Monoclonal Antibody Core Facility
Department of Neuroscience
Antigen Requirements
Successful hybridoma production depends on several factors that need to be adequately
addressed before a monoclonal project is initiated. The most important factors are the purity,
quantity and immonogenicity of the antigen used for immunization and hybridoma cells
screening.
1.
The antigen used in the immunization of the mice should not contain any detergents or
reducing agents and should be as pure as possible.
We cannot accept immunogens in buffers that have any of following conditions (for
immunization):
Contain any metal dyes
Contain KCl, NaCl, or MgCl2 at 1.0M
5% SDS
6M Urea
1% b-octylglucoside, a detergent
1% Triton 100, a detergent
1% Tween 20, a detergent
 20% Glycerol
0.1mM PMSF (Protease Enzyme inhibitor)
1ug/ml Leupeptin (Protease Enzyme inhibitor)
1ug/ml Pepstatin A (Protease Enzyme inhibitor)
0.1mM DIPF (Protease Enzyme inhibitor)
0.3M DTT (reducing agent)
3M Imidizole
2. About 6 mg of soluble antigen will be needed for immunization and the screening assays.
We can adjust project strategies if the antigen is difficult to isolate or not readily available.
3.
Immunogens must be stable and able to withstand specified storage condition for 6-12
months.
4. It is necessary to screen the clones on pure material, so if pure material is limited, crude
material can be used for the immunizations.
5. If peptide immunogens are used, it is important to select the peptide after considering the
following predictors of antigenicity: hydrophilicity, amphipathicity, surface accessibility,
and chain termination and sequence variability. The core can provide limited assistance into
the selection of peptides based on these predictors. It is preferable to add Cys at the NH2
terminus if the peptide is internal or it represents the very C-Terminus. For peptides
representing the very NH2 sequences, Cys should be added at the C-terminus of the peptide.
6. Molecules less than 8 kD are generally not good immunogens without further modification.
Haptens, peptides, and weak or nonimmunogenic antigens may require conjugation to
carried protein. The Core can do the conjugation of peptides to carrier proteins in order to
Standard Operating Procedure: Antigen Requirement
Monoclonal Antibody Core Facility
Department of Neuroscience
enhance their immunogenicity.
7. Protein tags are allowable. However, some untagged protein or with an alternate motif
must also be available.
Production of Monoclonal Antibodies from a Hybridoma Cell Line
Our Core Facility offers three methods for monoclonal antibody production from hybridoma cell
lines, including two in vitro methods (tissue culture and the integra system) and one in vivo
method (acites production). In the last few years, there has been a move in the scientific
community to seek alternative methods to avoid or minimize the use of animals in research.
Therefore, the majority of hybridoma cell lines will be grown in vitro, and the specific method
will depend on the amount, concentration, and purity of antibody required. The Core Facility will
resort to in vivo techniques only if other methods fail.
Tissue-Culture Method
The simplest approach for producing mAb in vitro is to grow the hybridoma cultures in batches
and purify the mAb from the culture medium. Fetal bovine serum is used in most tissue-culture
media and contains bovine immunoglobulin at about 50 ug/ml. The use of such serum in
hybridoma culture medium can account for a substantial fraction of the immunoglobulins present
in the culture fluids. To avoid contamination with bovine immunoglobulin, we will use Gibco
Hybridoma Serum Free Media, which is specifically formulated to support the growth of
hybridoma cell lines. In most cases, hybridomas growing in 10% fetal bovine serum (FBS) can
be adapted within four passages (8–12 days) to grow in less than 1% FBS or in FBS-free media.
However, this adaptation can take much longer, and in 3–5% of the cases the hybridoma will
never adapt to the low FBS media. After this adaptation, cell cultures are allowed to incubate in
commonly used tissue-culture flasks under standard growth conditions for about 10 days; mAb is
then harvested from the medium.
Integra System Method
This is a novel multi-chamber cell cultivation system based on membrane technology. This
system is easy to use and supports confluent cell densities which are ideal for high yield
monoclonal antibody production and recombinant protein expression. We use Integra CL350
flasks, which hold approximately 20 ml of supernatant yielding 10-30 mg of antibody.
Note: Hybridoma cell lines differ in their growth characteristics, such as doubling time,
maximum cell density, etc. For maximal yield, time must be taken to optimize growth conditions
for each specific cell-line.
