Study of immunoassay methods for erythropoietin in stimulants

Summary

As far as clinical application is concerned, the determination of EPO has gone through three stages: 1) in vivo assay; 2) in vitro assay; and 3) immunoassay. The immunoassay developed in the later stage is the most widely used clinical method to detect EPO.

Operation method

immunoassay

Principle

Immunoassays are based on the specificity and affinity of the antigen and antibody reactions, and are characterized by rapidity, sensitivity and simplicity. Radioimmunoassay (RIA) is one of the earlier developed methods, with high sensitivity and accuracy, and is the standard method for the determination of EPO concentration in the human body - it was basically adopted for the determination of normal EPO concentration in the human body at the very beginning.Since the 1990s, many scholars have further improved it by combining it with the new technology: Roberts-D et al. improved it by pre-treatment of blood samples. Since the 1990s, many scholars have further improved the method by combining it with new technologies: Roberts-D et al. improved the conventional RIA assay by pre-treatment of blood samples to increase the sensitivity of EPO detection. With the emergence of EPO-RIA kit, the operation of this method is easier, but because RIA method involves radioactive elements, which is unfavorable to the human body, the non-radioimmunoassay method with the same accuracy and sensitivity is more favored by scholars nowadays.

Materials and Instruments

Urine Sample
Antibody, polyacrylamide gel, pvdf membrane, electrophoresis buffer, membrane transfer buffer
Liquid phase analyzer Hydrophobic column UV spectrophotometer Electrophoresis instrument Enzyme plate Transfiltration instrument

Move

Radioimmunoassays mainly include enzyme immunoassay (including EIA and ELISA), fluorescence immunoassay, chemiluminescence immunoassay, and so on.

Enzyme immunoassay is not radioactive and is more flexible in the choice of color development mechanism and method (ELISA has been reported so far). However, since the enzyme method is based on the photometric method, it is limited by the limited absorbance and kinetic range of the colorant, and therefore the sensitivity is not high. Therefore, Rossi-R et al. used monoclonal antibody instead of polyclonal antibody in the assay, so that the sensitivity of ELISA was further improved, and the sensitivity of the detection of EPO content in body fluids reached 10-11M (comparable to that of the radioimmunoassay).

Fluorescence immunoassay (FIA) is the most sensitive of the non-radioimmunoassays, and it is often used to detect very low levels of biologically active substances. In recent years, the use of special fluorescent probes to establish an effective FIA method for the detection of EPO has also become the focus of many scholars, such as the extraction of phycobiliprotein from Spirulina as a fluorescent probe, etc. In addition, chemical radioimmunoassay (CRA) has been used for the detection of EPO.

In addition, chemical luminescence immunoassay (CLIA) is another active research field, which is characterized by high sensitivity and low background. The sensitivity of CLIA is generally higher than that of enzyme immunoassay. Currently, there are kits made using the principle of CLIA for sale.

Caveat

There are numerous difficulties in detecting exogenous EPO due to the physiological properties of EPO itself:1. Low levels of EPO in body fluids. The level of EPO in normal human body fluids is only nanograms per milliliter (ng/mL), and in blood it is 130-230 ng/mL.

2. EPO is structurally similar to rhEPO. EPO and rhEPO are structurally similar. They have identical amino acid sequences, with only a slight difference in the sugar group attached to the N-terminal end.

3. EPO has a short half-life in blood. The half-life of EPO in the blood is short. It is generally reported to be 5-6 hours by static injection.

4. The concentration of EPO varies greatly between individuals and among individuals themselves.

Common Problems

With the continuous development of immunoassay, some new analytical means and methods are emerging, especially about the conception of test strips, which makes the immunoassay more promising.

Sourced from Advances in Detection Technology of EPO

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