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Methode Type Competition ELISA
Pubmed 7 references available
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Catalog number from supplier Login to see New
Quantity 960 tests
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Purpose The Neoscreen ELISA 17a-Hydroxyprogesterone (17P) is a competitive solid phase ELISA in blood spot dried on WHATMAN903 filter paper. In the assay, 17P eluted from the blood spot competes with 17P conjugated with an enzyme (17O-HPE) for a limited amount of antiserum specific for 17P coated on microwells. The bound antigen forms an immune complex with the immobilized anti-17P. Unbound reactants (17P and 17P-E) are removed at the end of the incubation time. A substrate is then reacted with the enzyme bound on the wall of the microwells. The enzymatic reaction is terminated with an acid. The end product is measured at 450 nm. The 17P of the unknown sample is determined using a calibration curve generated with known concentration of 17P.
Detection Method Colorimetric
Components A. Neo-17P Calibrators/Controls – Dried Blood Spots. Six levels of 17P Antigen in dried blood spots (adjusted to 55% hematocrit) at approximate concentrations of 0(A), 10 (B), 25(C), 50(D), 100(E) and 200(F) ng/ml placed on WHATMAN type 903 filter paper. Three levels of controls in dried blood spots (adjusted to 55% hematocrit) at approximate concentration of 15, 40, 100 ng/mL were placed on S&S type 903 filter paper in circles C1 ,C2, and C3. A preservative has been added. Note 1: The exact values are printed on the outside of the aluminum pouch used for storage. Note 2: The Lot Specific calibrators, whole human blood based, were verified with the 17P blood spots supplied by CDC. Note 3: Do not use blood spots with appearance of caking, clotting or moisture. Note 4: Store at 2-8°C. Avoid excessive exposure to light and humidity. Stability: Refer to expiration date on bag. Note 5: Reseal bags after use ensuring that the supplied desiccant is added. Note 6: Control ranges are statistically determined using 20 measurements with 2 standard deviations statistics given. B. 17P Enzyme Conjugate (One vial of 25ml). One vial of 17P-horseradish peroxidase (HRP) conjugate in a protein-stabilizing matrix. A preservative has been added. Store at 2-8°C. C. Anti-17P Coated Plate (2 plates of 96 wells). Two 96-well microplates coated with purified rabbit anti-17P IgG and packaged in an aluminum bag with a drying agent. Store at 2-8°C. D. 10X Wash Concentrate (One bottle of 50ml). Phosphate saline wash buffer containing surface reactants. Dilute 1 part concentrate with 9 parts deionized water before using. Diluted wash buffer is stable at refrigerated temperature until the expiration of the kit. Store at 2-8°C. E. Substrate Solution – Color Developer. One bottle contains 30ml of tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. F. Stop Solution: One bottle contains 30ml of a strong acid (1N HCl). Store at 2-30°C. G. Product Instructions: Note A: Do not use reagents beyond the kit expiration date. Note B: Opened reagents are stable for 60 days when stored at 2-8°C. Note C: Do not use reagents that look cloudy or turbid. They may be contaminated. Note D: Do not exchange reagents between different batches.
Material not included 1. Laboratory Shaker capable of 150rpm rotation. 2. Dispenser(s) for repetitive deliveries of, 0. 050ml, 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Adjustable volume (20-200µl) and (200-1000µl) dispenser(s) for conjugate dilutions. 4. 1/8thinch hole punch. 5. Tweezers to pick up the punched spots. 5. Microplate washer or a squeeze bottle (optional). 6. Microplate Reader capable of absorbance readings at 450nm and 620nm. 7. Test tubes for making working enzyme conjugate. 8. Absorbent Paper for blotting the microplate wells. 9. Plastic wrap and microplate cover for incubation steps. 10. Vacuum aspirator (optional) for wash steps. 11. Timer. 12. External quality control.
Background Summary and Explanation of the Test: The results are used to screen newborns for CAH (congenital adrenal hyperplasia). CAH is a genetic disorder, 90% of which is caused by 21- hydroxylase deficiency. The incidence is roughly estimated to be 1 in 15,000 newborns and can reach as high as 1 in 1480 in native Alaskans. Early diagnosis is valuable to detect CAH in newborns afflicted with the disease, not clinically recognizable but which will lead to life threatening adrenal crisis in the neonatal period and to determine the cause of infants with ambiguous genitalia. Delayed diagnosis may also lead to further virilization in female children, acceleration of skeletal maturation and premature development of secondary sex characteristics in male children. Prompt treatment can save the life of infants and allow afflicted children to attain normal growth. 17P is a steroid produced in the adrenal cortex and the gonads. It is the immediate precursor to 11-desoxycortisol (CpS) which is converted to cortisol. Because CpS is produced by 21-hydroxylation of 17P, measurement of 17P is an indirect indicator of 21-hydroxylase activity. CAH occurs where there is a deficiency of this enzyme. The result is a decrease in the conversion of 17P to CpS which blocks the normal synthesis of cortisol. Due to the feed back mechanism, a decrease in cortisol causes an increase in ACTH secretion resulting in adrenal hyperplasia. As 17P is not being converted, increased concentrations of this steroid will be found. 17P concentration increases during pregnancy in the maternal and fetal blood. After birth, values decline rapidly to reach normal adult values in 2 to 7 days. Thus it is advisable not to collect samples before the 3rd day of life. Premature and sick term infants exhibit 2 to 3 fold 17P values with no CAH disorder. It is suggested that a different cut off be adopted to pre-term and sick infants. Intended Use: The Quantitative Determination of 17-hydroxylprogesterone concentration in Human Whole Blood by a Microwell Enzyme Immunoassay.
Application Notes Precautions: For In Vitro Diagnostic Use. Not for Internal or External Use in Humans or Animals. All products that contain human blood have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Sample Volume: DBS two Step (Sequential)

