MS344N RC R1
REFERENCES
1. 1 Anne M. Lewis, Christine Waterhouse, and Laurence S. Jacobs. (1980). Whole-Blood and Plasma
Amino Acid Analysis: Gas-Liquid and CationExchange Chromatography Compared. CLIN. CHEM. 26/2,
271-276
2. Hortin GL (2012). In: Burtis CA ed. Amino acids, peptides, and proteins. Tietz Textbook of Clinical
Chemistry and Molecular Diagnostics. 5th ed. Philadelphia, PA: Elsevier Saunders; 2012:chap 21
3.
4.
Naylor, E.W. (1993) in Laboratory Methods for Neonatal Screening (Therrell, B.L. Jnr. ed.) Apha p115
Snyderman, S.E., Norton, P.M., Roithan, E., and Holt, L.E. Jnr. (1964) Pediatrics 34: 454
Neonatal Maple Syrup Urine Disease (N-MSUD) ELISA
Catalog No.: MS344N (96 Tests)
INTENDED USE
The MSUD Screening Assay is an enzymatic colorimetric end-point method for the determination of L-
2020-03-09 leucine and other branched-chain amino acids in dried blood spot specimens taken from newborn human
infants as part of a newborn screening program. The test is intended as a screening method for measuring
the L-branched-chain amino acid (BCAA) concentrations in newborn blood spot specimens.
PRINCIPLE OF THE ASSAY
The MSUD Kit uses trichloroacetic acid (TCA) to extract Leucine and BCAAs from dried blood spot
samples. After extraction, the eluted sample is combined with the enzyme reagent Leucine
dehydrogenase. This enzyme reagent catalyzes the NAD-dependent oxidative deamination of Leucine
and L-BCAAs to α-ketoisocaproate acid. The NADH produced reacts with a color reagent in which a
tetrazolium salt gets reduced producing a distinct color.
MATERIALS PROVIDED 96 Tests
1. MSUD Calibrators: 6 Calibrators 0.5mL ea.
2. MSUD Controls 1-3: 2 Dried Blood Spots per level 6 DBS
3. Elution Reagent 12 mL
4. Reaction buffer: 1 bottle (ready to use) 8 mL
5. Color Reagent: 1 bottle (ready to use) 9 mL
6. Cofactor: 1 bottle (Lyophilized) 5 mL
7. Reaction Microplate 12x8x1
8. Elution Microplate 12x8x1
9. Leucine Dh Enzyme: 1 vial (ready to use) 120 uL
10. Cofactor Diluent 6 mL
MATERIALS NOT PROVIDED
1. 1/8” inch hole punch
2. Shaker capable of fixed speed
3. Distilled or deionized water
4. Precision pipettes
5. Disposable pipette tips
6. Reader capable of reading absorbance at 450 nm
7. Absorbance paper or paper towel
8. Graph paper
MS344N RC R1
WARNINGS AND PRECAUTIONS ASSAY PROCEDURE
1. For Research Use Only. Not for use in diagnostic procedures. 1. Equilibrate all reagents to room temperature.
2. For laboratory use. 2. Take a clean 96-well elution microplate and add dried blood spots (2 discs of 1/8inch diameter) of
3. Potential biohazardous materials: controls and samples to corresponding well.
The calibrator and controls contain human source components which have been tested and found 3. Add 100 µl of Elution Buffer to each well, mix the contents and place the plate on a plate shaker.
non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. 4. Incubate 30 minutes at room temperature on shaker at 900rpm (cover from light).
However, there is no test method that can offer complete assurance that HIV, Hepatitis B virus or 5. Reconstitute the lyophilized Cofactor with 5ml of Cofactor Diluent (protect from light).
other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as 6. Mix 60µl of Reaction buffer, 40µl of Cofactor and 1 µl of enzyme per test. Note Calculate the needed
recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in volume including standards and controls to make a master mix for all the wells. This mixture is stable
Microbiological and Biomedical Laboratories." 1984. for 5 hours.
