Immunoprecipitation

Single Diffusion

Agar plate impregnated with IgG is obtained and labeled. It is cut with a hexagonal array dots. Disposable graduated 1ml pipette is used and the lowest 2 graduation is cut off and the bulb of the pipette is compressed. Then, the tip of pipette is pressed into the gel and the compression of the bulb is released. The sample is added in duplicate. The plate is leaved at room temperature and within 30 minutes, result is obtained.

Rings of precipitation form around the wells with different diameter.

Double Diffusion

The agar plate is obtained and labelled. It is cut well. Then, the assay is conducted as for Section A; 50 micro lit of yellow and blue dye is added to appropriate wells. Section B; HS,HA,CA serums are added in appropriate wells.Section C; Unknown serum is added to centre well. Antisera of following order; Human Antiserum(HA),Chicken Antiserum(CA),Cat Antiserum(CatA) and Rabbit Antiserum(RabA)

Precipitation line form between HA and HS in section B. In section C, HA form precipitate line with unknown serum.

Immunoprecipitation assay is one method of determining the presence of unique biochemical in a chemical mixture or a biological sample. It identifies chemical by unique features of the chemical’s shape and surface charges. The assay used antibodies to detect the presence of specific components in a sample. The component’s presence is known when it becomes bound to an antibody. The binding of the antibody to the antigen result in visible detectable clump known as a precipitation bond.

This immunoprecipitation test is designed first by isolate the chemical or antigen that is interested in detecting. Then, the antigen is injected into a laboratory animal and the laboratory animal produces antibodies specifically designed to adhere to that antigen. The blood of the animal is purified to yield particular antibody. In this experiment, the serum that being used are human serum, chicken serum, unknown serum. The antiserum that being used are human antiserum, cat antiserum and rabbit antiserum. There are also dyes which are yellow and blue dye.

Antibody is a protein produced as a result of interaction with an antigen. The protein has the ability to combine with the antigen that stimulated its production. Meanwhile, antigen is a substance that can react to antibody. Not all antigens can induce antibody production; those that can are also called immunogens. In single diffusion test, the antibody is incorporated into the gel and the antigen is introduced into the wells in a gel. Rings of precipitate form around the wells, and the diameters of the rings are proportional to the amount of antigen placed in the well. In single diffusion test, sample from A give the biggest diameter follow by sample form B and the smallest is sample from C. This show that different amount of antibody in the sample of A, B and C.

Meanwhile, in double diffusion test, antigen and antibody are placed into separate wells; they diffuse into the gel in all directions; where they interact, a precipitation reaction occurs. Each antigen-antibody reaction forms an independent precipitation reaction called a precipitin boundary. In double diffusion test, It is divided into section A, section B and section C. In section A, colours can be seen travelling through the gel. In section B, a precipitation line form between HA(Human Antiserum) and HS(Human serum) which mean the test result is positive. In section C, the unknown serum is identified as HS(Human serum) as it form precipitation line with HA(Human Antiserum).