Download keyhole limpet haemocyanin, KLH

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Removal of cross-reactive anti-carrier (keyhole limpet
haemocyanin, KLH) antibodies from rabbit serum
using immobilized KLH
J. Porankiewicz-Asplund*, B. Nilsson*, A-S. Höglund† and L-G. Josefsson†
*AgriSera AB, Vännäs, Sweden; †Dept of Plant Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden
*[email protected]; †[email protected], [email protected]
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Fig 1. Immunohistochemical
staining of xylem part of
vascular tissues of Adonis
aestivalis. A. Tissue section
stained with rabbit preimmune serum. No staining
observed. B. Tissue section
stained with serum from
an immunized rabbit containing antibodies against
both KLH and coupled
peptide. The specific staining shown is due to the
anti-KLH portion of antibodies and is not characteristic for the specific
anti-peptide antibodies, as
evident from these and
other data (not shown).
C. Staining using serum
(flowthrough) passed over a
KLH-coupled affinity column
and thus depleted of antiKLH antibodies. The staining seen in panel B was
not present. Thus, passing
the rabbit immune serum
through the affinity column
removed in an efficient way
the cross-reacting anti-KLH
antibodies. Staining of plant
tissues with affinity purified
anti-KLH antibodies resulted
in the same staining pattern
observed in panel B (data
not shown), which confirms
the specificity of the reaction. All serum samples
were diluted 1:250 in PBS,
pH 7.4 before staining.
Polyclonal antibody production against peptides
conjugated to keyhole limpet haemocyanin (KLH) will
result in antibody production against both KLH and the
peptide. Presence of anti-KLH antibodies in serum may
cause non-specific background in immunohistochemical
staining of tissues from some plant species. Passing
serum through HiTrap™ NHS-activated column or a
column with CNBr-activated Sepharose™ 4B coupled
with KLH, will efficiently remove anti-KLH antibodies
in a single purification step. Thus it is a flexible and
convenient way of processing serum irrespective of the
peptide specificities. It is also an attractive alternative to
conventional purification of specific antibodies on
peptide affinity columns, at least for serum where the
titres of specific antibodies are high.
Introduction
Anti-peptide antibody production is an advantage
when the antigen (protein) of interest is difficult or
expensive to isolate in sufficient quantities, or when a
single epitope of the protein has to be detected. Since a
peptide molecule in itself is not immunogenic, it must
be chemically coupled to a larger molecule (carrier) for
successful immunization. Immunized animals will
elicit antibody responses, both to the coupled peptide
and to the carrier molecule. A common carrier protein
of high molecular weight and antigenicity is keyhole
limpet haemocyanin (KLH). Carrier-specific antibodies often correspond to a significant fraction of the
total antibody pool (10% or more) in the serum of the
immunized animal. Presence of anti-KLH antibodies
in serum may cause non-specific background, for
instance in immunohistochemical staining.
In the plant species, Adonis aestivalis, a specific and
distinct staining pattern has been observed that is the
result of an immunological cross-reaction between
anti-KLH antibodies and an as yet unidentified plant
antigen (Fig 1). The cross-reactive antigen is most
prominent in the xylem part of vascular tissues. Different parts of the plant as well as other plant species
have been tested with similar results (data not shown).
Life Science News 5, 2000 Amersham Biosciences
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Therefore, the possibility of cross-reactivity must be
seriously considered when planning immunohistochemical localization of plant proteins using antipeptide serum raised with KLH as a carrier.
S
Columns:
Sample:
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HiTrap NHS-activated coupled with KLH,
2 × 1 ml columns in series
10 ml of serum from a rabbit immunized
with a peptide-KLH conjugate
Sample:
pretreatment: Dilution of the serum 1:1 with PBS, pH 7.4
Binding buffer: 10 mM sodium phosphate buffer,
150 mM NaCI, pH 7.4
Elution buffer: 100 mM glycine, pH 2.5
Flow rate:
1 ml/min
System:
GradiFrac
However, the problem may easily be overcome by
removing anti-KLH antibodies from the immune serum
by passing the serum through an affinity column
coupled with KLH, for example a prepacked HiTrap
NHS-activated column or a column packed with CNBractivated Sepharose 4B. As an alternative one may
want to consider raising the anti-peptide antibodies
using another carrier protein.
A280 nm
anti-KLH IgG
2.00
Preparation of a KLH affinity column
KLH (8 mg) was dissolved in the coupling buffer
(200 mM sodium carbonate, 500 mM sodium
chloride, pH 8.3) and applied to two HiTrap
NHS-activated 1 ml columns connected in series.
The columns were prepacked with NHS-activated
Sepharose High Performance, which is designed for
covalent coupling of ligands containing primary amino
groups. Coupling of KLH protein was performed at room
temperature for 1 h with a calculated efficiency of approximately 90%. After deactivation of the excess amino
groups and extensive washing, the columns were ready
for use. KLH was also coupled to cyanogen bromide
(CNBr)-activated Sepharose 4B, following manufacturer’s
recommendations (Amersham Biosciences).
1.00
0
13
10
Time (h)
Fig 2. Removal of anti-KLH antibodies from immunized rabbit serum
on HiTrap NHS-activated affinity column coupled with KLH.
To confirm the removal of anti-KLH antibodies from
the immunized rabbit serum, an ELISA was
performed. Results indicated that the majority of the
anti-KLH antibodies were removed from the serum
after affinity purification (data not shown). It should
be considered that in this type of affinity purification,
not only specific anti-carrier antibodies will be
removed, but a pool of anti-peptide antibodies that
have an epitope composed of amino acids belonging to
both the peptide and the carrier protein will also be
removed.
Removal of anti-KLH antibodies from rabbit serum
in a single purification step
HiTrap columns can be used with a syringe or connected to a chromatography system. GradiFrac™
system (Amersham Biosciences) was used for
this purification. The anti-peptide serum was diluted
1:1 with PBS pH 7.4 before application on the KLHcoupled affinity column. The serum was recirculated
through the column for 2 h, or overnight, to allow
efficient binding of the KLH-specific antibodies to the
column. The flow-through fraction was collected
while specific, anti-KLH antibodies were eluted using
100 mM glycine pH 2.5, directly into 1 M Tris, pH 9.0
(Fig 2). After re-equilibration of the column with 20 ml
PBS, pH 7.4 it was ready for the next run.
ORDERING INFORMATION
HiTrap NHS-activated, 1 ml
5 × 1 ml
17-0716-01
HiTrap NHS-activated, 5 ml
1 × 5 ml
17-0717-01
NHS-activated Sepharose 4
Fast Flow
25 ml
17-0906-01
CNBr-activated Sepharose 4
Fast Flow
10 g
17-0981-01
CNBr-activated Sepharose 4B
Lab Pack
15 g
17-0430-01
Life Science News 5, 2000 Amersham Biosciences
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