ELISA stands for enzyme linked immunosorbent assay. In brief, it is an antibody test or a test of the immune system’s response to invaders such as viruses, bacteria, and allergies. The test is performed on an ELISA plate, sometimes referred to as a 96-well plate or microplate. The plate is read by the ELISA reader.
An ELISA reader quantifies and measures the colour changes between each of the 12 wells on the plate. ELISA readers and micro plate readers do spectrophotometry; they emit light at a single wavelength and measure the amount of light absorbed and reflected by an item, such as a protein. The spectrophotometer measures visible and ultraviolet light.
ELISA plate readers are also capable of measuring fluorescence and luminescence. When exposed to light, chemical dyes fluoresce or emit a single hue or wavelength. Reflection, absorption, and colour are used to detect and quantify the amount of a chemical.
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What is ELISA?
- In one of two methods, the ELISA is conducted in a 96-well polystyrene plate: either antigen is connected to the bottom of each well, or the capture antibody is affixed to the bottom of each well.
- Antigens are connected to the bottom of each well when the objective of the assay is to identify the presence of an antibody in a clinical sample.
- If the objective is to identify antigen in a clinical sample, the capture antibody is bound to the bottom of each well (also known as a sandwich ELISA).
- The ELISA is such a common and standardised assay that pre-coated, pre-blocked plates that are ready to receive the clinical or experimental sample upon opening the packaging can be purchased.
- The ELISA procedure continues beyond the introduction and incubation of the target sample.
- Next, a tagged detection antibody is added to determine whether or not binding between the capture and target antibodies and antigen happened.
- The detection antibody is tagged with a fluorescent, chemiluminescent, or chromogenic chemical that an ELISA reader can quantify.
What is ELISA reader?
- An ELISA reader is a device used to analyse the fluorescence, chemiluminescent, or chromogenic response of an ELISA in a 96-well plate.
- The gadget is designed to suit standard 96-well plates and will provide quantitative information regarding the magnitude of the response in each well.
- This allows up to 96 distinct circumstances to be studied simultaneously. The ELISA reader is both a fluorimeter (capable of inputting light at an excitation wavelength for the fluorophore being used as the label and recording the intensity of the emitted light at the emission wavelength) and a spectrophotometer (capable of recording the intensity of light emitted from chemiluminescent and chromogenic reactions across a spectrum of wavelengths).
- The software included with the majority of ELISA readers turns the raw intensity results into quantitative curves with dilution and error information.
- The software is loaded with calibration curves to ensure that the instrument always meets the highest quality and repeatability standards.
Purpose of an ELISA reader
- The purpose of ELISA readers is to measure antibody testing. Because they performed so well, the machine has been modified for different uses.
- They are utilised by scientists for protein and enzyme tests. They are also utilised for HIV detection and nucleic acid quantification.
ELISA Reader Principle
- The microplate reader is a spectrophotometer with a particular design. The microplate reader features filters or diffraction gratings that limit the wavelength range to that used in ELISA, often between 400 and 750 nm, in contrast to the traditional spectrophotometer, which permits readings on a broad spectrum of wavelengths (nanometres).
- Some ultraviolet-operating readers perform assessments between 340 and 700 nanometers. Numerous manufacturers employ an optical system that uses optical fibres to supply light to the sample-containing microplate wells.
- The diameter of the light beam travelling through the sample ranges from 1 to 3 mm. A detection system detects the light emitted by the sample, amplifies the signal, and calculates the absorbance of the sample.
- A reading system translates test results into data for interpretation. Some microplate readers employ dual-beam illumination systems.
- The method or test is carried out on plates containing a specific number of wells and containing test samples. Common are plates with 8 columns by 12 rows and 96 wells in total.
- Additionally, there are plates with an increased number of wells. The current tendency in specialised applications is to increase the number of wells (384-well plates) to reduce the amount of reagents and samples utilised and to boost throughput.
- Depending on the manufacturer, the optical sensors of the microplate reader may be positioned above the sample plate or directly beneath the wells of the plate.
- Modern microplate readers are equipped with microprocessor-controlled controls, links to information systems, and quality and process control programmes that, when run on a computer, enable total test automation.
Types of Elisa Plate Readers
By detecting technology, there are two primary categories of microplate readers: dedicated microplate readers and multimode microplate readers.
1. Dedicated Microplate Readers
- Microplate readers are only capable of measuring a single technology, such as absorbance, luminescence, or fluorescence.
