Counter immunoelectrophoresis

Practical Immunology And Serology Counter immunoelectrophoresis

1. Practical Immunology And Serology Counter immunoelectrophoresis By Ahmed Riyadh Abdul Rahman Al-Noor University College 1

2. 2 Counter immunoelectrophoresis * Counterimmunoelectrophoresis (CIEP) is a laboratory technique used to detect the binding of an antibody to its antigen . *CIEP is a modification of Ouchterlony immunodiffusion ,method that speeds up migration of an antigen and antibody in the diffusion medium, usually an agar or polyacrylamide gel by applying an electrical current. The effect is rapid migration of the antibody and antigen out of their wells towards one another to form a line of precipitation, or a precipitin line, indicate the formation of Ag- Ab complex in the zone of equivalence. *The technique is similar to the Ouchterlony method, the only difference being that the antigen/ antibody movement is facilitated by electrophoresis. It is thus also called ‘voltage facilitated double immunodiffusion ’.

3. CIEP depends on the movement of strongly negatively charged antigen towards the anode and of antibody towards the cathode through the agar under the electric field. The test is performed on a glass slide/plate in agarose gel, a pair of wells is punched out where one well is filled with antigen and the other with the antibody. Electric current is then passed through the gel, the migration of antigen and antibody is greatly facilitated under the electric field, and the line of precipitation as precipitin arcs (or lines) is made visible in 30–60 minutes, which indicates a positive reaction. 3 PRINCIPLE

4. Counter immunoelectrophoresis principle 4

5. Counter immunoelectrophoresis is mostly carried out with one or more of the following objectives: To rapidly check any antisera for the presence and specificity of antibodies for a particular antigen. To detect antigens and/or antibodies in serum for diagnosis of a particular disease. 5 Objective and Application

6. 6 It is a rapid and a highly specific method for detection of both antigen and antibodies in the serum, cerebrospinal fluid, and other body fluids in the diagnosis of many infectious diseases including bacterial, viral, fungal, and parasitic. The test was very popular in the past for detecting various antigens such as alpha-fetoprotein in serum and capsular antigens of Cryptococcus and Meningococcus in cerebrospinal fluid. Still today, it is commonly used for Hepatitis B surface antigen ( HBsAg ), fetoprotein, hydatid and amoebic antigens in the serum, and cryptococcal antigen in the CSF. It is a rapid sensitive method for detecting pneumococcal capsular antigens in sputum. CIEP has many uses

7. PROCEDURE 7 Prepare 10 ml of 1.0% Agarose (0.1 g/10 ml) in 1X Assay Buffer is heating slowly until agarose dissolves completely. Mark the ends of a glass slide as + ve and - ve so that when placed in the electrophoresis apparatus, the + ve mark is faced towards the anode and the negative mark faced towards the cathode. Place the glass plate or slide on a horizontal surface, pipette and spread (5) ml of agarose onto the glass slide, It is allowed to solidify for 15 minutes. Cut Wells in the gel according to the template using gel puncher. The distance between the two wells should not be more than 0.5 cm. Place the slide in the electrophoresis tank and fill the tank with 1X electrophoresis buffer till the buffer just covers the gel surface, Do not add excess of buffer. Add 10µl of antigen in each of the two wells towards the cathode (Negative electrode) and 10µl of positive control antiserum and test antisera in wells towards the anode (Positive Electrode). Connect the power cord to the electrophoretic power supply according to the convention. 50 V is applied and the electrophoresis is allowed to continue for about 45 minutes. Interpret the results after the completion.

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9. INTERPRETATION OF RESULTS   9 Precipitin line between the antigen and antisera wells indicate positive reaction or specific antigen-antibody reaction due to the presence of antibody specific to the antigen in the test sera (Indicates specificity). The absence of the precipitin line indicates no reaction or the absence of any antibody for the antigen in the test sera (Indicates non-specificity). The presence of more than one precipitin line indicates the heterogeneity of the antibody for the antigen.

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11. 11 Advantage The method is Much faster and more sensitive than double immunodiffusion . (takes 30 minutes). Limitations It is more expensive than agglutination based tests. It is believed to have decreased sensitivity, speed, and simplicity, than latex agglutination tests. Need of large quantity of Ag and Ab. Advantages and Limitations

12. 12 Any question?