Table Of ContentCHAPTER1
Production of Polyclonal Antisera
Jonathan A. Green and Margaret M. Munson
1. Introduction
All immunochemical procedures require a suitable antiserum or mono
clonal antibody raised against the antigen of interest. Polyclonal antibodies
are raised by injecting an immunogen into an animal and, after an appropri-
ate time, collecting the blood fraction containing the antibodies of interest.
In producing antibodies, several parameters must be considered with respect
to the final use to which the antibody will be put. These include (1) the speci-
ficity of the antibody, i.e., the ability to distinguish between different anti-
gens, (2) the avidity of the antibody, i.e., the strength of binding, and (3) the
titer of the antibody, which determines the optimal dilution of the antibody
in the assay system. A highly specific antibody with high avidity may be suit-
able for immunohistochemistry, where it is essential that the antibody re-
mains attached during the extensive washing procedures, but may be less
useful for immunoafbnity chromatography, as it may prove impossible to elute
the antigen from the column without extensive denaturation.
To produce an antiserum, the antigen for the first immunization is of-
ten prepared in an adjuvant (usually a water in oil emulsion containing heat-
killed bacteria), which allows it to be released slowly and to stimulate the
animal’s immune system. Subsequent injections of antigen are done with
incomplete adjuvant that does not contain the bacteria. The species used to
raise the antibodies depends on animal facilities, amount of antigen avail-
able, and the amount of antiserum required. Another consideration is the
phylogenetic relationship between antigen and immunized species. A highly
conserved mammalian protein may require an avian species in order to raise
From. Methods u-t Molecular B/ology, Vol 10. lmmunochemical Protocols
Ed. M. Manson 01992 The Humana Press, Inc , Totowa, NJ
1
2 Green and Manson
an antibody. Production of antibodies is still not an exact science and what
may work for one antigen may not work for another.
A simple, generally applicable protocol for raising polyclonal antiserum
to a purified protein of greater than 10,000 mol wt is described. This method
has been used to raise antibodies against a cytosolic protein, glutathione-S
transferase, and a membrane-bound glycosylated protein, gammaglutamyl
transpeptidase. The latter was first solubilized by cleavage from the mem-
brane with papain (‘1). Variations to this basic procedure are discussed in
Chapters 2, 3, and 5 of this vol. For proteins or peptides of low molecular
weight (~5-10 kDa) conjugation to a carrier protein is required for them to
elicit antigenicity (see this vol., Chapter 4). Variations on this basic technique
can be found in selected references (2-5).
2. Materials
1. Phosphate buffered saline (PBS), pH 7.4: 8 g of NaCl, 0.2 g of KH,PO,,
2.8 g of Na2HP0, lZH,O and 0.2 g of KC1 dissolved and made up to 1 L
in distilled water.
2. Antigen: Purified protein diluted to about 100 ug/mL in PBS.
3. Complete and incomplete Freund’s adjuvant.
4. Two glass luer lock syringes: 2 mL is the best size.
5. Three-way luer fitting plastic stopcock.
6. 19-g Needles, 0.‘7 mm x 22 mm Argyle medicut cannula.
7. Xylene.
8. Sterile glass universal tubes.
9. Up to four rabbits about 4-6 months old. Various strains can be used,
including half sandy lops or New Zealand whites (seeNote 1).
3. Method
1. Take up 1 mL of complete adjuvant in one of the syringes and 1 mL of
antigen solution containing approx 100 pg of the antigen in another.
Attach both to the plastic connector (Fig. l), making sure that the tap
on the connector is open in such a way that only the two ports connect-
ing the two syringes are open. Repeatedly push the mixture from syringe
to syringe until it becomes thick and creamy (at least 5-10 min) . Push all
the mixture into one syringe, disconnect this and attach it to a 19g needle
(see Notes 2 and 3).
2. Ensure that the rabbit to be injected is held firmly, but comfortably. For
the primary immunization, inject 500 uL deeply into each thigh muscle
and also inject 500 yL into each of two sites through the skin on the
shoulders.
Production of Polyclonal Antisera 3
Fig. 1. Preparation of emulsion for immunization. Two luer lock glass syringes
connected by a three-way plastic stqcock are used to form a stable emulsion of anti-
gen and adjuvant.
