NEONATAL TSH Screening ELISA. Cat. No.: MSUD Screening Assay Screening assay for the detection of Maple Sirup Urine Disease in Newborns
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1 GenWay Catalog Number: GenWay Biotech, Inc. Protein and Antibody Solutions NEONATAL TSH Screening ELISA Cat. No.: MSUD Screening Assay Screening assay for the detection of Maple Sirup Urine Disease in Newborns Package size : 500 tests Package size : 2000 tests 1/ Nancy Ridge Drive San Diego, CA Phone: Fax:
2 1. INTENDED USE The MSUD Assay is an enzymatic colorimetric method for the quantitative detection of Maple Sirup urine disease in newborn using dried blood spots specimens. This assay is for research use only. 2. INTRODUCTION Maple Syrup Urine Disease (MSUD) is a disorder in the body s ability to use three of the essential amino acids in protein. These three essential amino acids-leucine, isoleucine and valine-are often called the branched-chain amino acids (BCAAs). Protein in the body is made up of 20 amino acids, eleven of these are essential and nine are nonessential. The essential amino acids must be supplied in the daily diet. The nonessential amino acids can be obtained from the diet or produced in the body from other amino acids. In MSUD, the enzymes necessary to break down leucine, isoleucine and valine are either absent, inactive, or only partially active. Because of the enzyme deficiency in MSUD, the BCAAs and their byproducts, called ketoacids, become elevated. It is these elevations that cause an infant or child with MSUD to become symptomatic. 3. MSUD CLASSIFICATIONS Four general classifications are used to identify the types of MSUD: classic, intermediate, intermittent and thiamine-responsive. These terms refer to the amount and type of enzyme activity present in the affected child, which can vary considerably within each classification. Classic is the most common type of MSUD. In classic MSUD, little or no enzyme activity (usually less than 2% of normal) is present. Infants with classic MSUD will show symptoms within the first several days of life. They generally have poor tolerance for the BCAAs, so protein must be severely restricted in their diet. Intermediate MSUD is a variant of the classic type of the disease. Those with intermediate MSUD have a higher level of enzyme activity (approximately 3 to 8% of normal). They can usually tolerate a greater amount of leucine. However, when ill or fasting, the child with intermediate MSUD reacts just like a child with classic MSUD. Management is similar for the intermediate and classic types of MSUD. Intermittent MSUD is a milder form of the disease because of the greater enzyme activity present (approximately 8 to 15% of normal). Often the child does not have symptoms until 12 to 24 months of age, usually in response to an illness or surge in protein intake. During episodes of illness or fasting, the BCAA levels elevate, the characteristic maple syrup (or burnt sugar) odor becomes evident, and the child can go into a metabolic crisis. Thiamine-responsive MSUD is basically just what the name implies. Giving large doses of thiamine to the thiamine-responsive child will increase the enzyme activity which breaks down leucine, isoleucine and valine. In most cases only moderate protein restriction is needed for this more rare type of MSUD. 2/6
3 4. KIT CONTENTS 500 test 2000 tests Enzyme 4 x 5 ml 4 x 20 ml Coenzyme 4 x 5 ml 4 x 20 ml Dilution buffer 1 x 10.5 ml 1 x 42 ml Colour reagent 1 x 43 ml 1 x 175 ml Colour reagent booster 1 x 4.3 ml 1 x 17.5 ml Standards and controls 1 set 1 set Instruction for use MATERIALS REQUIRED BUT NOT PROVIDED 1. U - well microtitration plates (elution plates) 2. Elution buffer (TCA 3 % Trichloroacetic acid) 3. Water for injection (de-ionized water). 3. Flat-bottomed microtitration plates (assay plates) 4. Single or multichannel automatic pipettes to deliver volumes in the range of 15 to 75µl with an accuracy of +/- 1.5% over this range. 5. A microtitration plate reader capable of reading absorbances at 550 nm in KINETIC reading mode. 6. A hole puncher which produces 4.7 mm or 3.2 diameter discs. 7. A plate incubator set at 37 o C with an accuracy of +/- 1 C 8. Blood collection cards. The minimum preprinted information required (in accordance to Schleicher and Schuell 903) 9. Space for test results 10. Appropriate number of preprinted 1/2 (internal diameter) broken or dotted line circles on attached blood collection filter paper. 11. Manufacturer s and lot number of filter paper indicated on filter paper 12. Manufacturer or printer listed on the information section. Blood collection filter cards should be attached to at least 2 copies of the information section outlined above. Filter paper should be used according to the specification provided by the National Screening Association. 3/6
