Picture of an Ouchterlony double immunodiffusion plate, after immunodiffusion has taken place. In this, titre value of an antigen is quantified. The central well has an antibody, and the surrounding wells have decreasing concentration of the corresponding antigen.Ouchterlony patterns showing no identity between upper spotsOuchterlony patterns showing full identity between upper spotsOuchterlony patterns showing partial identity between upper spots
A gel plate is cut to form a series of holes ("wells") in an agar or agarose gel. A sample extract of interest (for example human cells harvested from tonsil tissue) is placed in one well, sera or purified antibodies are placed in another well and the plate left for 48 hours to develop. During this time the antigens in the sample extract and the antibodies each diffuse out of their respective wells. Where the two diffusion fronts meet, if any of the antibodies recognize any of the antigens, they will bind to the antigens and form an immune complex. The immune complex precipitates in the gel to give a thin white line (precipitin line), which is a visual signature of antigen recognition.[citation needed][2][3]
The method can be conducted in parallel with multiple wells filled with different antigen mixtures and multiple wells with different antibodies or mixtures of antibodies, and antigen-antibody reactivity can be seen by observing between which wells the precipitate is observed. When more than one well is used there are many possible outcomes based on the reactivity of the antigen and antibody selected. The zone of equivalence lines may give a full identity (i.e. a continuous line), partial identity (i.e. a continuous line with a spur at one end), or a non-identity (i.e. the two lines cross completely).[citation needed]
The sensitivity of the assay can be increased by using a stain such as Coomassie brilliant blue, this is done by repeated staining and destaining of the assay until the precipitin lines are at maximum visibility.[1]
Theoryedit
Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several antigenic determinants per molecule to which antibodies can bind). Antibodies have at least two antigen binding sites (and in the case of immunoglobulin M there is a multimeric complex with up to 10 antigen binding sites), thus large aggregates or gel-like lattices of antigen and antibody are formed. Experimentally, an increasing amount of antigen is added to a constant amount of antibody in solution. Initially at low antigen concentration, all of the antibody is contained in the precipitate. This is called the antibody-excess zone (i.e. prozone phenomenon). As more antigen is added, the amount of protein precipitated increases until the antigen/antibody molecules are at an optimal ratio. This is known as the zone of equivalence or equivalence point. When the amount of antigen in solution exceeds the amount of antibody, the amount of precipitation will decrease. This is known as the antigen excess zone.
Referencesedit
^ abHornbeck, Peter (February 2017). "Double-Immunodiffusion Assay for Detecting Specific Antibodies (Ouchterlony)". Current Protocols in Immunology. 116 (1): 2.3.1–2.3.4. doi:10.1002/cpim.18. PMID 28150863. S2CID 35908711.
^Moticka, Edward J. (2015). A Historical Perspective on Evidence-Based Immunology. Elsevier. p. 360. ISBN978-0-12-398381-7.
^Paul, William E., ed. (2008). "Immunodiffusion and the Ouchterlony Method". Fundamental Immunology (6th ed.). Lippincott Williams & Wilkins. pp. 176–177. ISBN9780781765190. p. 176 p. 177
Bailey, Graham S. (1996). "135: Ouchterlony Double Immunodiffusion". In Walker, John M. (ed.). The Protein Protocols Handbook. Springer Protocols Handbooks. Vol. VII: Immunochemical Techniques. Totowa, New Jersey: Humana Press. pp. 749–752. doi:10.1007/978-1-60327-259-9_135. ISBN978-0-89603-338-2.
Ouchterlony, Örjan (1948). "In vitro method for testing the toxin-producing capacity of diphtheria bacteria". APMIS. 25 (1–2): 186–191. doi:10.1111/j.1699-0463.1948.tb00655.x. PMID 18887479.
"Ouchterlony Double Diffusion - Patterns: Theory". Value @ Amrita. India: Amrita Vishwa Vidyapeetham University. 2012. from the original on 2012-11-13. Retrieved 2012-12-24.