- They lack the versatility of multimode microplate scanners, but they often offer greater sensitivity and dependability and are typically less expensive.
2. Multimode Microplate Readers
- Microplate readers that support multiple detection technologies called multimode.
- They are more sophisticated than specialised microplate readers, and some design sacrifices are required to integrate all detecting modes on a single device, but they offer a great deal of versatility.
- Historically, multimode microplate readers have been costly, but some instruments offer a high degree of modularity that enables the instrument to be built with only the functions that are actually required.
Microplate Readers by Wavelength Selection Collapse
When selecting a microplate reader, the wavelength selection system is also crucial, as it has a significant impact on numerous applications. Filters, monochromators, and no wavelength selection are the primary alternatives.
Filters
- Affordable microplate readers choose the desired wavelength for any given detection method using filters.
- Being the least expensive alternative does not imply that it is the worst: filters have a relatively high transmittance, which in many situations increases sensitivity.
- In addition, the wavelength change is extremely rapid, a trait that is useful in ratiometric assays.
- The biggest downside is that you need a distinct filter for each wavelength, which decreases flexibility and makes wavelength scanning impossible.
Monochromator
- Monochromators, on the other hand, offer complete flexibility not only in wavelength selection but also in bandwidth selection.
- However, as previously stated, sensitivity in the majority of apps is worse than when employing filters.
No wavelength selection
- Luminometers typically lack filters or other wavelength-selection mechanisms, thus all wavelengths of light can possibly reach the photomultiplier.
Detection Methods Or Detection Methods Used by An Microplate Reader
Following are common microplate reader detection methods:
1. Absorbance
- Microplate readers have long been utilised in the scientific community for absorbance detection.
- The world has implemented detection processes for more than three decades. They are utilised in numerous tests, including ELISA, protein, and nucleic acid assays.
- This microplate reader detection approach is also utilised for enzyme quantification or enzymatic tests.
- In this method of detection, a light source with a certain wavelength illuminates the biological specimen. In this instance, the wavelength is selected via an optical filter or monochromator.
- On the opposite side of the well in the plate, a light detector monitors the amount of initial light transmitted through the biological material.
- Typically, the amount of light transmitted is proportional to the concentration of the molecule of interest within the wells.
- Several additional conventional colorimetric analyses have been added to the wells of an elisa microplate reader in order to accurately assess the specimens. It is further downsized such that it may function quantitatively in a microplate reader, with performance adequate for biological research.
- Examples of such microplate reader absorbance analyses include ammonium, urea, orthophosphate, nitrate, nitrite, etc.
- In the analysis of all of these, colorimetric chemistry are also utilised for the most accurate sample analysis.
2. Fluorescence Intensity Detection
Throughout the last two decades of scientific research, microplate detection of fluorescence intensity has advanced significantly. Fluorescence intensity detection has a significantly broader variety of applications than microplate absorbance detection. However, fluorescence detection equipment is typically more expensive. Instrumentation for this type of microplate reader consists of:
- First optical system- Optical system’s first excitation The system lights the biological sample with a specific light wavelength. Typically, it is chosen using an optical filter or monochromator.
- The illumination system – As the light rays are transmitted through the specimen, the specimen emits light, which is known as fluorescence.
- Second optical system – The second optical system is the microplate reader’s emission system, which gathers and separates the emitted light from the excitation light. This separation is achieved through the use of a monochromator device. It also measures the signal using a photomultiplier tube, a light detector (PMT).
The primary advantages of fluorescence detection over absorbance detection using a microplate reader are:
- Sensitivity.
- The application variety has resulted in the availability of a wide variety of luminous labels.
Calcium imaging, which detects the fluorescence intensity of calcium dyes employed in the analysis process, is an example of this fluorescent technology.
3. Time – Resolved Fluorescence ( TFR)
- The time–resolved fluorescence measurement is comparable to the multiwell reader’s fluorescence intensity measurement.
- The only distinction between the two is the order in which the excitation and measurement processes occur.
- Excitation and emission of photons occur concurrently during the measurement of the sample’s fluorescence intensity.
- In the plate reader, the light emitted by the biological sample is measured during the excitation phase.
- The fluorescence optical system relies on lanthanides, which are particular fluorescent chemicals.
- After the conclusion of excitation, these molecules have the peculiar property of producing light over millisecond-scale intervals in the reader.
