Microalbumin ELISA. For the quantitative determination of albumin in human urine. For In Vitro Diagnostic Use.
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1 Microalbumin ELISA For the quantitative determination of albumin in human urine. For In Vitro Diagnostic Use. Catalog Number: Size: 24-MABHU-E01 96 Wells Version: ALPCO January 17, 2018 Page 1 of 9
2 I. INTENDED USE The Microalbumin ELISA test kit is used to measure albumin in human urine. For In Vitro Diagnostic Use. II. INTRODUCTION Under normal physiological conditions very little albumin is present in the urine (1). Approximately 99% of the filtered albumin is reabsorbed in the proximal tubule. In pathologic conditions when glomerular capillary wall permeability and/or filtration rate increase, albumin excretion along with other macromolecules in urine also increases (2). On the other hand, when proximal tubular reabsorptive capacity decreases, excretion of smaller macromolecules such as beta-2 microglobulin and lysozyme in urine increases. Thus, determining the clearance ratio of urinary albumin to beta-2 microglobulin can help localize the site of renal impairment (3). Detection of increased urinary albumin excretion is of particular importance in the study of incipient renal disease (4). Thus, a sensitive method of measuring minor increases in urinary albumin is of great significance to a) detect minimal renal impairment and b) to study its progression in developing end stage renal disease and cardiovascular disease amongst diabetics (5). A recent consensus report by Centers for Disease Control and the National Institute of Diabetes and Digestive and Kidney diseases recommended that albumin in the urine amongst diabetics be routinely monitored (6). Although microalbuminuria is tested primarily in diabetes mellitus research, it has also been used in studies of hypertension, pregnancy (preeclampsia, maternal morbidity, and fetal mortality), non-diabetic renal disease, and the renal effects of various drugs, hormones, and nephrotoxins (7-10). III. PRINCIPLE The Microalbumin assay procedure is a competitive ELISA in which enzyme labeled albumin competes with albumin in samples for limited antibody binding sites on the microplate. After incubation for a fixed time, separation of bound from free is achieved by simple decantation and plate washing. The enzyme activity on the plate is measured using an enzyme substrate and a chromogen. The absorbency of the color developed is read in an ELISA colorimetric reader. The measured absorbance is inversely proportional to the concentration of bound albumin in the microplate. The concentration of albumin in the urine is determined from a calibration curve. IV. WARNING AND PRECAUTIONS Materials contained within the kit are for in vitro diagnostic use only and are not for internal or external use in animals or humans. Some of the reagents in this kit contain Merthiolate. Merthiolate may be toxic if ingested. Care should be taken to avoid ingestion. Some of the reagents in this kit contain sodium azide. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide accumulation. Sodium azide is also toxic. Care should be taken to avoid ingestion. This kit contains components (urine) of human origin. Handle the kit and all samples as if capable of transmitting infection. V. REAGENTS AND MATERIALS Materials supplied in this kit are sufficient for 96 determinations. Calibrators: Page 2 of 9
3 Catalog nos , 12-12, 12-13, 12-14, and Buffered reagent containing five different concentrations of albumin. Concentration is indicated on the labels (approximately 0-50 µg/ml). Sodium azide (0.01%) added as preservative. Zero calibrator 4 ml, other calibrators 0.5 ml per vial. Controls: Catalog nos and Human urine containing two different concentrations of albumin. Values are listed on the vials. Sodium azide (0.01%) added as preservative; 0.5 ml per vial. Albumin-Enzyme conjugate reagent: Catalog no Buffered reagent containing albumin-horse radish peroxidase enzyme conjugate; 0.01% Merthiolate added as preservative; 5 ml. Albumin Antiserum coated microplate (96 wells): Catalog no well microplate containing goat anti-human albumin antibody adsorbed, packaged in a ziplock bag. 1 plate. This plate consists of twelve removable strips mounted in a frame. Each strip includes eight wells. TMB Peroxide solution: Catalog no ; buffered reagent containing