THE PRECIPITIN REACTION When an antiserum is mixed with a solution of the antigen to which it isspecifically directed, antibody molecules will begin adhering to the antigenmolecules. Several types of interactions stabilize Ag-Ab binding, which include:hydrophobic interactions, hydrogen bonds, van der Waals contacts, andelectrostatic interactions. The antibody and antigen surfaces generally havecomplementary shapes with respect to the placement of grooves and bumps,maximizing affinity and specificity. Since antibody is divalent, it is possible for these molecules to act as abridge, linking two antigen molecules together. Because most antigen molecules aremultivalent/have several epitopes (for example, numerous antigen molecules on aforeign cell surface), antibody bridging is possible in several directions, giving riseto a “lattice” of mutually bound molecules. Continuation of this process willultimately result in the formation of large aggregates of the two molecular species,which will ultimately settle out of solution and form a visible precipitate. Inimmunological jargon, this is known as the precipitin reaction. The precipitinreaction is a reaction in which antibody binds soluble antigen to form anaggregation of Ab-Ag complexes that can precipitate out of solution. There aretwo variations of this reaction: the qualitative (ring test) and the quantitativeprecipitin reactions. Examples of each type of reaction are included in theexperiments that follow. Reaction mechanisms: The precipitin reaction is ordinarily performed byallowing the reactants to interact 1-2 hours at 37 0C, followed by a prolongedincubation at 4 0C for 24-48 hours. This protocol recognizes the two-stage natureof the antigen-antibody reaction: (a) an early, rapid binding of the components toform small soluble complexes, followed by (b) a second, slow developing aggregationof the small complexes into large insoluble ones. The early reaction is favored athigh temperatures, while the late reaction proceeds to completion more favorablyat reduced temperatures. The interaction of reactants requires optimal conditions to occur. Theproper concentration of both antigen and antibody is necessary and is consideredthe most critical condition that must be met to produce insoluble aggregates.Optimal proportions (equivalent proportions) of each result in usage of all availableantigen and antibody in formation of the lattice. To produce optimal proportions ofeach or optimal mutual proportions (OMP), dilution is usually necessary. Dilution ofthe antigen is the most common procedure. When equivalent proportions of antigen and antibody are mixed andincubated, addition of more antigen or antibody to the supernatant will produce no
additional precipitation. The reason is that all reactants participated in the initialreaction. This is the ideal situation, and many tests have been designed to obtainthis result. When excess antibody is added to a reaction mixture, all of theepitopes on the antigen are covered by antibody resulting in inhibition of latticeformation. Excess antigen has a similar effect since there will not be sufficientantibody to crosslink with antigen to form the lattice, and once again, precipitationis inhibited. The problem is, therefore, to determine the optimal concentration of bothantigen and antibody. This is accomplished by serial dilution of antigen and additionof these dilutions to constant amounts of antibody. The amount of precipitationthat results can be measured visually or chemically, and the dilution of antigen thatgives the greatest amount of precipitation is considered to be equivalence. Quantitative precipitin reaction: Figure 1 (handout given in class)represents, a typical protein precipitation curve. To produce the curve, specificamounts (mg) of antigen were added to a series of tubes, each containing aconstant amount of antibody. After an incubation period, the precipitate wascollected and assayed. The amounts of precipitate were plotted against the amountof antigen added to produce the precipitation curve. To determine which tubecontains excess antigen or antibody, the supernatant fractions can be furtherstudied. To portions of each supernatant, more antigen or antibody could be addedand the tubes observed for additional precipitation. The tube that shows noadditional precipitation when either antigen or antibody is added is by definitionthe equivalence point or equivalence zone of the precipitation curve. Performance of the technique: In the experiment to follow, I will initiallyperform the qualitative precipitin reaction, which is called the ring test, followedby all groups performing the quantitative precipitin reaction. The ring test involvesoverlaying the antiserum with a solution of the