ELISA plate reader- Definition, Principle, Procedure, Uses

An ELISA plate reader is a specialized equipment that measures the fluorescence, chemiluminescence, or chromogenic response in an ELISA experiment performed on a 96-well plate. The ELISA reader is designed to work with conventional 96-well plates, which are the most popular format used in ELISA studies. It serves as both a fluorimeter and a spectrophotometer, allowing measurements of several sorts of signals generated throughout the experiment and offers quantitative data on the amplitude of the reaction in each well, allowing for the simultaneous investigation of up to 96 conditions.

• The ELISA plate reader functions as a fluorimeter by producing light at an excitation wavelength that is particular to the fluorophore that is being used as the label, and it measures the intensity of the light produced at the matching emission wavelength. This makes it possible to identify and measure the fluorescence signals that the assay produces.
• The ELISA plate reader functions as a spectrophotometer, detecting the intensity of light generated by chemiluminescent and chromogenic reactions across a range of wavelengths. This feature allows for the detection and measurement of signals generated by chemiluminescent or chromogenic labels, broadening the range of detection modalities available in ELISA tests.
• Software for data analysis and interpretation is typically included with ELISA readers. After processing the raw intensity readings from the ELISA reader, the program incorporates dilution and error information into quantitative curves. Furthermore, calibration curves are usually included in the software to guarantee that the instrument continues to operate at a high level and consistently.

Figure: ELISA plate reader

Overall, the ELISA plate reader is a specialized tool employed alongside a 96-well plate to scrutinize the fluorescence, chemiluminescence, or chromogenic signals generated during an ELISA assay. Functioning as both a fluorimeter and a spectrophotometer, it facilitates the measurement of diverse signals emitted by the assay. Supported by dedicated software, the device aids in data analysis, offering calibration curves to assure precise and dependable measurements. Widely indispensable, the ELISA reader plays a crucial role in quantifying and interpreting the outcomes of ELISA experiments across diverse scientific and medical applications.

What is ELISA?

Enzyme-linked immunosorbent Assay, or ELISA for short, is a popular laboratory method for identifying and quantifying antibodies or antigens in biological materials. It is essential in many areas, including immunology, biomedical research, and diagnostics. With the aid of ELISA, scientists may precisely identify and measure target compounds by taking advantage of the immune system’s capacity to recognize and attach to particular molecules.

Depending on the particular test goal, the plate can be set up in one of two ways:

• The target antigen is fixed at the bottom of each well while looking for antibodies in a clinical sample.
• In contrast, a capture antibody is attached to the bottom of each well, forming a structure similar to a sandwich, if the objective is to detect the presence of a particular antigen.

Applications for ELISA are found in many different scientific studies. Researchers can examine the presence, concentration, or activity of proteins and enzymes by using them in their studies. ELISA is also widely utilized in diagnostic testing, such as nucleic acid quantification and the identification of infectious agents like HIV. Furthermore, its significance lies in determining the existence of vaccine-induced antibodies or auto-antibodies linked to illnesses like rheumatoid arthritis.

Objectives of ELISA plate reader

• An ELISA reader’s main function is to make it easier to measure and analyze different tests, especially ones that involve antibody testing. These devices, which were first created for ELISA tests, have shown to be adaptable and have been altered for a variety of uses in the scientific and medical areas.
• When testing proteins and enzymes, ELISA readers are essential. They are used by researchers to examine the existence, concentration, and function of proteins and enzymes. ELISA readers give useful information that supports the comprehension of biological processes and the investigation of disease mechanisms by measuring the signals produced by certain protein-antibody or enzyme-substrate interactions.
• HIV detection is one of the major uses of ELISA readers. HIV infection can be diagnosed by using ELISA-based assays, which are frequently employed to find antibodies against the virus in patient samples. To reliably detect and diagnose HIV, ELISA readers precisely quantify the signals generated by the interaction of patient antibodies with HIV antigens.
• ELISA readers are also used in the measurement of nucleic acids. Scientists can determine the amount of nucleic acids, like DNA or RNA, in a sample by using particular tests. Numerous scientific fields, such as genetic analysis, gene expression investigations, and infectious disease diagnostic testing, depend on this knowledge. ELISA readers provide useful information for nucleic acid analysis by helping to quantify the signals produced by nucleic acid-specific probes.

ELISA plate reader principle

Measuring the energy difference in light before and after it passes through a test sample is the basis of an ELISA reader, also known as a microplate reader. Usually, the concentration of the test material has a linear relationship with the energy difference in light caused by the substance’s absorption.

A photoelectric colorimeter, often known as a spectrophotometer, is used to measure and identify the sample’s optical density or absorbance.

