Since the first commercial transitional electrophoresis system developed by the Swedish physical chemist Professor Tiselius in 1946, electrophoresis technology has developed extremely rapidly for more than half a century. Various instruments and equipment based on electrophoresis principle have been continuously introduced. Especially after the 1980s, many automated electrophoresis instruments have been adopted by clinical laboratories. Electrophoresis technology has become one of the important tools for basic medical and clinical medical research. At present, this technology has been widely used in the separation and identification of proteins, peptides, amino acids, nucleotides, organic matter, inorganic ions, and even viruses and cells. Especially after the support medium for electrophoresis is changed from mobile phase to solid phase support, various electrophoresis analysis devices are continuously introduced to meet the needs of different teaching, clinical and scientific research work. Nowadays, the combination of electrophoresis technology and mass spectrometry technology in post-genomics research is playing a huge role in bringing new vitality and vitality to the development of clinical testing.
First, electrophoresis analyzer
Electrophoresis analyzers can be divided into two categories: clinical laboratory routines, such as automatic fluorescent/visible double system electrophoresis, automatic vinegar membrane electrophoresis, automatic agarose electrophoresis and automatic agarose electrophoresis; research Mainly as a clinical sample, such as two-dimensional electrophoresis and two-dimensional electrophoresis 2 liquid chromatography 2 mass spectrometry, high performance capillary electrophoresis and high performance capillary electrophoresis 2 mass spectrometry, high performance capillary chip electrophoresis, DNA sequencing system.
1. Fully automatic fluorescence/visible dual system electrophoresis system: with fluorescence/visible dual system, fully automatic when using fluorescent reagent items such as creatine kinase (CK) and lactate dehydrogenase (LD) isoenzyme. After the sample, reagent, and agarose gel electrophoresis film are placed, the operator can complete the test off the machine and get the result. This is an automatic electrophoresis instrument. However, using visible light items such as protein electrophoresis, the intermediate person needs to return, and the electrophoresis film is placed in the dyeing system from the electrophoresis tank to complete the test. The biggest advantage is that the fluorescence system is fully automatic and sensitive, with high accuracy and high-pressure, low-temperature system. It takes only 20 minutes to complete electrophoresis analysis, and the speed is very fast.
2. Automatic vinegar membrane electrophoresis instrument: It is a visible light single system, using vinegar membrane electrophoresis tablets. The degree of automation is high, and only the samples, reagents, and electrophoresis tablets are placed, and the personnel can complete the test off the machine to obtain the results. However, because of the low sensitivity caused by the use of vinegar membrane, it is impossible to analyze urinary protein/cerebrospinal protein, and the analysis effect on isozyme is not satisfactory. Most of the laboratory is only used for serum protein electrophoresis analysis.
3. Fully automatic agarose electrophoresis instrument: a single system for visible light, using agarose gel electrophoresis film. The advantage is high sensitivity, which can be used for low-concentration protein tests such as urine protein and cerebrospinal fluid protein. The separation effect of isozymes is also quite good. However, the disadvantage is that the degree of automation is poor. When the electrophoresis is finished and the dyeing and decolorization is completed, the worker must take the electrophoresis sheet out of the machine, which is troublesome for the laboratory. However, because such instruments can do more projects and have higher sensitivity, they are still accepted by many laboratories.
4. Fully automatic electrophoresis analysis system: It combines the advantages of the above instruments, automatic spotting, electrophoresis, coloring (or dyeing, decolorization), and drying. A variety of electrophoresis tablets, including agar tablets, acetate tablets, polyacrylamide, etc., using visible light and fluorescent color dual system, is an ideal electrophoresis instrument.
