ouchterlony

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EVT 911108AM

All components are intended for educational research only. They are not to be used fordiagnostic or drug purposes, nor administered to or consumed by humans or animals.

Experiment Objective:

The objective of this experiment is to introduce theprinciples of antigen-antibody interactions by

using the Ouchterlony procedure.

270EDVO-Kit #

Antigen-AntibodyInteraction: TheOuchterlony Procedure

Storage: Some components require storagein the refrigerator. See page 2 for details

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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. Thisdocument, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.Copyright © 1989,1992,1995,1998, EDVOTEK, Inc., all rights reserved. EVT 911108AM

EDVOTEK • The BiotechnologyEducation Company ®

EDVO-Kit # 270: Antigen-Antibody Interaction: The Ouchterlony Procedure

Major Section Headings

Experiment Components

Page

Experiment Components 2Requirements 3Background Information 4Experimental Procedures 7Study Questions 9

Instructor's Guide General Information 11 Pre-Lab Preparations 11 Avoiding Common Pitfalls 14 Idealized Schematic of Results 15 Answers to Study Questions 16

This experiment is designed for 10 lab groups.Storage

A. Sample containing animal anti-serum RefrigeratorB Sample containing animal serum RefrigeratorC. Sample containing albumin antigen RefrigeratorD. Sample containing IgG antigen RefrigeratorE. Package containing powdered buffer Room temp

1 Package UltraSpec-Agarose™ Room temp1 Tube practice loading solution Room temp40 Transfer pipets40 Petri plates10 Well cutters1 Template for cutting wells40 Microtest tubes

Storage:Store components A-D

in the refrigerator.

All components of this experimentare intended for educational

research only. They are not to beused for diagnostic or drug

purposes, nor administered to orconsumed by humans or animals.

None of the experiment compo-nents have been prepared from

human sources.

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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This docu-ment, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.Copyright © 1989,1992,1995,1998, EDVOTEK, Inc., all rights reserved. EVT 911108AM



Requirements

• Micropipet and tips• Plastic container or Pyrex baking dish• Plastic wrap• Distilled Water• Pipets - 5 ml or 10 ml• Marking pen• Measuring spatula or toothpicks• Heat plate, Bunsen burner, or microwave• Paper towels• Waterbath (55°C)

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BACKGROUND INFORMATION

Antigen-Antibody Interaction: The Ouchterlony Procedure

Figure 1

Equivalence ZoneAb-Excess Zone Ag-Excess Zone

+ ±+ + ++ + + +

AntibodyPrecipitated

Antigen added

Excess Ab

Sup

erna

tant

s

Excess Ag

Figure 2

Antibody-Excess Zone Antigen-Excess ZoneEquivalence Zone

Antibody Antigen

The interactions of an antibody (Ab) with an antigen (Ag) is thefundamental reaction of immunology.

Ab + Ag → Ab-AgMost antigens are proteins. The exact identity of the groups that reactwith the antibody are usually not known. However, macromolecularantigens and antibodies frequently form complexes that become insol-

uble and precipitatefrom solution. Thisproperty makes itpossible to performqualitative and quan-titative assays on theant ibody-ant igensystem.

Precipitation occursbecause each anti-body can bind tomore than one anti-gen and each macro-molecular antigencan be bound by morethan one antibody.Experimentally, anti-gen is added to a con-stant amount of anti-body in solution. Ini-tially, the amount of

the antibody in solution is muchgreater than the amount of antigenin solution. This is called the anti-body-excess zone. As more antigenis added, the amount of protein pre-cipitated increases until the antigenand antibody molecules are at anoptimal ratio (roughly in equal pro-portions). This is called the equiva-lence zone, or equivalence point,where maximum precipitation oc-curs. When the amount of antigen insolution exceeds the amount of anti-body, the amount of precipitation

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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This docu-ment, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.Copyright © 1989,1992,1995,1998, EDVOTEK, Inc., all rights reserved. EVT 911108AM




Figure 3

Antigen 1 Antigen 2

Antibody

Figure 4:Reaction of Identity

Figure 5Reaction of Non-identity

Antigen 1 Antigen 2

Antibody

Antigen 1 Antigen 2

Antibody

Figure 6Reaction of Partial Identity

will decrease. This is known as the antigen-excess zone (Figures 1and 2).

When antibodies and antigens are inserted into different areas of anagarose gel, they diffuse toward each other and form opaque bands ofprecipitate at the interface of their diffusion fronts. Precipitationreactions of antibodies and antigens in agarose gels provide a methodof analyzing the various antibody-antigen reactions in a system.

Double diffusion in two dimensions is a simple procedure invented bythe Swedish scientist, Ouchterlony. Antigen and antibody solutions areplaced in separate wells cut in an agarose plate. The reactants diffusefrom the wells toward each other and precipitate where they meet atequivalent proportions. A single antigen will combine with its homolo-gous antibody to form a single precipitation line. When two antigensare present, each behaves independently of each other. Thus, thenumber of precipitin bands indicates there are at least that manyantibody-antigen systems present (see Figure 3).

Double diffusion in two dimensions is a useful technique for comparingantigens for the number of identical or cross-reacting determinants. Ifa solution of antigen is placed in two adjacent wells and the homolo-gous antibody is placed in the center well, the two precipitin bands thatform will join at their closest ends and fuse. This is known as a reactionof identity (Figure 4).

Background Information,continued

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Background Information,continued

Figure 7

Cross-reactingAntibody

HomologousAntibody

HomologousAntigen

Cross-reactingAntigen

Cross-reactingAntigen

HomologousAntigen

When unrelated antigens are placed in adjacent wells and the centerwell is filled with antibodies for each antigen, the precipitin bands willform independently of each other and will cross. This is known as areaction of non-identity (Figure 5).

If the antigen in one well and the antiserum in the center well comprisea homologous pair, and the antigen in an adjacent well is a cross-reacting antigen, the precipitin lines will fuse. However, an additionalspur will form which projects toward the cross-reacting antigen. Thisis known as a reaction of partial identity (Figure 6).

The spur represents the reaction between homologous antigen andthose antibody molecules that do not combine with the cross-reactingantigen. In other words, the homologous antigen contains determi-nants recognized by the homologous antibody which are not present onthe cross-reacting antigen. Since these non-cross-reacting antibodiesoften compose only a fraction of the total antibody involved in thehomologous precipitin reaction, the spur is usually less dense than theprecipitin band from which it projects (Figure 7).

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