Precipitin 1

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  • http://classes.midlandstech.edu/carterp/Courses/bio225/chap18/Slide14.GIF. precipitate should form near interface between the two solutions= zone of optimal mutual proportions \n
  • http://classes.midlandstech.edu/carterp/Courses/bio225/chap18/lecture2.htm\n
  • excess Ab: all epitopes on Ag are covered by Ab- cock blocks subsequent lattice formation\nexcess Ag: has similar effect... so all Ab already bound to Ag and doesn't really care if new Ag is added-not sufficient Ab to cross--link Ag to form lattice, more precipitation does not occur\n
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  • http://www.biology-online.org/dictionary/Ovalbumin\n
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  • http://www.medterms.com/script/main/art.asp?articlekey=5470\n
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  • 3) Deliver 50μl of PBS to wells 2 through 12 in row A\n Add 50 mL of the antigen (OA) solution (what you made in step 1) to wells 1 and 2 of row A. \n\n \n
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  • If you have excess Ab in your sample, it will diffuse (migrate) toward the Ag you plated. If you have excess Ag it will diffuse (migrate) toward the Ab you plated. This will allow you to confirm or deny your predicted OMP. By plating the samples from surrounding wells of your microtitter plate, you will be able to identify the correct OMP.\n
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  • http://www.therapak.com/catalog/img/lg/29854.jpg\n
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  • Observe turbidity- incubate 1h at 37°C- rank precipitate 1-5 (5=most precipitate)\n
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  • Precipitin 1

    1. 1. Precipitin Reaction Purpose: To understand thebiological basis of the precipitinreaction, and how to perform, and analyze the reaction.
    2. 2. Forces that stabilize Ab-Ag interactions
    3. 3. Complementary shape: maximizesaffinity and specificity
    4. 4. Complementary shape: maximizesaffinity and specificity
    5. 5. Antibodies are divalent so they can link Agtogether
    6. 6. Antigens aremultivalent so Abbridging ispossible, leading toformation of lattice
    7. 7. Precipitin reaction: continuation of latticeformation results in large aggregates whichsettle out of solution and form visibleprecipitate
    8. 8. Precipitin reaction: Ab binds soluble Ag to formaggregation of Ab-Ag complex (immune complex)that can precipitate out of solution
    9. 9. qualitativ e (ring test) tests for presence of Ab specificfor the Ag
    10. 10. Consideration ofconcentration of bothAg and Abisnecessaryto produceinsolubleaggregates
    11. 11. Optimal mutual proportions (OMP) aka equivalentproportions: usage of all available Ag and Ab information of latticeAt OMP, addition of more Ag or Ab shows noadditional precipitation. All reactants participatedin initial reaction
    12. 12. Methodology:Ab reacts with Ag for 1-2 h at 37*C to allowrapid binding of Ab-Ag; forms small, solublecomplexes
    13. 13. Incubate for 24-28 h at 4*C to allow slowdevelopment of small, soluble complexes fromstep 1 into large insoluble complexes
    14. 14. Hence want to determine OMP= optimalconcentration of Ab and Ag to allow precipitinreaction to occurSO dilute Ag, keep Ab constant, measure amountof precipitation, and determine dilution of Agthat gives greatest amount of precipitationDilution of Ag that gives greatest precipitation =OMP aka equivalence
    15. 15. • Change Ag amounts (mg), add to constant amount of Ab, measure amount of precipitation• Plot amounts of precipitate VS amount of Ag added• To determine if a particular SN has excess Ag or Ab, Add more Ag or Ab and see if additional precipitation occurs
    16. 16. Our AntigenOvalbumin:•Most prevalent protein inchicken eggs•Used as an allergen in testpatients•Used as molecular weight
    17. 17. Agglutination Precipitin ReactionQualitative (slide test) Qualitative (ring test)Ag – SRBC/Ab - α-SRBC Ag – OA /Ab – α-OAQuantitative (microtiter Quantitative (microtiter plate) assay) Result: Titer Results: OMP Serially diluted Ab Serially diluted AgMore sensitive (need . Less sensitive (need 8-16mg) 80-320mg) Ag is not soluble Ag is soluble
    18. 18. Serum: The clear liquid that can be separatedfrom clotted blood. Serum differs fromplasma, the liquid portion of normal unclottedblood containing the red and white cells andplatelets. It is the clot that makes thedifference between serum and plasma.Antiserum: antibody containing serum
    19. 19. Protocol1) Aliquot 120µl of OA into a clean, dryeppendorf tube and label accordingly. Setaside for now.  120µl OA
    20. 20. 2) Set up a 1:4 dilution (180µl of anti-OA serum +540µl of PBS) in a clean, dry eppendorf tube andlabel accordingly. Set aside for now. 540µl 180µl PBS α-OA
    21. 21. 50µl PBS50µl OA PBS OA
    22. 22. 1:2 serial of OA bubbledilution (Minimize 1 formation and change tips!!)   0.5 0.25 0.25 0.125 0.0625 0.03125 0.015625 0.0078125 0.00390625 0.00195312 5 0.00097656 25
    23. 23. When adding the serum solution, touch thedispensing tip to the upper surface of the wellabove the fluid level in the well. 50µl α- OA50µl α- OA 50µl PBS
    24. 24. Immunodiffusion plate = Immunodiffusion gelPurpose: To test if the well showingthe largest amount of precipitate isyour OMPExcess AbYour sample (centered on predictedOMP)
    25. 25. Day 2• Centrifuge plate (microtiter) 10 mins at 1200 rpm• Prepare 2 plates (petri dishes): 10mL of agar to each dish• Label plates• Incubate plates in fridge for 45-60 mins.• Remove plates
    26. 26. Day 2• Examine Day 1 plates• Determine well(s) that show largest amount of precipitate…OMP?*Be very careful to not to disrupt the precipitate (do not shake)!
    27. 27. Immunodiffusion plate =7µ Immunodiffusion gell 1 2 3 4 5 6 7 Ab Your Samples Ag
    28. 28. To plate your samples: 1 2 3 4 5 6 7 OMP Surrounding ? 7µl Surrounding wells wells
    29. 29. To plate your samples:7µl 1 2 3 4 5 6 7 Your Samples
    30. 30. MostExampl precipitat e OMP?e 1 2 3 4 5 6 7 8 9 10 11 12
    31. 31. Immunodiffusion plate =7µ Immunodiffusion gell 1 2 3 4 5 6 7 Ab 2 3 4 5 6 7 8 Your Samples Ag
    32. 32. Schedule a time to view your platetomorrowIf you know when you can make it, let me knowIf not, find time tomorrow to come find me inMH-317 I will go with you to view yourimmunodiffusion plate
    33. 33. Week 2 Day 1 Dilutions of Ag Final Volume= 750µl [OMP] 1 2 3 4 5
    34. 34. Week 2 Day 1 Add 750µl of antiserum 1 2 3 4 5
    35. 35. Week 2 Day 11 2 3 4 5
    36. 36. Week 2 Day 1 Excess OMP Excess Ag Ab 1 2 3 4 5
    37. 37. Week 2 Day 2 Centrifuge at 1500 rpm, 4°C for 10 min. Aspirate the SN_Resuspend pellet_Biorad 1 2 3 4 5
    38. 38. Calculations•Construct a standard curve (total proteinconcentration vs antigen concentration)•Calculate the amount of antibody protein atequivalence• OMP, [ppt] = [antigen] + [antibody]• Using the dilution of the original antiserum atequivalence, calculate the amount of antibodyprotein per ml of original undiluted antiserum 

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