The early electrophoresis technology was put forward by Professor Svedberg from the Department of Physical Chemistry, Uppsala University, Sweden. The phenomenon that charged colloidal particles move in an electric field is called electrophoresis.
In 1937, Professor ArneTiselius, a Nobel Prize winner, used some electrophoresis phenomena to invent the earliest interface electrophoresis, which was used in the study of protein separation, and created a new era of electric Shanghai swimming technology.
Since then, various electrophoresis techniques and instruments have been published one after another. With the continuous development of advanced electrophoresis instruments and electrophoresis techniques, it plays an important role in biochemical experiment technology. According to the principle of electrophoresis, there are three forms of electrophoresis separation systems: in principle, they are divided according to the principle of electrophoresis, namely moving interface electrophoresis, zone electrophoresis and steady-state electrophoresis or displacement (displacement) electrophoresis.
Electrophoresis at the free moving interface is a method that the moving rate of charged molecules is measured by observing the movement of the interface. This method has become history.
instead, zone electrophoresis with supporting medium is used.
The clinical application value of zone electrophoresis is different due to the different types of supports, particle sizes and electrophoresis methods.
solid support media can be divided into two categories: one is filter paper, cellulose acetate film, silica gel, alumina, cellulose, etc. The other is starch, agarose and polyacrylamide gel.
because of their fine porous network structure, they can not only produce electrophoresis, but also have molecular sieve effect, and small molecules will run faster than large molecules, thus improving the resolution.
Its biggest advantage is that it hardly absorbs protein, so there is no tailing phenomenon in electrophoresis.
Low concentration agarose electrophoresis is equivalent to free interface electrophoresis. protein can penetrate freely in electric field, with small negative force, clear separation and high transparency, and can penetrate the detection sensitivity of the * * * band with the wavelength of 2 ~ 7, nm. Therefore, the first type of support medium has been replaced by the second type of support medium.
the characteristic of stable electrophoresis or displacement electrophoresis is that the electrophoretic migration of molecular particles reaches a steady state after a certain period of time, such as isoelectric focusing and isokinetic electrophoresis.
zone electrophoresis is the most widely used technology in the field of clinical examination, which has important clinical significance, especially other new technologies, which have expanded its application scope and improved the detection technology. The present situation and development of this technology are reviewed.
1. Correct interpretation of electrophoresis results is helpful for clinical disease judgment.
Fresh serum can accurately describe the whole picture of protein of patients after electrophoresis. Generally, albumin is decreased and a globulin region is increased, suggesting different clinical significance.
In acute inflammation, the percentage of a1 and a2 areas increased. In nephrotic syndrome and chronic glomerulonephritis, albumin decreased, a2 globulin increased and β globulin also increased. In iron deficiency anemia, the β band may be increased due to the increase of transferrin, while albumin in chronic liver disease or cirrhosis is significantly decreased and R globulin is increased by 2-3 times, indicating that immunoglobulin polyclonal is increased, even β-r fusion bridging phenomenon can be seen, and thin and dense oligoclonal bands can be displayed in R region; Serum protein electrophoresis is the first choice for the detection of immunoglobulin M protein with uniform antibody-free activity produced by abnormal proliferation of single clone plasma cells.
A dense, deeply dyed and highly concentrated protein clonal proliferation zone can be found in a2-r zone of electrophoresis zone, which is called M protein zone. After scanning, high and narrow individual peaks will be formed. If some peaks are in R zone, the ratio of peak height to peak bottom width >: 2: 1, but the background staining is shallow due to the limitation of normal immunoglobulin synthesis.
A group of diseases caused by M protein, such as multiple myeloma, macroglobulinemia, heavy chain disease, free light chain disease, hemimolecular disease, benign idiogammia and double M proteinemia, are not uncommon at present.
serum protein electrophoresis is of great significance for early diagnosis, curative effect observation and prognosis judgment of this kind of diseases.
Second, the combination of electrophoresis technology and immune technology greatly expanded the scope of its clinical application.
Current was used to accelerate the diffusion of antigen and antibody and specify its running direction, thus speeding up the precipitation reaction.
There are many seeds of immunoelectrophoresis technology, such as convection immunoelectrophoresis (CIEP), rocket immunoelectrophoresis (RIE) and electroimmunodiffusion (EID).
On the basis of these immunoelectrophoresis methods, some new techniques have been continuously derived, such as immunoelectrophoresis (IEP). In 1969, Alper and Johnson recommended immunofixation electrophoresis (IFE), which is a mixed technique of agarose gel electrophoresis and immunoprecipitation. The test sample can be serum, urine, cerebrospinal fluid or other body fluids.
In 1976, serum immunofixation electrophoresis (IF) was recommended again for typing M protein.
