Chemiluminescence immunoassay (CLIA) is a detection and analysis technology that combines highly sensitive chemiluminescence assay technology with highly specific immune response, and can be used for the detection of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. CLIA first appeared in the mid-1970s, but it was not used in clinical detection because of the short duration and low sensitivity of the light signal. Subsequently, it was found that horseradish peroxidase (HRP), acridinium ester, amantadine and other derivatives and nanoparticles can enhance the luminescence intensity of CLIA, and this method has been developed as an advanced and widely used ultramicro activity detection method.
Chemiluminescence immunoassay mainly includes two aspects: Immune reaction and chemiluminescence system. The immune response is mainly the binding of antigens and antibodies, and the chemiluminescence system is designed to quantitatively detect this binding. Specifically, the chemiluminescent substance is labeled on the antigen (antibody), or the enzyme is acted on the chemiluminescent substrate, and under the action of catalysis and oxidation, the chemiluminescent substance is excited from the ground state to the excited state, and then back to the ground state and release photons. Due to the linear relationship between the strength of the light signal and the concentration of the object to be measured within a certain range, the optical detection system can quantitatively detect the light signal to determine the content of the object to be measured.
Compared with traditional immunoassay (radioimmunoassay, enzyme immunoassay, fluorescence immunoassay), CLIA has these advantages:
High sensitivity-up to 10^-16 mol/L, which can detect substances that cannot be detected by enzyme-linked immunoassay (ELISA), which is of great significance for early diagnosis.
Wide linear kinetic range-the luminous intensity between 4-6 orders and the measured substance concentration is linear relationship, obvious advantages.
Accurate quantitative detection-the intensity of the light signal has a linear relationship with the concentration of the substance to be measured, and the concentration of the substance to be measured can be accurately calculated according to the standard curve of the instrument, while ELISA is usually only used for qualitative or semi-quantitative analysis. Results are stable and the error is small-the sample itself emits light, no additional light source is required, the interference of external factors is avoided, the analysis results are stable and reliable.
Operation is simple-the optical signal lasts for a long time, and the vast majority of the analysis and determination only need to add a reagent, simplifying the experimental operation.
Direct chemiluminescence immunoassay is a new detection method for antibody or antigen labeling using chemiluminescent markers. Direct chemiluminescent agents participate directly in the luminescence reaction without enzyme catalysis in the process of luminescence immunoassay. They have specific groups on the chemical structure that produce light, which can directly label antigens or antibodies. At present, luminol and acridine esters are commonly used chemiluminescent markers. Luminol should not be used directly, and some compounds should be used to directly measure the enhancement or inhibition of luminol.
After the antibody is directly labeled with acridinium ester and the corresponding antigen in the specimen to be tested, the solid phase coated antibody-antigen-acridinium ester labeled antibody complex is formed. At this time, the oxidizer (H2O2) and NaOH are added to create an alkaline environment. Acridinium ester can be decomposed and luminous without the need of catalyst, and is received by the light collector and photomultiplier tube. The photon energy generated per unit time is recorded, and the integral of this splitting is proportional to the amount of antigen to be measured, and the amount of antigen to be measured can be calculated from the standard curve.
Chemiluminescent enzyme immunoassay (CLELA) using enzyme marker antigen or antibody immune response, immune response compound enzymes and effect on the bottom of the light on, under the effect of signal reagent, with luminous glow signal detector for determination, The concentration of the enzyme determines the intensity of chemiluminescence. HRP and alkaline phosphatase (ALP) are commonly used as marker enzymes for chemiluminescent enzyme immunoassay. Luminescent substrates are luminol and AMPPD.
ALP-AMPPD conjugates: The luminescence system composed of ALP and 1, 2-dioxocyclohexane is the most important and sensitive chemiluminescence system at present, among which the most representative is the ALP-AMPPD luminescence system. The reaction system uses alkaline phosphatase to label antibodies, and after immune reaction with the tested specimens and solid phase carriers in the reaction system, A solid-phase antibody-antigen-enzyme labeled hang-complex is formed, and AMPPD luminous agent is added. Alkaline phosphatase causes AMPPD to dephosphoric acid base group and emit light.