4. Optimal results will be obtained by strict adherence to the test protocol. Precise pipetting as well as 7. Transfer 40 µl of the eluted controls and samples to a Reaction Microplate, also transfer 40 µl of
following the exact time and temperature requirements is essential. each standard (liquid, ready to use) at the corresponding wells.
5. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit 8. Add 100 µl per well of the mixture prepared in step 7. Incubate 30 minutes at room temperature
reagents are handled. shaking at 900rpm (cover from light).
6. The components in this kit are intended for use as an integral unit. The components of different 9. Add 80 µl of Color Reagent ready to use, per well. Mix well and incubate 5-10 min on bench. Longer
lots should not be mixed. incubation time can contribute to background increase.
7. All blood samples of human origin should be regarded as a potential Biohazard. Handle all blood 10. Measure the absorbance at 450 nm, endpoint mode, single measurement. Calculate the slope and
samples as if capable of transmitting Hepatitis and HIV the sample values.
8. Reagents, standards and Controls contain sodium azide (NaN3) and ProClin as antimicrobial
preservatives. Users should be aware of their toxic properties if absorbed or ingested. Disposal of CALCULATION OF RESULTS
these reagents should be accompanied by copious flushing with water to avoid accumulation of A standard curve is constructed as follows:
explosive salts in plumbing systems. 1. Calculate the average absorbance values for each set of standards and patient samples.
9. Do NOT keep or use the reconstituted Enzyme, Coenzyme, or the combined Enzyme-Coenzyme 2. To construct the standard curve, plot the mean absorbance of each Leucine standards (vertical axis)
working solution for any longer than the specified periods of time. against its concentration in mg/dl (horizontal axis).
10. All the kit components used in an assay must be from the same kit. 3. Draw the best-fit curve through the plotted points.
11. Kit components and test specimens should be at room temperature before starting the assay. 4. Read the absorbance for each unknown sample from the curve to determine the corresponding
12. Do not use any reagents or solutions that have become cloudy or discolored. concentration of BCAA.
SPECIMEN COLLTION AND HANDLING OD450nm [mg/dl] 0 .4
Follow the guidelines in the NCCLS publication LA4T7 for collecting blood samples in the neonatal Std 1 0.0651 0.00
screening program, copies of which can be obtained from: NCCLS, 771 E. Lancaster Ave, Villanova, PA 0 .3
O D 450nm
19085. Use WHATMAN type 903 filter paper. For samples screening for CAH, collect samples 3 to 5 days Std 2 0.0841 1.30
after birth. Use disposable lancets with tips less than 2.5 mm to prick the medial or lateral sides of the Std 3 0.1048 2.70 0 .2
bottom of the heel. Allow a drop of blood to form with sufficient volume to fill a 5/8-inch diameter spot on Std 4 0.1410 5.50 0 .1
filter paper. Gently touch the drop of blood with the filter paper. DO NOT PRESS AGAINST THE SKIN.
Std 5 0.2111 11.00
DO NOT TOUCH SPOTTED AREA. Suspend spotted papers horizontally and allow drying at room
Std 6 0.3602 22.00
0 .0
temperature for a minimum of 3 hours. Avoid spots touching other surfaces and keep away from direct 0 5 10 15 20 25
light. The samples should be transported to the laboratory within 24 hours after collection in appropriate [ m g /d l L e u c in e ]
storage container. The laboratory should store the specimens at 2-8 °C protected from moisture and direct Expected Values
light. We recommend each laboratory to establish its own normal ranges, for the population it serves. Until
The dried blood spots are stable for at least 3 weeks at 2-8 °C protected from light and moisture. Reject then, literature values may be used as guidelines: BCAA normal range 3.26 - 8 mg/dl (ref. 1&2).
samples with the following conditions: REPORTABLE RANGE: Analytical Range = 1.3 - 22 mg/dl Samples that fall within the calibration curve
1. Specimens not collected on WHATMAN type 903 filter paper. should be reported as such.
2. Blood spots not completely saturated on both sides.
3. Blood spots with appearance of caking or clotting.