- The common fluorescent dye, fluorescein, emits within a few nanoseconds of being activated during the fluorescence intensity process.
- All of this results in the stimulation of lanthanides by a microplate reader’s pulsed light source.
- After the process of stimulation, the researchers take measurements.
- Time resolved fluorescence energy transfer is the primary application of TRF in the pharmaceutical sectors for drug screening applications.
4. Luminescence Detection Method
- The luminescence detection method results from a chemical and biological response within the microplate reader’s wells.
- Detecting luminescence is a less complex optical phenomenon than the aforementioned detection methods.
- It does not necessitate the use of a light source or the selection of specific excitation wavelengths.
- A typical luminescent optical system includes:
- A light-tight reading chamber and
- A PMT detector
- Some microplate readers assess the results using an analogue PMT detector. While the other microplate readers use a PMT detector with photon-counting capabilities.
- It is commonly believed that counting photons is the most sensitive method for measuring luminescence within the microplate reader’s wells.
- It permits the detection of tests containing several luminous marking enzymes as well as the creation of new luminescence assays.
Instrumentation of ELISA Plate Reader
- Source of light: As a source of illumination, xenon flash bulbs are typically employed.
- Filters and monochromators: Filters and monochromators are two competing technologies for wavelength selection crucial to the design of microplate readers. Monochromators convert multicoloured light to a single colour. The excitation and detection light pathways contain optical filters with specific wavelengths and bandwidths. Filters are more affordable than monochromators. These are positioned between the light source and the sample.
- Microplate: Microplates are built with wells that have a limited volume to which the sample will be treated. Well densities of 96, 384, and 1536 per plate are the most common for screening.
- Microplate Reader: Various technologies, including absorbance, fluorescence, and luminescence, are used to detect and quantify chemical, biological, or physical phenomena within a microplate’s well.
- Software for results: Various software can be installed and utilised to facilitate data analysis.
Other Parts of ELISA Plate Reader
The major components of an ELISA reader are listed and briefly described. The following are:
- Printer: When the ‘PRINT’ key is pushed, the printer is used to produce a printout of the currently displayed screen.
- Touch Screen: It displays the parameter’s options and text box, which comprise the touch zone.
- Keypad: Several keys, including ‘YES,’ ‘NO,’ ‘PRINT,’ ‘ENTER,’ ‘ESC,’ and ‘SHAKE,’ as well as navigation keys, are present on the keypad with their respective functions.
- Plate Carrier/Holder: This device transports the microtiter plate throughout the instrument. Stepper motor and timing belt are utilised for propulsion. It accurately puts each plate well below the optical path of each channel.
- Additionally, it includes an ON/OFF switch panel, USB cable connector, SMPS, Cooling Fan, and Serial RS232 output.
ELISA Plate Reader Operating Procedure
From a biological perspective, the ELISA technique microplate reader is essential. Following is the procedure for the elisa plate reader technique:
- The wells of the microplate reader are coated with antibodies or antigens. This contains samples of reactions.
- Incorporating the samples, colorimetric standards, and controls into the wells of the plate. They are incubated at temperatures between ambient temperature and 37 degrees Celsius. This is performed for a predetermined period of time in the microplate reader. The duration is determined by the criteria of the examination.
- The sample antigens attach to the antibody-coated well plate during incubation. The sample and antigens are capable of undergoing an additional binding procedure. According to the procedure of plate reading, the samples are analysed.
- After microplate reader incubation, the unbound antigen is washed and removed from the microplate reader using a washing buffer by the washer.
- The existence of a secondary antibody, known as the conjugate, is the subsequent step in microplate reader reading. This is added to the samples of the reaction. All of these steps are repeated until the microplate reader produces an accurate reading.
Precautions For Using The Microplate Reader
Working environment The microplate reader is a precise optical instrument; therefore, a proper working environment not only ensures its accuracy and stability, but also extends its service life.
- The device should be situated in an area devoid of significant magnetic fields and voltage interference.
- The device should be positioned in an environment with a decibel level below 40.
- To prevent the ageing of optical components, one should avoid direct sunlight.
- The operational ambient temperature and humidity should be between 15°C and 40°C and 15% and 85%, respectively.
- The voltage should be steady throughout operation.
- The operational environment has pure air, free of water vapour and smoke.
- Maintain a dry, clean, level work surface and adequate room for operation.
