Lab.

IX

Yarmouk University Biology Dept. B432 Lab.

Precipitation Double Diffusion Assay

Objectives After completing this laboratory exercise you should be able to: 1. Isolate mononuclear cells from peripheral blood. 2. Purify adherent cells including B cells and monocytes. 3. Separate T cells by sheep RBC rosette formation. Introduction Antigen-Antibody Interactions Another form of serological test is the precipitation test. In this test, antibodies are called precipitins. They react with dissolved antigens and form large complexes that become visible as a fine precipitate. Tests such as these can be performed in fluid or gel. The physical form of the antigen influences how one detects its reaction with an antibody. If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble antigen-antibody complexes. A precipitation reaction occurs as a result of the combination of antibodies in solution with soluble substances with which the antibodies react. If such a precipitation reaction occurs in vivo in the joints or the kidney, inflammation results because the immune complexes are filtered out in those areas, causing irritation. In a serum precipitin test, dilutions of antigen and antibody in aqueous solution are combined until a precipitation reaction occurs and visible particles accumulate. The precise "Zone of Equivalence" where there is a perfect concentration of both antibody and antigen in solution, is difficult to achieve. Many tubes of various antibody and antigen concentrations would need to be prepared in order to get just the right amount; the thin ring of precipitate visible only after centrifugation is much more likely than the tube full of precipitate shown at the top of the page. In practice, precipitation reactions are generally prepared as agar diffusion reactions, to make precipitates more visible. Immunodiffusion in Agar Gels

An agarose gel is used as a matrix for combining diffusion with precipitation. The reactants diffuse through the gel towards each other and precipitation results when the equivalence points have been reached. A single antigen will give rise to a single line of precipitation in the presence of its homologous antibody. When two antigens are present in a system, each behaves independently of the other. Therefore, if several bands of precipitation are detected, there are at least as many antigen-antibody combinations present. Serum antibodies are not homogeneous (you have antibodies that recognize all kinds of infectious organisms and allergens). It is difficult to obtain a single antigen even in the purest preparations, so there are several antigen-antibody reactions possible in a single mixture. Gel diffusion permits the examination of such multiple systems since the technique allows the separation of the reactions. The individual components in a system react independently of each other so precipitation tests in agar are termed "double immunodiffusion" tests or simply immunodiffusion. (Ouchterlony is used in the older terminology). The hazy white background of the solid agar matrix allows visualization of the precipitation reaction. The method is used clinically in immunology/rheumatology evaluations. In this picture, the Center contains the antiserum & wells 1 & 2 contain samples of homologous antigen that the serum recognizes.

Note the band of identity between wells 1 & 2. This technique has many different applications; examples include: 1. Determining the homogeneity of antigen-antibody systems 2. Diagnosing specific autoimmune disorders 3. Following the purification of an antigenic mixture 4. Elucidating the reactions among serologically related antigens

Figure 1: Pattern of identity Pattern of partial identity Pattern of nonidentity The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity. If a solution with antigens X and Y is placed in well 1, a solution with antigen X only is placed in well 2, and antiserum containing antibodies specific for both X and Y is placed in well 3, a reaction similar to that appearing in Fig. 2 will occur. Notice that there is a spur reaction towards the XY well. This indicates that the two antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. Such a reaction with spur formation indicates partial identity. If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines as in When antibodies are mixed with their corresponding antigens on the surface of large, easily sedimented particles such as animal cells, erythrocytes, or bacteria, the antibodies cross-link the particles, forming visible clumps. This reaction is termed agglutination. Agglutination is a serological reaction and is very similar to the precipitation reaction we learnt last week. Both reactions are highly specific because they depend on the specific antibody and antigen pair. The main difference between these two reactions is the size of antigens. For precipitation, antigens are soluble molecules, and for agglutination, antigens are large, easily sedimented particles. As you will see from this lab exercise, agglutination is more sensitive than precipitation reaction because it takes a lot of more soluble antigens and antibody molecules to form a visible precipitation. To make the detection of soluble antigen and antibody reaction more sensitive, a precipitation reaction can be transformed into an agglutination reaction by attaching soluble antigens to large, inert carriers, such as erythrocytes or latex beads.

Animals, Equipment, Materials and Reagents

Agarose. Distilled water. Microscopic slides. 60C oven. Phosphate buffered saline, pH 7.2. Templates and punchers for creating wells and well patterns. Micropipettes with tips. Sera of normal and immunized mice from students' immunization experiments (Lab. III). Soluble immunogen(s) used in students' immunization experiments (Lab. III). Large Petri plates (10cm in ). Large round filter papers (10cm in ). Normal saline (0.85% NaCl). Staining solution (0.25% Coomassie Brilliant Blue R250 in 5% acetic acid). Destaining solution (5% acetic acid). 500g weighting objects (ex.: water filled bottles). Hot air electric dryer.

Exercise
1234Prepare 1% (w/v) agarose in distilled water Carefully dispense 3mL of agarose solution on clean microscopic slides leveled on the bench. After agarose solidifies, place slides in a 60C oven and keep for at least 6 hours or until Prepare 1% (w/v) agarose in PBS, pH 7.2 as described in step 1.

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Carefully dispense 3mL of agarose solution on clean microscopic slides leveled on the bench. Using special templates and punchers, punch wells as designated in the well pattern below: Leave the slides in a humidity chamber Stain precipitin lines as follows:

Immerse slides in normal saline for 2-3 days with 2-3 daily changes of saline. Place in 2-3 changes of distilled water for half an hour each. Wrap slides with 3 filter papers and place a glass plate over them. Over the glass plate, place the 500g weighing object in a way that causes even pressure on all slides. Keep like this for 30 minutes. Unwrap slides and dry them with the hot air of an electric drier.

Stain precipitin lines as follows:

1. Remove filter papers from slide humidity chambers. Wash slides by immersing them in normal saline and place slide containing plate on a shaker. Washing should continue for 2-3 days with 2-3 daily changes of saline and continuous shaking. 2. Wash slides with 2-3 changes of distilled water for 1 hour and continuous shaking. 3. Immerse slides with protein staining solution and shake for 5-10 minutes. 4. Destain with 2-3 changes of destaining solution until excess dye gets out of gel. Be careful not to destain blue protein precipitin lines. 5. Wrap slides with 3 filter papers and place a glass plate over them. Over the glass plate, place a 500g weighing object in a way that causes even pressure on all slides. Keep slides like this for 30 minutes. 6. Remove weighing, glass plate and wrapping filter papers from slides and dry slides with the hot air of an electric drier. 7. Draw resulting precipitation patterns (if any) and analyze your results.