5. Two-dimensional electrophoresis and two-dimensional electrophoresis 2 liquid chromatography 2 mass spectrometry: the first direction of two-dimensional electrophoresis is isoelectric focusing, and the second direction is gradient sodium dodecyl sulfate (SDS) electrophoresis. The sample is separated by charge and mass twice to obtain information such as the isoelectric point and molecular weight of the molecule. This is the highest resolution and most informative technology in all electrophoresis technologies, and has become the basic tool for analyzing complex protein mixtures. Since the establishment of this technology by O'Farrel in 1975, there have been many improvements that have made this technology more sophisticated. The first direction of the ISO2DALT system is the use of tubular gels. Although the resolution is high, the amount of sample is large, and the salt is resistant to high salt, there is a cathode drift and the loss of basic protein, carrier ampholyte pH gradient instability, electric field and time. The shortcomings of influence and poor repeatability. In the late 1980s, Gorg et al. used solid-phase pH gradient isoelectric focusing as the first direction of two-dimensional electrophoresis, called IPG2DALT. Solid phase pH gradient isoelectric focusing has no cathode drift, stable pH gradient, high resolution and good repeatability. It is a popular two-dimensional electrophoresis technology. Compared with the traditional one-way electrophoresis method, it can analyze up to 5,000 to 10 000 protein spots in two-dimensional electrophoresis. Two-dimensional electrophoresis has an irreplaceable role in the separation of protein mixed samples. When new protein spots are found, they are cut and separated by liquid chromatography, combined with mass spectrometry, and finally the newly discovered protein components can be identified.
6. High performance capillary electrophoresis and high performance capillary electrophoresis 2 mass spectrometry: high performance capillary electrophoresis uses an elastic quartz capillary as a separation channel, driven by a high voltage DC electric field, depending on the difference in the degree of mobility and distribution between the components in the sample. And an electrophoretic separation analysis method that realizes separation. The separation efficiency is improved by using a capillary instead of a slab gel. The application of high performance capillary electrophoresis ranges from small molecules, inorganic ions to biomacromolecules, and even whole cells. From charged particles to neutral molecules, high performance capillary electrophoresis can be used for analysis. At present, the interface problem between capillary and mass spectrometry has been solved, and people can use the high-efficiency separation ability of capillary electrophoresis combined with the identification technology of mass spectrometry to exert its special function and discover unknown compounds. In particular, array capillary electrophoresis systems have been introduced, which will overcome the shortcomings of most commercial capillary electrophoresis systems that can only perform single capillary electrophoresis. This new high-throughput method will revolutionize genetic analysis in the post-genomic era. Sexual change.
7. Capillary electrophoresis chip: Capillary electrophoresis chip can be used for analysis of DNA length, sequence and genotyping, which is of great significance in clinical detection, especially in the diagnosis of genetic diseases. The DNA is now isolated by the use of a chip to separate fluorescently labeled oligonucleotides. The entire separation process is within 45 s and the chip is only 318 cm in length. Because it can carry high voltage (2 300 V / cm) and has a small sample volume, it is beneficial to obtain better separation. The separation of DNA samples by conventional capillary electrophoresis separation usually takes 10 to 30 minutes and the voltage can only be added to 500 V / cm. In addition, a chip for high-speed DNA separation has an effective length of only 315 cm, and a DNA fragment of 70 to 1 000 bp is isolated for less than 120 s. At present, high-speed, high-throughput capillary electrophoresis chips have been developed. In 2002, the author saw the DNA electrophoresis chip designed by Caliper at the 15th International Conference on Micro-separation and Analysis held in Sweden. There are 96 capillary electrophoresis arrays on a disc-sized chip, respectively, with 96 sample cells. Connected, radiating, and detecting the signal using a rotating confocal fluorescence detection system. This system can separate 96 different samples at the same time, and the separation detection process is within 8 min.
8. DNA Sequencing System: This system uses the principle of gel capillary to design a multi-channel capillary array, labeling 4 nucleotides with 4 different fluorescent dyes, synthesizing DNA single strands on the template, and then exonuclease The continuous hydrolysis and release of the base is carried out by the action of the laser to identify and record the released base. Can be used for DNA sequencing, automatic heterozygous detection, point mutation analysis, allele identification, SNP screening, heterozygous deletion detection, AFLP fingerprinting, microsatellite DNA instability analysis, gene expression, sequencing quality assessment, Sequence comparison, etc. In the mid-1990s, the sequencer was significantly improved, and clustered capillary electrophoresis replaced gel electrophoresis. The completion of the human genome framework in 2001 was due to the emergence of multi-channel, clustered capillary gel electrophoresis. At present, a few hospitals in China have begun to use 8-channel DNA sequencing to systematically carry out YMDD mutation detection of lamivudine in the treatment of hepatitis B virus (HBV).
Second, the clinical application of electrophoresis technology
With the advent of new electrophoresis technologies, various automated electrophoresis analyzers have been introduced and introduced into clinical laboratories. Electrophoresis technology is playing an increasingly important role in the diagnosis of clinical diseases, especially for various body fluid proteins and co-workers. The detection of enzymes and the like provides a new means.