appropriate specific antigen. Thistest will provide you with the information confirming that the antiserum containsantibody specific for the antigen in question. By mutual diffusion, the reactantsultimately establish a zone of optimal mutual proportions somewhere near theinterface between the two solutions, and a visible precipitate will form. In the next exercise you will be asked to ascertain the equivalence point ofyour antiserum-antigen solution using various ratios of the reactants in microtiterwells, accompanied by the use of gel diffusion reactions to assay the varioussupernatants that form (looking for excess antibody or antigen). Finally, once theproper concentrations have been established, you will be asked to complete theperformance of the quantitative precipitin reaction. You will be utilizing the Bio-Rad assay to determine the quantity ofprecipitate in each of the tubes you have set up, so that you can report the amount
of antibody protein present in your undiluted antiserum. This reaction must beperformed with extreme care to detail to obtain accurate results.Objectives:Understand the biological basis of the precipitin reactionUnderstand the purpose of each step of the precipitin reactionBe able to define and understand OMP.At OMP, [ppt] = [antigen] + [antibody]Precipitation curve: how is it constructed? Equivalence zone? Excess antigen andantibody zone?Be able to compare and contrast the precipitin and the agglutination reactionsMaterials:15ml conical tubes, eppendorf tubesPasteur pipettes with rubber bulbAntibody solution (anti-ovalbumin, αOA) diluted 1:4 using 1x PBSAntigen solution (OA): 1mg/mlNormal rabbit serum – 0.4mlDiluting solution, 1x PBSMicrotiter plate (round bottom)Immunodiffusion agar: 1% in PBSBio-Rad dyeSterile Petri dishAgar hole puncher with rubber bulbMoist chamberMicrotiter plate viewer with mirrorProcedureDay 1A. The Ring test (I will demonstrate this to class)Using a Pasteur pipette, introduce a small amount of antiserum directly into thebottom of a clean glass tube, withdrawing the pipette slowly and carefully so as notto leave any serum in the insides of the tube. Use one hand to insert or retrievethe pipette and the other to steady the tip as it is being moved in or out of thetube. Repeat the procedure in a separate clean glass tube using normal rabbitserum instead of antiserum and use a different Pasteur pipette.Next, carefully overlay an equal volume of antigen solution using a differentPasteur pipette, so that it floats in the surface of the antiserum. You should see a
visible line of demarcation at the interface. This maneuver is most easilyperformed by slanting the tube somewhat during the introduction of the antigensolution and dispensing it slowly.Observe the tubes for 20-25 minutes, watching for the development ofprecipitation at the interface. If precipitation occurs, it will do so within this timeinterval. Readings should not be taken after 30 minutes.If the reaction is negative, the amount of antibody might be too small compared tothe amount of antigen. If this occurs, reduce the antigen concentration in one tubeto 0.05% and in another to 0.01%. If neither of these tubes shows visibleprecipitation in the region of the interface, you may conclude that the antiserumcontains no antibodies appropriate to the antigen employed.B. Optimal proportions by gel diffusion1) Aliquot 120µl of OA into a clean, dry eppendorf tube and label accordingly. Setaside for now.2) Set up a 1:4 dilution (180µl of anti-OA serum + 540µl of PBS) in a clean, dryeppendorf tube and label accordingly. Set aside for now.3) Deliver 50µl of PBS to wells 2 through 12 in row A (or a row that has notpreviously been used) of your microtiter plate. Add 50 µL of the antigen (OA)solution (what you made in step 1) to wells 1 and 2 of row A. Now, beginning withwell #2, gently aspirate and expel with your micropipettor several times to mix thecontents of the well; then transfer 50µL from this well to the next well. (Minimizebubble formation and change tips!!) Continue this process of two-fold serialdilutions through well # 12, discarding 50 µL from the last well. Well #1 isundiluted antigen (1mg/ml); well #2 is a 1:2 dilution of Ag (0.5mg/ml), well #3 is a1:4 dilution of Ag (0.25mg/ml), etc.4) Add 50 µL of the 1:4 dilution of anti-OA to all wells of row A. When adding theserum solution, touch the dispensing tip to the upper surface of the well above thefluid level in the well. Well#1 of the next row will be used for your control. Add50µl of PBS + 50µl of diluted αOA. Mix the contents of all wells by using a rotarymotion with the plate. Cover the plate and incubate it at 370C for 1 hr. After thecompletion of incubation, transfer the microtiter plate to the refrigerator for 48hrs.
