apolipoprotein AⅡ

Apolipoprotein is a plasma lipoprotein fraction containing a variety of lipoproteins and several different specific apolipoproteins. There are more than 20 kinds of apolipoproteins that have been discovered so far. Among them, there are mainly aPOA1, AII, B-100, B48, CI, CII, CIII, D, E and the like. Basic Information Specialist classification: cardiovascular examination classification: biochemical examination Applicable gender: whether men and women apply fasting: fasting Tips: In the last meal before blood draw, avoid high-fat food and drinking, fasting 12 hours, extract forearm venous blood. Normal value 250 ~ 520mg / L. Clinical significance Reduced in coronary heart disease, diabetes, nephrotic syndrome, hereditary ApoA-I deficiency (Tangier disease), familial low alpha-lipoproteinemia, fisheye disease, severe malnutrition, active hepatitis, and liver function. Low results may be diseases: coronary heart disease, diabetes considerations (1) Immunoturbidimetry: 1 Regarding antiserum: The turbidimetric assay has higher requirements for antiserum than other methods. The turbidimetric method is preferably a polyclonal antibody. Anti-serum must be free of anti-serum. Must pay great attention to the apoA-II extracted from human serum to achieve immunopurity, chromatographic purity and electrophoresis purity, which is not possible in the general laboratory. The antiserum titer (titer) should not be less than 16. At present, in some domestic commercial reagents, the apoA-II antiserum has extremely low titer, so you must pay attention when purchasing the kit. If you do not have experience in identifying antisera quality before purchase, you should ask the qualified unit to identify it. 2 The upper standard specimen (serum) is diluted 200 times for manual operation (100 μl of sample), and if there is a precision sampler, it can be diluted 20 times (using 10 μl). In order to adapt to the conditions of different laboratories (such as different types of automated instruments), the proportion of antigen-antibody should be paid attention to when making appropriate modifications. It must be noted that there should be no antigen excess in the reaction system, and the linear upper limit should not be lower than 2.5g/L. In other words, the amount of antiserum must be sufficient, otherwise the results are low when the apoA-I is high in the specimen. At present, some domestic drug kits not only have low anti-serum titer, but the dosage of the specimens in the operation is too large (such as 3 ~ 5μl), the antibody is obviously insufficient, and the measured results are inevitably inaccurate. 3 In order to achieve accurate measurement, the calibration curve calculation results must be made in apoA-II and B turbidity measurement (end point method). A certain range (low sample dosage) concentration (X) is basically linear with turbidity (Y), and linear regression calculates a certain intercept on the Y-axis (A value <0.1), so the deviation of the calculation result is calculated by single point calibration. Larger, the measured results can not accurately reflect the level of high (high low, low high). Do not neglect the accuracy of the measurement because of the simplicity of the single point method. Regardless of the type of automated instrument, you must first try the calibration curve. If the test is repeated under the conditions of the instrument and the specific conditions, the intercept of the regression line is not obvious, then the single point calibration method can be used. When the amount of the sample is 3 to 5 μl, even if the amount of antiserum is increased, the concentration and the turbidity are not linear, and can only be calculated after the curve is linearly converted. 4 The main interference factor is the turbidity of the serum itself (such as high-fat serum), and the pretreatment methods such as ultracentrifugation or lipase hydrolysis are not practical. The effect of turbidity with surfactants is also limited, so a blank tube must be made in the assay. In addition to the two-point method used in automated analysis, it is a mistake to manually use a single reagent without subtracting the specimen blank. In order to reduce the effect of matrix effects on the turbidity response, fixed-value serum must be used as a calibrator. In addition, interference such as dust particles and turbid dish scratches must be excluded. 