After the serum protein is separated by electrophoresis on agarose gel medium, the fixative, various immunoglobulins and light chain antiserum are applied to the lane on the surface of the gel. After incubation, the fixative and antiserum permeate and diffuse in the gel, and if the corresponding antigen exists, an antigen-antibody complex is formed in a proper position.
After staining, the reference lane and antigen-antibody precipitation zone of protein electrophoresis were stained with amino black, and the monoclonal components were separated according to the electrophoretic movement distance, so that various immunoglobulins and their light chains could be typed.
the biggest advantage of this technology is that the sensitivity is 5-15mg/dl, the operation period is short, only a few hours, the resolution is high, and the results are easy to analyze.
At present, it is most commonly used for typing and identification of M protein, which has been included in the routine detection work in clinical laboratories.
3. Electrophoresis is used to analyze isoenzyme spectrum to improve the diagnostic rate
1. Serum lactate dehydrogenase isoenzyme (iso-LDH): There are electrophoresis, ion exchange column chromatography, immunization, inhibitor method and enzyme digestion method to determine LDH isoenzyme, but agarose gel electrophoresis is still the most widely used method so far.
after electrophoretic separation, five isozyme bands should be separated. ldh1 starts to increase at an average of 6 hours after the onset of acute biceps infarction, and LDH1/LDH2≥1 is the positive decisive level of myocardial injury, and LDH5 can be obviously increased in liver cancer. To determine the content of each band, the isoenzyme spectrum separated by electrophoretic scanning can be quantified, and its accuracy is obviously higher than that judged by naked eyes.
2. Serum creatine kinase isoenzyme (ISO-CK); The determination of CK and CH-MB is still used to confirm acute myocardial infarction.
in recent years, some units in China have measured CK=MB by immunosuppression method, and its principle is to resist the enzyme activity of M subunit. Because there is almost no CK=BB in normal serum, multiplying this value by 2 can be considered as roughly representing the activity of CK-MB.
This method is simple and rapid, but its disadvantage is poor specificity. If there is CK-BB or abnormal CK in the patient's serum, there will be false increase.
many authors have reported abnormal CD- isoenzymes, one of which is called giant CK(Maxro-CK), which is a compound of CK-BB and immunoglobulin, including IgG and IgA, and Marco-CK only accounts for .8%-1.6% in normal serum. The other is called mitochondrial CK(CK-MT), and CK-MT is bound in muscle, brain and liver. CK-MT does not appear in normal serum, nor in myocardial infarction. It can be detected in serum only when the tissue is extremely damaged and mitochondria and cell walls are extremely destroyed.
because neither Macro-CK nor atypical CK-MT can be inhibited by M antibody, when they appear, it will lead to a false increase of CK-MB higher than CK total activity.
the separation of CK-MB by electrophoresis is based on the different molecular structures of CK- isoenzymes, which are collected and sorted in electrophoresis buffer by Medical Education Network, with different charges. Different components of CK can be separated from cathode to anode, which are CK-MM, CK-MB and CK-BB respectively. The electrophoresis feature is that when abnormal 2 isoenzymes, such as giant CK ⅰ and giant CK ⅱ, appear, it is easy to find from the electrophoresis map.
Macro-CK is between CK-MM and CK-MB, while CK-MT is near the cathode and behind CK-mm.
In this way, CK-BB and various abnormal isoenzymes will not be mistaken for CK-MB and misdiagnosed, and the wrong reason of false increase of CK-MB can also be solved.
Therefore, CK isoenzyme electrophoresis is a very practical test technique, which has very important clinical significance.
3. Isozymes of CK subtype: In addition, isoelectric focusing electrophoresis or high-voltage electrophoresis of agarose gel is often used to determine CK-MB and CK-MM subtypes. Because the operation is more troublesome than general electrophoresis, routine determination is still not popular.
At present, the imported automatic electrophoresis instrument is provided with a kit, which can be used for CK subtype analysis. The reference value is CK-MM1 (57.7 4.7)%; CK-MM2 was (26.5 5.3); CK-MM3 was (15.8 2.5)%; The ratio of CK-MM3/CK-MM1 was .28 .5 (range .15-.39), and the positive decisive level was >: .5. MM3 was dominant in the blood of AMI on the first day, but MM1 was dominant after the second day.
CKMB was isolated by electrophoresis. CK-MB2 of CKMB1 and CKMB2 in normal human blood is extremely small, and the ratio is generally plausible. 4-6hCKMB2 subtype can be detected in the blood circulation after acute myocardial infarction, so the ratio of MB2/MB1 increased significantly, which was earlier than that of total CK-MB fragment.
When myocardial infarction is relieved, this ratio will gradually decrease.
It can also be used to observe the condition after thrombolytic therapy.