Day 2:1) After 2 days, centrifuge the microtiter plate for 10 minutes at 1200 rpm. In themeantime, prepare 2 immunodiffusion plates by pipetting 10 ml of immunodiffusionagar (found in the water bath in the back of the classroom) into each Petri dish.Label your plates and place them in the refrigerator. After approximately 45-60minutes, remove the plates and very carefully (no jagged edges!) punch holes intoONE of the plates according to the pattern seen in the handout (the second plateis simply a backup in case you screw up the first one). You can place the diagram onthe handout beneath your plate to show where each hole should be punched.2) Remove your microtiter plate from Day 1 and examine the bottom of themicrotiter plate wells with the mirror viewer and note the well or wells that appearto show the largest amount of precipitate. Be very careful not to shake orotherwise disturb these precipitates.3) Carefully remove about 7 µL of the supernatant from the well showing thelargest amount of precipitate from row A of your microtiter plate, and introducethe supernatant into the 4th hole from the left in the middle row of yourimmunodiffusion plate (see diagram on board). Do not overfill. If you have twowells whose precipitates appear to be equally large, put the supernatant from thehighest antigen concentration in agar hole # 4 and the other one in hole # 5. Finishdispensing supernatants from the microtiter plate A to the right and left of theones already completed, e.g. if well #7 was your best precipitate, put itssupernatant into hole #4, while supernatant from wells 4 to 6 will be put into theholes to the left (1 to 3), and the supernatants from well 8-10 will be dispensedinto holes to the right (5-7). After all holes are filled dispense 1:4 antiserum intoeach of the top row of holes and the OA antigen solution (1mg/ml) into each of thebottom row of holes. Put your plate in the refrigerator and let incubate for 24hours. Schedule a time to observe w/ TA for Thursday.Day3:The next day, observe your plates for lines of precipitate, and make drawings ofthese results. Any line appearing between the middle and upper holes wouldindicate excess antigen in the supernatant. Conversely, any precipitate formingbetween the middle and bottom holes would signify excess antibody in thesupernatant. Any holes showing no precipitate in either top or bottom areas wouldindicate no free antibody or antigen in the supernatant. Such a result designatesthe area of optimal mutual proportions of the antiserum and antigen (theequivalence point). At which dilution of Ag did you reach OMP?
C. The quantitative precipitin reaction (following week) You will work in larger groups for this week (in tables).Week 2 Day1:1) Prepare five 15ml conical tubes in a test tube rack and label with your table’sname and number. From the results obtained in the previous section, determine theantibody/antigen concentration, which will give you optimal proportions. Prepareserial dilutions of the antigen solution using 750µl as a final volume, arranged sothat the optimal tube will either be #2 or #3 in your row of tubes. After allantigen tubes have been prepared, pipette 750µl of the 1:4 diluted antiserum intoeach of the tubes. Mix the contents of each tube immediately after adding theantiserum. The total volume should now be 1.5mL in each of the five tubes.2) Observe the tubes for the development of turbidity. The middle tubes shouldshow this first and subsequently become the most turbid. The tubes on the left(antigen excess) and the right (antibody excess) will show some cloudiness, butshould be noticeably less so than the center tubes. Incubate all tubes for 1 hour at370C; then record the relative quantities of precipitate that you observe visually,using 5+ for the maximum precipitate, 4+, 3+, 2+, and 1+ (or 0 for none). (Thisshould be a chart in your results section in your lab notebook) Transfer all thetubes to the refrigerator for 48 hours.Week 2 Day 2:1) Centrifuge the tubes at 1500 rpm in a 40C centrifuge for 10 min, and thencarefully aspirate the supernatant from each tube showing a pellet of precipitate.Be very careful not to lose any of the precipitate during aspiration. Resuspend thepellet in 2ml of cold PBS with gentle mixing, then centrifuge and aspirate as beforeand again resuspend in 2ml of cold PBS.2) Perform the microtiter Bio-Rad assay on the contents of each tube. (Refer tothe Bio-Rad assay procedure).3) Construct a standard curve by plotting total protein concentration versus theantigen concentration for four tubes. In addition, you should be able to calculatethe amount of antibody protein at equivalence, because all of the added antigen(known) may be presumed to be in the precipitate (at OMP, [ppt] = [antigen] +[antibody]). Since you know the dilution of the original antiserum at equivalence,calculate the amount of antibody protein per ml of original undiluted antiserum.Show all calculations in your notebook.