Immunoprecipitation in Liquids and Gels

Immunoprecipitate. Maximum precipitation results when both reaction partners are present in an approximately equivalent ratio (Fig. 2.20). In antibody excess, or antigen excess, the amount of precipitate is considerably reduced.

Double diffusion according to Ouchterlony. This technique allows for a qualitative evaluation of whether certain antibodies or antigens are present or not, plus determination of the degree of relationship between antibodies and antigens. It also provides information on whether different antigenic de-

— Immunoprecipitation

Addition of specific

Labeled antigen mixture

Addition ofanti-lg

Addition of specific

Addition ofanti-lg

antibody

m %

4

Precipitate is pelleted by centrifugation

Specific complexes

Solubilization of precipitate

Solubilization of precipitate

Autoradiography

Fig. 2.20 To identify the unknown antigen, a known specific antibody is added to an antigen mixture which is radio-, or otherwise-, labeled. The immune complexes are precipitated with the help of co-precipitating reagents (e.g., antiimmunoglobulin antibodies). The precipitate is thoroughly washed to remove unbound antigen, then dispersed into solution once again (e.g., in SDS), after which the components are separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE). The labeled antigen is then rendered visible by means of autoradiography.

SDS-PACE

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All rights reserved. Usage subject to terms and conditions of license.

terminants are localized on the same, or on different, antigens; or whether different antibodies can bind to the same antigen (Fig. 2.21).

Radial immunodiffusion according to Mancini. This is a quantitative antigen assay based on a predetermined standard curve (Fig. 2.22).

Nephelometry. This method measures the amount of light scatter as a quantification of precipitation turbidity.

Immunoprecipitation Combined with Electrophoresis. Antigens are separated in an agarose gel by applying an electric current. The antibodies react by migrating in the gel, either without an electric field, or simultaneously within the electric field; and either in the same dimension as the antigens or in a second vertical step ("rocket" electrophoresis).

Immunoelectrophoresis according to Grabar and Williams. In the first instance serum proteins are electrophoretically separated within a thin agarose gel layer. A trough is then cut into the agar, next to the separated sample and parallel to the direction of migration along the entire migration distance, and anti-serum is applied to the trough. The antibodies diffuse into the gel, and precipitation lines are formed wherever they encounter their antigens. The

— Double Diffusion According to Ouchterlony

— Double Diffusion According to Ouchterlony

Precipitin Line And Mancini Test

a Identity band b Non-identity c Partial Identity

Fig. 2.21 This technique facilitates assignment of antigens (violet) to a certain test antibody (yellow), or vice versa. The antigens and antibodies are pipetted into troughs within the gel and diffuse through this medium (the numbers designate the epitopes present). Where they meet lines of precipitation (known as precipitin bands) develop, indicating immune complex formation. a The antibodies precipitate identical epitopes (epitope 1) of both antigens, resulting in formation of precipitin bands which flow together to form an arch, mutually inhibiting their migration. In b, three independent precipitin bands form, indicating that the antibodies differentiate three different epitopes on three different antigens. c Epitope 1 of both antigen samples forms precipitin bands which flow together. Anti-2 migrates beyond the line of confluence into the area in which it precipitates with free antigen 1, 2 and forms a spur.

a Identity band b Non-identity c Partial Identity

Fig. 2.21 This technique facilitates assignment of antigens (violet) to a certain test antibody (yellow), or vice versa. The antigens and antibodies are pipetted into troughs within the gel and diffuse through this medium (the numbers designate the epitopes present). Where they meet lines of precipitation (known as precipitin bands) develop, indicating immune complex formation. a The antibodies precipitate identical epitopes (epitope 1) of both antigens, resulting in formation of precipitin bands which flow together to form an arch, mutually inhibiting their migration. In b, three independent precipitin bands form, indicating that the antibodies differentiate three different epitopes on three different antigens. c Epitope 1 of both antigen samples forms precipitin bands which flow together. Anti-2 migrates beyond the line of confluence into the area in which it precipitates with free antigen 1, 2 and forms a spur.

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All rights reserved. Usage subject to terms and conditions of license.

Immunological Test Methods 123 — Radial Immunodiffusion According to Mancini -

Immunological Test Methods 123 — Radial Immunodiffusion According to Mancini -

Immunodiffusion Mancini

Fig. 2.22 Quantitative assay of an antigen using a monospecific anti-serum which is mixed with agar and poured into a plate. The antigen is then diluted to different concentrations, and pipetted into wells that have been previously punched into the plate. Antigen-antibody complexes precipitate in the form of a ring around the well, the diameter of which is proportional to the antigen concentration. The result is a standard curve from which unknown test antigens can be quantified. Analogously, antibodies can also be quantified by mixing antigens into the gel.

Fig. 2.22 Quantitative assay of an antigen using a monospecific anti-serum which is mixed with agar and poured into a plate. The antigen is then diluted to different concentrations, and pipetted into wells that have been previously punched into the plate. Antigen-antibody complexes precipitate in the form of a ring around the well, the diameter of which is proportional to the antigen concentration. The result is a standard curve from which unknown test antigens can be quantified. Analogously, antibodies can also be quantified by mixing antigens into the gel.

precipitate can then be stained and evaluated. This older method is still used to identify paraproteins, monoclonal immunoglobulins, etc. (Fig. 2.23).

Electrophoresis plus antibody reaction: Western blotting. This method involves electrophoresis of proteins in a gel, coupled with detection by specific antibodies. The separated proteins are transferred to nitrocellulose, where they are identified with the help of specific antibodies (Fig. 2.24). Polyclonal sera is normally used for this purpose as monoclonal antibodies only rarely bind to denaturated and separated proteins.

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Responses

  • bernd
    What does partial identity mean in ouchterlony test?
    7 years ago
  • Jukka-Pekk
    What do precipitin lines mean in ouchterlony?
    7 years ago
  • belinda
    How to interpret precipitin lines?
    6 years ago

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