BIOLABO
www.biolabo.fr TOTAL PROTEIN Biuret Method
MANUFACTURER:
BIOLABO SAS,
Les Hautes Rives Reagent for quantitative determination of total protein in human serum or plasma
02160, Maizy, France
REF 80016: R1 1 x 500 mL R2 1 x 500 mL
R3 1 x 5 mL
|
TECHNICAL SUPPORT AND ORDERS
Tel : (33) 03 23 25 15 50
IVD IN VITRO DIAGNOSTIC USE
Fax: (33) 03 23 256 256
CLINICAL SIGNIFICANCE (1) REAGENTS PREPARATION
The overall composition of a patient’s plasma or serum should be Fill to the top of the vial with demineralised water to complete content
studied first by determining its total protein content and then its of vial R1 (NaCl) and vial R2 (Biuret). Mix by swirling. Diluted reagents
composition by electrophoresis. are ready for use.
Decrease in the volume of plasma water (hemoconcentration), noted in
dehydratation (severe vomiting, diarrhea, Addison’s disease, or
diabetic acidosis), is reflected as relative hyperproteinemia. STABILITY AND STORAGE
Hemodilution (increase in plasma water volume) occuring with water On receipt, store Standard (vial R3) at 2-8°C.
intoxication or salt retention syndromes, during massive intravenous • Standard (vial R3): transfer the requested quantity, well recap the
infusions, and physiologically when a recombant position is assumed, vial and store at 2-8°C.
is reflected as relative hypoproteinemia. Hypoproteinemia due to low
• Stored and used as described, reagents (vial R1, R2, R3) are stable
levels of albumine in plasma is also common and has many causes.
in well recapped original vial, and without contamination, upon expiry
Mild hyperproteinemia may be caused by an increased in the
date stated on the label of the kit.
concentration of specific proteins (infection). Marked hyperproteinemia
may be caused by high levels of monoclonal immunoglobulins • Once opened, working reagents (ready for use) are stable stored at
produced in multiple myelomia and other malignant paraproteinemias. 18-25°C and away from light at least for 6 months.
Discard any reagent if cloudy or if the absorbance of the prediluted
PRINCIPLE (4) (5) mixture (1V/1V) of vials R1 and R2 is > 0.050 at 550 nm.
Colorimetric method described by Gornall and al. The peptide bonds of Don’t use working reagents after expiry date stated on the label of the
proteins react with Cu2+ in alkaline solution to form a coloured complex kit.
which absorbance, proportional to the concentration of total protein in
the specimen, is measured at 550 nm. The biuret reagent contains
sodium potassium tartrate to complex cupric ions and maintains their
SPECIMEN COLLECTION AND HANDLING (2)
solubility in alkaline solution. Serum or plasma. Analyse fresh or store at 2-8°C less than 72 h.
Total protein in serum is stable for:
REAGENTS 6 months at –20°C
indefinitely at -70°C.
Vial R1 SODIUM CHLORIDE
Sodium chloride 75 mmol/L
INTERFERENCES (3)
Vial R2 BIURET REAGENT Tests results with Procedure n°1:
Sodium hydroxide 370 mmol/L
Glucose: No interference up to 11 g/L
Na-K Tartrate 10 mmo/L
Potassium iodide 3 mmol/L Ascorbic Acid: No interference up to 250 mg/L
Copper II sulfate 3 mmol/L
Before dilution: Corrosive, R35: Causes severe burns Total Bilirubin: No interference up to 550 µmol/L
Once diluted: Xi, R36/37/38: Irritating to eyes, respiratory system and skin.
S36/37/39: Wear suitable protective clothing, gloves and eyes/face Haemoglobin: Positive interference above 150 µmol/L
protection
Lipemia: Positive interference above 0.150 abs
Vial R3 (measured at 600nm)
STANDARD Bovine Albumin 6 g/dL
Lipemia or hemolysis may cause falsely elevated results. It is
SAFETY CAUTIONS recommended to perform a specimen blank to prevent these
BIOLABO reagents are designated for professional, in vitro diagnostic interferences (see § MANUAL PROCEDURE: Procedure n°2).
use. For a more comprehensive review of factors affecting this assay refer
to the publication of Young D.S.
• Verify the integrity of the contents before use.
• Use adequate protections (overall, gloves, glasses).
• Do not pipette by mouth. MATERIALS REQUIRED BUT NOT PROVIDED
• Contents of vial R2 remains irritating after dilution (R34: causes 1. Basic medical analysis laboratory equipment.
burns). 2. Normal and pathological control sera.
• In case of contact with skin and eyes, thoroughly wash affected
areas with plenty of water and seek medical advice.
• Reagents contain sodium azide (concentration < 0.1%) which may
react with copper and lead plumbing. Flush with plenty of water
when disposing.
• Material Safety Data Sheet is available upon request.
• Waste disposal: Respect legislation in force in the country.
All specimens should be handled as potentially infectious, in
accordance with good laboratory practices using appropriate
precautions. Respect legislation in force in the country.
