Cromatest Total Protein Biochemistry Reagent 

PRINCIPLE

In the biuret reaction, a chelate is formed between the Cu²⁺ ion and the peptide bonds of the proteins in alkaline solutions to form a violet colored complex whose absorbance is measured photometrically. The intensity of the color produced is proportional to the concentration of protein in the sample.^1-2

REFERENCE VALUES³

Serum, plasma

Total serum protein is lower by 4 to 8 g/L with the subject supine than with the subject ambulatory.

Plasma

Plasma protein is 2 to 4 g/L higher due to the presence of fibrinogen in the sample.

It is recommended that each laboratory establishes its own reference range.

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REAGENT COMPOSITION:

R1:  Biuret reagent. Cupric sulfate 6 mmol/L, sodium-potassium- tartrate 21 mmol/L, potassium iodide 6 mmol/L, sodium hydroxide 0.75 mol/L. C R:3

CAL : Protein standard. Bovine serum albumin 7 g/dL (70 g/L). Secondary standard traceable to SRM 927

STORAGE AND STABILITY:

Store at 2-8ºC.

The Reagent and Standard are stable until the expiry date stated on the label.

REAGENT PREPARATION

The reagents are ready-to-use.

SAMPLES

Serum, EDTA or heparinized plasma, and exudates.

Total protein is stable in serum and plasma for 1 week at room temperature, for at least 1 month refrigerated at 2-8ºC, and for up to 2 months at –20ºC.

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QUALITY CONTROL

The use of a standard to calculate results allows to obtain an accuracy independent of the system or instrument used.

To ensure adequate quality control (QC), each run should include a set of controls (normal and abnormal) with assayed values handled as unknowns.

CLINICAL SIGNIFICANCE

The serum content of the soluble proteins, those circulating in extracellular and intracellular fluids, has been used as a marker to aid in clinical diagnosis. The main diagnostic tests are those measuring serum total protein and serum albumin.

Collectively, serum total protein including albumin is mainly involved in the maintenance of normal water distribution between tissues and the blood and responsible for maintaining the oncotic pressure of plasma and is used to transport many substances including macromolecules.

Hyperproteinemia o hyperalbuminemia, usually occurs during multiple myeloma caused by high levels of the monoclonal immunoglobulins, dehydration, excessive water loss, as in severe vomiting, diarrhea, Addison’s disease or diabetic acidosis. The hemoconcentration, decrease in the volume of plasma water, is reflected as a relative hyperproteinemia since concentration of all the individual plasma proteins are increased to the same degree.

Hypoproteinemia o hypoalbuminemia usually occurs in edema, malnutrition, nephrotic syndrome, malabsorption and severe liver cirrhosis. Since albumin is present in such high concentration low levels of this protein alone may also cause hypoproteinemia

ANALYTICAL PERFORMANCE

• Linearity: Up to 12 g/dL
• Precision

Replicates: 10 for each level.         Replicates: 10 for each level Instrument: CECIL CE 2021  for 8 days.

• Sensitivity. Using a 1:50 sample/reagent at 540 nm, 1 g of protein will produce a net absorbance of approximately 076.
• Correlation. This assay (y) was compared with a similar commercial method (x). The results were:

N = 25      r = 0.985       y = 0.962x + 0,2727

INTERFERENCES

• • • • Grossly hemolytic or lipemic samples result in a positive
      • Dextran’s used as plasma volume expanders for the treatment of low blood pressure, complex with copper and tartrate

      forming a precipitate.

   

MATERIALS REQUIRED

• Photometer or colorimeter capable of measuring absorbance at 540 ± 20 nm.
• Constant temperature incubator set at 37ºC.
• Pipettes to measure reagent and

PROCEDURE

1. Pipette into labelled tubes:
   

   TUBES	Blank	Sample	Standard
   Biuret	1.0 mL	1.0 mL	1.0 mL
   Sample Standard	– –	20 mL –	– 20 mL

   1. Mix and incubate the tubes 10 minutes at 37ºC.
   2. Read the absorbance (A) of the samples and the standard at 540 nm against the reagent

    

   The color is stable for at least 1 hour.

CALCULATIONS

If results are to be expressed as SI units apply: g/dL x 10 = g/L.

REFERENCES

1. Gornall, G., Bardawill, C.S., and David, M.M. J. Biol. Chem. 177 : 751 (1949).
2. Falkner, R., and Meites, S. Selected Methods of Clinical Chemistry, 9, 319, AACC., Washington, D.C. (1982).
3. Young, S., Pestaner, L.G. and Gibberman, P. Clin. Chem. 21, Vol. 5, 10-432D (1975).
4. N.W. Fundamentals of Clinical Chemistry, p. 940. W.B. Saunders Co. Philadelphia, PA. (1987).