"Ouchterlony Double Diffusion - Patterns: Procedure". Value @ Amrita]. India: Amrita Vishwa Vidyapeetham University. 2012. from the original on 2017-01-14. Retrieved 2012-12-24.
"Ouchterlony Double Diffusion - Titration: Theory". Value @ Amrita. India: Amrita Vishwa Vidyapeetham University. 2012. from the original on 2012-11-13. Retrieved 2012-11-23.
"Ouchterlony Double Diffusion - Titration: Procedure". Value @ Amrita. India: Amrita Vishwa Vidyapeetham University. 2012. from the original on 2017-03-16. Retrieved 2012-12-24.
"BSCI423: Lab 5. Precipitation". College of Computer, Mathematical, and Natural Sciences, University of Maryland, College Park. from the original on 2012-02-05. Retrieved 2012-11-23.
"Ouchterlony double immunodiffusion" (photograph). Retrieved 2017-05-15.[permanent dead link] Photograph of Ouchterlony double immunodiffusion plate with unstained precipitin lines of full identity and non-identity.
. Immunodiffusion principles and application. Archived from the original on 2019-12-11. Retrieved 2017-05-19. Photographs of Ouchterlony immunodiffusion patterns showing stained precipitin lines of full identity, partial identity and non-identity.
"Ouchterlony Assay". YouTube. Bio-Rad Laboratories. October 16, 2012.
April 12, 2024
ouchterlony, double, immunodiffusion, also, known, passive, double, immunodiffusion, immunological, technique, used, detection, identification, quantification, antibodies, antigens, such, immunoglobulins, extractable, nuclear, antigens, technique, named, after. Ouchterlony double immunodiffusion also known as passive double immunodiffusion is an immunological technique used in the detection identification and quantification of antibodies and antigens such as immunoglobulins and extractable nuclear antigens The technique is named after Orjan Ouchterlony the Swedish physician who developed the test in 1948 to evaluate the production of diphtheria toxins from isolated bacteria 1 Picture of an Ouchterlony double immunodiffusion plate after immunodiffusion has taken place In this titre value of an antigen is quantified The central well has an antibody and the surrounding wells have decreasing concentration of the corresponding antigen Ouchterlony patterns showing no identity between upper spotsOuchterlony patterns showing full identity between upper spotsOuchterlony patterns showing partial identity between upper spots Contents 1 Procedure 2 Theory 3 References 4 External linksProcedure editA gel plate is cut to form a series of holes wells in an agar or agarose gel A sample extract of interest for example human cells harvested from tonsil tissue is placed in one well sera or purified antibodies are placed in another well and the plate left for 48 hours to develop During this time the antigens in the sample extract and the antibodies each diffuse out of their respective wells Where the two diffusion fronts meet if any of the antibodies recognize any of the antigens they will bind to the antigens and form an immune complex The immune complex precipitates in the gel to give a thin white line precipitin line which is a visual signature of antigen recognition citation needed 2 3 The method can be conducted in parallel with multiple wells filled with different antigen mixtures and multiple wells with different antibodies or mixtures of antibodies and antigen antibody reactivity can be seen by observing between which wells the precipitate is observed When more than one well is used there are many possible outcomes based on the reactivity of the antigen and antibody selected The zone of equivalence lines may give a full identity i e a continuous line partial identity i e a continuous line with a spur at one end or a non identity i e the two lines cross completely citation needed The sensitivity of the assay can be increased by using a stain such as Coomassie brilliant blue this is done by repeated staining and destaining of the assay until the precipitin lines are at maximum visibility 1 Theory editPrecipitation occurs with most antigens because the antigen is multivalent i e has several antigenic determinants per molecule to which antibodies can bind Antibodies have at least two antigen