Chemiluminescence immunoassay experiment

Summary

Chemiluminescence (CL) refers to the phenomenon of light radiation that accompanies the process of a chemical reaction. Most chemiluminescence is the luminescence of an oxidation reaction in which O2 and H2O2 are used as oxidizing agents. The chemiluminescence reaction consists of two key steps, i.e., chemical excitation and luminescence. The chemical excitation needs to provide enough chemical energy to excite the chemiluminescent agent to jump to the electronically excited state after absorbing energy, and there is enough luminescence quantum yield when returning to the lower energy level ground state. Currently, there are three main methods used in chemiluminescence immunoassay experiments: chemiluminescence enzyme immunoassay, chemiluminescence labeling immunoassay and electrochemiluminescence immunoassay.

Principle

The basic principle of chemiluminescence immunoassay experiment is to label the antigen or antibody with enzyme or chemiluminescent substance, and the chemiluminescence reaction occurs after the immunoreaction by adding the luminescent substrate of enzyme reaction (the former) or luminescent initiator, such as alkaline H2O2 solution (the latter), and the chemiluminescence detector analyzes the acceptance of the light quantum yield by the photomultiplier tube (PMT), and displays the result of the antigen-antibody reaction with the intensity of the light signal. It has the advantages of high sensitivity, wide linear range, simple instrumentation and easy automation.

Operation method

Chemiluminescent enzyme immunoassay

Principle

The basic principle of chemiluminescence enzyme immunoassay (CLEIA) is a detection method based on ELISA, the operation procedure is the same as that of ELISA, competitive method for the determination of small molecule antigens, double antibody sandwich method for the determination of large molecule antigens, and indirect method for the detection of antibodies, etc. CLEIA is different from ELISA in the sense that the enzyme's catalytic substrate is the chemiluminescent agent, which produces chemiluminescence, and the light intensity of the light signal is detected by the chemiluminescence signal detector. The signal intensity is detected by the chemiluminescence signal detector, which is received by the light quantum reading system, and the photomultiplier tube converts the light signal into an electrical signal and amplifies it, resulting in a higher sensitivity and wider linear range than the spectrophotometric method.

Materials and Instruments

Equipment:
① MicroplateLuminometer (MPL1 MicroplateLuminometer)
② Mouse anti-human TSHa subunit monoclonal antibody pre-coated plate (luminescence-grade)
① HRP-labeled goat anti-human TSHa subunit monoclonal antibody
① HRP-labeled goat anti-human TSHβ subunit mAb (HRP-TSHg mAb)
① HRP labeled sheep anti-human TSHβ subunit mAb (HRP-TSHg mAb) ② TSH standard (0-20 μIU/ml)
② TSH standard (0-20 μIU/ml) ③ Wash solution (0.05% Tween20/PBS, pH 7.4)
③ Wash solution (0.05% Tween20/PBS, pH 7.4) ④ Chemiluminescent substrate A and B liquid

Move

The basic procedure of chemiluminescence enzyme immunoassay can be divided into the following steps:
A

. Preparation

Remove the reagents and antibody pre-coated slides from the refrigerator at 2-8 ℃.

B

. Sampling

Add 50 μl of sample and 50 μl of standard to each well, then add 100 μl of HRP-TSHs mAb to each well, mix well, and incubate for 60 minutes at room temperature.

C

. Plate Washing

Decant

the reaction solution in the wells of the plate, add about 300 μl of wash solution to each well, let it stand for about 20 seconds, remove the liquid, tap out the liquid in the plate, and wash the plate 4 times.

D . Addition of luminescent substrate solution

Before use, take equal amounts of chemiluminescent substrate A liquid and B liquid, mix well and add 100 μl to each well.

E . Measurement of RLU Measure

the luminescence intensity (RLU) of each well at 5-20 minutes after the addition of luminescent substrate solution.

F . Experimental results and analysis
(1) Quantitative methods can be used to establish the regression equation of TSH concentration - RLU and plot a standard curve using the quantitative software or according to the RLU of different concentrations of standards. The TSH concentration in the sample is quantified according to the standard curve established from the TSH concentration of the standard and the corresponding RLU.
(2) Determine the result of the specimen according to the normal value range of 1.6 (0.4-7.0) μIU/ml.

Caveat

1. In the absence of a catalyst, luminal and hydrogen peroxide may react slowly with chemiluminescence, resulting in some background luminescence. Therefore, it should be kept separately and mixed well before use.

2. HRP-Luminol-H2O2-Enhancer luminescence system emits blue light at a wavelength of 425 nm, and the luminescence intensity (RLU) varies with time, so the luminescence intensity of each well should be measured in the first 5-20 minutes.

3. Wash the wells thoroughly to prevent air bubbles in the wells from affecting the accuracy of the results and causing false positives.

4. The amount of luminescent substrate added manually should be accurate, and during the addition of luminescent substrate, the contact between the tip of the spiker and the plate wells or fingers should be avoided to prevent contamination of the substrate, and there should not be any air bubbles at the same time.

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