When an antiserum is mixed with the antigen it is directed against, antibody molecules will bind to antigen molecules via various interactions. Since antibodies are divalent, they can act as bridges between antigens, linking antigens together and forming aggregates. Continued bridging results in large precipitates that settle out of solution, known as the precipitin reaction. The precipitin reaction involves determining the optimal mutual proportions of antigen and antibody that result in maximum precipitation to identify the equivalence point.

1. THE PRECIPITIN REACTION
When an antiserum is mixed with a solution of the antigen to which it is
specifically directed, antibody molecules will begin adhering to the antigen
molecules. Several types of interactions stabilize Ag-Ab binding, which include:
hydrophobic interactions, hydrogen bonds, van der Waals contacts, and
electrostatic interactions. The antibody and antigen surfaces generally have
complementary 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 a
bridge, linking two antigen molecules together. Because most antigen molecules are
multivalent/have several epitopes (for example, numerous antigen molecules on a
foreign cell surface), antibody bridging is possible in several directions, giving rise
to a “lattice” of mutually bound molecules. Continuation of this process will
ultimately result in the formation of large aggregates of the two molecular species,
which will ultimately settle out of solution and form a visible precipitate. In
immunological jargon, this is known as the precipitin reaction. The precipitin
reaction is a reaction in which antibody binds soluble antigen to form an
aggregation of Ab-Ag complexes that can precipitate out of solution. There are
two variations of this reaction: the qualitative (ring test) and the quantitative
precipitin reactions. Examples of each type of reaction are included in the
experiments that follow.
Reaction mechanisms: The precipitin reaction is ordinarily performed by
allowing the reactants to interact 1-2 hours at 37 0C, followed by a prolonged
incubation at 4 0C for 24-48 hours. This protocol recognizes the two-stage nature
of the antigen-antibody reaction: (a) an early, rapid binding of the components to
form small soluble complexes, followed by (b) a second, slow developing aggregation
of the small complexes into large insoluble ones. The early reaction is favored at
high temperatures, while the late reaction proceeds to completion more favorably
at reduced temperatures.
The interaction of reactants requires optimal conditions to occur. The
proper concentration of both antigen and antibody is necessary and is considered
the most critical condition that must be met to produce insoluble aggregates.
Optimal proportions (equivalent proportions) of each result in usage of all available
antigen and antibody in formation of the lattice. To produce optimal proportions of
each or optimal mutual proportions (OMP), dilution is usually necessary. Dilution of
the antigen is the most common procedure.
When equivalent proportions of antigen and antibody are mixed and
incubated, addition of more antigen or antibody to the supernatant will produce no

2. additional precipitation. The reason is that all reactants participated in the initial
reaction. This is the ideal situation, and many tests have been designed to obtain
this result. When excess antibody is added to a reaction mixture, all of the
epitopes on the antigen are covered by antibody resulting in inhibition of lattice
formation. Excess antigen has a similar effect since there will not be sufficient
antibody to crosslink with antigen to form the lattice, and once again, precipitation
is inhibited.
The problem is, therefore, to determine the optimal concentration of both
antigen and antibody. This is accomplished by serial dilution of antigen and addition
of these dilutions to constant amounts of antibody. The amount of precipitation
that results can be measured visually or chemically, and the dilution of antigen that
gives 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, specific
amounts (mg) of antigen were added to a series of tubes, each containing a
constant amount of antibody. After an incubation period, the precipitate was
collected and assayed. The amounts of precipitate were plotted against the amount
of antigen added to produce the precipitation curve. To determine which tube
contains excess antigen or antibody, the supernatant fractions can be further
studied. To portions of each supernatant, more antigen or antibody could be added
and the tubes observed for additional precipitation. The tube that shows no
additional precipitation when either antigen or antibody is added is by definition
the equivalence point or equivalence zone of the precipitation curve.
Performance of the technique: In the experiment to follow, I will initially
perform the qualitative precipitin reaction, which is called the ring test, followed
by all groups performing the quantitative precipitin reaction. The ring test involves
overlaying the antiserum with a solution of the appropriate specific antigen. This
test will provide you with the information confirming that the antiserum contains
antibody specific for the antigen in question. By mutual diffusion, the reactants
ultimately establish a zone of optimal mutual proportions somewhere near the
interface between the two solutions, and a visible precipitate will form.
In the next exercise you will be asked to ascertain the equivalence point of
your antiserum-antigen solution using various ratios of the reactants in microtiter
wells, accompanied by the use of gel diffusion reactions to assay the various
supernatants that form (looking for excess antibody or antigen). Finally, once the
proper concentrations have been established, you will be asked to complete the
performance of the quantitative precipitin reaction.
You will be utilizing the Bio-Rad assay to determine the quantity of
precipitate in each of the tubes you have set up, so that you can report the amount