• The wavelength range in which the microplate reader works is normally 400–750 nm, while some models may go as far as 340–700 nm in the UV spectrum.
• Filters and diffraction gratings are used to control the light’s wavelength, allowing just the desired range of wavelengths to be transmitted or detected. This makes it possible to measure particular reactions or assay labels selectively.
• Optic fibers, which are a component of the microplate reader’s optical system, provide light to the sample-containing microplate wells. To ensure adequate coverage of the well region, the laser beam diameter that passes through the sample usually varies between 1 and 3 mm. Certain wavelengths of light are absorbed by the test material in the sample, resulting in an energy differential in the light that is transmitted.
• The microplate reader’s sensitive detecting mechanism then picks up the light that the sample emits or transmits.
• The signal is amplified by this apparatus and transformed into an electrical signal that is related to the absorbance of the sample.
• Beer’s Law, which asserts that absorbance is linearly proportional to concentration, relates the absorbance to the target substance’s concentration in the sample.
• A reading system that collects and stores the data from the detection equipment is included within the microplate reader. This reading system provides quantitative measures of the absorbance levels, which facilitate the understanding of the test results. The target substance’s concentration in the sample must be ascertained using these measurements.
• A double-beam light system, in which the light is divided into two beams, one of which travels through the sample and the other of which acts as a reference beam, is employed by certain sophisticated microplate readers. This arrangement compensates for possible fluctuations in the light source or optical components over time, enabling more precise and reliable observations.

In simple terms, an ELISA reader or microplate reader works by measuring the change in light energy before and after it goes through a test sample. By figuring out how much of the sample absorbs light at specific wavelengths, the reader helps to find the concentration of the target substance in the sample. This gives important information for a range of scientific and diagnostic purposes.

Instrumentation of ELISA plate reader

• Light source: Xenon flashlights are a common light source for ELISA plate readers. The broad spectrum light emitted by these lamps can be filtered or chosen to correspond with the required excitation wavelengths for various detecting techniques.
• Filters and monochromators:
  • Microplate readers rely on crucial wavelength selection technologies, including filters and monochromators, in their design.
  • Filters, optical devices positioned between the light source and the sample, selectively transmit particular wavelengths of light while blocking others. This enables the precise choice of excitation or emission wavelengths.
  • On the contrary, monochromators convert polychromatic light into monochromatic light by dispersing it into its component wavelengths and then selecting the specific wavelength needed.
  • Both filters and monochromators are indispensable components, exerting precise control over the wavelengths of light utilized for excitation and detection in ELISA assays.
• Microplate: Microplates are rectangular plates with many wells that are compatible with ELISA plate readers. The samples, reagents, and test components are kept in these limited-volume wells. The well densities of 96, 384, and 1536 wells per plate are the most often used for screening. The unique experimental requirements and throughput requirements determine the optimal well density.
• Detection methods: To monitor and quantify the chemical, biological, or physical phenomena taking place within the microplate wells, ELISA plate readers use a variety of detecting methods. The three most used types of detection are luminescence, fluorescence, and absorbance.
  • The amount of light absorbed by the sample is measured by absorbance detection, fluorescent molecules are detected by fluorescence detection, and light released by chemiluminescent reactions is captured by luminescence detection.
  • The equipment on the reader is set up to best record and process the signals produced by these various detection modes.
• Data analysis software: Typically, ELISA plate readers incorporate software that makes it easier to analyze the results. This software helps with understanding, management, and visualization of data. It is capable of producing standard curves, carrying out computations, and offering assay parameter readings that are quantitative. The program facilitates the data analysis process and improves the effectiveness and precision of result interpretation.

Procedure for operating ELISA plate reader

1. Preparation:
   • Put the capture antibody into the wells of the ELISA microplates.
   • To enable the antigen to attach to the capture antibody, add the antigen-containing sample to each well.
   • Only the target antigen bound to the capture antibody remains after the unbound material is removed from the microplate.
2. Detection:
   • Fill the wells with the enzyme-conjugated detection antibody.
   • A distinct site on the target antigen is where this antibody binds.
   • Only the particular antibodies bound to the target antigen will remain after washing the microplate to get rid of any unbound detection antibodies.
3. Substrate reaction
   • Load the microplate with a substrate solution.
   • The detecting antibody’s enzyme catalyzes a reaction with the substrate that changes color or produces a signal.
4. Measurement
   • Insert the microplate into the reader for ELISA plates.
   • The optical density (OD), or signal intensity, is measured by the microplate reader once it has identified the colored response in each well.
   • The reader produces an output with the assay’s results and additional parameters, typically in the form of an Excel file.

