What is Time-Resolved Fluorescence Immunoassay (TRFIA)?



I. Introduction

For nearly a hundred years, “specific reagents” have been the goal pursued by analytical chemists. The so-called “specific reagent” refers to a reagent that only reacts with one substance to be tested. In fact, the so-called “copper reagents”, “iron reagents”, “nitric acid reagents”, etc. currently used are all “well-known, but actually difficult to match”. In the 1940s and 1950s, the enthusiasm for synthesizing special-effect reagents had long since cooled. At a time when analytical chemists were disheartened, the dawn of this ideal was seen in the achievements of immunology and biochemistry: the immune system is simply a naturally occurring machinery for the synthesis of specific reagents. The immune reaction between antigen and antibody is highly specific, and the specificity of this recognition exceeds the recognition level of the enzyme to the substrate. The stability constant of the antigen-antibody complex is generally 109, and some are as high as 1010-1015. High stability. The characteristics of immune response make immunoassay a new interdisciplinary analytical technique, which is widely used in clinical body fluid analysis, drug analysis, environmental analysis, food analysis and biochemical research, especially in the identification of drugs, the identification of drug addicts and the identification of diseases. played an important role in diagnosis.

Time-resolved fluorescence immunoassay (TRFIA) is a new analytical technique developed since the 1980s. Compared with other immunoassay techniques, it has unique advantages. It overcomes the contamination problem caused by radioisotopes in radioimmunoassay (RIA); overcomes the disadvantage of enzyme instability in enzyme immunoassay (EIA); moreover, because TRFIA method can eliminate the interference of background fluorescence very well, This makes it several orders of magnitude more sensitive than ordinary fluorescence methods (FIA). It is precisely because of the unique advantages of TRFIA that it has become the most promising analytical method in immunoassay.

II, the principle of time-resolved fluorescence immunoassay

The principle of time-resolved fluorescence immunoassay is to use trivalent rare earth ions (such as Eu3+, Tb3+, Sm3+, Dy3+) as tracers, and through these rare earth ions, chelators with bifunctional structures and antigens form rare earth ions-chelator-antigen chelate. When the labeled antigen and the antigen to be tested compete for the antibody together to form an immune complex, since the antigen-antibody binding part of the immune complex contains rare earth ions, when some methods are adopted to separate the binding part from the free part, a time-resolved fluorescence analyzer is used. The fluorescence intensity emitted by the rare earth ions in the complex can be measured, thereby determining the amount of the antigen to be detected.

Under normal circumstances, the autofluorescence signal of rare earth ions in the immune complex is very weak. If an acidic enhancing solution is added, the rare earth ions are dissociated from the immune complex, and the β-diketone and tri-n-octane in the enhancing solution are dissociated. A kind of microcapsule is formed by components such as base phosphine oxide and Triton X-100. After the latter is excited by the excitation light, the rare earth ions can emit a long-lived extremely strong fluorescent signal, which enhances the original weak fluorescent signal by nearly 1 million times.

Fluorescence is measured by time-resolved technology, and gated technology is used, which is to measure the fluorescence of long-lived markers after the background fluorescence signal is reduced to zero.

III, the measurement method of time-resolved fluorescence analysis

(1) Dissociation enhancement measurement method
The dissociation-enhanced measurement method is a dissociation-enhanced rare earth ion fluorescence method, referred to as the DELFIA method. The Eu3+ or Sm3+ is chelated to the antigen, antibody or SA through the bifunctional group. After the immune reaction, part of the label is bound to the solid support, and the unbound label is washed away. Finally, use a low pH enhancement solution to dissociate Eu3+ or Sm3+ from the immune complex to form a new chelate of Eu3+-(NTA)3.(TOPO)3 or Sm3+-(NTA)3.(TOPO)3 , the fluorescence intensity is increased by a factor of nearly a million. Liquid phase detection was performed with an Arcus-type series instrument. This method has good repeatability and is especially suitable for the detection of large and small molecular active substances. Using DELFIA measurement, the sensitivity can reach 10-16-10-18mol DNA amount (ie 10pgDNA). The disadvantage of this method is that it cannot directly measure the fluorescence of solid-phase samples, and requires an external enhancement solution, which is easily interfered by exogenous Eu3+ or Sm3+, which affects the results and needs to be improved.

(2) Solid-phase fluorescence measurement
The solid-phase fluorescence method is also known as the DSLFIA method. It chelates Eu3+ or Sm3+ with antibody or antigen through chelating agent BCPDA with special bifunctional group. After the immunoreaction between antigen and antibody, the fluorescence intensity of Eu3+-BCPDA in the solid-phase immune complex can be directly measured. The whole process does not need to add an enhancement solution, and the fluorescence of solid-phase samples can be directly measured, which solves the problems of easy contamination and complicated operation caused by liquid-phase measurement. It has been used for nucleic acid probe detection and has made satisfactory progress, and has been supplied as a kit abroad.

