APOA, APOB Determination Experiment
APOA, APOB Determination Experiment
Rocket immunoelectrophoresis is an improvement of unidirectional immunodiffusion method, through the application of direct current (stabilized voltage and current) so that the anti-(APOA1 and B) in the agarose gel containing specific antibody diffusion, PH8.6 when the antigen to the anode, in the swim graph and the antibody in the gel reaction, and gradually formed similar to the rocket precipitation fronts, and the height of its fronts (or frontal area) and the concentration of the antigen is proportional to the antigen, so can be used as a quantification of the antigen. Therefore, it can be used for antigen quantification. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.
Operation method
APOA APOB assay experiment
Principle
Rocket immunoelectrophoresis is an improvement of unidirectional immunodiffusion method, through the application of direct current (stabilized voltage and current) so that the anti-(APOA1 and B) in the agarose gel containing specific antibody diffusion, PH8.6 when the antigen to the anode, in the swim graph and the antibody in the gel reaction, and gradually formed similar to the rocket precipitation fronts, and the height of its fronts (or frontal area) and the concentration of the antigen is proportional to the antigen, so can be used as a quantification of the antigen. Therefore, it can be used for antigen quantification. Move I. Experimental equipment: Caveat l. Antigen sheet dilution and antiserum dosage calculation, should be rocket peak clear standard curve slope moderate and into a straight line is appropriate, this method at the same time to determine apoA1 and apoB, should be adjusted to the amount of two antisera, so that the two fronts have a difference in the height of the front apoB front is not less than 1cm. 2. different kinds of sources of serum (such as rabbit and sheep) under the condition of equal potency, the results will be different. 2. different kinds of sources of serum (such as rabbit and sheep) in the case of equal potency of the test, the results will be different, apoA1 determination of rabbit antiserum is better, with rabbit serum when the front of the progressive shape of the tip of the thin, and sheep serum produced by the peak of coarse l, the tip of the front of the rounded, the advantage of the peak in the term of the virtual shadow. 3. under certain conditions electrophoresis different dilution, the reference serum front height will not have obvious changes, the standard curve slope is basically the same, such as the specimen peak height exceeds the range of the specimen curve, should be adjusted specimen dilution multiples measured after the inter-plate CV is usually less than 5%. 4. Rocket electrophoresis results can be stained or directly observed with the naked eye visible rocket peaks. The former with a sample mmol / less antiserum, but if the appropriate increase in the amount of specimen and antiserum. Not coloring is more convenient.5 "rocket peak measurement can be calculated area or peak height, area is the peak height multiplied by the peak width (peak width at half-height) measurement accuracy of the best can reach 0.1mm, need to use mechanical or electronic amplification equipment. For more product details, please visit Aladdin Scientific website.
l. Voltage stabilized rectifier for voltage, electrophoresis tank with water cooling device (domestic electrophoresis apparatus can generally be used).
(domestic electrophoresis apparatus can generally be used)
2. Horizontal table.
3. 10cmX7cm glass plate and mylar film of the same size (choose a thin film for the projector that is not stained).
film for projectors that are not stained).
4. gauze and filter strips for bridging.
5. several pieces of coarse filter paper.
6. agarose gel punch.
II. experimental reagents:
1. barbiturate buffer, free from strength 0.05 PH8.6
2. 10g/l agarose (standard electrically internal soluble) prepared with barbiturate buffer, heated to dissolve, precipitate, divided into 10ml/tube, cooled and stored at 4℃.
3. 0.15mmol/l NaCl solution.
4. free (or goat) anti-human apoA antiserum (potency not less than 1:16) and apoB antiserum (potency
(potency not less than 1:32).
5. reference serum.
6. Staining dye: Kaomas Brilliant Blue R-250, 0.259g dissolved in 45ml of methanol, add 10ml of glacial acetic acid and 45ml mixed, dissolved and ready for use.
7. Decolorizing solution: 50ml of glacial acetic acid and 100ml of glycerol per liter.
IV. Experimental operation:
l. Pouring plate: 10ml of 1g/dl agarose per plate, melted in boiling water bath, mixed well, cooled to 50-55℃.
Add 50ul of human apoA antiserum and 8ul of apoB antiserum (the dosage depends on the potency of the antiserum), mix rapidly.
After mixing, then pre-set on a glass plate on a horizontal stage and cooled at 4℃, 20min after punching holes, the holes are in the negative of the plate, and the holes are in the negative of the plate, and the holes are in the negative of the plate.
After 20 min, the wells were punched in the negative end of the plate with a diameter of 3 mm, a spacing of at least 5 mm, and a capacity of 5 ul. The plate was placed in an electrophoresis tank and bridged with filter paper.
2. Dilution of antigen: use 0.15mmol/lNacl solution to dilute the reference serum into 1:100, 1:150, 1:200, 1:300 and 1:400 (for the standard curve), serum specimen is diluted according to 1:20O, put in the refrigerator at 4℃ for no more than 3 days.
3. Sampling: in the low-current state (10mA / plate) will be diluted to a fixed value of serum and samples were sucked 5ul (accurate), added to the agarose gel spiking wells, each plate should be made a series of standards.
4. Energized: steady current stabilization 24mA/plate, end voltage 6-8V/cm, cooling with running water to keep the agar warm 15C electrophoresis for 3-4h.
5. de-hybridized protein and gelatin membrane: after electrophoresis, the agarose plate was immersed in 0.15mmol/lNaCL for 3Omin, and the gelatin membrane was placed on the polyester membrane, and the multi-layer filter paper was gently pressurized to absorb the water in the cavity, and after finishing, the filter paper was placed on the polyester membrane, and the gelatin membrane and the polyester membrane were placed on the polyester membrane. The adhesive film and polyester membrane in the instrument to dry naturally, or blow dry with a hot hair dryer, after drying the adhesive film will naturally separate from the filter paper and polyester membrane.
6. Apply color: Soak the agarose film in the staining solution for 20-30min.
7. Decolorization: Soak the dyed gel film with decolorizing solution until the rocket sample is clear and the background is basically colorless, which can be done in water.
In the water with two pieces of glass in the film will be clamped, drying can be stored for a long time, but also can be used in running water immersion decolorization to background
clean.
Third, calculation: measure five concentrations of serum and each specimen of the front height (from the center of the hole to the tip of the front count), apoA1 peak is higher than the apoB peak, the two were connected to the antigen concentration and the front height as a standard (or a straight line, but there is an intercept on the axis) in the curve to find out the concentration of apoA1 and apoB in each specimen.
The range of this method is: apoA1 0.4-2.5g/l apoB 0.3-2.0g/l