Immunoelectrophoresis (IEP) Lab 7 PDF

Immunoelectrophoresis (IEP) – Lab. 7  Immunoelectrophoresis Is a lab test that measures proteins called immunoglobulins (Igs) in the blood There are many types of Igs such as IgG, IgM, and IgA. Some can be abnormal (due to cancer) A normal (negative) result means no Igs were seen in...

Immunoelectrophoresis (IEP) – Lab. 7  Immunoelectrophoresis Is a lab test that measures proteins called immunoglobulins (Igs) in the blood There are many types of Igs such as IgG, IgM, and IgA. Some can be abnormal (due to cancer) A normal (negative) result means no Igs were seen in the blood sample  Immunoelectrophoresis (IEP) Techniques IEP is a combination of two techniques Electrophoresis Immunodiffusion The Principle of Immunoelectrophoresis (IEP) Techniques A patient sample is applied and the proteins in the serum are separated according to their electrical charge. After that , an antiserum containing antibodies against protein of interest is applied parallel to the electrophoretic margin. The antigen and antibodies are allowed to diffuse toward each other and a precipitation form where they meet  Materials - humidity chamber - - phosphate saline Ph Immunoelectrophoresis 7.2 chamber - Patient serum sample - power supply - antihuman globulin - disposale wicks -Coomassie blue stain - 26 x76mm slide - human IgG - agarose - barbital buffer PH 8.2 - micropipette  Procedure (Methods) Prepare 1% agarose in barbital buffer PH8.2  Procedure (Methods) Make two holes toward the margin of the slide  Procedure (Methods) Transfer the slide (gel) to the electrophoresis tank and fill it’s compartment with barbital buffer pH 8.2. Fill one well with human IgG and the other well with human serum connect the electricity Human serum cathode (-) anode (+) Human IgG  Procedure (Methods) Connect the electricity cathod (-) anode (+) Start the electrophoresis run at 110 Volt for 45 minutes  Procedure (Methods) Make a central trough in the agar using a blade and fill it with 200µl of antihuman serum and incubate 48 hours at room temperature in a humid chamber. Precipitation will appear If the precipitation is not clear, wash the slide (agarose gel) with phosphate buffer saline and stain with Coomassie blue stain  Applications of IEP IEP is used to examine serum abnormalities such a multiple myeloma, macroglobulinemia, and recurrent infection IEP is used to purify antigen and/or antibody