CLSC 4441

Lab # 4
Analytical Methods for Total Protein

I. Objectives

1. Using the chemistry lab supply inventory list, determine which reagents or kits
should be used to measure protein in serum/plasma or urine.

2. Describe the principles of each method for measurement of protein. State the
reagents used and purpose of each.

4. State the specimen requirements and proper handling of serum/plasma, CSF and
urine specimens for the Total Protein assay.

5. Correlate each test with its purpose (diagnosis, monitor treatment, detect organ
damage).

5. List factors which affect the quality of the sample for protein assays and
describe or recognize the effects of these variables on the assay value.

6. Discuss sources of and recognize errors in the methods for Total Protein in
serum/plasma and urine.

7. Analyze patient samples for protein with serum/plasma and urine methods and obtain
results that can be reported to the physician.

8. Correlate the patients urine and serum results with diagnoses of liver disease,
Multiple Myeloma and early diabetic nephropathy.

9. Discuss differences and similarities between the methods for protein quantitation in
urine and serum.

10. Evaluate the patient serum/plasma and urine results and recommend additional tests
that would be needed. Justify your selections.

Rev 2012

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II Reading Assignment

1. Protein methods on Elsevier web site in Student Resources at

http://evolve.elsevier.com .
2. Lecture notes on protein analysis
3. Package inserts for Stanbio Total Protein and Microbumin test (stored online in
Blackboard Course Documents folder for Analytical Methods for Total Protein)

III. Laboratory Exercise

A. Manual Serum/Plasma Total Protein Assay- Each student will

perform his/her own assays.

1. Principle - see package insert

2. Procedure- follow instructions on package insert. Double reagent and

sample volumes to get minimum volume of 2.0 mL for bench spectrophotometers.

3. Perform Total Protein analysis on serum samples A, B, and C. Record the results in
your lab report.

B. Urine Protein Methods Students will perform these tests in

groups of four, due to the small amount of reagents.

1. Urine protein test principles- see package inserts.

2. Test urine samples A, B, and C using urine protein reagents. Record the results on
your lab report.

C. Correlation of Serum and Urine Total Protein Results with Disease

1. The urine and serum sample results are grouped in pairs. Serum A and Urine A are
from Patient A, Serum and Urine B from Patient B, and so on. Each patients
results represent a disease that would cause abnormal protein levels. The three
diseases these results correlate with are liver disease, Multiple Myeloma and early
diabetic nephropathy.

2. Correlate each patients protein results with the disease state they represent.

In your laboratory report, discuss:

1. How each disease state causes abnormal serum and urine protein level.
2. Which patients results require urine and serum immunofixation electrophoresis?

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 Explain your answer.

D. Principles of Serum and Urine Protein Methods

Using the package inserts for each protein test, fill in information on the principle of
each reagent. Write out the description and chemical equation (if listed on
package insert). Knowledge of the test principle can help you to solve problems with
control or patient results if you know the problem must be with the reagent and not the
analytical instrument or the sample.

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 CLSC 4441
Lab # 4- Analytical Methods for Total Protein
Report

A. Manual Total Protein Assay

Body fluid tested (urine or serum) ____Serum_____________

(2) Spectrophotometer used: ___Turner 3_________________

Tube # Abs Concentration Expected Conc

g/dL

Standard .315 10

Standard .297 10

Ser-T-Fy I .141 4.6 3.5-5.1 g/dL

Ser-T-Fy II .250 8.2 5.9-9.1 g/dL

Patient A .447 14.6

Patient B .082 2.7

Patient C .053 1.7

(1) Is assay valid?__Yes__ Explain __both controls are within expected ranges

(0.5) Linearity of method: ___1.5-10.0 g/dL _________________________________

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 (1) Interferences and their effect on results (false increase, false decrease, none)

Gross hemolysis (red serum/plasma)- falsely increased

Lipemia- could falsely increase result

B. Urine Protein Results

(1.5) Method (Name)___Pyrogallol Red _____________________

Urine Protein Expected Reference Interpretation

Sample Abs. Result Range Interval
mg/dL mg/dL

Standard .204 100

Standard .204 100

Control II .930 467 25-500 Within range;

mg/dL assay valid

Sample A .909 470 22-120 Elevated

Sample B .004 2.1 22-120 decreased

Sample C 0.450 221 22-120 Elevated

(1) Are assay results valid? _yes_______ Explain ___control result within range
(Normally you should run both a normal and an abnormal control) _____

(1) Sensitivity of _Pyrogallol Red __________ protein assay ___1.4 mg/dL

(1) Specificity of _ Pyrogallol Red _____________ protein assay ____measures

albumin and globulin proteins with approximately equal sensitivity______

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(1.5) Method (Name) __Diascreen Urine Reagent Strips ____

Urine Result Expected Reference Interpretation

Sample mg/dL Range Interval

Control II 100 25-500 mg/dL Within range;

assay results
should be valid
Less than 30
Sample A 300 mg/dL Elevated;
increased
protein in urine