Made in France Latest revision : www.biolabo.fr Revision : 26/07/2011
CALIBRATION (6) MANUAL PROCEDURE
• Standard (vial R3) enclosed in the Kit or BIOLABO Multicalibrator,
Procedure n°1 (without Specimen blank)
REF 95015 traceable to SRM927d.
Let stand reagents and specimens at room temperature.
• Or any calibrator traceable to a reference method or material.
The calibration frequency depends on proper instrument functions and Pipette into well identified test
Blank Standard Assay
on the preservation of the reagent. tubes:
It is recommended to calibrate in the following cases: Reagent R1 1,02 mL 1 mL 1 mL
1. When changing vial of reagent.
2. After maintenance operations on the instrument.
3. When control values are out of range, even after using a new vial of Reagent R2 1 mL 1 mL 1 mL
fresh serum.
Standard 20 µL
QUALITY CONTROL
• BIOLABO EXATROL-N Level I REF 95010. Specimen 20 µL
• BIOLABO EXATROL-P Level II REF 95011. Mix well. Let stand for 10 minutes at room temperature.
• Other assayed control sera referring to the same method. Record absorbance at 550 nm (530-570) against reagent blank.
• External quality control program.
It is recommended to control in the following cases: Procedure n°2 (with Specimen blank)
• At least once a run.
• At least once within 24 hours. Pipette into well
Blank Specimen Standard Assay
• When changing vial of reagent. identified test
Blank
• After maintenance operations on the instrument. tubes
If control is out of range, apply following actions:
Reagent R1 1,02 mL 2 mL 1 mL 1 mL
1. Repeat the test with the same control.
2. If control is still out of range, prepare a fresh control serum and
repeat the test. Reagent R2 1 mL 1 mL 1 mL
3. If control is still out of range, use a new vial of calibrator or a fresh
calibrator and repeat the test. Standard 20 µL
4. If control is still out of range, calibrate with a new vial of reagent.
5. If control is still out of range, please contact BIOLABO technical Specimen 20 µL 20 µL
support or your local Agent.
Mix well. Let stand for 10 minutes at room temperature.
EXPECTED VALUES (2) Record absorbance at 550 nm (530-570) against reagent blank. Read specimen
blank against reagent R1.
In serum or plasma
Total Protein (g/dL) Notes:
Specific procedures are available upon request for automated
In cord 4.8-8.0 instruments. Please contact BIOLABO technical support.
Premature 3.6-6.0 Specimen blank is recommended for cloudy, lipemic or hemolysed
Newborn 4.6-7.0 serum.
1 week 4.4-7.6 Caution: Target values of control sera or multicalibrator may have
been obtained with or without specimen blank.
7 months-1 year 5.1-7.3 Bichromatic analyser: The 2nd wavelength is 600 or 700 nm.
1 year-2 years 5.6-7.5
> 3 years 6.0-8.0
CALCULATION
Adult, ambulatory 6.4-8.3
Adult, recombent 6.0-7.8 Calculate the result as follows:
> 60 years Lower by 0.2 Without specimen blank:
Each laboratory should establish its own normal ranges for the Abs (Assay)
Result = x Standard concentration
population that it serves.
Abs (Standard)
PERFORMANCES With specimen blank:
Within run Low level Normal Between run Low level Normal level Result = Abs (Assay)- Abs (Specimen blank) x Standard concentration
n = 20 level n = 20 Abs (Standard)
Mean g/dL 4.88 5.45 Mean g/dL 6.92 7.45
S.D. g/dL 0.06 0.06 S.D. g/dL 0.11 0.13 REFERENCES
rd
C.V. % 1.2 1.2 C.V. % 1.6 1.7 (1) TIETZ N.W. Text book of clinical chemistry, 3 Ed. C.A. Burtis, E.R.
Ashwood, W.B. Saunders (1999) p. 477-530.
Detection limit: approximately 0.21 g/dL (2) Clinical Guide to Laboratory Test, 4th Ed., N.W. TIETZ (2000) p. 916-917.
Sensitivity for 1 g/dL: approximately 0.028 Abs. at 550 nm. (3) YOUNG D.S., Effect of Drugs on Clinical laboratory Tests, 4th Ed. (1995) p.
3-498 à 3-511
Comparison study with commercially available reagent (Biuret method) (4) GORNALL A. C., BARDAWILL C. J., DAVID M. M., J. Biol. Chem. 1949,
93 specimens (sera) within 3 g/dL and 11 g/dL are assayed with 2 177, 751
(5) TIETZ N.W. Text book of clinical chemistry, 3d Ed. C.A. Curtis, E.R.
methods Silverman L . M., Christensen R. H. (1995) p. 523-524.
Y (g/dL) = 0.9758 x + 0.14819 r = 0,9879 (6) SRM: Standard reference Material ®
LINEARITY
The assay is linear up to 10 g/dL. Above, dilute the specimen with
saline solution and reassay taking into account dilution factor to
calculate the result. Linearity limit depends on specimen/reagent ratio
IVD REF LOT →
Manufacturer Use by In vitro diagnostic Temperature limitation Catalogue number See insert Batch number Store away from light sufficient for dilute with
Made in France Latest revision : www.biolabo.fr Revision : 26/07/2011