3. Repeat these injections biweekly for a further four weeks, but make the
emulsion with incomplete adjuvant.
4. Ten days after the last injection, test-bleed the rabbits from the marginal
ear vein. Hold the animal firmly and gently swab the rear marginal vein
with xylene to dilate the vein. Then cannulate the vein with an Argyle
medicut cannula and withdraw the needle, leaving the plastic cannula
in place. Draw blood out of the cannulawith a syringe until the required
amount has been collected. Transfer the collected blood into a sterile
glass universal container.
5. Remove the cannula and stem the blood flow by sustained pressure on
the puncture site with a tissue.
6. Allow the collected blood to clot by letting it stand at room temperature
for 2 h and then at 4°C overnight. Separate the serum from the blood by
detaching the clot carefully with a spatula from the walls of the con-
tainer and pouring the liquid into a centrifuge tube. Then centrifuge
the clot at 2500gfor SO min at 4°C and remove any expressed liquid.
Add this liquid to the clot-free liquid collected previously and centrifuge
the whole pooled liquid as described above. Finally, remove the serum
from the cell pellet with a Pasteur pipet (see Note 4).
7. At this stage, test the antiserum using an appropriate assay (see Note 5).
If the antibody has the requirements for the use to which it will be put,
up to three further bleeds on successive days may be performed. If the
4 Green and Manson
Fig. 2. Ouchtorlony double-diffusion technique. The antigen is placed in the center
well, cut in an agaroze gel, and different antisera in a range of dilutions are placed in
the surrounding wells. Antigen and antiserum diffuse toward each other and form a
white precipitin line where an antibody recognizes the antigen.
antiserum is unsatisfactory, i.e., the reaction is very weak, inject the rab-
bit again one month after the test bleed, and again test-bleed 10 days
after this injection.
8. Store antibodies in small, preferably sterile, aliquots at a minimum of
-2OOC. Repeated freezing and thawing should be avoided. For long-term
storage, aliquots may be freeze-dried and reconstituted when needed
(see Note 6).
4. Notes
1. The production of antibodies in animals must be carried out in strict
accordance with the legislation of the country concerned.
2. Emulsions containing antigens are just as immunogenic to humans as to
the experimental animal. Great care should be exercised during all the
procedures.
3. A stable emulsion has been produced when a drop of the preparation
does not disperse when placed on water.
4. Serum should be straw colored; a pink coloration shows that hemolysis
has taken place. This should not affect the performance of the antibod-
ies during most assay procedures.
5. This can be done by the Ouchterlony diffusion technique (seeFig. 2 and
ref. 6), by ELBA (see this vol., Chapters 29, 30, and 3.5), or by Western
blot, either using the purified protein or a more complex mixture of
Production of Polyclonal Antisera 5
proteins containing the antigen of interest separated on an SDS/PAGE
gel (see this vol., Chapters 24-28).
6. Some freeze-dried antisera are dimcult to reconstitute, or occasionally
may lose activity. Test a small sample before drying the whole batch. Any
cloudiness after reconstitution is denatured lipoprotein and can be clari-
fied by centrifugation and does not tiect antibody binding.
References
1. Cook N D and Peters T J (1985) Punficauon of ~lutamyl transferase by phenyl
boronate affinity chromatography Btochrm Brophys. Actu 828, 20.5-212.
2 Catty, D. and Raykundalia, C (1988) Production and quality control of polyclonal
anttboches, m Anttbodres wol 1-A A-actacul Approach (Catty, D , ed.), IRL, Oxford
3. Mayer, R J. and Walker, J 1-I (eds ) (198’7) I mmunochemrcal Methods zn Cell and Molecu-
lar Brology. Academic, London
4. Harlow, E. and Lane, D. (1988) Antrbodtes. A Lubvrutory Manual Cold Spring Harbor
Laboratory, New York
5. Langone, J J and Van Vunalus, I-I. (1983) Methods rn Enzymology, vol. 93. Academic,
New York.
