Commonly used detection methods in the determination of glycated hemoglobin
Shenzhen Poweray Biotechnology Co., Ltd
Ion exchange chromatography (HPLC, LPLC): analysis based on charge differences. The combination of glucose and the N-terminal valine (Val) of the β-chain of hemoglobin (Hb) reduces the isoelectric point, resulting in less positive charge of glycated Hb than unsaccharified Hb, and less adhesion to the resin. The buffer of ion concentration elutes Hb from the cation exchange column at different times, and then calculates the ratio of HbA1c to total Hb according to the area under each peak.
High precision, some products can have CV less than 1%
Non-specific problems exist, some products will still be affected by mutant Hb, carbamylated and acetylated Hb
High-throughput high-performance liquid chromatography (HPLC) and low-pressure system (LPLC) methods can be used
Reference method of American Standardization Program for Glycated Hemoglobin (NGSP)
Electrophoresis (Elec): Developed in the 1980s, the same as ion exchange chromatography is also based on the saccharified and unsaccharified Hb charge different separation. This method is easy to operate and is not affected by temperature and pH. At that time, it was more precise than other methods at the time, but it required batch sample analysis, and the analysis time was longer. It has been rarely used clinically.
Affinity chromatography (HPLC-af chr): analysis based on structural differences. It is designed by the special reaction of m-aminophenylboronic acid-dependent glycated hemoglobin 1,2-cis-diol group and immobilized boric acid anion. Both glucose linked to Val and Lysine (?-Lys) of Hbα and β chains will combine with boric acid to form a reversible complex. Unsaccharified Hb will be eluted first and then separated and complexed with sorbitol Substances, and elute all saccharified Hb.
The test result is "total" glycated Hb, and the result has a good correlation with HPLC through calibration
No interference from temperature, uremia, HbF or Schiff base
The indoor precision is high, and the results between the laboratories are different
Immunity: Immunization principle of antigen and antibody. The application of monoclonal antibody technology has led to the rapid development of this method. The earliest British company Dako developed a monoclonal antibody that recognizes the 8 amino acid antigen sites of glucose attached to the N-terminus of the β chain of Hb. It is carried out by the principle of enzyme immunity (EIA) Detection.
Has better precision and better correlation with other methods
The turbidimetric method can be used for quantitative determination on an automatic biochemical analyzer
The antigen is similar to the 6-amino acid peptide end of the β-chain N-terminus of the IFCC primary reference substance
If 6 amino acids are used as the antigen, when the 6th amino acid variation of Hb occurs [HbS (β6glu→val) and HbC (β6glu→lys)], it will not be recognized, which may lead to bias in the test results between manufacturers
Factors such as different antigenic determinants and different affinities for monoclonal antibodies of various manufacturers' products will also affect the comparability of the results
Enzyme method (En): The biochemical reaction method measured on the automatic biochemical analyzer has only been introduced in recent years. The principle is to cut off HbA1c under the action of protease? Glycated dipeptide at the N-terminus of the chain. The glycated dipeptide generates hydrogen peroxide under the action of fructosyl peptide oxidase. In the presence of peroxidase, it reacts with the color reagent to produce a color reaction. The HbA1c is calculated by measuring the absorbance. Sub-concentration. This method has good precision and has good correlation with HPLC and immunoassay.
Quick inspection (POCT: Mic chr, Flu im)