1. Serum protein electrophoresis: After the fresh serum is electrophoresed and stained with cellulose acetate membrane or agarose, 5 bands of albumin, α1, α2, β and γ globulin are common. The serum protein electro-ice map is a valuable method for understanding the whole picture of serum proteins in patients, and can be used as a preliminary screening test. Acute inflammation or acute phase reaction is often characterized by deepening of α1 and α2 zones; pregnancy type α1 zone is increased with β zone increase; nephrotic syndrome, chronic glomerulonephritis exhibits albumin decline, α1, β Elevated globulin; iron deficiency anemia can increase β-zone due to elevated transferrin, while albumin is significantly reduced in chronic liver disease or cirrhosis, gamma globulin is increased 2 to 3 times, showing immunoglobulin The protein (Ig) is highly polyclonal, and even the β-γ bridge can be seen. It can also display a fine and dense oligoclonal region in the γ region; the monoclonal Ig abnormality (M proteinemia) is in the electric ice zone with α～γ. The region presents a dense and deeply stained, highly concentrated protein clonal proliferative zone (M-protein zone).
2. Urine protein electrophoresis: The main purpose of urine protein electrophoresis is to assist clinical judgment of the severity of renal disease without damage. When renal biopsy is not available, urine protein electrophoresis results are a good aid in clinically determining the major damage to the kidney. After urinary protein electrophoresis, the medium and high molecular protein bands mainly reflect glomerular lesions, showing a low molecular protein band visible in renal tubular lesions or spilled proteinuria (such as this week protein); mixed proteinuria can be seen Various molecular weight bands indicate that both glomeruli and renal tubules are involved. The diagnosis of patients with atypical clinical symptoms and the diagnosis of microalbuminuria and the dynamic analysis of the condition of various kidney diseases are also of great value.
3. Cerebrospinal fluid protein electrophoresis: If the oligoclonal zone is detected in the cerebrospinal fluid (CSF) specimen, and the corresponding blood specimen is not detected, it is reflected by the central nervous system itself, which has important clinical significance. However, in order to ensure correct comparison and analysis, patient serum and CSF should be analyzed simultaneously on the same day to demonstrate Ig from different sources. Central synthetic Ig is an important signal of central nervous system disorders, mainly used for the diagnosis and differential diagnosis of central nervous system diseases such as multiple sclerosis, dementia, myelitis, subacute leukoencephalitis and neurosyphilis.
4. Hemoglobin and glycosylated hemoglobin electrophoresis: The application of electrophoresis to identify the type and content of Hb in patients' blood is of great significance for the clinical diagnosis and treatment of anemia. Increased HbA2 is an important feature of β2 mild thalassemia. HbA2 reduction is seen in iron deficiency anemia and other Hb synthetic disorders (commonly known as α2 thalassemia). Electrophoresis found that abnormal Hb such as HbC, HbD, HbE, HbK and HbS can be diagnosed as corresponding Hb molecular diseases. Electrophoresis under acidic conditions separates the different components of glycated hemoglobin, HbA1 a, HbA1 b and HbA1 c. The formation of HbA1 c is related to glucose in RBC, and can specifically reflect the glucose level in the body 6 to 8 weeks before the measurement. In addition, glycated hemoglobin may explain the false increase in HbA1 c caused by increased HbF in some patients.
5. Immunofixation electrophoresis: It can classify various types of Ig and its light chain, and is most commonly used for the classification and identification of clinical routine M proteins. Generally used for the diagnosis and differential diagnosis of monoclonal Ig proliferative disease, monoclonal Ig disease, this week protein and free light chain disease, multi-component monoclonal Ig disease, heavy chain disease, CSF oligoclonal protein identification, polyclonal Ig disease .