6. Ouchterlony, 0 and Ntlsson, L A (1978) Immunodtffuslon and tmmunoelectro
phorests, in HandbookofExp~mentalImmunology, 3rd Ed. (Weir, D. H., ed ). Blackwell,
Oxford, UIS, pp 19 1-19 44
CHAETER2
Efficient Elution of Purified Proteins
from Polyvinylidene Difhoride
Membranes (Immobilon)
After Transfer from SDS-PAGE
and Their Use as Immunogens
Boguslaw Szewczyk and Donald E Summers
1. Introduction
The great analytical power of sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDSPAGE) makes it one of the most effective tools of pro
tein chemistry and molecular biology. In the past, there have been many
attempts to convert the technique from analytical to preparative scale be-
cause, by SDSPAGE, one can resolve more than one hundred protein spe-
cies in 5-6 h. The number of papers that describe preparative elution from
polyacrylamide gels is immense (for example, see refs. I-5). In spite of the
numerous variations in the procedure of elution, none of the available meth-
ods is entirely satisfactory. Some of the methods are very laborious, and oth-
ers lead to loss of resolu tion or poor recovery.
In general, the elution of proteins above 100 kDa from polyacrylamide
gels always presents considerable problems. Another of the serious limita-
tions of elution from gels is owing to the elastic nature of preparative polyac-
rylamide gels. The precise excision of a protein band from a complex mixture
is difficult and the slice may contain portions of other protein bands located
close to the band of interest. To overcome some of the limitations of elution
from gels, Parekh et al. (6) and Anderson (7) attempted to elute proteins
From Methods in Molecular Biology, Vol 10. lmmunochemrcel Protocols
Ed.. M. Manson 01992 The Humana Press, Inc., Totowa, NJ
7
8 Szewczyk and Summers
from nitrocellulose replicas of SDSPAGE gels. Binding of proteins to nitro-
cellulose is, however, so strong that the dissociating reagents (acetonitrile,
pyridine) partly or completely dissolve the membrane. When such prepara-
tions are used for immunization, they may cause adverse effects in animals.
We have found that when a polyacrylamide gel replica is made on Immobilon
membrane and not on nitrocellulose, then the conditions for elution are
much milder. Often, there is no need for concentration of the sample or for
the removal of elution agents prior to immunization. Furthermore, elution
from Immobilon is nearly independent of protein mol wt and recoveries of
70-90% are routinely obtained. We have also shown that, following elution
using the technique described herein plus the use of E. co11t hioredoxin to
catalyze protein renaturation, one can recover significant enzymatic activity
for some large complex enzymes such as E. coli RNA polymerase (8) and influ-
enza A virus RNA polymerase (9). Proteins are first separated by SDSPAGE,
and then are electroblotted to Immobilon membranes and stained with amido
black or Ponceau S. The protein bands of interest are excised and are then
eluted from the membrane with detergentcontaining buffers at pH 9.5.
2. Materials
1. Transfer buffer: Tris-glycine (25 mMTris/l92 mMglycine), pH 8.3.
2. Methanol.
3. Protein stains:
a. 0.01% Amido black in water.
b. 0.5% Ponceau S in 1% acetic acid.
4. Elu tion buffers:
a. 1% Triton X-100 in 50 mMTris-HCl, pH 9.5.
b. 1% Triton X-100/2% SDS in 50 mMTris-HCl, pH 9.5.
5. Immobilon (polyvinylidene fluoride) membrane from Millipore Corp.,
Bedford, MA.
6. Whatman 3MM filter paper.
7. Scotch Brite pads.
8. SDSPAGE apparatus.
9. Transfer apparatus (e.g., Trans Blot Cell from BioRad Laboratories,
Richmond, CA).
10. Glass vessels with flat bottom (e.g., Pyrex baking dishes)
11. Rocker platform.
12. Microfuge.
13. Small dissecting scissors.
Elution ofProteins from Immoblin 9
3. Method
1. Apply a mixture of proteins containing immunogen to be purified (see
Notes 1 and 2) to an SDSPAGE gel and run the gel (seevol. 1, Chapter 6
and this vol., Chapter 24).
2. Prepare transfer buffer (about 4 L for Bio-Rad Tram Blot Cell) and five
glass dishes, one of them large enough to accommodate the gel holder.
