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.

Comparative Analysis

	HPLC	VitaKitTM
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

Time-Method Comparison

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.

Detection Limits

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)
Conjugate diluent
Reaction buffer
Enzyme substrate – TMB
Stop solution

Sample Flowchart

1. Prepare the sample, pipette 1mL of milk, add KOH
2. Mix as prescribed
3. Add hexanes, centrifuge for separate the layers, remove 200 uL of the hexanes layer to a second vial
4. Bring the components of the VitaKit™ to room temperature
5. Pipette 10 uL of the calibrators, controls, and unknown samples into the desired wells of the provided 96-well plate 
6. Shake to evaporate the hexanes
7. Add reaction buffer, shake as prescribed
8. 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
9. Was the plate with distilled water 
10. Add the developer, TMB, and incubate up to 5 minutes
11. When colour has developed sufficiently, add the stop solution to prevent over-developing which will skew the results
12. Read using a microplate reader set to 450nm
     

Analysis:
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