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The didecamer consists of 20 subunits and occurs
in two different forms named KLH1 and KLH2. The
subunits are biochemically distinct. Analysis of
KLH by native-PAGE gives two characteristic
bands, one corresponding to KLH1 with an
apparent molecular mass of about 390 kDa (kilo
Dalton) and the other corresponding to KLH2 with
an apparent molecular mass of 350 kDa. There are
eight functional units in the KLH subunits, KLH1
and KLH2. The molecular mass of the functional
units is about 50 kDa. Based on this information
it is expected that the molecular masses of the
KLH1 and KLH2 subunits are approximately 400 kDa.
The functional unit (FU) within the subunit
contains a binuclear copper binding site that
binds molecular oxygen. The copper is in the
cuprous [Cu(I)] state, and oxygen binding
generates light absorbance in the near
ultraviolet around 340 nm and imparts the
characteristic blue color to the molecule. The
deoxygenated molecule is colorless.
The peptide sequence surrounding the two
copper-binding sites is highly conserved, with
three copper-liganding histidines in both cases.
The active site geometry and molecular
architecture of the mollusk hemocyanin differ
from those of arthropod hemocyanin. Removal of
second copper from mollusk hemocyanin by cyanide
ions is both slower and more difficult compared
to arthropod hemocyanin, where all the copper
come off readily. Further differences between
the two hemocyanin subunits are revealed by
hydrogen peroxide treatments.
Native
KLH
The high molecular weight, native KLH, has been
extensively over the last several years, and is
still being used. Native KLH has been
manufactured and supplied by many chemical
suppliers.
However, most such KLH are not of the required
quality and consistency suitable for use in
human clinical trials. Typically, the endotoxin
content in such products is very high as the
hemolymph sera production is done by cutting
open the animals or extraction from dead animals
shipped frozen. The product characteristics are
also not very good and could lead to
precipitation during reconstitution of
lyophilized KLH or short shelf-life of one year
or less for liquid preparation. It is well
documented that either freezing or
lyophilization of native KLH leads to loss in
activity.
Characterization of native KLH is also an issue
as the large molecular weight of 8-32 MDa does
not allow use of standard biochemical methods
for routine quality control, which is further
complicated by the use of excessive metal ions
like calcium and magnesium, presumably to
stabilize the molecule.
To achieve the desired safety, quality and
consistency of native KLH, we use the non-lethal
hemolymph sera collection procedure from live
animals and instituted necessary animal handling
and quarantine techniques. Quality control
methodologies for routine production of this
critical raw material have been developed. In
addition to this, we established aquaculture
developmental activities to spawn, culture and
grow the animals. The hemocyanin production
process is thus environmentally friendly.
Moreover, the non-lethal technology allows for
the development and institution of appropriate
mariculture/aquaculture techniques to ensure
future stable source of the raw material.
The end result of these technology developments
in the animal handling, bleeding and
manufacturing process was the production of low
endotoxin and high concentration native KLH of
consistent high quality in a phosphate buffer
containing no extraneous metal ions for product
stabilization, however with improved stability.
The native KLH is supplied as High Purity Grade
and Research Grade. The GMP grade material is
ideally suited for use as a carrier protein in
the manufacture of vaccines for human use. The
Research Grade material is suited for use in
vaccine product development activities and also
for routine immunological studies, antibody
production, production of activated KLH and
other developmental activities. The native KLH
formulations currently available from gentaur
are listed in the table above.
General Conjugation Procedures
- Using Hemocyanin and Immunocyanin Products
Why Conjugate?
Substances of low molecular weight (haptens) and
some high molecular weight proteins alone will
not generate antibodies or are not highly
immunogenic when injected into an animal.
Therefore, the haptens are usually conjugated to
a large protein molecule known as a "carrier."
The carrier facilitates the production of
antibodies in the following ways:
-
The increased size of the hapten-carrier
complex is sufficiently large to be
recognized and engulfed by the antigen
presenting cells of the immune system.
-
The protein carrier molecules contain
sequences that are T cell helper epitopes
that stimulate the proliferation of helper T
cells. Interaction of helper T cells with B
cells is essential for a strong immune
response.
-
Since carrier proteins are foreign to the
animal, they enhance the response from the
immune system.
Choosing a Carrier Protein
The choice of the carrier protein depends on the
intended use of the hapten-carrier protein
complex. For routine animal studies for research
investigation and possibly development of
immunological assays, there are a number of
proteins that have been used as carriers,
including Keyhole Limpet Hemocyanin (KLH),
Bovine Serum Albumin (BSA), ovalbumin, and
Tetanus toxoid. KLH and BSA are probably the
most commonly used.
However, for human clinical studies the most
widely used carrier proteins are Keyhole Limpet
Hemocyanin, Tetanus toxoid or Diphtheria toxoid.
The critical parameters in the selection of the
protein are elucidation of the necessary immune
response for the intended clinical use and the
toxicity of the carrier protein and the
conjugate, i.e. minimal secondary complications.
These are generally evaluated during the early
research and pre-clinical studies. KLH has been
the most widely used protein due to the
consistent yield of enhanced immune response
with a wide variety of haptens and also protein
molecules.
KLH is one of the largest foreign proteins that
is most different from the human and generates
the best immune response. When immunized with a
hapten-KLH conjugate, antibodies are also
generated to the KLH. Therefore, it is necessary
to use a hapten-conjugate that is different from
the KLH used for your assays. For example, a KLH
conjugate should be used for immunizations and a
BSA conjugate for assays. More recent
investigations suggest that biosyn hemocyanin
from abalone and horse shoe crab may be used as
an additional carrier protein.
GMP KLH has many advantages over other
commercial grade material that can be procured
from other vendors. The major distinction lies
in:
-
Environmentally friendly process used in the
extraction of hemocyanin from animals.
-
a proprietary methodology and has built-in a
test procedure for harvesting and handling
of live animals, unlike the use of dead or
frozen animals or lethal extraction
procedure used by most commercial KLH
suppliers.
-
GMP hemocynin and final products are
therefore of high purity and low endotoxin
quality.
-
GMP KLH subunits, Immunocyanin, are of low
molecular weight (400 kDa) and are a
completely characterized product.
-
GMP Immunocyanin in water is a unique
formulation and overcomes one of the major
drawbacks of using KLH in clinical and
commercial grade conjugate vaccine products.
-
GMP products are extensive tested for safety
and lack of viruses including hepatitis A,
B, C, vibrio species, rota virus and Norwalk
viruses.
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