3. Pour methanol (around 50 mL) into one of the dishes, and 100-200 mL
of transfer buffer into the other dishes.
4. Place the gel holder and Scotch Brite pads in the biggest dish, and six
sheets of Whatman paper in another dish. The size of the Whatman
sheets should be slightly smaller than the size of the Scotch Brite pads.
5. Using gloves, cut some Immobilon to a size slightly bigger than the size
of the resolving gel. Put the sheet into a dish with methanol for 1 min
and then place it in one of the dishes containing transfer buffer.
6. Put a wetted Scotch Brite pad on one side of the gel holder and then
three sheets of Whatman paper saturated with transfer buffer on top of
the pad.
7. On completion of electrophoresis, carefully remove the upper stacking
gel because it may stick to the membrane.
a. Place the lower resolving gel on Whatman paper in the gel holder. Pour
a few milliliters of transfer buffer on top of the gel.
9. Place a sheet of Immobilon membrane on the gel. Roll over the mem-
brane with a glass rod to remove air bubbles from between the gel and
the membrane.
10. Next place three Whatman sheets prewetted with Tris-glycine buffer on
top of the Immobilon and finally a prewetted second Scotch Brite pad.
Close the holder.
11. Place the holder in the transfer tank bearing in mind that the mem-
brane should face the anode.
12. Begin electroblotting. Apply 20V for overnight runs. It is not necessary
to use methanol in the transfer buffer as it does not improve the binding
of proteins to this membrane. (see Note 3)
13. After transfer, stain the membrane with amido black solution for 20-30
min or with Ponceau S for 5 min (see Notes 4 and 5).
14. Destain the membrane with distilled water.
15. Excise the band(s) of interest with small dissecting scissors and place it
in an Eppendorf tube (see Note 6).
10 Szewczyk and Summers
16. Add 0.2-0.5 mL of elution buffer/cm* of Immobilon strip. Two buffers
that we used are:
a. 1% Triton X-100 in 50 mMTris-HCI, pH 9.5.
b. 2% SDS/l% Triton in 50 mMTris-HCI, pH 9.5.
The first buffer is less effective (50-75% of total protein eluted) than the
second one, but the eluted protein can be injected into animals without
the necessity of Triton X-100 removal. On the other hand, the 2% SDS/
1% Triton X-100 mixture leads to the complete elution of bound pro
tein from Immobilon, but SDS has to be removed before injections (see
Notes 7-9).
17. Mix well by vortexing the Immobilon in eluant for 10 min. Spin down (5
min) the Immobilon. Use the supernatant directly for injections (elu-
tion with Triton X-100 only) or after protein precipitation with acetone
(if SDS and Triton were included in the elution buffer). Protein precipi-
tation is carried out in a dry ice bath. Add 4~01. of cold acetone to 1 vol.
of protein solution. After 2 h at -20°C, pellet the protein, solubilize, and
inject into animals by standard procedures (see Note 10 and this vol.,
Chapters 1, 3,5, and 6).
4. Notes
1. The method was used to obtain a variety of sera against bacterial, viral,
and eukaryotic proteins. The amount of immunogen needed to stimu-
late high levels of antibodies varies for different proteins, but generally,
50-500 pg of protein is sufficient to induce the formation of high levels
of specific antibodies.
2. The Immobilon matrix should not be overloaded with protein to pre-
vent its deep penetration into the membrane. The protein band excised
from a single electrophoretic lane (about 1 cm in length) should not
contain more than 10-20 ltg of protein.
3. Transfer of proteins from the gel to Immobilon should not be done at
elevated temperatures (above 30°C), as the force of protein binding to
Immobilon apparently increases with temperature. Therefore, it is ad-
visable to make transfers in a cold room at 4°C or use precooled transfer
buffer.
4. Depending on the supplier and batch of amido black, the sensitivity of
protein detection with this reagent may vary. If the sensitivity of staining
is not satisfactory, it is advisable to dilute the amid0 black solution 5-10
times with water rather than to increase its concentration.
5. Staining with Ponceau is done in 1% acetic acid. This may lead to partial
denaturation of proteins bound to Immobilon. In this case, 2% SDS/