tetramethylbenzidine and peroxide; Merthiolate (0.0 1%) added as a preservative; 5 ml. Stop solution: Catalog no One vial containing stop solution; 10 ml. VI. STORAGE AND STABILITY Store the test kit in the refrigerator (2-8 o C) and protect from moisture. Do Not freeze. Reagents are stable until the expiry date shown on kit label. VII. SAMPLE COLLECTION An accurately timed (3 hour, overnight, or 24 hour) and measured urine collection is required so that the albumin can be expressed as albumin excretion rate (µg/min.). Due to alterations in renal hemodynamics, urine should not be collected during heavy exercise or prolonged upright position. The urine should not be acidified or any additive added to it. Store urine samples at 2-8 C for up to two weeks. Do not freeze. If urine samples appear turbid, clarify by centrifugation. Do not assay any urine sample with blood contamination (11-13). VIII. TEST PROCEDURE Materials supplied: Material Quantity Catalog No. Calibrator 1 One vial Calibrator 2 One vial Page 3 of 9
4 Calibrator 3 One vial Calibrator 4 One vial Calibrator 5 One vial Control I One vial Control II One vial Enzyme conjugate reagent One vial Antiserum Coated Microplate (96 wells) One plate TMB Peroxide solution One vial Stop solution One vial Materials required but not supplied: EIA microplate reader capable of reading at 450 nm Refrigerator (2-8 C) Distilled or deionized water ul precision pipet with disposable tips Volumetric pipet, 10 ml Container, 10 ml Assay Procedure: All components and samples must be at ambient temperature before use. 1. Prepare a plate with enough 8-well strips to assay calibrators, controls, and samples in duplicate. 2. It is preferable that TMB Peroxide solution not be pipetted directly from the vial because the solution can be easily contaminated with carryover of HRP conjugate into the vial. It is therefore suggested that the desired quantity is transferred to a clean test tube and pipetted out of that. Discard any solution that is left over. 3. Pipet 10 ul of each calibrator, control, and unknown sample to the appropriate wells. 4. Add 50 ul diluted Albumin-Enzyme conjugate reagent to every well. 5. Mix well by a slow shaking of the wells for a few seconds and incubate for 30 minutes at room temperature. 6. After the incubation, add distilled or deionized water to the wells and decant. Repeat this procedure four more times. Tap the wells gently and blot the rims to remove all residual droplets. 7. Add 50 ul TMB Peroxide solution to each well. 8. Mix well by a slow shaking of the wells for a few seconds and incubate for 15 minutes at room temperature. 9. Add 100 ul Stop solution to each well to stop the color development. Mix well by a slow shaking of the wells for a few seconds. 10. Determine the absorbance for all the wells in an ELISA colorimetric analyzer at 450 nm against air blank within 5 minutes of the addition of the Stop solution, with a reference wavelength approximately 620 nm. Page 4 of 9
5 Table 1. Assay Protocol Summary Tube # Maximum binding (B0) Calibrator/ Control/ Unknown (µl) Enzyme Conjugate (µl) TMB Peroxide (µl) Stop Soln. (µl) 1,2 Calibrator ,4 Calibrator ,6 Calibrator ,8 Calibrator ,10 Calibrator ,12 Control ,14 Control ,16 Unknown Pipet 10 ul Calibrator, Control, or unknown sample into the wells, in duplicate - Add 50 ul diluted Enzyme conjugate reagent - Incubate 30 minutes at room temperature - Decant and wash 5 times - Add 50 ul TMB Peroxide solution - Incubate 15 minutes at room temperature - Add 100 ul Stop Solution and read absorbance within 5 minutes Calculation of Results: 1. Use the data reduction program (logit-log) in the reader to determine the albumin concentration in the unknown sample. 2. Alternatively, calculate percent conjugate bound (%B) for each calibrator, control, and sample relative to the maximum binding (B0, Zero calibrator) wells as follows: %B/B0= Absorbance (calibrator, control, or unknown) Absorbance (blank) Absorbance (Zero calibrator) Absorbance (blank) x 100 Using logit-log graph, plot % bound on the vertical axis against concentration on the horizontal axis for each of the calibrators and draw a smooth line through the points. Albumin concentrations for the unknown may then be estimated from the line by interpolation. Albumin excretion in urine is more reliability expressed as albumin excretion rate. Determine the total volume of accurately timed urine. Convert µg albumin/ml to µg albumin/min. by: Albumin (µg) x Total Volume (ml) 1 ml x Total Time (min.) Quality Control: The reliability of test results should be monitored by the routine use of control reagents of known albumin concentrations. Two quality control pools are supplied with the kit and should be analyzed with each assay. The results should be charted from assay to assay and the overall performance checked periodically. Page 5 of 9
