the purpose of ELISA reader-

To detect and quantify ELISA reaction.
To measure the absorbance of sample present in microplate well.
To read the colour change formed after enzyme-substrate reaction.
To measure the intensity of colour in each well.
To determine the amount of antigen or antibody present in the sample.
To compare the optical density (OD) value of test sample with standard or control.

To read many samples at same time by using 96 well microplate.
To use in direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA.
To quantify biological molecules such as protein, antibody, hormone, glycoprotein, DNA and RNA.

To use in other laboratory tests like cell viability test, cell proliferation test, MTT assay, XTT assay, enzyme activity test and reporter gene assay.

Principle of ELISA Plate Reader

ELISA plate reader is based on the principle of absorbance or colorimetric detection.

It is based on Beer-Lambert law. In this method, the enzyme present in ELISA reaction converts a colourless substrate into coloured product. The intensity of this colour depends on the amount of target antigen or antibody present in the well.

In ELISA plate reader, a particular wavelength of light is passed through the sample present in microplate well. The light source, optical filter and detector are used for this reading.

Some amount of light passes through the coloured solution and reaches the detector. Some amount of light is absorbed by the coloured sample. This absorbed light is measured as optical density (OD).

More colour formation causes more absorption of light and gives higher OD value. Less colour formation causes less absorption of light and gives lower OD value.

The OD value is directly proportional to the concentration of target molecule present in the sample. The unknown concentration is calculated by comparing the OD value with standard curve.

Types of Elisa Plate Readers

The following are the types of ELISA plate readers-

1. Dedicated ELISA plate reader

It is a single-mode microplate reader. It measures only one type of signal, mainly absorbance. It is commonly used for routine ELISA test. It is simple, low cost and reliable.

2. Multimode microplate reader

It is a reader which can measure different types of signals in one instrument. It can measure absorbance, fluorescence and luminescence. It is used in laboratories where different types of assays are performed.

3. Filter-based ELISA plate reader

It uses optical filters to select a particular wavelength of light. These filters allow only required wavelength to pass through the sample. It is commonly used in standard ELISA because few fixed wavelengths are needed.

4. Monochromator-based ELISA plate reader

It uses diffraction grating to separate light into different wavelengths. In this reader, wavelength can be selected by software. It does not need separate physical filters.

5. Hybrid microplate reader

It is an advanced type of reader which has both filter-based and monochromator-based system. Filter system gives high sensitivity for routine ELISA. Monochromator system gives flexibility for different assay development.

Detection Methods Used

The following are the detection methods used for ELISA plate readers-

1. Absorbance or Colorimetric detection

It is the most common method used in ELISA plate reader. In this method, enzyme reacts with substrate and produces colour change. The reader measures the amount of light absorbed by the coloured sample.

2. Fluorescence intensity detection

In this method, the sample is excited by a specific light source. After excitation, the sample emits fluorescent light. This emitted light is measured by the plate reader. It is more sensitive than normal colorimetric method.

3. Luminescence or Chemiluminescence detection

In this method, light is produced by chemical reaction in the sample. External excitation light is not required. The reader detects the light produced from chemiluminescent ELISA reaction.

4. Time-resolved fluorescence detection

It is a sensitive fluorescence method. In this method, there is a short time gap between excitation and measurement. This helps to reduce background fluorescence and gives better signal.

Instrumentation of ELISA Plate Reader

The following are the instrumentation of ELISA plate reader-

Light source- It is used to produce light for reading the sample. Xenon flash lamp, halogen lamp, LED and laser are used as light source.

Wavelength selection system– It is used to select particular wavelength of light. Optical filter, monochromator, prism, diffraction grating and UV-visible spectrometer are used in this system.
Reference channel- It is used to measure the original light intensity before light passes through the sample.
Sample compartment- It is used to hold the microplate during reading. Most commonly 96 well plate is used. Some readers also use 384 well and 1536 well plates.

Detector– It is used to measure the light passing through the sample. Photomultiplier tube (PMT), silicon photodiode and CCD detector are used as detector.
Data analysis software- It is used to control the reading process. It converts absorbance into optical density (OD) value and calculates concentration by using standard curve.

ELISA Plate Reader Operating Procedure

The following are the operating procedure of ELISA plate reader-

The ELISA protocol is selected from the reader software or touchscreen. The required absorbance wavelength is selected. Commonly 405 nm, 450 nm or 492 nm wavelength is used.
The plate layout is entered in the software. Standards, blanks and unknown samples are marked in their proper wells.
The ELISA steps are completed in the microplate. Antibody is attached to the well, sample is added, washing is done, detection antibody is added, again washing is done and substrate is added for colour development.

