VitaKits™ permit the rapid analysis of vitamins A & D in fortified products. The technology is based on proprietary monoclonal antibodies against these vitamins developed by SciMed. As an ELISA-method, these assays require only a microplate reader, and can be completed with only minimal training.
Technology Behind VitaKit™
VitaKit™ technology can be divided into two major innovations: a new vitamin extraction procedure, and the ELISA assay:
1. Vitamin extraction
Current methods of extracting the fat-soluble vitamins (vitamins A & D) require an overnight saponification using a large amount of potassium hydroxide, and then require a technician-intensive, all-day extraction. The VitaKit™ line revolutionizes this by using less than 1% of the reagents previously used, and the process is complete in less than 1 hour. Additionally, SciMed has automated much of the process, permitting technicians to handle a greater number of samples, as opposed to the current maximum of 5.
2. ELISA Assay
SciMed has developed proprietary monoclonal antibodies (Mabs) against vitamins A & D, which permit the measurement of these vitamins using ELISA. Currently, a technician must use an expensive HPLC, and run only one sample at a time, leading to long analysis time. With the ELISA method, it is simply a matter of loading the sample onto the included 96-well plate, then loading the antibody, and finally measuring the absorbance using a much less expensive ELISA reader. Using the VitaKit™ methodology, a technician can analyze as many samples concurrently as can fit on a plate!
Due to the reduced human-interaction, the ELISA methodology for vitamin analysis has been documented to be much more accurate than the HPLC method. The error permissible in HPLC analysis is 50%! As demonstrated by our validation studies, SciMed has been able to reduce this error to as low as 4%.
The VitaKit™ line has been validated by a blind study involving some of the major North American dairy processors, including: Fonterra, Lucerne, Parmalat, Saputo, and Nestle.
Why Use a VitaKit™? Save Time & Money!
- Ability to process up to 41 separate samples, all done at once by 1 technician;
- Total time of less than 2 hours, including sample extraction and data processing;
- Concurrent analysis of Vitamin A & D. No more preparative HPLC;
- Can be used with minimal training and experience.
Who Can Use a VitaKit™?
- Analytical laboratories involved in food quality assurance testing;
- In-house by food processors to get immediate results for quality control;
- Product development researchers seeking higher quality processed food for the discerning customer;
- Regulatory and inspection agencies to ensure end-user (customer) safety.
|Time||2-4 days of dedicated technician time||2 hours|
|Capacity||5 samples per technician||Up to 41 samples at once, with 1 technician|
|Equipment||Expensive HPLC and glassware||Inexpensive ELISA reader|
|Accuracy||Wide variance in results between labs & technicians||Validated externally to be narrower and more specific|
|Cost||Up to $350 / sample||As low as $38 / sample|
|Traditional HPLC Method||VitaKitTM A & D|
|Day 1||Begin saponification by adding KOH||8am - Saponification using KOH, Extraction with hexanes, separation using centrifuge|
8:45 - Load vitamin onto 96-well plate, complete remaining assay steps
9:45 - Read plate using ELISA reader
10:00 - Coffee Break!
|Day 2||Complete the saponification Extract vitamin using hexanes and separatory funnel Evaporate hexanes|
|Day 3-4||Load vitamins onto the HPLC Measure vitamin A directly on HPLC Purify vitamin D, evaporate solvent Measure vitamin D|
VitaKit™ A vs. VitaKit™ D
VitaKits™ A & D are based on different versions of ELISA methodology. VitaKit™ D is a competitive assay, so the 96-well plate comes pre-coated with a known amount of vitamin D. Alternatively, VitaKit™ A is a sandwich-style assay, so the 96-well plate comes pre-coated with one monoclonal antibody. Hence, there is a difference in the final output in terms of graph-style (see below for sample graphs). This is also the reason for diluting samples for vitamin A analysis more than samples for vitamin D analysis. Both antibodies are bound to horseradish peroxidase, which is activated by TMB and absorbs at 450nm; a typical procedure for an ELISA assay.
VitaKit™ A: 0.014 – 0.45 I.U./mL
VitaKit™ D: 0.063 – 0.75 I.U./mL
Each Kits Contains
Coated 96-well microplate
5 standards used to construct standard curve
2 controls used to verify the standard curve
Required antibodies (conjugate)
Enzyme substrate – TMB
- Prepare the sample, pipette 1mL of milk, add KOH
- Mix as prescribed
- Add hexanes, centrifuge for separate the layers, remove 200 uL of the hexanes layer to a second vial
- Bring the components of the VitaKit™ to room temperature
- Pipette 10 uL of the calibrators, controls, and unknown samples into the desired wells of the provided 96-well plate
- Shake to evaporate the hexanes
- Add reaction buffer, shake as prescribed
- For VitaKit™ D, add the antibody-enzyme conjugate; for VitaKit™ A wash the plate first with distilled water, then add the antibody-enzyme conjugate; shake as prescribed
- Was the plate with distilled water
- Add the developer, TMB, and incubate up to 5 minutes
- When colour has developed sufficiently, add the stop solution to prevent over-developing which will skew the results
- Read using a microplate reader set to 450nm
This is what your final analysis will look like after you have read the microplate (standards only):
Determining the value of your samples is a simple as finding the best-fit line for the curve, and inputting your optical density. The values of your vitamin concentrations will then be determined. Note that the VitaKit™ best measures vitamin concentrations between the upper and lower limits of the standards supplied with the kits – beyond that may lead to inaccurate results. If necessary, dilute your sample before applying it to the VitaKit™ so that it will fall in this range.
Feature Article: Rapid Analysis of Vitamin A and D in Milk
|KTSP-71051||VitaKit A ELISA||Sufficient for 96 determinations; Ready to use kit; For in vitro quantification use.||$531.00|
|KTSP-72051||VitaKit D ELISA||Sufficient for 96 determinations; Ready to use kit; For in vitro quantification use.||$531.00|
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