Reagent Preparation All reagents are packaged ready to use except the wash concentrate. Dilute the entire bottle of wash concentrate. Add 1 part concentrate with 9 parts deionized water and stored the diluted wash buffer at 2-8 °C. Note the dilution date. Diluted wash buffer is stable at refrigerated temperature until the expiration of the kit.
Sample Collection Follow the guidelines in the NCCLS publication LA4T (7) for collecting blood samples in the neonatal screening program. Copies of which can be obtained from: NCCLS, 771 E. Lancaster Ave, Villanova, PA 19085. Use Schleicher and Schuell (S&S) Filter paper. For samples screening for CAH, collect samples 3 to 5 days after birth. Wash the heel with warm water to enhance blood flow. Wipe dry. Swab the area with alcohol and allow to dry. Use disposable lancets with tips less than 2. 5 mm to prick the medial or lateral sides of the bottom of the heel. Wipe off the first drop of blood with sterile gauze. Allow another drop of blood to form with sufficient volume to fill a 5/8 inch diameter spot on filter paper. Gently touch the drop of blood with the filter paper. (DO NOT PRESS AGAINST THE SKIN). Make 3 spots with 1 application per spot. DO NOT TOUCH SPOTTED AREA. Suspend spotted papers horizontally and allow to dry at room temperature for a minimum of 3 hours. Avoid spots touching other surfaces and keep away from direct light. Once dried, cover each spotted paper with a sturdy paper overlay and enclose the dried specimen in a high quality bond envelope. Mail or transport (13) to the laboratory within 24 hours after collection. The receiving laboratory should store the specimens at 2-8 °C protected from moisture and direct light. Information on the neonates should include name, date of birth, sex, time and date of collection and birth weight. Also indicate if the infant was pre-term or postdate, and to what degree and whether the infant is a twin. Record the hematocrit of the infant if such information is known. In addition, the name and address of the hospital, patient identification number, place of collection, the name, address and phone number of the physician should also be included on the collection sheet. Blood spots are stable for at least 3 weeks at 4-37 °C protected from light and moisture. Reject Samples with the following conditions: 1. Specimens not collected in S& S paper. 2. Blood spots not completely saturated on both sides. 3. Blood spots with appearance of caking or clotting. 4. Blood spots with appearance of moisture.
Calculation of Results A dose response curve is used to ascertain the concentration of Neo-natal 17P unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding NNT4 concentration in ng/ml on semi-log graph paper (average the duplicates of the serum references before plotting). 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of 17P for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in ng/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated).
Restrictions For Research Use only
Handling Advice Before proceeding with the assay, bring all reagents and patient samples to room temperature (20 - 27°C). 1. Assemble the required number of microwells for each calibrator, control and patient sample to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and Store at 2-8°C. 2. Punch out 1/8” blood dot out of each calibrator, control and specimens into the assigned wells. (NOTE: Do not punch blood dots from areas that are printed or that are near the edge of the blood spot). 3. Add 0. 100 ml (100µl) of 17P Enzyme Reagent to all the wells. 4. Shake the microplate gently for 20-30 seconds to mix. (NOTE: Make sure that all blood dots are fully submerged in the liquid and not stuck to the walls of the microwells). 5. Cover with a microplate cover and rotate for 60 minutes at ambient temperature using a laboratory rotator set @ 150rpm. 6. Incubate overnight (at least 17 additional hours) without shaking. 7. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. NOTE: Make sure all the blood dots are removed at this point. There should be no dots left in the microwells. 8. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat four additional times for a total of Five washes. An automatic or manual plate washer can be used. Follow the manufacturer’s instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat four additional times. 10. Add 100 µl of substrate solution (color developer) to each well. Cover the microplate and incubate for 30 minutes at ambient temperature. 11. Add 0. 100ml (100µl) of stop solution to each well and gently mix until a uniform color is obtained. NOTE: Always add reagents in the same order to minimize reaction time differences between wells. 12. Read the absorbance in each well at 450nm using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 15 minutes of adding the stop solution. 13. Plot O. D. versus log dosage (in ng/mL) serum equivalent using semi-log paper. Note: Calibrators and controls are provided in the kit and should be assayed in duplicates. Each Anti-17P plate is limited to 38 patient samples when calibrators and controls are included. Should there be more than 38 samples needed to assay at the same time, it is advisable to assay one set of calibrator and control in singlets per plate and take the average O. D. from each plate for calculation. Assay no more than 3 plates at a time. This means the maximum patient sample load is 129 samples.
Storage 4 °C/-20 °C
Background publications New: "An update of congenital adrenal hyperplasia." in: Annals of the New York Academy of Sciences, Vol. 1038, pp. 14-43, 2005 (PubMed).

