CHEMISTRY REAGENT KITS
                        ------->TOTAL PROTEIN (Biuret) End-Point

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CLINICAL SIGNIFICANCE
Decreased levels are associated with malnutrition and hypoalbumlnaemla. Increased levels are observed in dehydration and hyper-globulinaemia.

PRINCIPLE
In the presence of alkaline cupric sulphate, the proteins produce a VIOLET colour. Intensity of the colour is proportional to protein concentration.

REAGENTS SUPPLIED
Cat.No.A190

Composition

1. Biuret Reagent 100mL
    Cupric sulphate 6mMols/L
    Sodium and Potassium     tartarate 21mMols/L
    Sodium Hydroxide 750mMols/L
    Potassium lodide 6 mMols/L
3.Standard (Albumin) 2.5 mL
    Value of Albumin     Standard 8.0Gms/dL

Preparation of Working Reagent
All reagents are ready to use.

STABILITY
Biuret (Reagent No. 1)
18 months at 18-25°C away from light. Protein Standard (Reagent No.3) 18 months at 2-8°C. Do not freeze the Standard.

SAMPLE
Serum or plasma which has no sign of haemolysis. Common anticoagulants have no interference on this assay. Avoid haemolysed serum or plasma. (All samples should be handled as potential infective agents as no laboratory methods make conclusive findings for its safety. Therefore, adequate protective laboratory measures should be taken while handling such materials).

SYSTEM PARAMETERS

Reaction End Point
Temperature 30° + 5°C
Wavelength 550 + 20nM
Standard Concentration 4.0 Gms/dL
Absorbance Range 0-2°A
Cuvette Path Length 1 cM
Reagnet Volume 1000µL 500µL
Sample Volume 50µL 20µL
Reaction Time 10 Mins at RT/5 Mins at37 C
Protein Standard Value 8.0Gms/dL
Linearity 15.0Gms/dL
Max. limit of blank rgt. 0.4°A
Final Colour Stability 30 Minutes

MANUAL METHOD
1
    Pipette into 3 Test Tubes B
(ml)
S
(ml)
T
(ml)
    Reagent No. 1 3.0 3.0 3.0
    Standard Reagent No 3 - 0.1 -
    Sample - - 0.1

2.Mix well Incubate for 10 minutes at RT or 5 minutes at 37C.
3.Read at 550 nM (550+20 nM) or GREEN filter against Blank.
4.The final colour is stable for approximately 30 minutes.

MICRO METHOD FOR DISCRETE ANALYSERS
Pipette into clean dry test tubes labeled as Blank (B), Standard (S), and Test (T)
    For Cuvette Volume 1.0mL 0.5mL
    Reagent 1 1000µl 500µl
    Sample or Standard 50µl 20µl

Mix well. Incubate at 37C for 5 minutes or at RT for 10 minutes. Read at 550 nM (550+20 nM) against blank. The final colour is stable for apx. 30 mins.
NOTE : Progrmame the analyser using system parameters. A specific programme data sheet may be provided for each analyser upon request.

RESULTS
Compute   O.D.Test
Total Proteins in Gms/dL=        O.D.STD   xC
*C = Concentration of Standard as printed on
label.

This kit contains two different standards for Albumin and Proteins assays. Reagent No. 3 Standard for Total Proteins only. Reagent No. 4 Standard for Albumin only. MAKE SURE  THE STANDARD  USED IS APPROPRIATE BEFORE REPORTING THE RESULTS.

EXPECTED VALUES
6.5to7.5Gms/dL
As with all diagnostic methods, the final diagnosis should not be made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations

LINEARITY
Colour development gradually increases on prolongation of time more than 30 minutes specified in the procedure. This assay is linear up to 15. 0Gms/dL proteins. For values higher than 15.0 Gms/dL repeat the test with serum diluted in 0.9% sodium chloride solution. Multiply the results by the dilution factor applied i.e. multiply by 2 for a 1:1 dilution.
NOTE: This method is not useful for Microprotein/ C.S.F.Protein assay. (For Microprotein  assay,  BIOLAB  offers C.S.F.ProteinKit-Turbidometry method).

QUALITY CONTROL
To ensure adequate quality control, each kit should be tested against a standard control sera. It should be realised that the use of quality control material checks both instrument and reagent function together. Factors which might affect the performance of this test include proper instrument function, temperature control, cleanliness of glasswares and accuracy of pippetting. It is appropriate to establish each laboratory's accuracy constant and interpretvalues accordingly.

WARNING
This reagent system is for in vitro use only. This reagent system is contains preservatives and components that have not established for safety if contacted on broken skin or eye or taken orally. In case of such incidents wash off with plenty of water, or consulta physician.

BIBLIOGRAPHY
GORNALL A.C., BARDAWILL C.J., DAVID M.M. -J.Biol.Chem., 1949,177,751. N. TIETZ, Fundamentals of Clin. Chem., W.B. Saunders Company, Philadelphia P.A.