Application of ELISA plate reader

• Diagnosis of disease: A key tool in the diagnosis of many diseases is the ELISA plate reader. The detection of infectious disorders like rotavirus, AIDS, Ebola, and Lyme disease is one of its common uses. It facilitates the identification of particular antibodies or antigens associated with various illnesses, supporting precise patient monitoring and diagnosis.
• Antibody testing: In antibody testing, the ELISA plate reader is a frequently used tool. It makes it possible to identify and measure particular antibodies in biological material samples. This is important for several research, therapeutic, and diagnostic applications, including the diagnosis of autoimmune illnesses, tracking the effectiveness of vaccines, and studying immunological responses.
• Nucleic acid analysis: The quantification of nucleic acids, including DNA and RNA, is another use for the ELISA plate reader. It makes it possible for scientists to precisely measure the quantity of nucleic acids, which is essential for several molecular biology investigations, such as genotyping, gene expression analysis, and PCR.
• In assays for proteins and enzymes, the ELISA plate reader is widely utilized. It makes it possible to measure the amounts of proteins, the activity of enzymes, and the characteristics of different biochemical reactions. Scientists can measure enzymatic kinetics, examine protein interactions, and determine how medications or other substances affect the activity of enzymes.

Advantages of ELISA plate reader

• The sample amount needed for the ELISA plate reader is usually quite tiny, ranging from two to 100 microliters. This is beneficial because it enables researchers to reduce sample waste and preserve priceless samples. It is especially helpful when working with expensive or scarce samples.
• Sample analysis, both quantitative and qualitative, is simpler with the use of ELISA plate readers. Signals like luminescence, fluorescence, or absorbance that are correlated with the target analyte’s concentration can be measured for intensity. This makes it possible to precisely quantify the analytes in a sample. Additionally, by signaling the presence or absence of particular analytes based on predefined threshold values, ELISA plate readers can also provide qualitative information.
• Multiple samples can be analyzed simultaneously by ELISA plate readers. ELISA assays typically use a standard microplate format with 96 wells, which enables a high sample throughput. Because of this, scientists can process a lot of samples in one run, which reduces processing time and boosts productivity.
• Numerous ELISA plate readers come with automation capabilities like robotic arms and plate handling devices. By limiting manual handling and lowering the possibility of human mistakes, these features enable automated sample loading, assay incubation, and data gathering. Moreover, ELISA plate scanners frequently include intuitive software that makes data management, analysis, and interpretation easier.
• Superior precision, sensitivity, and speed can be obtained with an ELISA plate reader that is well-designed and calibrated. To identify and quantify the signals produced by the assay, the reader’s optical systems and detectors are optimized. Accurate and dependable outcomes are guaranteed by this. Quick data processing and reading are possible with ELISA plate readers, which means that analysis can be completed rapidly.

Limitations of ELISA plate reader

• In comparison to other laboratory equipment, ELISA readers are usually huge, heavy devices that are less portable. To ensure accurate results, they must be handled carefully and in a steady environment due to their intricate design and fragile optical components. This makes them less useful in situations where portability is essential, as in the field.
• ELISA readers may be costly to maintain as well as initially acquire. These instruments cost more because of their sophisticated optical systems and cutting-edge technology.
• When biological and chemical components are measured in microplates, prolonged air exposure can change their optical properties. In assays that rely on accurate absorbance measurements or color-based methods, this can have an impact on measurement accuracy.
• The assay’s additional capture agents, such as antibodies, must have specificity for ELISA readers to function. Yet, cross-reactivity or interference from other molecules in the sample is always a possibility, which could result in results that are either falsely positive or falsely negative.

Precautions

• To avoid interfering with the readings, keep the microplate reader away from strong magnetic fields and interference voltages.
• Avoiding direct sunlight can help prevent the optical components from deteriorating and may improve the accuracy of the readings. Make sure the instrument is situated in a well-ventilated environment.
• In order to limit any external interference that can affect the measurements, the instrument should be placed in an environment where noise levels are less than 40 dB.
• Be cautious not to drop samples or reagents on the internal surfaces of the device or inside it. Spills should be cleaned up right once to avoid contaminating the area and harming the instrument.
• When taking measurements, do not turn off the power. During the measurement procedure, abrupt power outages can interfere with measurements and possibly result in data loss. It is advised to finish the measurement and appropriately turn off the device according to the manufacturer’s instructions.

References

• https://www.biocompare.com/Lab-Equipment/20131-Microplate-Reader-ELISA-Plate-Reader/
• https://microbiologynote.com/elisa-plate-reader-microplate-reader-or-assay-reader/
• https://hudsonrobotics.com/elisa-microplate-reader-principle-and-uses/
• https://www.protocols.io/view/microplate-reader-operating-procedure-6qpvrox62vmk/v2?step=5
• https://www.sepmag.eu/blog/elisa-reader