(3) Direct fluorescence measurement
The direct fluorescence method uses the bifunctional chelating agent diethylenetriaminepentaacetic acid-p-aminosalicylic acid (DTPA-PAS) to couple with the antigen or antibody. After the immune reaction, an appropriate amount of Tb3+ is added to directly measure the liquid phase fluorescence intensity. There is no need to prepare Tb3+ label in advance, which simplifies the operation steps, but the sensitivity is low, only 10-9mol/l. In order to improve the detection sensitivity, an enzyme amplification system can be introduced to hydrolyze 5-fluorosalicylic acid phosphate with SA-alkaline phosphatase to generate 5-fluorosalicylic acid, which can react with Tb3+-EDTA-HCl under high pH conditions. Forms complexes with high fluorescence intensity, which can be directly measured.

(4) Homogeneous fluorescence measurement method
Homogeneous fluorescence measurement method is to use special bifunctional chelator W1174 to label small molecule active substances with Eu3+. When combined with antibody, the immune complex can significantly enhance or quench the fluorescence signal of Eu3+, so it is not necessary to carry out the measurement before measurement. separation, the fluorescence intensity in the liquid phase can be directly measured. This method omits the cumbersome steps of washing, separation and adding enhancement solution, and has the advantages of rapidity and convenience, but the disadvantage is that a special chelating agent is required.

(5) Synergistic fluorescence measurement method
The synergistic fluorescence measurement method uses some non-fluorescent rare earth ions, such as Cd3+, Y3+, La3+, Lu3+, etc., which can greatly enhance the fluorescence signal of the fluorescent rare earth ions. In the immunoassay system, when the pH value is 6.0-8.0, their fluorescence intensity is the largest, which is beneficial to improve the detection sensitivity. In addition, this method can simultaneously measure the content of different components in a sample. However, a special enhancement solution is required, and the purity of rare earth ions such as Cd3+, Y3+, La3+, Lu3, etc., has a significant impact on the measurement results.

IV, the application of time-resolved fluorescence immunoassay

Time-resolved fluorescence immunoassay can be used to detect biologically active substances, especially in the immunoassay of biological samples, showing more and more unique advantages. In the detection of endocrine hormones, tumor marker detection, antibody detection, viral antigen analysis, drug metabolism analysis and the analysis of various in vivo or exogenous ultra-trace substances, the application of TRFIA method is more and more common. In recent years, this technology has been applied to nucleic acid probe analysis, cell activity analysis, and biological macromolecular analysis, and it has developed rapidly.

(1) Application of TRFIA method in endocrinology
Endocrine hormones are some active small molecules, which can react with appropriate antibodies and are immunoreactive, but cannot produce antibodies and are not immunogenic. They belong to haptens. For the determination of these haptens, the competitive time-resolved fluorescence immunoassay is generally used. The determination of this aspect mainly includes the determination of serum progesterone, estradiol, testosterone, thyroid hormone, prostaglandin and so on.

(2) Application of TRFIA in Oncology
For some complete antigens, most of them are proteins with both immunoreactivity and immunogenicity, mainly including thyroid stimulating hormone, serum insulin, serum carcinoembryonic antigen, serum alpha fetus Protein, hepatitis B surface antigen, etc., are mainly determined by non-competitive TRFIA method.

(3) Application of TRFIA method in immunology
The activity of some immune cells (such as NK, LAK, T killer cells, etc.) can be detected by TRFIA method. For example, Blomberg used this method to measure the activity of NK cells; Granberg used TRFIA to measure the activity of killer T lymphocytes; Volgmann measured the activity of LAK cells; Maley used TRFIA Cell-mediated, complement-mediated and antibody-dependent cellular activities were measured by the method; Lovgren and Blomberg used this method to simultaneously measure the activity of two target cells; These assays yielded good results.

(4) Application of TRFIA method in microbiology
Due to the high sensitivity and specificity of TRFIA technology, the application of TRFIA method in the detection and analysis of microorganisms has become increasingly extensive and in-depth. At present, TRFIA has been widely used in hepatitis B virus, encephalitis virus, influenza virus, respiratory syncytial virus (RSV), paramyxovirus, rubella virus, potato virus, rotavirus, human immunodeficiency virus (HIV), hemorrhagic fever Antigen antibodies to viruses and Treponema pallidum and detection of antibodies to certain bacteria and parasites.

V. Outlook

Time-resolved fluorescence immunoassay has the advantages of high sensitivity, strong specificity, good stability of the reagents used, wide detection limit, especially the elimination of high background in conventional fluorescence assays, etc. It has great potential for development in non-radioimmunoassay. Analytical method. Relevant research papers account for about 10%-15% of the immunoassay literature. Find, design and synthesize ideal lanthanide chelate probes; improve labeling technology and simplify separation technology; use multiple labeling methods for amplification to further improve sensitivity and improve the programming of detection methods to further improve the signal-to-noise ratio. Become the development direction of time-resolved fluorescence immunoassay.

In Vitro Diagnostic (IVD) Research and Development