Four methods for determining total protein compared by using purified protein fractions from human serum

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Accurate quantification of low protein concentrations is crucial in clinical diagnostics.
• Existing protein determination methods exhibit varying degrees of proportional bias.
• Human serum protein fractions offer a standardized model for evaluating assay performance.

Purpose of the Study:

• To evaluate the proportional bias of four common protein determination methods.
• To compare the accuracy of these methods across different human serum protein fractions.
• To identify the most reliable method for quantifying low protein concentrations, particularly globulins.

Main Methods:

• Utilized human serum protein fractions (Cohn Fractions II, III, IV, V) as standards.
• Assigned protein concentration values using the biuret method, calibrated with human serum albumin monomer.
• Evaluated four methods: Coomassie Brilliant Blue dye-binding, Lowry et al., ultraviolet absorption, and immunonephelometry.

Main Results:

• All four methods accurately quantified Fraction V (albumin).
• Ultraviolet absorption and Lowry methods overestimated globulin fractions (II, III, IV).
• Coomassie Brilliant Blue and immunonephelometry underestimated globulin fractions.
• The Lowry method demonstrated the least proportional bias for globulin fractions.

Conclusions:

• Method selection for protein quantification depends on the specific protein fraction being analyzed.
• The Lowry method is recommended for determining low globulin concentrations due to minimal proportional bias.
• Further validation is needed for diverse clinical samples to confirm method reliability.