What is ELISA Microplate Reader?
The microplate reader, that is, enzyme-linked immunosorbent assay, is a special instrument for enzyme-linked immunosorbent assay. In fact, it is a special photoelectric colorimeter or spectrophotometer in disguise, and its basic working principle is basically the same as the main structure and photoelectric colorimeter. It can be simply divided into two categories, semi-automatic and fully automatic, but their working principles are basically the same. Its core is a colorimeter, that is, the colorimetric method is used to analyze the content of antigen or antibody. ELISA determination generally requires the final volume of the test solution to be less than 250μL, and the test cannot be completed with a general photoelectric colorimeter, so there are special requirements for the photoelectric colorimeter in the microplate reader.
Principle
The microplate reader is actually a special-purpose photoelectric colorimeter or spectrophotometer in disguise, and its basic working principle is basically the same as the main structure and photoelectric colorimeter. The figure shows the work of a single-channel automatic sampling microplate reader. Schematic diagram. The light wave emitted by the light source lamp is transformed into a monochromatic light by the filter or monochromator, and enters the specimen under test in the plastic microporous pole. Part of the monochromatic light is absorbed by the specimen, and the other part is transmitted through the specimen Illuminated to the photodetector, the photodetector converts the different light signals of the specimen to be tested into corresponding electrical signals. The electrical signals are processed by pre-amplification, logarithmic amplification, and analog-to-digital conversion. Enter the microprocessor for data processing and calculation, and finally display the results by the display and printer. The microprocessor also controls the movement of the mechanical drive mechanism in the X direction and Y direction to move the microplate through the control circuit, so as to realize the automatic sampling detection process. Other microplate readers use manual movement of the microplate for detection, so the mechanical drive mechanism and control circuit in the X and Y directions are omitted, so that the instrument is smaller and the structure is simpler.
The microplate is a transparent plastic plate specially used to place the sample to be tested. The plate has multiple rows of small holes of uniform size, and the corresponding antigen or antibody is embedded in the hole. Each small hole can hold a few ten milliliters of solution.
Light is an electromagnetic wave. The wavelength of 100nm-400nm is called ultraviolet light, the light between 400nm and 780nm can be observed by the human eye, and the wavelength greater than 780nm is called infrared light. People can only see colors because the light hits the object and is reflected back by the object. Green plants are green because most plants absorb red-orange light and blue-violet light, but they do not absorb and reflect green, so plants appear green. The principle of microplate reader measurement is to detect the absorbance of the analyte at a specific wavelength.
Structure
There are 40-well plates, 55-well plates, 96-well plates, and so on. Different instruments use different orifice plates, which can be tested one by one or row by row.
The monochromatic light used by the microplate reader can be obtained either through a coherent filter or the same monochromator as a spectrophotometer. When using a filter as a filter device, it is the same as an ordinary colorimeter. It can be placed in front of the microplate or behind the microplate. After the condenser, the diaphragm reaches the reflector, it is reflected by the reflector at 90° and passes through the colorimetric solution vertically, and then sent through the filter. To the photocell.
From the working block diagram and light path diagram of the microplate reader, it can be seen that it has the following differences with ordinary photoelectric colorimeters:
(L) The container for the colorimetric solution to be tested no longer uses a cuvette, but a plastic microplate. The microplate is usually made of transparent polyethylene material, which has a strong adsorption effect on antigen and antibody, so it is used It acts as a solid phase carrier.
⑵Because the plastic microplate containing the sample is multi-row porous, the light can only pass through vertically, so the light beam of the microplate reader passes through the solution to be tested and the microplate vertically. The light beam can be from top to bottom or It can pass through the colorimetric fluid from bottom to top.
⑶ Microplate readers usually not only use A, but sometimes also use optical density OD to express absorbance. Microplate readers can be divided into two types: single-channel and multi-channel, and single-channel is divided into two types: automatic and manual. Automatic type of instrument There is a mechanical drive mechanism in the X and Y directions, which can send the small holes of the microplate L one by one under the beam for testing, and the manual type uses the manual movement of the microplate for measurement.
On the basis of the single-channel microplate reader, a multi-channel microplate reader has been developed. This type of microplate reader is generally automated. It does not have multiple beams and multiple photodetectors, such as a 12-channel instrument with 12 beams or 12 light sources, 12 detectors and 12 amplifiers, under the action of the mechanical drive device in the X direction, 12 samples are detected in a row. The detection speed of the multi-channel microplate reader is fast, but its structure More complex prices are also higher.
Application range of microplate reader
Hematology test Platelet-related antibody test PAIgA, PAIgG, PAIgM
Determination of D-Dimer D-Dimer
Determination of serum fibrin degradation products FDP
Determination of T3, T4 with triiodothyronine and tetraiodothyronine
Immunological test C-reactive protein determination CRP
Determination of immunoglobulin IgD, IgE
Determination of circulating immune complexes CIC
Determination of rheumatoid factor IgG, IgA, IgM RF
Anti-thyroglobulin antibody, microsomal antibody determination TG, TM
Tumor cancer immunological detection Alpha-fetoprotein determination AFP
Determination of carcinoembryonic antigen CEA
Determination of prostate specific antigen PSA
Determination of pancreatic cancer, biliary tract cancer, gastric cancer CA19-9
Determination of ovarian cancer CA125
Determination of breast cancer CA15-3
Determination of cervical squamous cell carcinoma SCC
Detection of multiple bone marrow cancer
Determination of thyroid cancer hTG
Infectious disease immunological test, hepatitis A serological test, anti-HAV-IgM
Hepatitis B Serology Test Two and a half pairs
Hepatitis C serology detection Anti-HAV-IgG, anti-HCV-IgM
Serological detection of hepatitis D Anti-HDV-IgG, anti-HDV-IgM
Serological detection of hepatitis E Anti-HEV-IgG
Detection of antibodies to hemorrhagic fever with renal syndrome HFRS-IgG
Detection of Japanese encephalitis virus antibody IgM
Detection of human immunodeficiency virus antibodies (ie, regional AIDS) HIV β 2 M
Prenatal and postnatal care function test Toxoplasma virus test TOXO
Rubella virus detection RV
Cytomegalovirus detection CMV
Detection of herpes simplex virus Anti-HSV (Ⅰ, Ⅱ)
Others can also do genetic testing and animal residues, pesticide residues inspection items, etc.
