Technique / Immunology / Antibody

epitope mapping (4)

Production of monoclonal antibodies.
(Curr Protoc Immunol. 2006 Sep;Chapter 2:Unit 2.5.)
Yokoyama WM, Christensen M, Santos GD, Miller D.

Washington University School of Medicine, St. Louis, Missouri, USA.

This unit describes the production of monoclonal antibodies beginning with
immunization and cell fusion and selection. Support protocols are provided for
screening primary hybridoma supernatants for antibodies of desired specificity,
establishment of stable hybridoma lines, cloning of these B cell lines by
limiting dilution to obtain monoclonal lines, and preparation of
cloning/expansion medium. An alternate protocol describes cell fusion and
one-step selection and cloning of hybridomas utilizing a semi-solid
methylcellulose-based medium (ClonaCell-HY).

Antibody protocols from Nature Protocols.
(Nat Protoc.)
Nat Protoc. 2007;2(10):2574-81.

Production of a site- and phosphorylation state-specific antibody.

Goto H, Inagaki M.

Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi
464-8681, Japan.

Protein phosphorylation plays important roles in various aspects of cellular
events. Visualization of site-specific phosphorylation in cells is of great
importance not only to analyze spatial and temporal distribution but also to
investigate biological function. Now, site- and phosphorylation state-specific
antibodies are widely utilized as the most powerful tools for these analyses.
This protocol details a method to produce the polyclonal version of such an
antibody by immunizing a synthetic phosphopeptide corresponding to a protein
phosphorylated at targeted site(s). This protocol is also applicable to the
production of other types of antibodies, which specifically recognize the
site-specific modification, such as acetylation, methylation and proteolysis. The
protocol can be completed in 2-3 months.

2: Nat Protoc. 2007;2(7):1763-9.

Purification of antibodies using the synthetic affinity ligand absorbent
MAbsorbent A2P.

Chhatre S, Titchener-Hooker NJ, Newcombe AR, Keshavarz-Moore E.

Department of Biochemical Engineering, University College London, Torrington
Place, London WC1E 7JE, UK.

A protocol for the purification of polyclonal antibodies from ovine serum using
the synthetic protein A absorbent MAbsorbent A2P is described. Clarified serum is
loaded directly onto the affinity column without prior adjustment and albumin and
unwanted serum components are washed from the column using a sodium octanoate
buffer before elution of bound antibodies. MAbsorbent A2P was shown to bind
approximately 27 mg ml(-1) of polyclonal immunoglobulin under overloading
conditions, with eluted IgG purities of >90% and minor levels of albumin
(approximately 1%). The anticipated time required to complete the purification
protocol is 6-7 h. Although the protocol is similar to methods utilized for
antibody purification using chromatography with protein A derived from the cell
wall of the microorganism Staphylococcus aureus or protein G from Streptococcus
as the affinity ligands, affinity absorbents based on synthetic ligands offer a
number of advantages to compounds derived from biological sources, in particular
robustness, relatively low cost, ease of sanitization and, in principle, lack of
biological contamination.

Antibody technique books
(Biowww bookshelf)
Antibody method and protocol books

Figure. Preparation of hybridomas that secrete monoclonal antibodies against a particular antigen
(Molecular Biology of the Cell)
III. Methods 8. Manipulating Proteins, DNA, and RNA Isolating Cells and Growing Them in Culture

Antibody-antigen interactions
(Molecular Biology of the Cell)
V. Cells in Their Social Context 24. The Adaptive Immune System B Cells and Antibodies

Figure. Procedure for producing a monoclonal antibody
(Molecular Cell Biology)
6. Manipulating Cells and Viruses in Culture 6.2. Growth of Animal Cells in Culture

Storage of Antibodies
best way of store antibodies for Westerns

Hybridoma stability
fusions with sp2/0 to make a hybridoma

antibody generation from rat
not got any response after the second boost in rats

Method to freeze hybridoma
hybridomas from transgenic mouse spleen

Raising Antibody problems
problem in raising antibody against 35Kd recombinant protein in Rabbit

Hybridoma production troubleshooting
mouse spleen and sp2 cell fusion problem

preabsorbing chicken antibodies
chicken primary antibody that preabsorbed against it's antigenic peptide


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