binding sites and in the case of immunoglobulin M there is a multimeric complex with up to 10 antigen binding sites thus large aggregates or gel like lattices of antigen and antibody are formed Experimentally an increasing amount of antigen is added to a constant amount of antibody in solution Initially at low antigen concentration all of the antibody is contained in the precipitate This is called the antibody excess zone i e prozone phenomenon As more antigen is added the amount of protein precipitated increases until the antigen antibody molecules are at an optimal ratio This is known as the zone of equivalence or equivalence point When the amount of antigen in solution exceeds the amount of antibody the amount of precipitation will decrease This is known as the antigen excess zone References edit a b Hornbeck Peter February 2017 Double Immunodiffusion Assay for Detecting Specific Antibodies Ouchterlony Current Protocols in Immunology 116 1 2 3 1 2 3 4 doi 10 1002 cpim 18 PMID 28150863 S2CID 35908711 Moticka Edward J 2015 A Historical Perspective on Evidence Based Immunology Elsevier p 360 ISBN 978 0 12 398381 7 Paul William E ed 2008 Immunodiffusion and the Ouchterlony Method Fundamental Immunology 6th ed Lippincott Williams amp Wilkins pp 176 177 ISBN 9780781765190 p 176 p 177 Bailey Graham S 1996 135 Ouchterlony Double Immunodiffusion In Walker John M ed The Protein Protocols Handbook Springer Protocols Handbooks Vol VII Immunochemical Techniques Totowa New Jersey Humana Press pp 749 752 doi 10 1007 978 1 60327 259 9 135 ISBN 978 0 89603 338 2 Ouchterlony Orjan 1948 In vitro method for testing the toxin producing capacity of diphtheria bacteria APMIS 25 1 2 186 191 doi 10 1111 j 1699 0463 1948 tb00655 x PMID 18887479 Ouchterlony Orjan 1949 Antigen antibody reactions in gels APMIS 26 4 507 515 doi 10 1111 j 1699 0463 1949 tb00751 x PMID 18143039 Ouchterlony O 1958 Diffusion in gel methods for immunological analysis Progress in Allergy 5 1 78 PMID 13578996 Ouchterlony O 1962 Diffusion in gel methods for immunological analysis II Progress in Allergy 6 30 154 doi 10 1159 000313795 PMID 14482809 Ouchterlony O Nilsson L A 1986 Weir Daniel Mackay ed Handbook of Experimental Immunology Vol 1 Immunochemistry 4th ed Oxford England Blackwell Scientific pp 32 1 32 50 ISBN 9780632014996 Retrieved 2012 11 23 at Google Books Ouchterlony Analysis PDF Medical Immunology 544 University of California Irvine College of Medicine Fall 2011 Archived from the original PDF on 2012 02 07 Retrieved 2012 11 23 Ouchterlony Double Diffusion Patterns Theory Value Amrita India Amrita Vishwa Vidyapeetham University 2012 Archived from the original on 2012 11 13 Retrieved 2012 12 24 Ouchterlony Double Diffusion Patterns Procedure Value Amrita India Amrita Vishwa Vidyapeetham University 2012 Archived from the original on 2017 01 14 Retrieved 2012 12 24 Ouchterlony Double Diffusion Titration Theory Value Amrita India Amrita Vishwa Vidyapeetham University 2012 Archived from the original on 2012 11 13 Retrieved 2012 11 23 Ouchterlony Double Diffusion Titration Procedure Value Amrita India Amrita Vishwa Vidyapeetham University 2012 Archived from the original on 2017 03 16 Retrieved 2012 12 24 BSCI423 Lab 5 Precipitation College of Computer Mathematical and Natural Sciences University of Maryland College Park Archived from the original on 2012 02 05 Retrieved 2012 11 23 External links editSimulator Archived 2007 05 22 at the Wayback Machine at University of California Irvine Ouchterlony double immunodiffusion photograph Retrieved 2017 05 15 permanent dead link Photograph of Ouchterlony double immunodiffusion plate with unstained precipitin lines of full identity and non identity Diffusion Patterns Immunodiffusion principles and application Archived from the original on 2019 12 11 Retrieved 2017 05 19 Photographs of Ouchterlony immunodiffusion patterns showing stained precipitin lines of full identity partial identity and non identity Ouchterlony Assay YouTube Bio Rad Laboratories October 16 2012 Retrieved from https en wikipedia org w index php title Ouchterlony double immunodiffusion amp oldid 1212613553, wikipedia, wiki, book, books, library,