6 IX. LIMITATIONS Samples with greater than 50 µg/ml should be diluted with Zero calibrator and reassayed. Hematuria or blood contaminated samples will interfere with the assay. Acidified urine samples or sample with significant bacterial growth may give false results. Frozen samples may give lower values. Samples stored for more than two weeks may give lower values. Urine collected during heavy exercise or prolonged upright position may give false results. Typical Standard Curve Well No. Concentration Average Absorbance % B/B0 1, , , , , Control I Control II X. PERFORMANCE CHARACTERISTICS Precision: The reliability of the Microalbumin ELISA test kit procedure was assessed by examining its reproducibility on samples selected to represent a range of albumin levels. a. Intra-assay: Within assay variation was determined from eight single determinations of albumin in three different sample pools. Data are summarized below: No. of Determination Mean Value SD %CV b. Inter-assay: Between assay variation was determined from six separate assays in eight single determinations of albumin on three sample pools. Data are summarized below: No. of Determinatio n Mean Value SD %CV Page 6 of 9
7 Sensitivity: Ten Zero calibrator (maximum binding) wells were processed along with a calibration curve. Mean and standard deviation were calculated for the absorbency of the ten Zero calibrator wells. Apparent sensitivity was then calculated from the average of 2, 3, 4, and 5 standard deviations and was found to be 0.24 µg/ml. Spiking Recovery: Recovery studies were done by spiking four samples with different concentrations of albumin. The results are summarized below: Base line Amount added Amount recovered % Recovery A B C D Parallelism: Five samples were serially diluted with the kit s Zero calibrator. The observed and expected values are summarized below: Sample Dilution Observed Expected %Recovery A : : : : : : B : : : Page 7 of 9
8 1: C : : : : : D : : : E : : : : Specificity: The antiserum is highly specific for human albumin. No cross-reactivity to bovine serum albumin, myoglobin, hemoglobin, and alpha fetoprotein was noted at significantly high concentrations. Method Comparison: The Microalbumin ELISA procedure was compared to an RIA procedure of a commercial microalbumin kit on 53 samples. The concentration of the samples range from 0.1 to 62.5 mg/ml. Linear regression yielded the following statistics: y (commercial kit) = x (Microalbumin ELISA) covariance: correlation:0.961 R-squared:0.923 XI. EXPECTED VALUES Normal range study was conducted on 29 human timed urine samples using the Microalbumin ELISA test kit. The following results were obtained: Mean: mg/min Range: mg/min The normal range limits suggested by this study should be regarded as guidelines only. It is important that each laboratory establishes its own normal range to conform with the characteristics of the population that is being tested. XI. REFERENCES 1. Mogensen, C.E.: N Engl J Med. 310, 356, Mogensen, C.E. and Christensen, C.K.: N Engl J Med. 311, 89, Ratge, D. and Wisser, H.: J Chromatography. 230, 47, Mogensen, C.E.: Kidney Int. 31, 673, Bernard, A. and Lauwerys, R.: Toxicol Lett. 46, 293, Page 8 of 9
9 6. Herman, W., Hawthorne, V., Hamman, R., Keen, H., De Fronzo, R., Newman, J., De Stefano, F., Striker, G., Hirschman, G., Agodoa, L.: Am J Kidney Dis. 13, 2, Parving, H.H., Mogensen, C.E., Jensen, H.E., and Ervin, P.E.: Lancet. 1, 1190, Gosling, P. and Beevers, D.G.: Clin Sci. 76, 39, Yudkin, J.S., Forrest, R.D., and Jackson, C.A.: Lancet. 2, 530, Lauwerys, R.R. and Bernard, A.M.: Br J Ind Med. 43, 433, Hutchison, A.S. and Paterson, K.R.: Diabetic Med. 5, 527, Vittinghus, E. and Mogensen, C.E.: Kidney Int. 21, 725, Elving, L.D., and Bakkeren, J.A.M., Jansen, M.J.H., de Kat Angelino, C.M., de Nobel, E., and Van Munster, P.J.J.: Clin Chem. 35, 308, Page 9 of 9
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