TOTAL PROTEIN PROCEDURE

Intended for the Quantitative Determination of Total Protein in Serum

SUMMARY AND EXPLANATION

Proteins form the major portion of solutes in plasma (1). The serum proteins may be distinguished by two general fractions - albumin and globulins (2). Albumin represents the most abundant protein constituent, while the globulins are a heterogeneous group comprising immunoglobulins, complement, enzymes, coagulation factors, hormones and specific transport proteins.

Total protein determinations are useful in detecting hyperproteinemis due to hemoconcentration as seen in dehydration, for example, and the various hyperglobulemic conditions occuring with multiple myeloma, Waldenstrom's marcoglobulinemia, infections, certain hepatic diseases and other pathological states associated with an increase of one or more of the many proteins found in serum (3). Total protein assays are useful in detecting hypoproteinemia observed in malnutrition, protein-losing kidney diseases, edemia, hemorrhage and malignant and other wasting conditions (3) (4).

Historically, total proteins were first determined by the Kjeldahl method using acid digestion of the proteins to produce nitrogen. The basis for the modern day determination using the biuret reagent was first reported in 1939 by Kingsley (5). The EAGLE TOTAL PROTEIN PROCEDURE is a modification of the biuret reaction as described by Kingsley (6).

PRINCIPLE

A purple-violet chromogen results when curpric ions in an alkaline medium complex with the unshared electrons of the nitrogen and oxygen atoms of the protein peptide bonds. The amount of color produced is proportional to the protein concentration and is measured spectrophotometrically at 550 nm.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 3000 provides:

TOTAL PROTEIN COLOR REAGENT - (Cat. No. 3001)

REACTIVE INGREDIENTS:

0.5% (w/v) potassium iodide, 0.9% (w/v) potassium sodium tartrate, and 0.3% (w/v) cupric sulfate.

PRECAUTIONS:

TOXIC. May be harmful is swallowed. Do not pipet by mouth.

STORAGE AND STABILITY:

Store at 15 - 30° C. Stable until expiration date if sealed tightly.

DETERIORATION:

T he reagent should be a clear, pale blue solution. Turbidity or presence of a black precipitate would indicate deterioration.

TOTAL PROTEIN CALIBRATOR - (Cat. No. 3002)

REACTIVE INGREDIENTS:

8.0 g/dL bovine albumin in saline. Preservatives added. Consult vial label for assigned value.

PRECAUTIONS:

Do not pipet by mouth.

STORAGE AND STABILITY:

Store at 15 - 30° C. After opening, store at 2 - 8° C. Stable until expiration date if sealed tightly.

DETERIORATION:

Turbidity would indicate deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 550 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Serum is the specimen of shoice. Plasma may be used but slightly higher values should be expected because of the fibrinogen content.

2. Grossly hemolyzed samples should not be used.

3. Note the ambulatory or recumbent status of the patient. The normal total protein of ambulatory patient sera is about 0.5 g/dL greater than for recumbent patient sera (1).

SAMPLE STORAGE:

Serum Total Protein is stable for one week at 25°C or one month at 4°C (7). Avoid microbial contamination.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Hemolysis should be avoided (7). The color produced by 1 mg of hemoglobin is equivalent to 1.9 mg of serum protein. Lipemic samples and lypophilized control sera should have serum blanks. Bromsulfophthalein will give falsely high results. Bilirubin up to 25 mg/dL does not interfere. Young et al, (8) have reviewed drug effects on serum total protein levels.

PROCEDURE

MATERIALS PROVIDED:

TOTAL PROTEIN REAGENT (Cat. No. 3001) and TOTAL PROTEIN CALIBRATOR (Cat. No. 3002).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.02 mL micropipettor

2. 1.0 mL pipettor or dispenser

3. Test tubes, rack and timer

REACTION CONDITIONS:

Wavelength 550 nm

Filter Selection 530 - 570 nm

Reaction Type Endpoint

Incubation Temperature 15 - 30° C

Incubation Time 5 minutes

Sample Volume 0.02 mL

Reagent Volume 1.0 mL

Total Volume 1.02 mL

Low Normal 6.0 mg/dL

High Normal 8.3 mg/dL

Calibrator Value (See Vial)

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE: For instruments requiring a total volume of 1.0 mL, increase TOTAL PROTEIN REAGENT volume to 2.0 mL and Sample volume to 0.025 mL. Proceed as outlined.

