Cross-electrophoresis experimental technology

related topics(a) Principle
cross-immune electrophoresis is a
combination of
agar, flat-panel electrophoresis and rocket electrophoresis. First, the
antigen
sample is electrophoretically separated in the agar gel, and then the separated antigen components and the direction of the original swimming direction is 90 degrees angle to the agar gel containing
antibodies
, so the antigen sample of the various antigen components and its corresponding antibodies in turn form a number of conical precipitation lines, according to the location and area (or height) of the sediment line can determine the amount of the antigen. This test can be used to identify antigens, i.e. compared with standard antigen antibody systems, the quality and quantity of antigens in the sample to be tested can be known. This method can also identify antibodies, the unknown antibody-containing samples and standard antigen reaction, the results obtained with standard antigen, antibody reaction results compared, so as to determine the antibody content and its titration.(ii)
reagents
materials
1. Barbie buffer 0.05mol/L pH8.2. 2． 1%
agar
or high-quality agar is made with barbie buffer. 3． pH7.2 PBS liquid 4. Antigen 5. Antibodies 6. Electrophoresis, glass plate,
straw,
, droppers, etc.(iii) Operating methods
1. Take 10cm × 10cm clean glass plate, add 1% dissolved agar sugar 10ml, spread well, condensation. 2． Punching, 2cm from the extremes of the yin, 2.2cm from the edge of the glass plate, punching. 3． Add a certain concentration of antigens to the hole and place the antigen hole in the negative pole for electrophoresis. Voltage 10V/cm, swim 1h. 4． After electrophoresis, cut unless the antigen electrophoresis part of the agar (about 6cm×10cm). 5． Take the above 1% dissolved agar 10ml into a 60c water bath, add the appropriate concentration of preheated
serum
antibody quickly mixed, spread into the cut agar part. After cooling, seal the indicing with antibody-free agar. 6． Place the glass plate at a 90-degree angle with the original electrophoresis direction, so that the antigen gel after the swim is placed at the negative pole, electrophoresis. The voltage is 3V/cm and the swim is 10h to 20h. 7． Rinse, dye, decolor, dry.(IV) the results were
compared with the standard antigen antibody conical precipitation line, and the results were determined and analyzed.(v) Precautions
1. The most appropriate dilution of antigen antibodies must be explored in advance. 2． The length of the second electrophoresis time is based on observing the appearance of the conical precipitation line. Electrophoresis time is different, short can be 2h that appears.