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What is an ELISA reader?

An enzyme-linked immunosorbent assay (ELISA) reader, also known as an ELISA detector, is a specialized instrument designed for use in enzyme-linked immunosorbent assays. In essence, it is a type of modified, specialized spectrophotometer or colorimeter. Its basic operating principle and main structural components are fundamentally the same as those of a conventional spectrophotometer. ELISA readers can be broadly categorized into two types: semi-automatic and fully automatic. However, their underlying working principles are essentially identical; at their core lies a colorimeter—a device that uses colorimetric analysis to determine the concentration of antigens or antibodies.

What is the enzyme-linked immunosorbent assay?

The enzyme-linked immunosorbent assay, often abbreviated as ELISA, is a type of labeling technique that has evolved from fluorescent antibody technology and isotopic immunoassay. It is a modern technique that is sensitive, specific, rapid, and capable of automation.

The basic principle of enzyme-linked immunosorbent assay (ELISA) is to conjugate an antigen or antibody with an enzyme using a coupling agent, thereby producing an enzyme-conjugated antigen or antibody. This enzyme-conjugated antigen or antibody can specifically react with the corresponding antigen or antibody immobilized on a solid-phase carrier or present in tissues, forming a stable immune complex that retains its biological activity. When a suitable substrate is added, the substrate is catalyzed by the enzyme, resulting in a characteristic color reaction. The intensity of the color is directly proportional to the concentration of the corresponding antigen or antibody.

Since this technology is based on the antigen-antibody reaction and the highly efficient catalytic action of enzymes, it boasts high sensitivity and specificity, making it a remarkably robust immunological assay technique.

The principle of an enzyme-linked immunosorbent assay (ELISA) reader.

An enzyme-linked immunosorbent assay (ELISA) reader is an instrument that operates on the principle of enzyme labeling. It is similar to a modified spectrophotometer or colorimeter, and its basic operating principle as well as its main structural components are essentially the same as those of a conventional spectrophotometer.

The light waves emitted by the light source are filtered through a filter or monochromator to become a beam of monochromatic light, which then enters the plastic micro孔 array containing the sample to be measured. Part of this monochromatic light is absorbed by the sample, while the remainder passes through the sample and strikes a photoelectric detector. The photoelectric detector converts these varying-intensity light signals from the sample into corresponding electrical signals. After undergoing signal processing—including pre-amplification, logarithmic amplification, and analog-to-digital conversion—the electrical signals are sent to a microprocessor for data processing and computation. Finally, the results are displayed on a monitor and printed out by a printer.

The microprocessor also controls the mechanical drive mechanism to move the microplate in the X and Y directions via control circuitry, thereby automating the sample-loading and detection process. In contrast, some other ELISA readers rely on manual movement of the microplate for detection, eliminating the need for mechanical drive mechanisms and control circuits in the X and Y directions. As a result, these instruments are more compact and have a simpler structure.

A microplate is a transparent plastic plate pre-coated and specifically designed to hold samples for analysis. The plate features multiple rows of uniformly sized, identical small wells, each containing a corresponding antigen or antibody. Each well on the microplate can accommodate a few tenths of a milliliter of solution. Common specifications include 40-well plates, 55-well plates, 96-well plates, and others. Different instruments are equipped with microplates of varying specifications, allowing for either single-well or row-by-row detection.

Enzyme readers measure the absorbance of the analyte at a specific wavelength. With the advancement of detection methods, single-unit desktop enzyme readers equipped with multiple detection modes are called multifunctional enzyme readers. These readers can detect absorbance (Abs), fluorescence intensity (FI), time-resolved fluorescence (TRF), fluorescence polarization (FP), and chemiluminescence (Lum).

From a principle standpoint, microplate readers can be categorized into grating-type microplate readers and filter-type microplate readers. Grating-type microplate readers can select any wavelength within the range of the light source’s spectrum, whereas filter-type microplate readers, depending on the selected filters, can only detect specific wavelengths.

Microplate Reader Structure

The monochromatic light used in enzyme readers can be obtained either through coherent filters or by means of a monochromator identical to that found in spectrophotometers. When using filters as filtering devices, just as in conventional colorimeters, the filters can be placed either in front of or behind the microplate; the effect is the same in either case. The light emitted by the source lamp passes through a condenser lens and an aperture, then reaches a mirror. After being reflected at a 90° angle by the mirror, the light travels vertically through the colorimetric solution and then passes through the filter before reaching the phototube.

Microplate readers can be categorized into two types: single-channel and multi-channel. Single-channel readers, in turn, come in two varieties: automatic and manual. Automatic models are equipped with mechanical drive mechanisms in the X and Y directions, enabling them to sequentially move the tiny wells of a microplate one by one beneath the light beam for testing. Manual models, on the other hand, rely on manual movement of the microplate to perform measurements.

Building upon the single-channel microplate reader, multi-channel microplate readers have been developed. These multi-channel readers are generally automated. They are equipped with multiple light beams and multiple photodetectors—for example, a 12-channel instrument features 12 light beams or 12 optical paths, 12 detectors, and 12 amplifiers. Under the mechanical drive in the X-direction, samples are scanned sequentially in rows of 12. Multi-channel microplate readers offer fast detection speeds, but their structure is more complex and they come at a higher price.

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