Turbidimetric inhibition immunoassay is an analytical technique that combines precipitation in liquid phase with optical instruments and automated analytical techniques. The basic principle is: when the antigen and antibody react in a special dilution system and the ratio is appropriate (generally stipulates that the antibody is excessive), the soluble immune complex formed is under the action of the polymerization promoter (polyethylene glycol, etc.) in the dilution system. , precipitated from the liquid phase to form microparticles, causing turbidity in the reaction solution. When the antibody concentration is fixed, the amount of the formed immune complex increases with the increase in the amount of antigen in the test sample, and the turbidity of the reaction solution also increases. By measuring the turbidity of the reaction solution and comparing it with a series of standard substances, the content of the antigen in the test sample can be calculated.
Classification of Immunoturbidimetry
According to the light path, immunoturbidimetry can be divided into immunotransmission turbidimetry and immune scattering turbidimetry.
Immunoturbidimetry
After the antigen-antibody is combined, an immune complex is formed, and the complex aggregates and appears turbid within a certain period of time. When light passes through the solution, it can be absorbed by immune complexes. The higher the amount of immune complexes, the more light is absorbed. The amount of light absorbed is proportional to the amount of immune complexes within a certain range. Compared with general immunochemical quantitative methods such as one-way agar diffusion test and rocket electrophoresis, this method is more sensitive, quicker and simpler, but requires the number and molecular weight of immune complexes to reach a certain height, otherwise it is difficult to detect.
Immune Scattering Nephelometry
Light of a certain wavelength is irradiated along the horizontal axis, and when it passes through the solution, it encounters the particles of the antigen-antibody complex, which refracts and deflects the light. The angle of light deflection is closely related to the wavelength of the emitted light and the size and number of the particles of the antigen-antibody complex. The intensity of scattered light is proportional to the content of the complex, that is, the more antigens to be tested, the more complexes are formed, and the stronger the scattered light is. The intensity of scattered light is also closely related to various physical factors, such as the time of adding antigen or antibody, the intensity and wavelength of the light source, and the measurement angle. Nephelometric method is further divided into rate nephelometric method and end-point nephelometric method.
Rate nephelometry is a dynamic assay. By continuously and dynamically monitoring the scattered light intensity generated by the antigen-antibody complex particles formed in a unit time, it can be found that the antigen-antibody reaction is the fastest and the scattered light is the strongest in a certain unit time, that is, the rate peak. Its peak size is positively correlated with antigen concentration.
End-point nephelometry is to detect the total amount of antigen-antibody complexes formed when the antigen-antibody reaction is in equilibrium, and the process generally takes more than 10 minutes.
Latex-enhanced immunoturbidimetry (LETIA)
Due to the size and number of antigen-antibody complexes, the immunoturbidimetric method has poor sensitivity and a limited detection range. The latex-enhanced immune turbidimetric method is a relatively stable and accurate immune turbidimetric derivatization technology that appeared later. The volume of the conjugate increases, and after the light passes through, the intensity of the transmitted light and scattered light changes more significantly, thereby improving the sensitivity of the test.
Common Interfering Factors about Immunoturbidimetry
1. Lipemia: The lipid blood sample contains a large number of chylomicrons, which have the characteristics of light scattering and will produce turbidity;
2. Jaundice: the background interference produced by bilirubin and its derivatives at a specific wavelength, bilirubin is unstable when exposed to light and heat, and is prone to generate biliverdin, bilirubin and other derivatives;
3. Hemolysis: interference of the absorbance value of hemoglobin itself;
4. Endogenous interfering substances: RF, heterophilic antibodies, human anti-animal antibodies, autoantibodies, M protein, etc.;
5. Incomplete centrifugation and repeated freezing and thawing of specimens;
6. Reagents fall into dust;
7. Dilution cup or cuvette is not clean;
8. Hook effect: when antigen is added to a solution with a constant amount of antibody, the amount of immune complexes generated increases with the increase of the amount of antigen, and when it reaches the peak, it decreases with the increase of the amount of antigen;
9. Drug interference, etc.
Immunoturbidimetry Solution:
1. Properly dilute the sample to be tested and the antigen standard/calibrator;
2. Calibrate the instrument;
3. Immunoprojection turbidimetry can reduce interference by setting dual wavelengths, sample blanks, and automatic sample dilution:
4. Use polyethylene glycol to pre-treat the sample, and add team serum or blocking agent before detection to reduce endogenous interference.
Clinical application
Since Klett and Bloor used turbidimetry to detect fat particles in blood and milk in 1915, Hyland and Behring introduced turbidimeter in 1976, and Stenberg proposed immunoturbidimetry in 1977. Immunoturbidimetry has been developed for a century. . At present, in vitro diagnostic reagents using immunoturbidimetry are widely used in clinical practice.
Epilogue about Immunoturbidimetry
Immunoturbidimetry is obviously the principle of immunity, why is it called the representative technology of biochemical diagnosis? With the gradual progress of automation technology, the diagnostic technology composed of clinical chemistry and immunology has been continuously developed. Immunoturbidimetric detection items are generally completed on a biochemical analyzer, so they are classified as biochemical diagnosis.
Axispharm nanoparticle-enhuanced immunoturbidmetric assays have increased sensitivity with high degree of automation. Our test kits have high efficient in the measurement and suitable for a large clinical analysis. In our latex enhanced agglutination tests, coating of high affinity antibodies onto a latex particle provides better sensitivity, precision, working range, with greater calibration stability and signal change. We also offer Latex-based kits and related development services with our automatic platforms.