5 Some commercial kits (including some imported products) with inaccurate calibration sera values ​​are an important source of error. (2) Rocket electrophoresis method: 1 The dilution ratio of the antigen and the amount of antiserum should be selected so that the rocket peak is clear, the slope of the calibration curve is moderate, and the line is suitable. The method simultaneously measures apoA-II and apoB, and the amount of the two antisera should be adjusted so that the peak heights of the two are different, and the apoB peak height is not less than 1 cm. 2 Different kinds of antiserums (such as rabbits and sheep) are tested at the equivalent price, and the results will be different. The apoA-I assay is preferably rabbit antiserum. When the rabbit serum is used, the peak shape is sharp, and the peak produced by the sheep serum is thick, the peak tip is round and blunt, and sometimes a ghost image appears before the peak. The slope of the calibration curve for the calibration serum is also different. However, no matter what antiserum is used, the quantitative results are not much different. 3 Electrophoresis under certain conditions, the peak height of the calibration serum of different dilutions will not change significantly, and the slope of the calibration curve is basically the same. If the peak height of the specimen exceeds the calibration curve range, the specimen dilution factor should be adjusted and retested. The inter-board CV is typically less than 5%. 4 Rocket electrophoresis results can be observed by staining or direct visual observation of the rocket peak. The former uses less specimens and saves antiserum, but if the specimens and antiserum are appropriately increased, it is more convenient to not stain. The agarose used should be standard electroosmotic or hypotonic. Adding proper amount of dextran or polyethylene glycol to the gel will make the rocket peak clearer. 5 The measurement of the rocket peak can calculate the area or peak height, and the area is the peak height multiplied by the peak width (the width at the half height of the peak). The measurement accuracy is preferably up to 0.1 mm. Mechanical or electronic amplification equipment must be used. The peak height in the range of the standard curve is preferably 1 to 4 cm. 6 This method is applicable to the analysis of a small amount of specimens. Also suitable for apoAII, CI, CII, CIII, D, E and Lp (a) determination. Inspection process Rocket electrophoresis: 1 pouring plate: 10g/L agarose 10ml per plate, melt in boiling water bath, mix well, add 50μl apoA-II antiserum and 50μl apoB antiserum when cold to 50~55°C (the amount depends on antiserum titer) ), quickly mix and pour on the glass plate preset on the water platform, cool and then placed at 4 ° C, after 20 min, punch holes, the hole is at the cathode end of the plate, the hole diameter is 3 mm, the hole spacing is at least 5 mm, and the hole capacity is 5 μl. Place the plate in the electrophoresis tank and bridge with filter paper. 2 diluted antigen: the calibration serum was diluted to 1:100, 1:150, 1:200, 1:300, and 1:400 (for calibration curve) with 0.15mol/L NaCl solution, and the serum sample was diluted 1:200. Put the refrigerator at 4 °C for no more than 3 days. 3 Loading: In the low current state (10 mA / plate), dilute the fixed serum and specimens to absorb 5 μl (accurate), and add to the agarose gel sample well. Each board has to do a series of standards. 4 power: steady flow 24mA / board, terminal voltage 6 ~ 8V / cm, cooling with running water to maintain agarose 15 ° C, electrophoresis 3 ~ 4h. 5 deproteinized protein and dry film: the agarose plate after electrophoresis was immersed in 0.15mol/L NaCl for 30min, and the film was placed on the polyester film, and the glue was absorbed by the multi-layer filter paper under gentle pressure. Moisture, then dry the filter paper, film and polyester film together, or dry it with a hot air blower. After drying, the film will naturally separate from the filter paper and polyester film. 6 staining: The agarose film was immersed in the staining solution for 20 to 30 minutes. 7 Decolorization: Soak the dyed film with a decolorizing solution until the rocket peak is clear, and the background is basically colorless. It can be clamped in two pieces of cellophane in water and can be stored for a long time after drying. It can also be soaked in running water to remove the background. Not suitable for the crowd Generally no crowds. Adverse reactions and risks 1. Infection: Pay attention to aseptic operation when collecting blood samples, and stop bleeding after blood collection to avoid contamination of water and liquid to avoid local infection. 2, bleeding: after the blood is given a full compression time, especially coagulopathy, bleeding tendency, to avoid local subcutaneous oozing, bruising and swelling.