3. of antibody protein present in your undiluted antiserum. This reaction must be
performed with extreme care to detail to obtain accurate results.
Objectives:
Understand the biological basis of the precipitin reaction
Understand the purpose of each step of the precipitin reaction
Be able to define and understand OMP.
At OMP, [ppt] = [antigen] + [antibody]
Precipitation curve: how is it constructed? Equivalence zone? Excess antigen and
antibody zone?
Be able to compare and contrast the precipitin and the agglutination reactions
Materials:
15ml conical tubes, eppendorf tubes
Pasteur pipettes with rubber bulb
Antibody solution (anti-ovalbumin, αOA) diluted 1:4 using 1x PBS
Antigen solution (OA): 1mg/ml
Normal rabbit serum – 0.4ml
Diluting solution, 1x PBS
Microtiter plate (round bottom)
Immunodiffusion agar: 1% in PBS
Bio-Rad dye
Sterile Petri dish
Agar hole puncher with rubber bulb
Moist chamber
Microtiter plate viewer with mirror
Procedure
Day 1
A. The Ring test (I will demonstrate this to class)
Using a Pasteur pipette, introduce a small amount of antiserum directly into the
bottom of a clean glass tube, withdrawing the pipette slowly and carefully so as not
to leave any serum in the insides of the tube. Use one hand to insert or retrieve
the pipette and the other to steady the tip as it is being moved in or out of the
tube. Repeat the procedure in a separate clean glass tube using normal rabbit
serum instead of antiserum and use a different Pasteur pipette.
Next, carefully overlay an equal volume of antigen solution using a different
Pasteur pipette, so that it floats in the surface of the antiserum. You should see a

4. visible line of demarcation at the interface. This maneuver is most easily
performed by slanting the tube somewhat during the introduction of the antigen
solution and dispensing it slowly.
Observe the tubes for 20-25 minutes, watching for the development of
precipitation at the interface. If precipitation occurs, it will do so within this time
interval. Readings should not be taken after 30 minutes.
If the reaction is negative, the amount of antibody might be too small compared to
the amount of antigen. If this occurs, reduce the antigen concentration in one tube
to 0.05% and in another to 0.01%. If neither of these tubes shows visible
precipitation in the region of the interface, you may conclude that the antiserum
contains no antibodies appropriate to the antigen employed.
B. Optimal proportions by gel diffusion
1) Aliquot 120µl of OA into a clean, dry eppendorf tube and label accordingly. Set
aside for now.
2) Set up a 1:4 dilution (180µl of anti-OA serum + 540µl of PBS) in a clean, dry
eppendorf 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 not
previously 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 with
well #2, gently aspirate and expel with your micropipettor several times to mix the
contents of the well; then transfer 50µL from this well to the next well. (Minimize
bubble formation and change tips!!) Continue this process of two-fold serial
dilutions through well # 12, discarding 50 µL from the last well. Well #1 is
undiluted antigen (1mg/ml); well #2 is a 1:2 dilution of Ag (0.5mg/ml), well #3 is a
1:4 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 the
serum solution, touch the dispensing tip to the upper surface of the well above the
fluid level in the well. Well#1 of the next row will be used for your control. Add
50µl of PBS + 50µl of diluted αOA. Mix the contents of all wells by using a rotary
motion with the plate. Cover the plate and incubate it at 370C for 1 hr. After the
completion of incubation, transfer the microtiter plate to the refrigerator for 48
hrs.

