two-way immunodiffusion assay (BIDA)
two-way immunodiffusion assay (BIDA)
The diffusion of antigen and antibody in a gel and the precipitation reaction is called immunodiffusion. An antigen and its corresponding antibody are placed in adjacent wells of a gel (e.g., agar) plate, so that they diffuse into each other, and when they diffuse to the point where they meet at the right concentration ratio, a milky-white precipitation line appears, which is known as a bi-directional immunodiffusion test. This method was developed by Ouchterlony and is therefore also known as the Ouchterllony technique. Source: Laboratory Microbiology (Third Edition)
Operation method
basic program
Principle
The two-way immunodiffusion test not only qualitatively identifies and determines the potency of an antigen or antibody, but also analyzes the purity of the antigen or antibody and simultaneously compares two different sources of antigen or antibody to analyze the similarities and differences in the components they contain. If two or more pairs of antigen-antibody systems are present in two wells, a corresponding number of separated precipitation lines can be produced (Fig. 1). Thus, the purity of antigen or antibody can be analyzed using this method. Figure 1 The number of precipitation lines exhibited by a two-way immunodiffusion plate A. Single antigen-antibody system B. Multiple antigen-antibody system The position of the precipitation line formation is related to the antigen and antibody concentration, the denser the concentration of the antigen, the further the precipitation line is from the antigen wells, and the denser the concentration of the antibody, the farther the precipitation line is from the antibody wells (Fig. 2); therefore, when the concentration of the antibody is fixed, and the antigen diluted, the position of the precipitation line can be determined according to the known concentration of the antigen. Therefore, when the concentration of antibody is fixed and the antigen is diluted, the concentration of unknown antigen can be determined from the position of the precipitation line of known concentration of antigen; on the other hand, the potency of antibody can be determined by fixing the concentration of antigen. Figure 2 Precipitation lines in a two-way immunodiffusion plate at different distances from the antigen wells Ab: antibody, surrounding wells are antigen In addition, observe whether the two lines formed by the antigen and antibody in two neighboring wells are crossed or connected, which can be used to determine whether the two antigens have a common component (Figure 3). If the same pure antigen a is placed in two neighboring wells and the corresponding antibody is placed in the central well, the two precipitation lines will join and fuse with each other at their adjacent ends; if two different antigens a and b are present, the lines will cross each other; and if the two antigens have partly the same components, a and ab, the lines will have a projection in addition to a connection. Figure 3 Types of Precipitation Lines in a Two-Way Immunodiffusion Plate A. Same antigen in two adjacent wells; B. Different antigens; C. Partially identical antigens
Materials and Instruments
Rabbit anti-horse serum Horse serum Bovine serum Goat serum Human serum Move 1. Dissolve 1% Ion Agar in a boiling water bath. 2. 2. Cool to about 50-60°C. Pipette 3.5-4 ml onto a slide (which must be placed horizontally) so that it spreads evenly over the slide without loss. 3. 3. After the agar has solidified, punch holes with a square punch or a single-hole metal tube according to Fig. 4, and then pick the agar out of the holes with a hypodermic needle, making two square punches for each agar plate. 4. Number the wells on the bottom surface of the agar plate with a marker. 5. 5. Using a capillary buret, add rabbit anti-horse serum antibody to the center wells of the two squares, adding bovine serum to hole 1, equine serum to hole 2, sheep serum to hole 3, and serum to hole 4 around the first square. The holes around the second square are filled with the same antigen as in the first square, but the concentration is changed to 1:20. 6. 6. Place the slides in a culture plate with wet filter paper or in an enamel box with a lid. 7. Place the slide in the incubator at 37 ℃ for 24-48 h. Remove the slide and observe the results. Caveat The added serum and antiserum must not spill out of the well. For more product details, please visit Aladdin Scientific website.
1% Ion agar
Square punch or single-well metal tube (approx. 3 mm pore size) Straws Capillary burette 2.5 x 7 cm Slides, syringe needles Petri dishes or enamel cases with lids containing wet filter paper or wet gauze
Figure 4 Bidirectional diffusion type
Hole diameter is about 3 mm; the distance from the center hole is 5-6 mm.