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Retinol Binding Protein (RBP) Urine

The DetectX® Urinary Retinol Binding Protein (RBP) kit is designed to measure RBP present in urine samples.

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Summary

The DetectX® Urinary Retinol Binding Protein (RBP) kit is designed to measure RBP present in urine samples. Please read the complete kit insert before performing this assay. A RBP standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. Standards or diluted samples are pipetted into a clear microtiter plate coated with an antibody to capture rabbit antibodies. A RBP-peroxidase conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of the RBP polyclonal antibody to each well. After an hour incubation the plate is washed and substrate is added. The substrate reacts with the bound RBPperoxidase conjugate. After a short incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450 nm wavelength. The concentration of the RBP in the sample is calculated, after making a suitable correction for the dilution of the sample, using software available with most plate readers

Test Principle
  1. Use the plate layout sheet on the back page of the kit insert to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to foil pouch with desiccant. Seal the ziploc plate bag and store at 4°C.
  2. Pipet 50 µL of samples or standards into wells in the plate. Pipet 75 µL of Assay Buffer into the non-specific binding (NSB) wells. Pipet 50 µL of Assay Buffer into wells to act as maximum binding wells (Bo).
  3. Add 25 µL of the DetectX® RBP-peroxidase conjugate to each well, using a repeater pipet.
  4. Add 25 µL of the DetectX® RBP Antibody solution to each well, except the NSB wells, using a repeater pipet.
  5. Gently tap the sides of the plate to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and shake at room temperature for 1 hour.
  6. Aspirate the plate and wash each well 4 times with 300 µL wash buffer. Tap the plate dry on clean absorbent towels.
  7. Add 100 µL of the TMB Substrate to each well, using a repeater pipet.
  8. Incubate the plate at room temperature for 30 minutes without shaking.
  9. Add 100 µL of the Stop Solution to each well, using a repeater pipet.
  10. Read the optical density generated from each well in a plate reader capable of reading at 450 nm.
  11. Use the plate reader’s built-in 4PLC software capabilities to calculate RBP concentration for each sample.
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References
  1. Blaner WS. “Retinol binding protein: the serum transport protein for vitamin A.” Endocr Rev. 1989 Aug;10(3):30816.
  2. Wolf G. “Multiple functions of vitamin A.” Physiol. Rev. 1984 Jul;64(3):873-937.
  3. Petersen PA. “Characteristics of a Vitamin A-transporting Protein Complex Occuring in Human Serum.” J. Biol. Chem. 1971, 246:34-43.
  4. Goodman DS, Blaner WS. in The Retinoids, eds. Sporn MB, Roberts AB, Goodman DS, (Orlando: Academic Press, 1984) vol.2, 1-39.
  5. Olson, JA. “Vitamin A, retinoids and carotenoids.” Modern Nutrition in Health and Disease, eds. Shils ME, Olson JA, Shike M, (Philadelphia: Lea & Febiger, 1994) 8th ed. 287-307.
  6. Peterson PA, and Berggard I. “Isolation and Properties of a Human Retinol-transporting Protein.” J. Biol. Chem. 1971 246:25-33.
  7. Bernard AM, and Lauwerys RR. “Retinol Bining Protein in Urine: A More Practical Index than Urinary B2microglobulin for the Screening of Renal Tubular Function.” Clin. Chem. 1981 27:1781-2.
  8. Camara NO, Matos AC, Rodrigues DA et al. “Early Detection of Heart Transplant patients with Increased Risk of Cyclosporin Nephrotoxicity.” The Lancet 2001 357: 856-857.
  9. Hosaka B, Park SI, Felipe CR, Garcia RG, Machado PG, Pereira AB, Tedesco-Silva H and Medina-Pestana JO. “Predictive Value of Urinary Retinol Binding Protein for Graft Dysfunction after Kidney Transplantation.” Transplant Proc. 2003 35:1341-1343.
  10. Abahusain MA, Wright J, Dickerson JWT and de Vol EB. “Retinol, a-Tocopherol and Carotenoids in Diabetes.” Am J Clin Nutr. 2004;79:218-25.
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