Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and
reaction with antibodies. Rocket immuno-electrophoresis (RIEP) Aim: To perform rocket immuno-electrophoresis (RIEP) for the determination of arious concentration of antigen in gi en unknown sample. Principle: Rocket Immunoelectrophoresis (RIEP) also known! as electroimmuno diffusion is a simple! "uick and reproducible method for determining the concentration of antigen (#g) in an unknown sample. $arious concentrations of antigen are loaded side b% side in small circular wells along the edge of an agarose gel that contains the specific antibod% (#b). &n electrophoresis! the antigen begins to migrate towards the anode and interacts with antibod% molecules to form a soluble antigen-antibod% comple'. (owe er! as the samples electrophoresis farther through the gel! more antibod% molecules are encountered that interact with the antigen and when the )e"ui alence point) is reached! the #g-#b comple' precipitates. This precipitin line is seen in the form of a rocket. (igher the amount of antigen loaded in the well! farther the antigen will tra el through the gel. (ence! with increasing antigen concentration! a series of rockets of increasing heights are seen that is proportional to amount of antigen in the well. Therefore! a direct measurement of the height of rocket will reflect upon the antigen concentration. # standard graph of antigen concentration ersus peak height is then constructed and from the peak height of the unknown sample! concentration of antigen is determined. *aterial Re"uired+ ,onical flask! measuring c%linder! alcohol! distilled water! micro pipette! micro tips *aterial pro ided+ -tandard antigen+ .-# #+ /./012 mg3ml .+ /.12 mg3ml ,+ 0mg3ml #ntiserum+ 4oat anti .-#
Procedure+ 0. Prepare 0/ ml of 0./5 agarose (/.0 g30/ ml) in 06 electrophoresis buffer b% heating slowl% till agarose dissol es completel%. Take care not to scorch or front the solution. 1. #llow the molten agarose to cool to 227,. 8. #dd 0 ml of antiserum to 0/ ml of agarose solution. *i' gentl%! ensure uniform distribution of antiserum. 9. Pour the mi' onto a glass plate placed on a horizontal surface and allow it to gel3solidif%. 2. Place the glass plate on the template holder pro ided (in ET--1) and fi'es the RIEP template. :. Punch 8 mm wells with gel puncher towards one edge of the plate. ;. Place the glass plate in the electrophoresis tank< ensure that the wells are towards the cathode. =. >ill the tank with 06 electrophoresis buffer till it co ers the gel. ?. ,onnect the power cord to the electrophoretic power suppl% according to the con ention+ Red+ anode and .lack+ cathode. 0/. #dd 0/3;0 each of the gi en standard antigen and test antigen to the wells. @oading of wells should be carried out "uickl% to minimize diffusion from the well. 00. Electrophoresis the samples at 0// olts! till the rockets are isible or the d%e front reaches the edge. This generall% takes 0 to 003A hours. Electrophoresis can be continued for an additional 02 minutes after the d%e has run out of the gel. This ensures better isibilit% of the precipitation peaks. 01. -top electrophoresis< remo e the glass plate from the electrophoresis tank. 08. &bser e the precipitation peak or rocket formed against a dark background. If the rockets are still not clear< incubate the plate in a moist chamber at room temperature for 0 hour to o ernight. 09. *easure the rocket height from the upper edge of the well to the tip of the rocket. Record %our obser ations.
02. ,onstruct a standard graph b% plotting the height of the rocket on B-a'is (linear scale) against the concentration of antigen on 6-a'is (log scale) on a semi-log graph sheet. 0:. Cetermine the concentration of antigen in the test sample b% reading the concentration against the rocket height from the standard graph. Observation: Result and discussion: Initiall% the gel is prepared in gi en 0'P.- buffer add and ade"uate amount of anti serum to that and spread it on a plate and allow it to settle than make wells and to each well add the ade"uate "uantit% of the standard and test antigen then allow the gel to run through electrophoresis at 0// $olts up to 0 hour. # pattern of rocket is obser ed. Put the result in the table and plot the graph using the obser ed result. >rom the graph calculate the concentration of unknown. >rom the graph concentration of unknown test sample 0 and1 is found to be /.=0 and /.;2 mg3ml respecti el%. Counter current immunoelectrophoresis #I*+ To check antisera for the presence and specifit% of antibodies for a particular antigen b% ,ounter current immunoelectrophoresis. IDTR&CE,TI&D+ ,ounter current immunoelectrophoresis (,,IEP) is a rapid ersion of &uchterlon% double diffusion (&CC) techni"ue. The techni"ue is used to check antisera for the presence and specificit% of antibodies for a particular antigen. PR&,ECERE+ 0. Prepare0/ ml of 0.25 agarose (/.02g30/ml) in 06 reser oir buffer b% adding dr% agarose to the buffer and heating slowl% to dissol e the agarose completel%. 1. *ark the end of the slide that will be towards positi e electrode during the electrophoresis.
8. Place the slide on a le eled tabletop and "uickl% pipette ;ml agarose onto 2/6;2mm slide! spreading while releasing the agarose. 9. #llow solidif%ing for 02min. 2. Place the gel plate on the template holder and fi' it for ,,IEP. Punch 8mm wells with the gel punch at position indicated for ,,IEP. :. Place the slide in electrophoresis tank and fill the tank with buffer. ;. #dd 0/ l of antigen in the four wells towards F e electrode and 0/ l of positi e control antiserum and three test antisera in wells towards positi e electrode. =. #ppl% 2/ and allow the electrophoresis to continue for about 92 min. ?. &bser e for precipitin line between the antigen and antisera wells. &bser ation+ (ere! pattern shows precipitin line between antigen and antisera wells. IDTERPRET#TI&D+ Precipitin line indicates the presence of antibod% the antigen in the test sera. The presence of more than one precipitin lines indicates the heterogenicit% of the antibod% for the antigen in the test sera. The absence of the precipitin line indicates the absence of an% antibod% for the antigen in the test sera
