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.