1. Dispense 1.0 mL of Total Protein Reagent into tubes labeled Reagent Blank, Calibrator, Sample 1, etc.

2. Place 0.02 mL specimen into appropriately labeled tube. Mix well. Use D-H2O as sample for Reagent Blank.

3. Permit the test samples to stand at room temperature (15-30° C) for 5 minutes.

4. Zero instrument at 550 nm using the Reagent Blank.

5. Read and record absorbance values for Calibrator, Control and Unknowns.

NOTE: For a direct read-out instrument, set read-out at concentration of Standard and read the Unknown concentrations directly.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 60 minutes after color development.

SERUM BLANK:

In the case of lipemic specimens and lyophilized control sera, a serum blank should be performed. A blank value for a particular lot of control serum needs to be determined only once. This value can be used in subsequent assays providing reaction conditions and reagent lots do not change.

1. Dispense 1.0 mL of 0.9% sodium chloride solution into a tube.

2. Add 0.02 mL specimen. Mix well.

3. Zero instrument at 550 nm with 0.09% sodium chloride solution.

4. Read and record the serum blank absorbance.

5. Subtract the serum blank absorbance from the Unknown absorbance obtained in STEP 5 of MANUAL PROCEDURE. Use this value in calculating the Unknown concentration.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0-15 g/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use TOTAL PROTEIN CALIBRATOR (Cat. No. 3002) which is provided in the reagent set or other suitable calibration material for this purpose.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for Unknowns. EAGLE DIAGNOSTICS offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentrations:

Unknown (mg/dL) =

Unk. Abs.

------------- X Cal. Conc. (mg/dL)

Cal. Abs.

EXAMPLE:

An 8.0 g/dL Calibrator had Abs. = 0.370 while the Unknown Abs. = 0.250. The total protein concentration of the Unknown is:

0.250

--------- X 8.0 g/dL = 5.4 mg/dL

0.370

LIMITATIONS

The bluret reaction is not sufficiently sensitive to permit determination of proteins in fluids of low concentration such as urine and spinal fluid.

In some cases, a decrease in one fraction of the total proteins may be offset by an increase in the other fractions. Therefore, the total protein determination may not reflect either the nature or the extent of an existing abnormality. In such cases, the determination of albumin and calculation of the Albumin/Globulin (A/G) Ratio are suggested to assist in differentiation. After determining total protein and albumin, the A/G may be calculated as follows:

ALBUMIN (g/dL)

------------------------------------------------------------ = A/G

TOTAL PROTEIN (g/dL) - ALBUMIN (g/dL)

EXPECTED VALUES (9)  

Ambulatory 6.4 - 8.3 g/dL

Recumbent 6.0 - 7.8 g/dL

A/G Ratio 1.1 - 2.5

The range represents the 95% confidence interval for specimens obtained from a clinically normal population. It is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 15 g/dL.

PRECISION:

Normal and abnormal control sera were assayed 20 times each for within run precision and for 10 working days to establish run to run precision.

WITHIN RUN

MEAN / ST. DEV. / %CV

Normal 6.9 / 0.11 / 1.6

Abnormal 5.1 / 0.13 / 2.5

RUN TO RUN

Normal 7.0 / 0.16 / 2.3

Abnormal 5.0 / 0.15 / 3.0

SPECIFICITY:

A comparison of this TOTAL PROTEIN PROCEDURE with another widely used commercial method showed a 99% correlation with samples in the normal and abnormal range.

SENSITIVITY:

This procedure has a sensitivity of 0.02 g/dL per 0.001 absorbance unit.

REFERENCES

1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd ed., W.B. Saunders Co., Philadelphia, 1976, p. 298.

2. Cantarow, A., and Trumper, M., Clinical Biochemistry, 6th ed., W.B. Saunders Co., Philadelphia, 1962, p. 137.

3. Hoffman, W.S., The Biochemistry of Clinical Medicine, 4th ed., Year Book Medical Publishers, Chicago, 1966, p. 41.

4. Rheinhold, J. G., Standard Methods of Clinical Chemistry, Vol. 1, Academic Press, New York, 1953, p. 88.

5. Kingsley, G. R., J. Biol. Chem., 131, 197 (1939).

6. Kingsley, G.R., J. Lab. Clin. Med., 27, 840 (1942).

7. Henry, R.J., Clinical Chemistry Principles and Techniques, 2nd ed., Harper & Row, Hagerstown, 1974, p. 413.

8. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. Vol. 21, p.368D (1975).

9. Tietz, N.W., Clinical Guide to Laboratory Tests, 2nd ed., W.B. Sanders Co., Philadelphia, 1990, p. 470.