Sample B Less than 30 Normal

Negative mg/dL
Less than 30
Sample C 300 mg/dL mg/dL Elevated

(1) Sensitivity of ___Diascreen___________ assay _____5-10 mg/dL _________

(1) Specificity of __Diascreen_______ assay _reagent square reacts with albumin

(6) C. Correlation of Patient Results with Disease

Patient Disease Justification

Multiple Serum and urine protein results are elevated

myeloma
Sample A

Liver The serum protein is below the reference interval and the
disease urine protein results are negative. This indicates that protein is
not being lost in the urine, which would rule out kidney
Sample B disease. This patient could have liver disease, malnutrition, or
be losing protein through the GI tract.

Kidney Serum protein is low and urine protein result is elevated,

disease indicating loss of serum proteins in urine. This kidney disease
could be caused by diabetes, hypertension, or other causes.
Sample C

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1. Discuss how each disease state causes abnormal serum and urine protein levels.

Multiple Myeloma a single B cell clone is stimulated to reproduce itself,

resulting in large numbers of B cells that all secrete the same Ig molecule. This causes
the increase in serum protein. The excretion of large numbers of Ig molecules causes
kidney damage.

Liver disease patients with advanced liver disease (cirrhosis, chronic hepatitis,
inherited diseases) lose a significant number of hepatocytes and are no longer able to
make the amounts of protein required by the body.

Malnutrition patients dont eat enough protein in their diets, which can
contribute to low serum protein values.

GI disease Malabsorption or removal of portions of the intestine can interfere

with absorption of proteins from the diet, which can cause low serum protein levels.

Kidney Disease
Hypertension the increased blood pressure over time can damage the blood vessels
and glomeruli of the kidneys.

Diabetes- protein glycosylation can cause damage to the kidneys.

2. Which patients results require urine and serum immunofixation electrophoresis?

Explain your answer.

Patient A requires urine and serum IFE, but you would need to perform serum
and urine protein electrophoresis first. The source of the elevated protein needs to
be identified, and serum protein electrophoresis will accomplish this. If there is a
monoclonal spike, the urine protein electrophoresis should show evidence of a
monoclonal spike as well, indicating the presence of Bence-Jones proteins. The
SPE and UPE results would then be confirmed using serum and urine IFE.

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(5) D. Test Principles - Write out description and chemical equation (if listed on
package insert)

1. Serum/Plasma Total Protein-

Biuret reacts with the peptide bonds of the sample proteins to form a violet colored
complex. The intensity of violet color is proportional to the concentration of protein in
the sample.

Protein Molecules contain a large number of peptide bonds. When treated with copper
ions (cu2+) in alkaline solution, a color complex is formed between the copper and the
carbonyl and imine groups of these peptides.

Acceptable sample types for test (circle correct ones)

Serum Plasma Urine Cerebrospinal fluid

The Stanbio method is designed for use with serum only.

2. Urine Protein Method (Name) ____Pyrogallol Red _____________-

This test is a dye-binding method which uses pyrogallol red-molybdate complex, a red
colored complex with maximum absorbance at 470 nm . When the complex combines
with protein in acidic conditions, a blue-purple color forms that shifts the maximum
absorbance to 610 nm. The absorbance of this product is directly proportional to the
concentration of protein in urine or serum.

3. Urine Protein Method (Name) ____Diascreen urine reagent test strips _ -

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 This test is based on the color change of the indicator, tetrabromophenol blue
in the presence of protein. A positive reaction is indicated by a color change from
yellow, which is negative to green-gold for a trace reaction through green and then to
greenish-blue for elevated levels.

3. Urine Protein Method (Name) _Microbumin tablets ____

Test is based on the protein error of indicators principle. At a constant pH,

certain indicators will have one color in the presence of protein and another
when protein is absent. When a drop of urine containing albumin is placed on a
Microbumintest tablet, the albumin is absorbed onto the tablet. Following
addition of two drops of water, the urine is washed through the tablet, but the
albumin remains on the surface of the tablet, where it reacts with the indicator to
produce a bluish-green color.

The pH indicator is bromphenol blue.

(2) Compare the measurement principles for the serum protein tests and the urine
protein tests.

Both the Diascreen and Microbumin urine tests use similar dyes and are based on
the protein error of indicators principle. These reagents react primarily with albumin
and could give negative results if the urine only contains globulin proteins, as in the
case of Multiple Myeloma (would just contain Bence-Jones proteins).

The Pyrogallol Red Urine/CSF total protein reagent is a dye-binding method that is
based on the ability of proteins to shift the maximum absorbance of the dye. This
reagent reacts with both albumin and globulins.

The serum test is based on the reaction of biuret reagent with the peptide bonds of the
proteins in the sample.

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My Documents/Old Computer/ My Documents/CLSC4441/lab anskeys/2012/proteinanalysis2012anskey

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