4 6. PRECAUTIONS - SAFETY AND OPERATING All reagents must be stored according to the instructions on the label and instructions for use. The assay-performance can be seriously affected if the instructions as outlined in this package insert and on the labels are not strictly adhered to. The reagents contain 0.15% sodium azide as anti microbial preservative. This substance has a toxic effect if absorbed or indigested. They should be discarded with an adequate water flow. All blood samples of human origin must be regarded as potential biohazards and normal laboratory precautions must be taken whilst handling these samples. Do not use kit components after the expiry date stated on the label. Do not mix components of different lot numbers. Do not use any solutions that have become turbid or discoloured. 7. SAMPLE COLLECTION Collect from the infant's heel according to the NCCLS Approved Standard LA4-A2 (S&S 903) "Blood Collection on Filter Paper for neonatal Screening Programs" Second edition (1992) NCCLS, Villanova, PA After the sample is taken and the blood has dried, the cards must be stored at 2-8 C. Spots not stored under these conditions gradually loose the enzyme activity due to heat inactivation, causing potential risk of misclassifying samples as screen-positive (34, 35). 8. ASSAY PROCEDURE 1. Take a clean 96-well (preferably U bottom) microplate (elution microplate). 2. Add one disk cut from a dried blood spot (3/16 inch or 2 X 1/8inch diameter) per well. Remember to add controls, standards and one blank well. 3. Warm up all reagents (except the Color Reagent) to room temperature. 4. Add 100 microliters of Elution Buffer (TCA 3%) in each well, mix well the contents of each well and place the plate on a plate shaker. 5. Wait 30 minutes at room temperature (20-26 o C). 6. While waiting reconstitute one Enzyme and one Coenzyme Vial with distilled water. Each vial should be reconstituted with 20 ml of distilled water. Stable for one week refrigerated. Mix 2 parts of Enzyme solution with 2 parts of Coenzyme solution and 1 part of Dilution buffer. You need 100 microliters of this Enzyme-Coenzyme-Dilution buffer mixture for each sample. Please note that you should only mix the quantity you need for the day s run. The Enzyme-Coenzyme mixture should be discarded if not used within 5 hours. The following table gives the volumes required from each of the three components to run specific number of tests (volumes in ml). We highly recommend the addition of the Dilution buffer just before using the mixture. 4/6
5 # tests Enzyme Coenzyme Dilution buffer Total volume Transfer 40 microliters of the TCA eluant in a new microplate at the corresponding wells. Add 100 microliters of the mixture prepared in step 6, per well. Mix well, avoiding the formation of foam. Wait for 30 minutes at room temperature (20-26 o C) 8. Take the Color Reagent and the Color Reagent Booster out of the refrigerator and mix one part of Color Reagent Booster with 10 parts color reagent just before using it. Do not pre-warm the mixture. Return the original bottles back to the refrigerator the soonest possible. Avoid exposure to light. Prepare only the quantity you will need for the day. 9. Add 80 microliters of Color Reagent mixture per well. Mix well avoiding the formation of foam. 10. Wait for 10 minutes and measure the microplate at 550 nm, endpoint mode, single measurement. There is no need to wait longer than 20 minutes. 11. Calculate the slope and the sample values. Please note the following: The Color Reagent contains a Tetrazolium salt, which is light sensitive. Do not leave the vial out of the refrigerator longer than needed. Avoid exposure to direct sunlight. This assay is to be performed at room temperature (20-26 o C). At higher temperatures (over 29 o C) an abnormally high blank may be observed. The Dilution Buffer and / or the reconstituted Enzyme-Coenzyme-Dilution buffer mixture may appear cloudy. Cloudiness does not affect the assay Please note that the Dilution buffer contains potassium hydroxide which is irritant. Avoid contact with skin or the eyes. In case of accidental contact rinse with plenty of water. 5/6
6 9. RECOMMENDATIONS MSUD is a serious disorder with life-threatening consequences unless addressed promptly. However, MSUD is manageable, just as diabetes is manageable. Careful monitoring and adherence to dietary restrictions are essential. Even minor illnesses and infections must be taken seriously and require special care. Treatment for MSUD has improved greatly over the past few years. It is of utmost importance that all children who have MSUD receive the benefit of the most up-to-date treatment. WARNING: Most of you are aware of the dangers of giving children or teenagers aspirin for colds, chickenpox or the flu. There is evidence to link aspirin in such illnesses with a serious complication known as Reye Syndrome. Children with MSUD are very susceptible to a Reye Syndrome-like illness with the accompanying brain edema. It is very important that children with MSUD are not given aspirin or products containing aspirin. Make a practice to read the ingredient list on any over-the-counter medications. FOR RESEARCH USE ONLY 6/6
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