IV. Detection of oligoclonal bands in cerebrospinal fluid by enzyme immunolabeling antibody technology
It has been reported that two-dimensional electrophoresis and silver staining were used to separate and identify protein of CSF, which was complicated and time-consuming. Now, protein in CSF was separated by high-resolution agarose gel electrophoresis, and the "oligoclonal bands" (OCB) were identified by the reaction between antigen and specific IgG antibody labeled with horseradish peroxidase. After the enzyme immuno-labeling amplification technology and chromogenic step, the protein concentration can be detected when it reaches 31-125ul/L, which can improve the detection sensitivity by 1 times, so that the cerebrospinal fluid does not need to be concentrated, and the loss of protein during the concentration process is avoided.
can be used to confirm and distinguish OCB immunoglobulin and its types.
if OCB is detected in cerebrospinal fluid samples, but no bands are detected in corresponding samples, it is positive, which truly reflects the immunoglobulin synthesized by the central nervous system itself, which has important clinical significance.
It is a qualitative test, and OCB is a very important marker in multiple sclerosis.
However, the patient's serum and CSF should be analyzed simultaneously on the same day to identify immunoglobulins from different sources.
Central synthetic immunoglobulin is an important signal of central nervous system diseases, which is mainly used for differential diagnosis of central nervous system diseases, such as multiple sclerosis, dementia, myelitis, paraneoplastic encephalitis, neuromeisu and so on.
5. Lipoprotein (A) was separated by antigen-antibody reaction in solid phase, which was used to detect independent risk factors of heart and brain.
This technology used antigen-antibody reaction to identify lipoprotein separated by electrophoresis.
After agarose gel electrophoresis and staining, different lipoprotein bands can appear in serum.
Because of the different isoelectric points of lipoproteins in the gel, not only can the A, pre-β and β zones be distinguished, but also because the medium contains anti-lipoprotein (a)LP(a) antibodies and cations, and the anti-LP(a) binds with LP(a) in the patient's serum to form a complex, while the cations inhibit the swimming speed of other lipoproteins, so that LP(a) is separated from other lipoproteins, so that LP can be distinguished very clearly. After scanning the positive band, the area and percentage content of the band can be obtained. This method makes the electrophoresis technology perfect and greatly reduces the disadvantages of manual operation, which can not only be semi-quantitative, but also save the film for comparison.
To improve the sensitivity and specificity of detecting LP (a), an independent risk factor for cardiovascular and cerebrovascular diseases.
VI. Non-concentrated urine protein electrophoresis according to the molecular weight to distinguish the types of urine protein
Urine protein electrophoresis can separate various protein in urine to distinguish the types of urine protein, and can help clinical judgment of the location of kidney injury without operation.
SDS-PAGE disc electrophoresis is used to separate urine proteins in some domestic laboratories. This method is limited, time-consuming, complicated to operate, requires high samples, and urine needs to be pre-concentrated, which is not suitable for clinical laboratories.
SDS=AGE electrophoresis does not need preconcentration, and urine protein electrophoresis shows medium and high molecular weight protein regions with mainly reactive glomerular lesions; It presents low molecular weight protein bands, which can be seen in renal tubular lesions and overflow proteinuria; Mixed proteinuria can see large, medium and small molecular weight zones, indicating that glomeruli and renal tubules are involved.
The scanner can scan the protein profile in urine after electrophoresis, and find out the percentage, so as to show the degree of glomerular or renal tubular injury. Its electrophoresis map and scanning pattern can be used as national data for permanent analysis and comparison.
The biggest progress of this technology is that urine does not need to be pre-concentrated, the operation is simple, the results are clear, the test can be completed in only three hours, and a complete qualitative standard is available, which is easy to quantify and analyze, and it is of great value for the diagnosis, differential diagnosis, guiding treatment and judging healing of kidney diseases.
recently developed capillary electrophoresis is a new type of zone electrophoresis, also known as capillary zone electrophoresis.
it is to fill a buffer in a capillary, inject a sample at one end of the capillary, apply a DC high voltage at both ends of the capillary to separate the sample, and the separated samples are detected by a detector at one end of the capillary in turn.
Capillary electrophoresis is also widely used in laboratory medicine. According to the source of the detected samples, it can be divided into urine samples, plasma, serum, cerebrospinal fluid, red blood cells, other body fluids or tissues, and living experimental animals.
From the separation object, it includes protein, polypeptide, amino acid, sugar, enzyme, DNA, oligonucleotide, virus, small and biologically active molecules, ions, drugs and their metabolites.
it can be divided into clinical disease diagnosis, clinical protein analysis, clinical drug analysis, metabolic research, pathological research, isozyme analysis, PCR product analysis, DNA fragment and sequence analysis, etc.
Because of its incomparable efficiency and rapidity, more and more scientists pay attention to it.
Electrophoresis technology in China started not too late, but for various reasons, most of them are real.