The prepared microplate is placed in the tray of ELISA plate reader. 96 well plate or 384 well plate can be used.
The reading is started by the software. The selected wavelength of light passes through each well and absorbance is measured as optical density (OD).
The software subtracts blank value and prepares standard curve. The concentration of unknown sample is calculated from the standard curve.

The final result is saved and exported. It may be exported in Excel, PDF, CSV or XML format.

Precautions For Using The Microplate Reader

The following are the precautions for using the microplate reader-

Internal filters and mirrors should not be cleaned directly. Any wrong cleaning or movement may cause photometric error.

External parts should be cleaned carefully. The plate carrier should be cleaned by using non-abrasive solution and lint-free lens paper.
Contamination should be avoided because it is a common cause of instrument failure.
Reagent injector should be flushed regularly, if present in the reader. The fluid line should be washed with deionized water daily.

The injector line should be flushed with 70% ethanol weekly to prevent drying of salts and blockage of nozzle.
Condensation should be controlled during long kinetic assay. Condensation on plate lid may disturb the reading.
Plate should be covered during incubation to reduce evaporation of liquid.

Temperature difference should be avoided because it may cause edge effect in the wells.
Well to well contamination should be prevented by avoiding wicking of liquid along the wall of wells.
Reader performance should be checked routinely by using validation plate.

Software and firmware should be updated when required.
Plate carrier offset and other routine maintenance should be checked properly.

Applications of ELISA Plate Reader

The following are the applications of ELISA plate reader-

It is used to perform ELISA and other immunoassay tests.
It is used to detect and quantify antigen, antibody and disease marker.

It is used for protein quantification by Bradford assay, BCA assay and Lowry assay.
It is used to measure concentration and purity of DNA and RNA sample.
It is used in cell viability and cell proliferation assay.

It is used in MTT assay and XTT assay for checking cell health and survival.
It is used to measure enzyme activity and enzyme kinetic reaction.
It is used to monitor bacterial and yeast growth by measuring turbidity.

It is used in reporter gene assay for measuring gene expression.
It is used in clinical diagnosis like blood typing, infectious disease detection and autoimmune disease screening.
It is used in drug discovery and vaccine development.
It is used for high-throughput screening of many samples.
It is used in food and water quality testing.
It is used in food allergy testing and environmental monitoring.

Advantages of ELISA Reader

The following are the advantages of ELISA plate reader-

It can measure many samples at same time by using microplate.
It commonly uses 96 well plate. Some readers also use 384 well and 1536 well plates.
It gives high-throughput result than single sample spectrophotometer.
It uses small amount of sample and reagent.
It saves costly reagent and biological sample.
It can read micro-volume assay, usually 5 to 50 microliter.

It can be attached with automated liquid handling system and robotic system.
It reduces manual pipetting error.
It also reduces chance of sample contamination.
It gives rapid and consistent result.
It reduces the time required for analysis of many samples.
It can be used for small experiment as well as large screening work.

It gives flexibility to test many compounds or many samples according to need.

Limitations of ELISA Plate Reader

The following are the limitations of ELISA plate reader-

Standard ELISA plate reader is mainly used for colorimetric detection only.
It measures only the colour change formed in the assay.

Dedicated ELISA reader cannot detect fluorescence and luminescence.
It cannot be used for many different types of assay in single instrument.
Traditional ELISA reader uses optical filters with fixed wavelength.
If other wavelength is required, new filter has to be purchased and changed manually.
It cannot perform full spectral scanning.
It is not suitable for finding absorbance peak of new dye or new assay.

The filter gives broad band of light.
It has lower spectral resolution than advanced microplate reader.
It may not separate nearby wavelengths properly.
It is less useful when two spectra are overlapping.

References

eCFR. (2026, April 29). 21 CFR Part 11 — electronic records; electronic signatures. Retrieved from [URL].
PSC Software. (n.d.). 21 CFR Part 11 compliance: Key requirements & pitfall. Retrieved from [URL].
Jackson, T. (2025, February 27). 5 major detection methods in multimode microplate readers. Thermo Fisher Scientific. Retrieved from [URL].

Agilent Technologies, Inc. (2021, October 25). Agilent BioTek multimode microplate readers. Fisher Scientific. [PDF].
Agilent Technologies, Inc. (2025, June 18). Agilent BioTek Synergy Neo2 hybrid multimode reader. BioSPX. [PDF].
Biocompare. (n.d.). Agilent BioTek Synergy Neo2 hybrid multimode reader. Retrieved from [URL].

