Detection principle of ELISA

Light is an electromagnetic wave. The wavelength of 100nm to 400nm is called ultraviolet light. The light between 400nm and 780nm can be observed by the human eye, and light above 780nm is called infrared light. People can only see colors because light is reflected by objects when it is irradiated on them. The reason why green plants are green is that they absorb the red spectrum in the light. The principle of the microplate reader is to detect the absorbance value of the object under a specific wavelength.
　　Detection unit:
　　The energy difference before and after the light passes through the object is the energy absorbed by the object. At a specific wavelength, the concentration of the same object under test is quantitatively related to the absorbed energy.
　　The detection unit is expressed by OD value. OD is the abbreviation of optical density, which indicates the optical density absorbed by the object under test. OD = log (1/trans), where trans is the transmittance value of the object under test. According to the Bouger-amber T-beer rule, the OD value and light intensity have the following relationship:
E＝OD=logΙ0/Ι, where E represents the absorbed optical density, Ι0 is the light intensity before the test object, and Ι is the light intensity from the test object.
　　The OD value is calculated by the following formula:
　　E＝OD=C×D×E
　　C is the concentration of the test object,
　　D is the thickness of the test object, and
　　E is the molar factor
　　Each substance has its own specific wavelength when measured at a specific wavelength, at which the substance can absorb the most light energy. If other wavelength bands are selected, the test results will be inaccurate. Therefore, when measuring the test object, we choose a specific wavelength for detection, which is called the measurement wavelength
.
　　However, each substance still has a certain nonspecific absorption of light energy. In order to eliminate this nonspecific absorption, we select a reference wavelength to eliminate this inaccuracy. At the reference wavelength, the absorption of the test object light is the smallest. The difference between the absorbance values ​​of the detection wavelength and the reference wavelength can eliminate nonspecific absorption.
　　Anthos ELISA instrument test value calculation
　　The detector in the instrument receives the light energy transmitted through the object to be tested and converts it into a binary digital signal with a maximum value of 4095. The instrument defines the light transmittance value without light source as 0% and the light transmittance value without the object to be tested as 100%. In actual testing, the light transmittance value of the object to be tested is between 0% and 100%. The calculation of transmittance value
is as follows:
　　T = (Meas-Min) / (Max-Min)
　　where T is the transmittance value, Meas is the binary value of the detection, Min is the binary value detected in the case of 0%, and Max is the binary value detected in the case of 100%. For example:
　　MaX = 3600 Mn = 20 Meas = 30
　　T = (30-20) / 3600-20) = 0.0028
　　OD = 1og (1/T) = 1og (1/0.0028) = 2.552
　　Anthos microplate reader centering
　　The instrument will automatically center the microplate well. The centering is to eliminate the uneven thickness caused by the convexity and concavity at the bottom of the microplate well, which brings inaccurate detection. When testing each microplate reader, the instrument actually measures 35 points, and the average of the 5 middle points is selected as the OD value of this well.
　　Reference channel of light source
　　The reference channel is used to calibrate the effects caused by unstable voltage or bulb wear.
　　Uses and other tips for the ELISA reader
　　It is used for the determination of ELISA reagents and is widely used in various laboratories, including clinical laboratories.
　　Quality control
　　Quality control is an important factor in reagent testing. Please perform quality control according to the requirements of the reagent instructions.
　　Blank correction
　　Some test kits have blank wells set to air in their instructions, while most other blank wells are set with reagents. Please follow the instructions of the kit.
　　Interpretation of test results
　　Since there are quite a few factors that affect the test results, such as different ELISA plates and the volume of test reagents, which will cause different OD values, only the test results of reagents reacted with the same ELISA plate can be compared and analyzed. Please follow the instructions of the kit for clinical interpretation of the results.