6. Isoenzyme electrophoresis: clinically used for isozyme or isozyme subtype analysis. (1) Lactate dehydrogenase (LD / LDH) isoenzyme: Five isozyme bands (LD1 to LD5) can be isolated by agarose gel electrophoresis (AGE). It is mainly used for the diagnosis and differential diagnosis of acute myocardial infarction (LD1 > LD2) and skeletal muscle disease (LD5 elevation). In malignant tumors and cirrhosis, LD5 was significantly elevated, or an abnormal LD6 zone appeared in the chest and ascites. (2) Creatine kinase (CK) isoenzyme: Three kinds of CK isoenzymes can be isolated by AGE method. When an abnormal isozyme such as Creatine Kinase 1 (CK1), Giant CK2, etc. occurs, it is easy to find from the electropherogram. The increase in CK2MB in the early stage of myocardial infarction and peak in a short time is also an indication of myocardial reperfusion. Increased CK2BB is seen in glioma, small cell lung cancer, and gastrointestinal malignancies, which are often associated with increased CK2Mt. (3) CK isoenzyme subtype: refers to CK2MM subtype (CK2MM1, CK2MM2, CK2MM3) and CK2MB subtype (CK2MB1, CK2MB2), often using agarose gel IEF or high pressure electrophoresis. Agarose gel high-pressure electrophoresis can be used for routine rapid analysis of CK isoenzyme subtypes for clinical diagnosis and differential diagnosis of early myocardial injury. It is mainly used for the early diagnosis of acute myocardial infarction, and can also be used to determine the condition of myocardial reperfusion and thrombolytic therapy. (4) Alkaline phosphatase (ALP) isoenzyme: A routine rapid analysis of ALP isoenzymes can be performed by the AGE method. Extrahepatic obstructive jaundice, metastatic liver cancer, liver abscess and cholelithiasis have a high detection rate of bile ALP, accompanied by an increase in liver ALP, while intrahepatic cholestasis, acute hepatitis, primary liver cancer, etc. ALP is increased, and most do not have bile ALP. Hyperthyroidism, malignant bone injury, rickets, fractures, bone damage caused by acromegaly, etc., all cause an increase in bone ALP isoenzyme. The positive predictive value of bone ALP and high molecular ALP isoenzyme for bone metastasis or liver metastasis of malignant tumors was higher than that of total ALP. Intestinal ALP occurs in malignant tumors such as gastrointestinal tumors and lung cancer. (5) γ2 glutamyl transpeptidase (γ2GT/GGT) isoenzyme: γ2GT isoenzyme can be separated into γ2GT1～γ2GT4 by CAE or AGE method, γ2GT2 and γ2GT3 are common in normal people, severe hepatobiliary diseases and liver cancer are often used. There is γ2GT1, and γ2GT4 is closely related to the increase of bilirubin.
7. Lipoprotein electrophoresis: Lipoprotein electrophoresis detection of various lipoproteins (including cholesterol and TG) is mainly used for the classification of hyperlipidemia, the risk assessment of coronary heart disease, and the occurrence and development of atherosclerosis and related diseases. Research on the observation of the effects of diagnosis, treatment and treatment (including therapeutic lifestyle changes, diet and lipid-lowering drugs).
Third, the status quo and prospects
Compared with foreign countries, there are obvious gaps in the clinical application of electrophoresis technology in China. For electrophoresis projects, serum proteins are mainly used in China, and foreign projects include serum protein, urine protein, CK, LD, ALP, GGT isoenzyme, and various lipoprotein cholesterol/triglycerides. For electrophoresis reagents, the agarose gel electrophoresis tablets accounted for more than 95% in the past few years in Europe and America. 40% of the domestic market still uses vinegar membrane electrophoresis tablets. Analysis of these gaps has subjective factors as well as objective reasons. At present, many clinical laboratories in China still use backward electrophoresis equipment or manual electrophoresis methods, which can not obtain the analysis results in time and accurately, and also make many new test items can not be carried out and applied to clinical routine analysis, affecting clinical related diseases. The diagnosis and treatment and the development of laboratory medicine. There are also some large-scale hospitals. Although advanced automatic electrophoresis systems have been purchased, due to insufficient publicity, clinicians know less about electrophoresis testing projects and do not open inspection application forms.
From the requirements of current clinical laboratories, the electrophoresis apparatus must have the following functions:
(1) Fully automatic: personnel must be off-machine, and there is no manual action from the sample to the report.
(2) Fast speed: All items should be completed within 60 minutes and have emergency items such as CK, LD equivalent enzyme and subtype items.
(3) Complete project: In addition to traditional items such as serum protein, lipoprotein, hemoglobin, CK, LD isoenzyme, etc., it is also necessary to complete urinary protein, cerebrospinal fluid protein, immunofixation (monoclonal antibody), ALP and GGT isoenzyme, The latest testing items such as cholesterol (high density / very low density / low density lipoprotein), lipoprotein (a).
(4) High sensitivity: It is necessary to use a highly sensitive electrophoresis sheet. For example, the agarose gel system can obtain electrophoresis results with high accuracy and high resolution.
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