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 ASK THE DOCTOR by Dr. Chad Larson 
[Special
Report ] Vendor
Comparison: Not All Nattokinases Are The Same! |
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The following article is that of Dr. Sumi, his endorsement
of NSK-SD and Elisa testing
There is only one Nattokinase that has been shown scientifically
to be identical to the Nattokinase first discovered and studied
by Dr. Sumi. Amino acid sequence data has shown that NSK-SD
has an identical protein structure to the Dr. Sumi enzyme
- all 275 amino acids of both proteins line up exactly (BBRC
193 (3), 1340-1347).
Unfortunately, the current assay for Nattokinase that measures
fibrinolytic units (FU) with cross-linked fibrin as substrate
is not specific for Nattokinase but appears to be a more general
protease assay. Not only do all the Nattokinase products currently
on the market show activity with this assay, but many other
commercial proteases also have considerable activity. In fact,
I have not found a protease that does not have some activity
using the Nattokinase assay. In order to develop a simple
and reliable procedure to distinguish between authentic Nattokinase
as first described by Dr. Sumi and other proteases that may
have activity in the Nattokinase assay, but differ in structure
from the Dr. Sumi enzyme, we have utilized the ELISA technique.
The ELISA (Enzyme Linked ImmunoSorbant Assay) technique
is one of the most widely used techniques in modern biology.
It is the foundation of nearly all antibody based clinical
assays currently in use. ELISA assays take advantage of the
highly specific interaction of an antibody and its corresponding
epitope the molecular structure that induced the formation
of the antibody. When a protein is used as an antigen to produce
antibodies, each antibody will recognize an epitope corresponding
to 10 or fewer amino acids. Thus, a protein antigen the size
of NSK-SD can produce many different antibodies, each of which
will recognize a specific structural feature (epitope) on
the surface of the protein. This collection of very specific
antibodies can then be used to define the NSK-SD protein structure
by ELISA assay: only proteins identical or very nearly identical
to NSK-SD in structure will react with all the antibodies
that react to NSK-SD while proteins that are very different
in structure compared to NSK-SD will react with very few of
the antibodies that react with NSK-SD. The extent of the reaction
of a protein with antibodies can be measured quantitatively
by labeling the antibodies directly, or indirectly. In the
ELISA described below, the antibodies are labeled by conjugation
with horse radish peroxidase (HRP), which catalyzes a colorimetric
reaction that is easily measured.
In the ELISA assay described schematically in Figure 1 (coming
soon), equal amounts of NSK-SD or Vendor Nattokinase are immobilized
on the surface of separate microplate wells. The structural
features or epitopes of the proteins are depicted by geometric
shapes. (For simplicity, only three epitopes are indicated.)
Next, antibodies labeled with HRP are added to the wells and
allowed to interact with the bound protein. As indicated,
NSK-SD interacts with antibodies to all three epitopes. In
contrast, Vendor Nattokinase proteins interact with antibodies
to fewer epitopes because they have fewer structural features
in common with NSK-SD. Once the antibodies have reacted with
the bound protein, the wells are washed to remove all antibodies
that have not interacted with the bound protein. The amount
of bound antibody is then measured by addition of a reagent
that will change color in the presence of HRP (O.D. 450).
The intensity of the color is a quantitative measure of the
amount of bound antibody.
Figure 2 (coming soon) presents the actual ELISA data for
the vendor comparison. It can be seen that none of the vendor
Nattokinase proteins interact with the same intensity with
the NSK-SD labeled antibodies as does NSK-SD. For instance,
while Nattokinase from Vendors A and B show some similarity
to NSK-SD, Nattokinase from Vendors C and D are quite different
from NSK-SD. The differences seen between the Nattokinase
proteins cannot be due to differences in the concentration
of the protein (amount of inert material present in the product)
because the protein concentration was determined by a protein
determination assay (Brandford) and not simply the weight
of the product. Also, the results cannot be explained by differences
in the purity of the proteins because the proteins were of
comparable protein purity as judged by SDS gel electrophoresis.
The ELISA assays were carried out with standard blocking and
washing procedures and with appropriate controls to insure
that the binding of labeled antibody was not the result of
non-specific binding of antibody to the wells and that excess
unbound antibody was removed.
In conclusion, since NSK-SD is identical to the Nattokinase
described by Dr. Sumi, and since the ELISA data presented
above demonstrates that NSK-SD and the "Nattokinase" from
the Vendors in this study are quite different in structure
then the Vendors "Nattokinase" used in this study must be
different from the Nattokinase described by Dr. Sumi. This
is an important point because it means that all of the efficacy
data developed by Dr. Sumi cannot be assumed to apply to a
"Nattokinase" that can be demonstrated by ELISA assays to
be very different in structure (and therefore function) from
the Nattokinase described by Dr. Sumi. In addition, it means
that the vast amount of safety and efficacy data developed
with NSK-SD also only applies to NSK-SD and not the structurally
and functionally different Vendor "Nattokinases" of this study.
In future articles to appear in this space we will present
the extensive safety and efficacy data we have established
with NSK-SD. |
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