Ascites Production
Clones are grown and then injected into mice for ascites production. Cells are injected into a
pristane primed mouse. Ascites fluid generally develops within 10 to 14 days and is harvested by
gentle aspiration of the abdominal cavity. Each mouse is tapped up to 2 times. The amount of
ascites production and the presence of specific antibody in ascites are variable. Antibody
concentrations will typically range between 1 and 10 mg/ml.
Standard Operating Procedure: Antigen Requirement
Monoclonal Antibody Core Facility
Department of Neuroscience
Note: In vitro technologies are preferred for production of monoclonal antibodies and is
explicitly recommended by the National Institutes of Health.
Choosing an Immunoassay
When choosing an immunoassay, one must consider the following:
1. the type of antibody (monoclonal or polyclonal), number of different antibodies and
amount of antibodies available;
2. the purity and amount of the available antigen;
3. the nature of the sample (media, plasma, tissue extract);
4. the number of anticipated samples (tens, hundreds, or thousands);
5. special equipment required for the assay (washers, spectrophotometers, gamma counters,
etc.).
Solid-phase ELISA
Our standard solid-phase ELISA uses antigen-coated microtiter plates, a secondary antibody (for
example, goat anti-mouse IgG conjugated with horseradish peroxidase), and a chromogenic
substrate. The output signal (absorbance of the chromogenic substrate following oxidation)
depends on the formation of primary antibody-antigen complexes. Under certain conditions, the
assay can be linear with respect to antibody concentration, and therefore useful for comparing
the relative concentrations of antibodies in different samples. One factor to consider with this
assay is that an antigen bound to a surface might react differently than an antigen in solution.
Some epitopes are highly dependent on the solution versus the surface properties of the antigen.
This is particularly true for small linear peptides.
Antigen Capture ELISA
This solid-phase assay uses antibody-coated microtiter plates (with antibody either directly
bound or bound via another protein (i.e., protein A)). We first capture the antigen from solution
and then detect antigen-antibody complexes using either biotin-labeled antigen with horseradish
peroxidase-conjugated streptavidin, or a second antibody positioned at a site distinct from the
first antibody. As with any solid-phase assay, critical parameters include the type of plate, the
method of blocking nonspecific protein binding sites on the plate, the relative concentration of
assay components, and the number of washes and composition of the wash buffer. We generally
use biotinylated-antigen in this format.
Competitive ELISA for antigen in solution
This assay measures the amount of free antibody remaining in an equilibrium mixture of
antibody and antigen in solution.
Immunoblotting (Western Blot)
Standard Operating Procedure: Antigen Requirement
Monoclonal Antibody Core Facility
Department of Neuroscience
Antibodies can recognize both linear epitopes and nonlinear epitopes determined by the
conformation of the antigen. For this reason, immunoblotting assays often detect a
different set of antigenic determinants than other immunoassays, and antibodies or antigens
screened in this method can behave differently from other assay methods. This assay is
useful for detecting antigens in complex mixtures and for estimating molecular weight.
RadioImmune Assay (RIA)
The RIA is simple in principle. The concentration of the unknown, unlabeled antigen is
obtained by comparing its inhibitory effect on the binding of radioactively labeled antigen
to a specific antibody with the inhibitory effect of known standards. RIA is an in vitro test,
i.e., the ingredients, which are labeled antigen, specific antibody, and standards or
unknowns, are incubated together in test tubes. At the end of 1-2 hours of incubation, the
antibody-bound and free fractions of radioactive antigen are separated, the radioactiveity in
each fraction is determined, and a calibration curve is drawn from the data on standards.
The concentration of unknown sample is then determined from the calibration curve.
Peptide Selection and Design
The first step in the process is the selection of the appropriate peptide sequence. At this step, the
ultimate use for the antibody must be considered. If the antibody is needed to probe a specific
protein domain then the choice is simple. For example, if one is studying proteolytic processing
of an N-terminal precursor, antibodies against the N-terminal region of interest would be raised.
Likewise, if the goal is to monitor the phosphorylation state of a specific sequence, antibodies to
the phosphorylated sequence can be used.