EquipNet. (n.d.). BioTek Instruments Synergy Neo2 multi-mode reader fluorescence measuring equipment. Retrieved from [URL].
BioSPX. (n.d.). BioTek Synergy Neo2. Retrieved from [URL].
Cardonnel, C. (n.d.). Critical considerations for choosing the right microplate reader [Webinar]. Molecular Devices. Retrieved from [URL].
Molecular Devices UK Ltd. (2025, September 24). Choosing the best curve fit option for you. News-Medical.Net. Retrieved from [URL].

Berthold Technologies GmbH & Co.KG. (n.d.). ELISA microplate readers. Retrieved from [URL].
BMG LABTECH. (n.d.). ELISA plate readers. Retrieved from [URL].
Revvity. (n.d.). EnVision XCite multimode plate reader. Retrieved from [URL].
Brendan Bioanalytics. (n.d.). Five parameter logistic and four parameter logistic curve fitting of asymmetric assays. [PDF].

Dorge, R. (2026, April 1). Global microplate systems market size, share 2026-2035. Custom Market Insights (CMI). Retrieved from [URL].
Revvity, Inc. (2023). Guide to selecting a microplate. [PDF].
Bolus, T. (2023, July 17). GxP-regulated industry assessments of microplate readers. Molecular Devices. Retrieved from [URL].
Beckman Coulter. (n.d.). High-throughput IgG quantitation platform for clone screening during drug discovery and development. Retrieved from [URL].

Berthold Technologies GmbH & Co.KG. (n.d.). How sensitive is your microplate reader? Retrieved from [URL].
LabX. (n.d.). Maintaining and calibrating microplate instruments: A guide for optimal performance. Retrieved from [URL].
PerkinElmer / Gulf Scientific. (n.d.). Meet the plate reader family. [PDF].

Wu, E. (n.d.). Microplate reader buying guide: Choose the best model. AELAB. Retrieved from [URL].
Agilent Technologies, Inc. (n.d.). Microplate reader FAQs. Retrieved from [URL].
Strategic Market Research. (2025, October). Microplate reader market size ($1.09 billion) 2030. Retrieved from [URL].

BMG LABTECH. (n.d.). Microplate reader software. Retrieved from [URL].
Danaher Life Sciences. (n.d.). Microplate reader software. Retrieved from [URL].
Berthold Technologies GmbH & Co.KG. (n.d.). Microplate readers. Retrieved from [URL].
Revvity. (n.d.). Microplate readers. Retrieved from [URL].
Lab Manager. (n.d.). Microplate technology buying tips. Retrieved from [URL].

The Lab World Group. (n.d.). Molecular Devices Spectramax iD5 microplate reader. Retrieved from [URL].
D’Amico, D. (2024, December 17). Monochromator or filter-based plate reader? How to choose. Thermo Fisher Scientific. Retrieved from [URL].
Revvity / BMS. (n.d.). Multimode plate reader. [PDF].
National Center for Biotechnology Information (NCBI). (n.d.). Nonlinear calibration model choice between the four and five-parameter logistic models. Retrieved from [URL].

Promega Corporation. (n.d.). Plate reader | luminescence | fluorescence | absorbance detection. Retrieved from [URL].
Wikipedia. (2026, March 12). Plate reader. Retrieved from [URL].
Biocompare. (n.d.). Recent advances in microplate reader throughput and accuracy. Retrieved from [URL].
Thermo Fisher Scientific Inc. (n.d.). SkanIt microplate reader software. Retrieved from [URL].

Molecular Devices, LLC. (2020). SpectraMax iD5 multi-mode microplate reader. [PDF].
Agilent BioTek. (n.d.). Synergy Neo2 multi-mode microplate reader. iBioTech.cz. [PDF].
Anonymous. (n.d.). The architecture of modern microplate instrumentation: A technical treatise on detection modalities, operational optimization, and regulatory frameworks in bioanalytical research. [Markdown].

Kalstein. (n.d.). The best microplate readers of 2024: Driving clinical research and best medical practices. Retrieved from [URL].
LabX. (n.d.). The best microplate readers of 2026: Top picks for research, clinical & industry labs. Retrieved from [URL].
Revvity. (n.d.). The EnVision Nexus multimode plate reader features sensitive HTRF detection technology. [PDF].

Molecular Devices, LLC. (2021). The ultimate guide to microplate reader solutions. [PDF].
Baker, V. (2024, August 20). Understanding the different types of microplate readers and their uses. Marshall Scientific. Retrieved from [URL].

ELISA Plate Reader (Microplate Reader) – Principle, Parts, Uses