Determination method of non-steroidal anabolic hormone drug residues (5)

(8) Immunoassay (IA)

The immunoassay method is based on the specific reaction of antigen and antibody, and is known for its high specificity and high sensitivity.
It can simplify the analysis process and is suitable for the analysis of trace components in complex matrices
.
Mainly include chemiluminescence enzyme immunoassay (CLEIA), radioimmunoassay (RIA), radioreceptor analysis (RRA), enzyme-linked immunosorbent assay (ELISA), time-resolved fluorescence immunoassay (TRFIA) and immunosensor

1) Chemiluminescence enzyme immunoassay (CLEIA)

CLEIA uses enzymes involved in a certain chemiluminescence reaction, such as horseradish peroxidase (HRP) or alkaline phosphate (ALP) to label antigens or antibodies, and the corresponding antigen in the sample to be tested After (antibody) reacts, a solid-phase coated antibody-antigen-to-be-tested-enzyme-labeled antibody complex is formed.
After washing, the substrate (luminescent agent) is added, and the enzyme catalyzes and decomposes the substrate to emit light, which is received by the light quantum reading system.
The photomultiplier tube converts light signals into electrical signals and amplifies them, and then transmits them to the computer data processing system to calculate the concentration of the object to be measured
.

Zhu et al.
established a CLEIA detection method for ZER in milk and urine
.
Add 15μL of β-glucuronidase /arylsulfatase to 5mL sample for 16h at 37℃, homogenize for 3min, add 10mL acetonitrile , centrifuge at 3500r/min for 10min, and blow dry the supernatant in a water bath at 40℃.

2) Radioimmunoassay (radioimmunoassav, RIA)

RIA uses an isotope-labeled and unlabeled antigen to competitively inhibit the reaction with an antibody.
After the reaction, the amount of unlabeled antigen is obtained by separating and measuring the radioactivity
.
Since it was first used to detect DES residues in animal urine in the 1970s, RIA has been widely used to detect DES residues in animal muscles, liver, urine, and milk, and to study the metabolism of DES in animal tissues

vanPeteghem et al.
used RIA to determine DES residues in meat
.
The sample was hydrolyzed by enzyme, extracted with ether, cleaned up by SPE column, and determined by RIA

3) Radio receptor assay (RRA)

RRA is a competitive radioanalysis method that uses receptor protein as a specific coupling agent to determine the analyte (ligand)
.
Receptor is a kind of protein in the cell, which can specifically bind to the ligand and has a high affinity

4) Enzyme-link immunosorbent assay (ELISA)

ELISA uses the specific reaction of antigen and antibody to connect the analyte to the enzyme, and then produces a color reaction between the enzyme and the substrate.
It is used for quantitative determination with high sensitivity and safe operation.
It does not require expensive instruments and can meet large-scale rapid determination.
Demand
.

Goldstein et al.
used ELISA to determine the content of DES in animal tissues, cells, and sub-cells.
The detection sensitivity was 10 ng/mL, which was similar to the results measured by RIA at the same time.

Sawaya et al.
did an additive test on sheep urine and chicken.
The added value of urine was 0.
5-5ng/mL.
The urine was diluted with sodium acetate buffer and purified by C ₁₈ column.
The DES in sheep urine was measured by ELISA.
The recovery rate is 90%～106%; the added value of chicken meat is 0.
2～1.
5ng/mL, the sample is repeatedly extracted with tert-butyl methyl ether and purified by _C18 column.
The recovery rate of DES in chicken meat is 49%～68%.

.
Li et al.

Wang Hejia and others established an ELISA method for the detection of ZER residues in cow urine
.
The bovine urine sample was centrifuged at 6000r/min for 10min, the supernatant was passed through a 0.

5) Time-resolved fluoroimmunoassay (time-resolved fluoroimmunoassay, TRFIA)

TRFIA is a non-isotopic immunoassay technology that uses lanthanide elements to label antigens or antibodies.
According to the luminescence characteristics of lanthanide chelates, time-resolved technology is used to measure fluorescence, and the two parameters of wavelength and time are simultaneously detected for signal resolution.

6) Immunosensor (immunosensor, IS)

The immunosensor method is a biosensor that combines high-sensitivity sensing technology with specific immune response to monitor the antigen-antibody reaction.
It has the high selectivity of immunoassay and the high sensitivity of electrochemical analysis, which is easy to implement.
The portability, miniaturization and automation of the residue detection instrument have the characteristics of fastness, sensitivity, high selectivity, and easy operation
.
Liu et al.
established composite materials based on hollow silica-gold nano-multi-wall carbon nanotubes (MSN-GNPs-MWCNTs) and horseradish peroxidase antibody-Prussian blue-multi-wall carbon nanotubes (HRP-Ab-GNPs).
-PB-MWCNTs) Electrochemical immunosensor method for detecting DES in milk
.
MSN-GNPs-MWCNTs composite material as a fixed matrix can enhance the electrical activity and stability of the electrode, and HRP-Ab-GNPs-PB-MWCNTs as a marker is used to improve the catalytic activity of the electrode
.
The LOD of the method is 0.
12ng/mL, and the recovery rate is 92%-107%
.
Wang Chuanxian and others established an electrochemical immunosensor method to detect DES in animal tissues and milk powder samples
.
Nano gold (AuNP) was modified on the electrode.
Then the graphene (Gr)-chitosan (CS) composite was decorated on the surface of the glassy carbon electrode, and the modified electrode was characterized by cyclic voltammetry
.
Using [Fe(CN) ₆ ] ^3- / ^4- as the oxidation-reduction probe, based on the change in the current response of the [Fe(CN) ₆ ] ^3- / ^4- probe caused by the DES antigen-antibody reaction , the response to DES can be realized Detection
.
The mass concentration of DES is in the range of 0.
5～1500.
0ng/mL, which has a good linear relationship with the peak current, the correlation coefficient is 0.
985, and the LOD is 0.
1ng/mL
.
The recoveries of DES in pork, beef, duck and milk powder were 71.
2%～117.
7%, 80.
5%～110.
1%, 80.
8%～117.
8% and 72.
3%～117.
2%, respectively
.

Feng et al.
used nanoporous PtCo alloy as the antibody carrier to prepare an immunosensor, and coupled the ZER antibody to a glassy carbon electrode.
They found that the enzyme-free rice porous PtCo alloy immunosensor has strong point catalytic activity for antigen-antibody reactions, and can detect cattle.
The detection limit of ZER in urine is 13 pg/mL
.
Feng et al.
also converted nano-montmorillonite into sodium-montmorillonite (Na-Mont) for the curing of thionine (TH), horseradish peroxidase (HRP) and secondary anti-ZER antibody (Ab ₂ )
.
The modified particles (Na-Mont-TH-HRP-Ab ₂ ) were used as a marker for the immunosensor to detect ZER
.
The immunosensor was prepared by using a nanoporous gold membrane (NPG) immobilized with a primary antibody (Ab1) and modified with a glassy carbon electrode (GCE) on the surface
.
In the concentration range of 0.
01-12ng/mL, the correlation coefficient r of ZER is 0.
9996; LOD is 3pg/mL
.

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