5. Day 2:
1) After 2 days, centrifuge the microtiter plate for 10 minutes at 1200 rpm. In the
meantime, prepare 2 immunodiffusion plates by pipetting 10 ml of immunodiffusion
agar (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 45-60
minutes, remove the plates and very carefully (no jagged edges!) punch holes into
ONE of the plates according to the pattern seen in the handout (the second plate
is simply a backup in case you screw up the first one). You can place the diagram on
the 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 the
microtiter plate wells with the mirror viewer and note the well or wells that appear
to show the largest amount of precipitate. Be very careful not to shake or
otherwise disturb these precipitates.
3) Carefully remove about 7 µL of the supernatant from the well showing the
largest amount of precipitate from row A of your microtiter plate, and introduce
the supernatant into the 4th hole from the left in the middle row of your
immunodiffusion plate (see diagram on board). Do not overfill. If you have two
wells whose precipitates appear to be equally large, put the supernatant from the
highest antigen concentration in agar hole # 4 and the other one in hole # 5. Finish
dispensing supernatants from the microtiter plate A to the right and left of the
ones already completed, e.g. if well #7 was your best precipitate, put its
supernatant into hole #4, while supernatant from wells 4 to 6 will be put into the
holes to the left (1 to 3), and the supernatants from well 8-10 will be dispensed
into holes to the right (5-7). After all holes are filled dispense 1:4 antiserum into
each of the top row of holes and the OA antigen solution (1mg/ml) into each of the
bottom row of holes. Put your plate in the refrigerator and let incubate for 24
hours. Schedule a time to observe w/ TA for Thursday.
Day3:
The next day, observe your plates for lines of precipitate, and make drawings of
these results. Any line appearing between the middle and upper holes would
indicate excess antigen in the supernatant. Conversely, any precipitate forming
between the middle and bottom holes would signify excess antibody in the
supernatant. Any holes showing no precipitate in either top or bottom areas would
indicate no free antibody or antigen in the supernatant. Such a result designates
the area of optimal mutual proportions of the antiserum and antigen (the
equivalence point). At which dilution of Ag did you reach OMP?

6. 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’s
name and number. From the results obtained in the previous section, determine the
antibody/antigen concentration, which will give you optimal proportions. Prepare
serial dilutions of the antigen solution using 750µl as a final volume, arranged so
that the optimal tube will either be #2 or #3 in your row of tubes. After all
antigen tubes have been prepared, pipette 750µl of the 1:4 diluted antiserum into
each of the tubes. Mix the contents of each tube immediately after adding the
antiserum. 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 should
show this first and subsequently become the most turbid. The tubes on the left
(antigen excess) and the right (antibody excess) will show some cloudiness, but
should be noticeably less so than the center tubes. Incubate all tubes for 1 hour at
370C; 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). (This
should be a chart in your results section in your lab notebook) Transfer all the
tubes 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 then
carefully aspirate the supernatant from each tube showing a pellet of precipitate.
Be very careful not to lose any of the precipitate during aspiration. Resuspend the
pellet in 2ml of cold PBS with gentle mixing, then centrifuge and aspirate as before
and again resuspend in 2ml of cold PBS.
2) Perform the microtiter Bio-Rad assay on the contents of each tube. (Refer to
the Bio-Rad assay procedure).
3) Construct a standard curve by plotting total protein concentration versus the
antigen concentration for four tubes. In addition, you should be able to calculate
the 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.