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.
2.
Application
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:
<20
mg/l
<30 mg/die |
normal
no indication of microalbuminuria |
20-200
mg/l
30-300 mg/die |
pathological:
microalbuminuria |
> 200
mg/l
> 300 mg/die |
albuminuria
|
10. Limitations
With hemolytic specimen slightly lower
relative fluorescence counts may be found. This test is intended for measuring
microalbuminuria
only.
11.
References
(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
(4)
US
5,182,2314
EP 0,413,678
DE
590-07-613.2-08
FR 94/44 DU
02-11-94
UK 15-4-41899
JP
Hei-2-214459
(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.
Research
Diagnostics Inc
San Jose, 95123 CA Snell ave 658
USA
or 408-780-0908
email: margaret@cellular-research.com
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