TOTAL PROTEIN (Biuret Method)
INTENDED USE:
The reagent kit is intended for in vitro quantitative determination of Total Protein in serum/plasma.
CLINICAL SIGNIFICANCE:
Proteins are conjugations of amino acids, enzymes, hormones and several other kinds of molecules, structural entities in the body. They are involved in the maintenance of the normal distribution of water between blood and the tissues. Considering many of albumin and globulin in the blood has many diagnostic and analytical values as several fractions are involved in many (diseases). Decreased levels are found mainly in malnutrition, hepatic synthesis, protein losses as in hemorrhage or excessive protein catabolism.
PRINCIPLE:
Proteins, in an alkaline medium, bind with the cupric ions present in the biuret reagent to form a blue-violet colored complex. The intensity of the color formed is directly proportional to the amount of proteins present in the sample.
REACTION:
Total Protein + Cu²⁺ → Violet complex.
CONTENTS:
Reagent 1: Biuret Reagent
Reagent 2: Protein Standard 6 g/dl
MATERIALS REQUIRED BUT NOT PROVIDED:
Clean Dry Glassware
Laboratory Glass Pipettes or Micropipettes & Tips
Colorimeter or Bio-Chemistry Analyzer
SAMPLES:
Serum, Heparinized/EDTA Plasma. Proteins are reported to be stable in the sample for 6 days at 2–8°C.
PREPARATION OF REAGENT & STABILITY:
All reagents are stable till the expiry date mentioned on the label at standard room temperature.
If stored at 2–8°C, this is stable till expiry when not in use.
All reagents are ready to use form.
GENERAL SYSTEM PARAMETERS:
Wavelength: 546 nm (530–570 nm)
Temperature: 25°C
Light Path: 1 cm
Assay Type: End Point
Reaction Time: 5 min
Standard Concentration: 6 g/dl
Zero setting: Reagent Blank
Sample Volume: 10 µl
Reagent Volume: 1.0 ml
PROCEDURE:
Pipette into clean dry test tube labeled as Blank (B), Standard (S) and Test (T):
| Addition sequence | Blank (B) | Standard (S) | Test (T) |
|---|---|---|---|
| Biuret Reagent | 1.0 ml | 1.0 ml | 1.0 ml |
| Distilled Water | 10 µl | — | — |
| Standard | — | 10 µl | — |
| Sample | — | — | 10 µl |
Mix and incubate for 5 minutes at Room Temperature. Measure the absorbance of Standard and Sample (Aₛ) against the Blank (Aᵦ) at 546 nm.
CALCULATION:
Total Protein Conc. (g/dl) =
(Aₛ / Aₛₜ) × 6
NORMAL VALUE:
Serum: 6.0–8.0 g/dl
It is recommended that each laboratory establish its own normal range.
LINEARITY:
This procedure is linear up to 10 g/dl. Samples above this concentration should be suitably diluted and results should be multiplied by dilution factor.
QUALITY CONTROL:
For accuracy it is necessary to run known controls with every assay.
LIMITATION & PRECAUTIONS:
Storage condition mentioned on the kit must be adhered to.
Do not mix reagents from different lot numbers.
Maintain clean dry glassware for accurate results.
Samples showing hemolysis are to be avoided.
Avoid contamination of reagents with even traces of saliva (as causes false high absorbance due to dirt).
BIBLIOGRAPHY:
Henry, T.R., Clin. Chem., Am. J. Clin. Path., 16:40
Henry, T.R., Cannon, D.C., Winkleman, J.W., Clinical Chemistry, Principles and Techniques, Harper & Row, 2nd Edition, 1974.
CODE NO / PACK SIZE / REAGENT 1 / REAGENT 2
217 / 1 × 100 ml / 1 × 5 ml
217A / 1 × 50 ml / 1 × 3.0 ml