Fragments of the article:
Removal of anti-carrier (KLH) antibodies from serum using HiTrap
NHS-activated SepharoseTM or cyanobromide activated Sepharose 4B
Joanna Porankiewicz-Asplund and Berit Nilsson
AgriSera AB, Vännäs,Sweden
Anna-Stina Höglund and Lars-Göran Josefsson
Swedish University of Agricultural Sciences, Uppsala, Sweden
Published in Life Science News. To read whole article,
please go to:
http://www.apbiotech.com/lsn_home/lsn_index.html
LSN (life Science News) Archive
Issue 5, year 2000.
Anti-KLH antibodies may cross-react with antigens present in stained
tissues. Anti-peptide antibody production is of the advantage when the
antigen (protein) of interest is difficult or expensive to isolate in
sufficient quantities, or when the single epitope of the protein has to be
detected. Since peptide molecule itself is not immunogenic, it must be
chemically coupled with a larger molecule for successful immunisation.
Immunised animal will elicit antibody response to both, coupled peptide
and a carrier molecule. Carrier-specific antibodies can correspond to a
significant fraction of an antibody pool (10% and more). In plant species
Adonis aestivalis specific staining pattern has been observed, as a result
of cross-reaction between anti-KLH antibdies and a plant antigen.
Additionally, other plant species has been tested with a similar result
(data not shown). This problem can be overcome by removal of anti-KLH
antibodies by affinity column or raising anti-peptide antibodies using
other carrier protein. %). However, possibility of cross-reactivity should
be seriously considered when planning immunohistochemical localisation of
plant proteins using anti-peptide sera raised with KLH as a carrier.
Preparation of the affinity column
KLH (8 mg) has been dissolved in the
coupling buffer (200 mM sodium carbonate, 500 mM sodium chloride, pH 8.3)
and applied on the two 1 ml HiTrap NHS-activated columns connected
together. NHS-activated Sepharose TM High Performance packed in the
column, is designed for covalent coupling of ligands containing primary
amino groups. Coupling of KLH protein has been performed at room
temperature for 1 hour with calculated efficiency around 90 %. After
deactivation of the excess amino groups, which did not couple the ligand,
column is ready for use. Coupling of KLH to cyanobromide activated
Sepharose 4B has been done following Amersham Pharmacia recommendations.
Removing of the anti-KLH antibodies from anti-peptide serum in a single
purification step
HiTrap column can be conveniently used with syringe or connected to the
chromatography system. Serum is diluted at least 1:1 with PBS pH 7.4 and
recirculated in the system for few hours or over night to allow efficient
binding of KLH-specific antibodies. Flow through is collected. Specific,
anti-KLH antibodies are eluted using 100 mM glycine pH 2.5. The column is
reequilibrated and ready for the next run.
This type of affinity purification will remove majority of specific
anti-carrier antibodies, as well as the antibodies which bind part of the
peptide and the carrier protein.
As a control anti-KLH antibodies can be used
to detect if the signal is not coming from antibodies directed against carrier protein - KLH.