Precautions for the use of microplate reader
- Add liquid with a pipette, and the pipette tips cannot be combined.
- To prevent cross-contamination, plates must be washed with clean water.
- If the kit instructions are followed precisely, the response time will be correct.
- Do not spill samples or reagents on the exterior or interior of the instrument, and clean the device thoroughly after use.
- Do not turn off the electricity during the measurement.
- For the deviation in measurement results caused by a problem with the kit, the parameters must be updated in real time to produce the optimal effect.
- After use, replace the dust cover’s cover.
Uses of ELISA reader
Using cytokine and pro-inflammatory indicators, ELISA can be utilised to determine the presence of COVID-19.
- Inflammation: The reader is able to identify cytokine and chemokine cascades resulting from anti-inflammatory reactions.
- Cell therapy: ELISA can be utilised for the process development of cell therapy.
- Preclinical research: The automatic reader can provide more data and faster outcomes for preclinical research.
- Cancer: An automated ELISA can be utilised in biomarker research for cancer.
- Bioprocess development: The device can detect host cell proteins (HCPs) for biotherapeutic synthesis during bioprocess development.
- Neuroscience: An ELISA can provide information about low abundance biomarkers in plasma, serum, and cerebrospinal fluid.
- Quantification of viral titer: An automated reader is capable of quantifying the viral titer of both adeno-associated virus capsids and lentivirus.
Advantages of ELISA Reader
- Spectrophotometers demand more sample every measurement. The molecule must be dissolved in solution for usage with a spectrophotometer or ELISA plate reader. Depending on the manufacturer and model, 400 microliters to four millilitres are required for a spectrophotometer. An ELISA plate reader requires between two and one hundred microliters; ELISA plate readers use far less of a sample to provide a response.
- ELISA plate readers measure greater numbers of samples in less time. A spectrophotometer simultaneously measures one to six samples. An ELISA plate typically measures 96 wells in the same period of time.
ELISA Plate Reader Limitations
- These are not portable, fragile, or costly.
- Biological and chemical contents in the microplates may experience significant changes in their optical properties as a result of their prolonged exposure to air throughout this extensive measuring procedure (for example, colour and absorbance). As a result, the full-spectrum measurement of the microplate reader cannot accurately measure the test substance.
- During the measurement process, the microplate reader provides only numeric data describing the absorbance of various wavelengths; it cannot generate a spectrogram in real time.
ELISA Plate Reader Examples with Buy Link
1. Labomed EMR-500 ELISA Microplate Reader, Touch Screen
- Eight-channel photometer for sequential and simultaneous microplate reading
- Cut-off, single and multi-standard, O.D. bi-chromatic, and dynamic reading analysis modes
- 0.001 to 3.500Abs linear range
- 405/450/492/630nm optical filters
- Automatic focusing provides accurate readings
2. Accuris Instruments MR9600-630 Filter for Smart Reader 96 Microplate Absorbance Reader, 630 nm
- The Accuris Smart Reader 96 is a welcome addition to any lab that is routinely measuring concentration or absorbance in 96 well plates.
- The instrument’s 7 inch touch screen, intuitive software and graphical interface makes it easy to use as a standalone instrument (a separate computer and additional software is not required).
- The Smart Reader 96 comes complete with 4 popular filters: 405, 450, 492, 630nm and twenty six additional filters are available separately to cover a full range from 340 to 750nm.
- The integrated filter wheel allows up to 8 filters to be loaded at once, and filters can be selected during programming.
- Use the touch screen, or connect a mouse for programming and operation.
- The menu interface allows the user to choose the wavelength filter, set shaking parameters, enter details of the plate layout, and also choose the details for the reading protocol and data calculations.
3. Accuris Instruments MR9600 SmartReader 96 Microplate Absorbance Reader, 115V, Fluid_Ounces, Degree C
- Intuitive, 7 inch color touch screen control panel
- Filter based system with wavelength range from 340 to 750Nm
- Operates as a stand alone system, with usb flash drive drive for data transfer
- Absorbance range: 0.0 to 4.0000 abs
- 8 channel vertical optical path, with zero dispersion
Reference
- Yang J, Wu Y, Wang H, Yang W, Xu Z, Liu D, Chen H-J, Zhang D. An Improved Automated High-Throughput Efficient Microplate Reader for Rapid Colorimetric Biosensing. Biosensors. 2022; 12(5):284. https://doi.org/10.3390/bios12050284
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