Westgard, Barry, Hunt et al.: "A multi-rule Shewhart chart for quality control in clinical chemistry." in: Clinical chemistry, Vol. 27, Issue 3, pp. 493-501, 1981 (PubMed).

Pang, Murphey, Levine et al.: "A pilot newborn screening for congenital adrenal hyperplasia in Alaska." in: The Journal of clinical endocrinology and metabolism, Vol. 55, Issue 3, pp. 413-20, 1982 (PubMed).

Zachmann, Tassinari, Prader: "Clinical and biochemical variability of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency. A study of 25 patients." in: The Journal of clinical endocrinology and metabolism, Vol. 56, Issue 2, pp. 222-9, 1983 (PubMed).

Hirschfeld, Fleshman: "An unusually high incidence of salt-losing congenital adrenal hyperplasia in the Alaskan Eskimo." in: The Journal of pediatrics, Vol. 75, Issue 3, pp. 492-4, 1970 (PubMed).

Hofman, Klaniecki, Smith: "Direct solid-phase radioimmunoassay for screening 17 alpha-hydroxyprogesterone in whole-blood samples from newborns." in: Clinical chemistry, Vol. 31, Issue 7, pp. 1127-30, 1985 (PubMed).

Hughes, Riad-Fahmy, Griffiths: "Plasma 17OH-progesterone concentrations in newborn infants." in: Archives of disease in childhood, Vol. 54, Issue 5, pp. 347-9, 1979 (PubMed).

Catalog No. ABIN649073

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