If the goal is to raise antibodies that will recognize the protein in its native state, the problem
becomes more complex. Anti-peptide antibodies will always recognize the peptide. However, the
same antibody may not recognize the sequence within the folded intact protein. Sequence
epitopes in proteins generally consist of 6-12 amino acids and can be classified as continuous
and discontinuous. Continuous epitopes are composed of a contiguous sequence of amino acids
in a protein. Anti-peptide antibodies will bind to these types of epitopes in the native protein
provided the sequence is not buried in the interior of the protein. Discontinuous epitopes consist
of a group of amino acids that are not contiguous but are brought together by folding of the
peptide chain or by the juxtaposition of two separate polypeptide chains. Anti-peptide antibodies
may or may not recognize this class of epitope depending on whether the peptide used for
antiserum generation has secondary structure similar to the epitope and/or if the protein epitope
has enough continuous sequence for the antibody to bind with a lower affinity.
When examining a protein sequence for potential antigenic epitopes, it is important to choose
sequences which are hydrophilic, surface-oriented, and flexible1. The peptide sequence must be
selected from an accessible region of the protein. The trans-membrane region of a protein is not
usually exposed and should thus be avoided. Similarly, any region that undergoes
posttranslational modification (e.g. glycosylation), should also be avoided. Additionally, it has
been shown that epitopes have a high degree of mobility2. Because the C-termini of proteins are
Standard Operating Procedure: Antigen Requirement
Monoclonal Antibody Core Facility
Department of Neuroscience
often exposed and have a high degree of flexibility, they are usually a good choice for generating
anti-peptide antibodies directed against the intact protein. If the protein is an integral membrane
protein and the C-terminus is part of the transmembrane segment, this sequence will be too
hydrophobic and not a good choice. The N-terminus is also frequently exposed and on the
surface of the protein making it an ideal candidate for antibody generation. Peptide amides and
N-terminally acetylated peptides are straightforward to prepare but must be made during
synthesis, not after. Other concerns arise when posttranslational modifications are suspected.
Unless these issues are the topic of the investigation, initial attempts at antibody preparation
should avoid regions rich with probable disulfide bonds or modified residues. Algorithms can be
effective at predicting protein characteristics such as hydrophilicity/hydrophobicity, secondary
structure (i.e. alpha-helix, beta-sheet and beta-turn) and exposed, immunogenic internal
sequences. Hydrophilicity plots as described by Hopp and Woods3 assign an average
hydrophilicity value for each residue in the sequence. The highest point of average hydrophilicity
for a series of contiguous residues is usually at or near an antigenic determinant. A slightly
different algorithm described by Kyte and Doolittle4 evaluates the hydrophilic and hydrophobic
tendencies of the sequence. This profile is useful for predicting exterior vs. interior regions of the
native protein. Secondary structure can be identified by the use of algorithms developed by Chou
and Fasman5or Lim6. Surface regions or regions of high accessibility often border helical or
extended secondary structure regions. In addition, sequence regions with beta-turn or
amphipathic helix character have been found to be antigenic7. Many commercial software
packages such as MacVectorTM, DNAStarTM, and PC-GeneTM incorporate these algorithms.
To be successful, none of the algorithms should be used alone. Combined use of the predictive
methods may result in a success rate as high as 86% in predicting antigenic determinants7.8.
Once the protein region of interest has been identified, the length of the peptide must be selected.
There are two differing schools of thought relating to peptide length. One suggests that long
peptides (20-40 amino acids in length) are optimal because this increases the number of possible
epitopes. The other suggests that smaller peptides are sufficient and their use ensures that the
site-specific character of anti-peptide antibodies is retained. Clearly, any peptide selected must
be chemically synthesizable and should be soluble in aqueous buffer for conjugation to the
carrier protein. Peptides longer than 20 residues in length are often more difficult to synthesize
with high purity because there is greater potential for side reactions, and they are likely to
contain deletion sequences. On the other hand, short peptides (<10 amino acids) may generate
antibodies that are so specific in their recognition that they cannot recognize the native protein or
do so with low affinity. The typical length for generating anti-peptide antibodies is in the range
of 10-20 residues. Peptide sequences of this length minimize synthesis problems, are reasonably
soluble in aqueous solution and may have some degree of secondary structure.
References
1. Van Regenmortel, M.H.V., 1986, Trends in Biochemistry, 11:36-39.
2. Westof, E., 1984, Nature, 411: 123-12
3. Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A., 78: 3824-382
4. Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol., 157: 105-132.
