Imunoclone® ELISA From American Diagnostica, Inc.

The mechanism of molecular polymerization in the process of heamostasis is widely observed for clinical and research purposes. There is a wide variety of blood components such as Factor V, Factor VII, TF, HPS, etc, which take part in primary and secondary heamostasis and the study of these multiple factors can yield beneficial information pertaining to diagnosis or prognosis of many disease states. ELISA kits for the quantification of these factors in human blood samples are available from a variety of vendors. Unfortunately, no single vendor of ELISA kits stands out as the standard leader in the field or offers kits for all specificities. Vendors offer a small variety of specificities of ELISA kits with some overlap between companies. Although larger companies like Invitrogen or R&D Systems offer ELISA kits for some of the specificities, American Diagnostica Inc. makes ELISA kits for quite a few of the coagulation factors (as well as ELISA kits for other targets). Out of the large and varied field of coagulation ELISA kits, American Diagnositica stands out as one of the more prominent vendors because this is what they focus on.

The Imuclone® line of kits from American Diagnostica Inc. is a planar based colorimetric quantitative ELISA kit. Strips of microwells are coated with monoclonal capture antibodies after which a horseradish-peroxidase conjugated monoclonal antibody is used for detection. With the kits that I have used, the colorometric signal is produced by reaction with 3, 3’, 3, 5 – tetramethylbenzidine (TMB) or by 2-2’azino-bis (ABTS) substrates of which the absorbances are read at 450 nm or 405 nm, respectively, on a microplate reader. Some kits may be used with specimens that are either citrated or treated with EDTA, but some kits are only usable with one type of sample, so I suggest that careful attention be paid to sample compatibility when considering using these kits.

The buffer components vary from kit to kit, which is understandable due to the varying specificity requirements of each. However, all the kit types were complete with all the reagents needed to perform the given assay, with few exceptions. Some reagents were not provided with resuspension buffers, which needed to be formulated in the lab. Also, one kit required Hydrogen Peroxide, which was not provided. The well strips, which are provided in 12 strips of 8 wells, are convenient because the reagents for the assay can be used incrementally, instead of at once with a 96 well plate.

The kit procedures were relatively easy to follow and should be understandable for a beginner level operator/technician. I was able to achieve good results upon initial uses of the kit, without having to repeat initial tests to learn how to use the kit. Disturbingly, there seemed to only a general homology between product literature and manuals between kits. Different kits seemed to have different formats for the technical literature and performance specifications. Despite this, each corresponding assay procedure seems to work well.

The technical results of the assays seemed to be accurate. The standard curves were linear and tested samples of commercially available normal human serums yielded concentration values in ranges that would be expected. A number of the assay kits have technical specifications, with values of about 5% coefficient of variation (%CV) for interassay specs and 5 – 10 % CV for intra-assay specs (depending on specificity). However, some of the kits offer no specs; additionally, none of the kits offered a linear recovery or accuracy specification. Another drawback of the kits is the extremely limited linear range of detection, which is less than one log of concentration. This requires the sample to be diluted to the exact correct level in order to obtain an accurate measurement, which is tedious and expensive to determine empirically.

The cost of the kits ranged from around $600 to over $800. This makes the average per sample cost around $7.50. Considering the wells used for standard curves, the per sample price will be about $10 per sample. This price is comparable to other products available.

I am on the fence whether this is a good value for laboratory usage. The kit is not without its drawbacks and technical problems. This must be weighed against the general unavailability of a reliable source for coagulation factor assay kits. The kits did satisfy the general requirements of what I was seeking to accomplish, which was to verify accuracy of multiplexed coagulation factor microarrays. In summary, the kits did function as expected, but not an optimal solution for quantifying samples by ELISA. I would not be opposed to trying other vendors.