When a certain voltage or current is applied to the electrode, a chemical reaction occurs between the electrode electrochemical reaction products or between the electrode reaction products and their components in the solution to produce an excited state. When the excited state transition back to the ground state, energy is released. This process is called electrochemiluminescence (ECL). Electrochemiluminescence immunoassay is an electrochemical luminescent agent labeled with tripyridine ruthenium antibody, tripropylamine (TPA) as electron donor, and the specific chemiluminescence reaction occurs due to electron transfer in the electric field.
In the electrochemical luminescence immunoassay system, the magnetic particle is a solid phase carrier coated with antibody, and the antibody is labeled with tripyridine ruthenium. After the immune reaction between the test specimen and the corresponding antibody in the reaction system, the magnetic particle is coated with antibody-antigen to be measured, and the tripyridine ruthenium labeled antibody complex is formed. At this time, the complex is inhaled into the flow chamber, and the TPA buffer is introduced. When magnetic particles flow over the electrode surface, unbound labeled antibodies and specimens are caught by an electromagnet mounted below the electrode and washed away by the buffer. At the same time, the electrode is pressurized to start the electrochemical luminescence reaction, so that the tripyridine ruthenium and TPA carry out electron transfer on the electrode surface, resulting in electrochemical luminescence, and the intensity of light is proportional to the concentration of the antigen to be measured.
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CLIA has gradually received attention in various fields, with strong specificity, high sensitivity, high accuracy, simple operation, rapid results and other advantages, and is widely used in life science, environmental science, clinical medicine, physiology and other fields.
Thyroid hormone testing: CLIA is often used for thyroid hormone assessment in a clinical setting. HRP is used to label thyroid hormone-specific antibodies such as thyroxine (T4) and triiodothyronine (T3). This enzyme catalyzes a luminescent reaction that accurately quantifies hormone levels in serum samples.
Reproductive hormone detection: ALP-labeled antibodies sensitively measure reproductive hormones such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are critical for assessing fertility and menstrual cycle disturbances.
Pituitary hormones and corticosteroid hormones: Cortisol levels are measured using antibodies that bind to HRP. The chemiluminescence signals produced reflect cortisol concentrations and help diagnose diseases such as Cushing's syndrome.
Anemia factor testing: When diagnosing anemia, CLIA measures specific markers, such as erythropoietin (EPO). EPO can bind to biotin, which binds to streptavidin coated surfaces in assays, improving the sensitivity and reliability of the detection of this key hormone involved in red blood cell production.
Tumor marker assessment: Tumor markers such as prostate-specific antigen (PSA) can be detected with CLIA. Biological conjugates, such as avidin-biotin complexes, amplify the signal. Through the use of biotinylated antibodies against PSA, this method allows for early detection of prostate cancer, thereby aiding timely intervention.
Biomarkers of infection: For infectious diseases, CLIA can effectively detect biomarkers such as C-reactive protein (CRP). The test uses HRP-conjugated antibodies against CRP to quickly and reliably indicate inflammation or infection to guide treatment decisions.
Diabetes monitoring: In diabetes management, CLIA helps quantify insulin levels. Insulin antibodies can bind to ALP to sensitively detect insulin in plasma, which is essential for monitoring blood sugar control in patients with diabetes.
Cardiac biomarkers: CLIA is critical for measuring cardiac markers such as troponin I. The test utilizes biological conjugates such as HRP labeled antibodies to quickly and accurately assess myocardial damage, which is critical for the diagnosis of acute coronary syndrome.
Allergy testing: The use of biotinized allergen proteins linked to streptavidin enables accurate quantification of allergen-specific IgE, which is critical for diagnosing allergy conditions.
Therapeutic drug monitoring: Drug-specific antibodies that bind to ALP accurately measure drug concentrations in plasma, ensuring effective treatment while minimizing side effects.