Immunoturbidimetry
Turbidimetric inhibition immunoassay is an analytical technique that combines liquid-phase precipitation with optical instruments. The basic principle is straightforward: when an antigen and antibody react in a specific dilution system, an excess of antibody forms a soluble immune complex. A polymerization promoter, like polyethylene glycol, then causes the complex to precipitate into microparticles, creating turbidity in the reaction solution. As the antibody concentration remains fixed, more antigen leads to increased immune complexes and greater turbidity. By measuring this turbidity and comparing it with standards, we can determine the antigen content in the sample.
Classification of Immunoturbidimetry
Immunoturbidimetry is divided into two types based on the light path: immunotransmission turbidimetry and immune scattering turbidimetry.
- Immunotransmission Turbidimetry:
In this method, the immune complex forms and becomes turbid over time. Light passing through the solution gets absorbed by the complexes. More complexes result in greater light absorption. This method is quicker and more sensitive than traditional immunochemical methods but requires sufficiently large immune complexes for detection. - Immune Scattering Nephelometry:
Here, light of a specific wavelength is directed horizontally through the solution. When it hits particles from the antigen-antibody complex, it refracts and scatters. More antigen leads to more complexes and stronger scattered light. This method includes:- Rate Nephelometry: Continuously monitors scattered light intensity over time to find the peak reaction rate, correlating peak size with antigen concentration.
- End-Point Nephelometry: Measures the total amount of complexes formed at equilibrium, generally taking over 10 minutes.
- Latex-Enhanced Immunoturbidimetry (LETIA):
Standard immunoturbidimetric methods may lack sensitivity due to complex sizes. LETIA improves sensitivity by using latex particles, which increase the volume of complexes. This change significantly enhances the intensity of transmitted and scattered light.
Overall, turbidimetric inhibition immunoassay provides an effective and efficient approach for analyzing and detecting antigens in various biochemical applications.
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 first used turbidimetry to detect fat particles in blood and milk in 1915, Hyland and Behring introduced the turbidimeter in 1976. Then, in 1977, Stenberg proposed immunoturbidimetry. Over the last century, immunoturbidimetry has significantly evolved. Today, in vitro diagnostic reagents that utilize immunoturbidimetry are widely employed in clinical practice, demonstrating its importance in modern diagnostics.
Epilogue about Immunoturbidimetry
Immunoturbidimetry is based on immunity principles, making it an important technology in biochemical diagnosis. As automation improves, diagnostic methods that combine clinical chemistry and immunology also evolve. Researchers and clinicians frequently use biochemical analyzers for these tests, reinforcing their importance in diagnostics.
AxisPharm’s nanoparticle-enhanced immunoturbidimetric assays offer greater sensitivity and a high level of automation. Our test kits efficiently measure and are suitable for large-scale clinical analysis. In our latex-enhanced agglutination tests, we coat high-affinity antibodies onto latex particles. This approach improves sensitivity, precision, working range, and provides better calibration stability and signal change.
Ref:
Whicher JT, Price CP, Spencer K. Immunonephelometric and immunoturbidimetric assays for proteins. Crit Rev Clin Lab Sci. 1983;18(3):213-60.