Ricin Detection and Analysis Method

Immunolabeling Assay

The immunolabeling method is a detection method that biologically labels antigens or antibodies and uses the characteristics of RT antigen sites to specifically bind to antibodies. It is the most commonly used technical means to detect RT. Commonly used immune antibody labeling methods include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), colloidal gold labeling method and immunoPCR method.

Figure 1. Ricin detection methods in relation to biological steps that occur during ricin intoxication.(Source: Bozza WP, et al. 2015)

• Radioimmunoassay

Radioimmunoassay (RIA) is an in vitro radionuclide detection method that combines the measurement method of radionuclides with the basic principles of immune response. It is highly sensitive and accurate. Radioimmunoassay is one of the earliest methods to detect low-concentration RT, which uses radioactive I125-labeled RT. Based on rabbit antiserum in buffer, it can be used to quantify RT as low as 100pg. Although radioimmunoassay has good sensitivity, its main disadvantages are the long incubation time, difficulty in dissolving isotopes, and the serious impact of radioactive elements on the environment. These limitations make it less popular compared to ELISA. The radioimmunoassay method can detect RT in the sample on the order of 100pg, and is suitable for detecting RT content in the blood of patients with RT poisoning. However, the disadvantages of this method are that the operation and processing are relatively cumbersome, the equipment requirements are higher than those of the enzyme-linked immunoassay, and the requirements for the professionalism of personnel and the processing of radioactive elements limit the wide application of this method.

• ELISA Method

With the further development of biomarker technology, researchers have successfully established the ELISA method. The ELISA method is based on antibodies with different antigenic epitopes of RT and detects toxins through the reaction between antigens and antibodies. This method has the characteristics of wide adaptability, easy operation, short detection cycle and high specificity and sensitivity. In addition, the sandwich ELISA method proposed in recent years has further reduced the detection limit.

ELISA is a relatively mature detection method. Experimenters do not need to undergo special training or use special equipment. At the same time, it ensures the speed and accuracy of biological sample poisoning detection, providing technical support for the rapid detection of RT poisoning. Although the assay is less sensitive than reported amplification or chemiluminescent immunoassays, the assay is sufficient to support in vivo RT distribution analysis and on-site RT contamination monitoring.

• Colloidal Gold Labeling Method

The colloidal gold labeling method is a new immunolabeling technology that uses colloidal gold as a tracer marker or chromogenic reagent for antigen-antibody reactions. Because it does not have problems such as endogenous enzyme interference and radioactive isotope contamination, and it uses different particles Small and large colloidal gold can also be double or even multiple labeled to make the positioning more accurate, and the colloidal gold biosensor can be used to read the signal and fit the detection curve to complete quantitative detection. In recent years, relevant researchers have tested contaminated samples containing RT, and the detection sensitivity, specificity and stability are good. Some researchers have established an immunochromatographic method for rapid detection of RT. The method is to use rabbit anti-RT polyclonal antibodies as labeled antibodies, label colloidal gold particles, and coat RT antibodies (RCA) on nitrocellulose membranes as detection lines, coated with goat anti-rabbit IgG antibody as a quality control line, and used a colloidal gold biosensor to read the signal, fit the detection curve, and complete quantitative detection. Simulate the addition of RT to common foods to evaluate the detection ability of the method. The results show that the colloidal gold immunochromatography test strip can complete the detection within 15 minutes, with a sensitivity of 0. 925 μg/ml, a linear range of 0. 925~14. 8 μg/ml, and good specificity and stability. Take animal serum, milk, apple juice, orange juice, biscuits, jelly and milk powder to simulate contamination samples respectively, treat them with sample diluent and then add RT. The results show that the test strip can still detect 0.925 μg/ml for the above simulated contaminated RT.

• Immuno-PCR Method

Immuno-PCR (IPCR) is a micro-antigen detection technology based on the highly specific reaction of antigen-antibody binding and the extremely high sensitivity of PCR reaction. IPCR was established by Sano in 1992. This method is essentially an improved ELISA that uses PCR instead of enzyme reaction to amplify and display the antigen-antibody binding rate. Just by introducing PCR, the sensitivity of the existing antigen detection system can be improved by at least several order of magnitude, it is the most sensitive method in RT detection at this stage. IPCR can represent a simple method to detect mature proteins in the immune systems and tissues of animals and humans and can assess the pharmacokinetic behavior of immunotoxins. Comparing the IPCR method with traditional immunological methods, PCR and polymerase chain reaction post-analysis require more time for detection, use more expensive reagents, and increase reagent consumption, making this technology less attractive. However, these limitations are offset by higher sensitivity, enabling a wider range of applications.

Biosensing

Biosensors combine biotechnology and electronic technology to convert the chemical signals of biologically sensitive substances generated by the specific combination of the object to be measured and the recognition element into electrical signals, optical signals, etc., so as to achieve the purpose of analysis and detection. This method does not require special markers and has the characteristics of good selectivity, simple operation, and real-time online monitoring.