5. Chou, P.Y. and Fasman, G.D., 1974, Biochemistry, 13: 222-245.
6. Lim, V.I., d1974, J. Mol. Biol., 88: 873-894.
Standard Operating Procedure: Antigen Requirement
Monoclonal Antibody Core Facility
Department of Neuroscience
7. Parker, J.M.R. and Hodges, R.S., 1991, Peptide Res., 4: 347-354.
8. Parker, J.M.R. and Hodges, R.S., 1991, Peptide Res., 4:355-363.
Peptide Conjugation Guidelines
There are 3 factors to consider when determining peptide conjugation:
1. Carrier Protein:
Attachment of the peptide (antigen) to a carrier protein is a key factor in eliciting an
immune response. Many different carrier proteins can be used for coupling synthetic peptides,
and are chosen based on immunogenicity, solubility and availability of useful functional group.
The two most commonly used carriers are keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA). The higher immunogenicity of KLH often makes it the preferred choice.
Another advantage of choosing KLH over BSA is that BSA is used as a blocking agent in many
experimental assays. Because antiserum raised against peptides conjugated to BSA will also
contain antibodies to BSA, false positive may result. Thyroglobulin is very antigenic too. There
is a background consideration in vertebrates. Glutaraldehyde is a bifunctional coupling reagent
that links two compounds through their amino groups. Glutaraldehyde provides a highly flexible
spacer between the peptide and carrier protein for favorable presentation to the immune system.
Unfortunately, glutaraldehyde is a very reactive compound and will react with Cys, Tyr and His
to a limited extent. The result is a poorly defined conjugate. The glutaraldehyde method is
particularly useful when a peptide contains only a single free amino group at its amino terminus.
If the peptide contains more than one free amino group, large multimeric complexes can be
formed, which are not well defined, but are highly immunogenic.
2. Location of peptide segment within the native protein:
If the peptide segment is located at the N-terminal region of the native protein,
conjugation should be done at the C-terminus of the peptide segment. If the peptide segment is
located at the C-terminal region of the native protein, conjugation should be done at the Nterminus of the peptide segment. This will present the peptide (antigen) in a similar state as the
native protein.
NOTE: if the peptide segment is located internally of the native protein, conjugation can be done
at either end of the peptide segment.
3. Conjugation Chemistry:
a. EDC: peptide attachment to a carrier protein via the carboxyl groups within the peptide
sequence (Aspartic Acid, Glutamic Acid, and C-terminal carboxyl group)
b. Activated EDC: peptide attachment to a carrier protein via the amino groups within the
peptide sequence (Lysine and N-terminal amino group)
c. MBS: peptide attachment to a carrier protein via the thiol group of a cysteine residue
within the peptide sequence.
Standard Operating Procedure: Antigen Requirement
Monoclonal Antibody Core Facility
Department of Neuroscience
Plasmid Preparation for Mouse Immunization
In order to be able to carry out successfully the antibody production using intro-muscular
injection, the following purification steps must be followed:
Reagent List:
Use LB media and not “Terrific Broth”;
Use recommended bacterial strains (e.g. DH5);
Do not overload the Qiagen column (e.g. the material from 500 ml of LB grown culture
goes onto one Mega column);
Use the buffers provided with the Qiagen kit;
1 x PBS: sterile and endoxin free;
100 X TE: sterile and endotooxin free.
Procedure:
1. DNA is eluted from a Qiagen column (the procedure is carried out precisely following
the Qiagen protocol and using Qiagen buffers). After iospropanol precipitation, ethanol washing
and vacuum drying, the DNA is dissolved with 1X endotoxin free TE (Sigma). In order to
maximize recovery of precipitated plasmid DNA, leave tube on bench for 24 hours.
2. Add NaCl to the DNA-TE solution to a final concentration of 0.1 M.
3. Then add 2 volumes of 100% ethanol. Precipitate at -20C for 30 minutes and recover
the pellet by centrifugation.
4. Wash once with 70% ethanol in water (no EDTA) and recover by centrifugation.
5. Dry the pellet and resuspend in 1x sterile PBS. From the size of the Qiagen column
used, one can estimate the amount of DNA to be recovered. Add the PBS to obtain a solution of
about 1.5 to 2 mg/ml.
At this point, it is best to leave DNA-PBS overnight in a 15 ml round bottom tube to
increase contact between the dried DNA and PBS. If necessary, DNA-PBS can be warmed to
37ºC to speed up to dissolution.
6. Read OD 260 and 280; the 260/280 ratio should be greater than 1.7 for good DNA
preparation. Dilute the DNA to 1 mg/ml.
7. The DNA is ready for injection.
Standard Operating Procedure: Antigen Requirement