MICROFLUORALä Microalbumin Test
A Quick, Quantitative, Fluorescence Test for the Detection of Microalbuminuria
Size of the Kit: 5 x 96 Determinations
Storage: 2 - 8° C
1. Clinical Significance
Persistent microalbuminuria, defined as a urinary excretion rate of 20 - 200 micrograms/ml, indicates a high probability of damage of the glomerular filtration capacity of the kidney and is of great diagnostic relevance
· in diabetic patients for early diagnosis of nephropathy
· in hypertensive patients as indicator of end-organ damage associated with lower life expectancy,
· in pregnancy as predictor of delivering preeclampsia, and
· is probably also associated with cardiovascular disease in non-diabetic subjects.
All patients with potential disease involvement of the kidneys should be screened for early diagnosis.
Microfluoral test is a quick low-cost method with high accuracy for rapid quantification of microalbuminuria in large sample numbers.
3. Test Principle
The assay is based on Albumin Blue 580 (AB580) and performed in a 96-well microplate and measured in a Fluorescence Microplate Reader at 590-600 nm excitation and 630-645 nm emission. AB580 binds to albumin by forming a strongly fluorescent complex without interfering with urinary proteins or frequently used drugs. Sample and reagent are pipetted into the wells of a microplate. The plate can be measured without further incubation. Fluorescence intensity directly reflects albumin concentration. A set of standards gives an almost linear standard curve from 0 to 200 mg/l. Detection limit is 2mg/l albumin.
4. Material Required
· Precision pipettes
· Sterile pipette tips
Microplate mixer or 8-channel multipipette
· Fluorescence microplate reader with excitation filter 590-600 nm and emission filter 630-645 nm
5. Contents of Test Kit
12x8-well microplates, 5 pcs.
P Positive control, 500 µl (ready-to-use). Contains human albumin*.
S1 Human albumin* standard 200 mg/l, 500 µl (ready-to-use).
S2 Human albumin* standard 100 mg/l, 500 µl (ready-to-use).
S3 Human albumin* standard 30 mg/l, 500 µl (ready-to-use).
S4 Human albumin* standard 2 mg/l, 500 µl (ready-to-use).
Dil Dilution buffer**, 2x 50 ml
Dye AB580 concentrate*** (50x), 2.5 ml
* Human albumin was found negative for HIV I und II, Hepatitis B and C. Nevertheless, standards and control should be handled as potentially infectious material.
** Standards, control and buffer contain sodium azide as preservative.
*** Contains Isopropanol, flammable and irritant.
6. Specimen and Sample Storage
Random urine is the simplest approach to obtaining a specimen for analysis, but gives the most variable results. The preferred specimen is 24-hr urine without preservative. The albumin measurement is multiplied by the volume expressed in L to obtain mg albumin excreted in 24 hr. Refrigerated samples are stable for up to two weeks. Samples may be stored at -20°C for up to 6 months.
Warm up kit to room temperature. Just before use prepare the amount of AB580 working reagent necessary and mix well:
For 1x 8-well microplate strip: 30µl AB580- concentrate ad 1500µl dilution buffer
For 1x 96well microplate: 360 µl AB580 concentrate ad 18ml dilution buffer
Attention: Use polypropylene vials only!
7. Test Procedure
1. Place 25 µl standard S1 to S4, positive control and specimen in appropriate wells of the microplate.
2. Add 150µl AB580 working reagent.
3. Mix well, e.g. by suspending with an 8-channel multipipette or by mixing in a microplate mixer.
4. Measure fluorescence at 590-600 nm excitation and 630-645 nm emission in a fluorescence microplate Reader.
9. Calculation and Interpretation of Results
Read Relative Fluorescence Counts (RFC) of specimen from the standard curve.
Example of a standard curve:
With hemolytic specimen slightly lower relative fluorescence counts may be found. This test is intended for measuring microalbuminuria only.
(1) Microalbuminuria, a marker for organ damage, C. E. Morgensen (ed.), Science Press, London. (1993).
(2) Mathiesesen E. R., Ronn B., Jensen T., Storm B., Deckert T.; Relationship between blood pressure and urinary excretion in the development of microalbuminuria. Diabetes 1990, 39: 245-9.
(3) Kessler, M.A., Hubmann, M.R., Dremel, B.A. & Wolfbeis, O.S. Non-immunological assay of urinary albumin based on laser-induced fluorescence (1992). Clin. Chem. 38: 2089-92
FR 94/44 DU 02-11-94
(5) Kessler, M.A., Meinitzer, A., Petek, W. & Wolfbeis, O.S. (1996). Determination of microalbuminuria and borderline elevated albumin excretion using the AB580 fluorescence assay on the Roche Cobas FaraäII. Clin. Chem., in press.
(6) Kessler, M.A., Meinitzer, A., Petek, W. & Wolfbeis, O.S. (1996). Microplate fluorescence assay for determination of microalbuminuria. Eur Clin Laboratory, Febr. 1996.
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