TITAN IV Immunoelectrophoresis Procedure - Helena Laboratories

TITAN IV Immunoelectrophoresis 

Procedure 

Cat. No. 9050 and 9061 

Helena 

Laboratories 

Titan IV Immunoelectrophoresis (IEP) is intended for 

semiquantitative determinations by immunoelectrophoresis. 

SUMMARY 

Immunoelectrophoresis (IEP) combines two techniques, 

electrophoresis and immunodiffusion. In this two-part procedure, 

proteins in a serum or urine sample are first separated 

according to charge by electrophoresis. Then, antisera 

complimentary to the proteins under study are applied to the 

plate and allowed to diffuse. When a favorable antigen to 

antibody ratio exists, a precipitin arc will form on the plate. 

IEP is used for the diagnosis and differential diagnosis of 

monoclonal gammopathies when using serum or urine 

specimens. 1-4 The method is also used for a number of other 

purposes, including screening for circulating immune 

complexes, characterization of cryoglobulinemia and 

pyroglobulinemia, recognition and characterization of antibody 

syndromes, and recognition and characterization of the various 

forms of dysgammaglobulinemias. IEP is a reliable and accurate 

method for routine protein evaluations, detecting both structural 

abnormalities and concentration changes. 1-6 

The most common application of IEP is the diagnosis of 

monoclonal gammopathies. A monoclonal gammopathy is a 

condition in which a single clone of plasma cells produces 

elevated levels of a single class and type of immunoglobulin. 

The elevated immunoglobulin is referred to as a monoclonal 

protein, M-protein, or paraprotein. Monoclonal gammopathies 

may indicate a malignancy such as multiple myeloma or 

macroglobulinemia. The class (heavy chain) and type (light 

chain) must be established since the patient’s prognosis and 

treatment may differ depending on the immunoglobulin involved. 

Differentiation must also be made between monoclonal and 

polyclonal gammopathies. 1-6 

A polyclonal gammopathy is a secondary disease state caused 

by disorders such as liver disease, collagen disorders, 

rheumatoid arthritis, and chronic infection. It is characterized by 

the elevation of two or more (often all) immunoglobulins by 

several clones of plasma cells. Polyclonal increases are usually 

twice normal levels. 1-6 

PRINCIPLE 

The patient’s serum or urine sample and normal human serum 

control are electrophoresed on the agarose plate, separating the 

immunoglobulins according to their electrophoretic mobility. 

Antisera are then applied to troughs in the plate and allowed to 

diffuse into the agarose support medium. When a favorable antigen-to-antibody 

ratio exists, a precipitin arc will form on the 

plate. Proteins are thus differentiated not only by their 

electrophoretic mobility, but also by their diffusion coefficient and 

antibody specificity. 

Diffusion is halted by rinsing the plate in 0.85% saline. Unbound 

protein is washed from the plate by the saline, and the 

antigen/antibody precipitin arcs are stained with a protein 

sensitive stain. The precipitin arcs formed by the patient sample 

and the control are compared for a semi-quantitative protein 

analysis. 

REAGENTS 

1. Titan IV IEPlate, Cat. No. 9050, 9061 

Ingredients: Each IEP plate contains 1.5% agarose (w/v), in 

barbital-sodium barbital buffer with 0.1% sodium azide added 

as a preservative. 

WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. This 

product contains sodium azide. Refer to the sodium azide 

warning. 

Preparation for Use: The plates are ready for use as 

packaged. 

Storage and Stability: Plates should be stored flat, in the 

protective packaging, at 2°C to 8°C and are stable until the 

expiration date indicated on the label. DO NOT FREEZE 

PLATES OR EXPOSE THEM TO EXCESSIVE HEAT. 

Signs of Deterioration: The plates should have a smooth, 

clear agarose surface. Discard the plates if they appear 

cloudy, show bacterial growth, or if they have been exposed to 

freezing (a cracked or bubbled surface) or excessive heat (a 

dried, thin surface). 

2. Antisera for Assay of Immunoglobulins 

Antiserum to Human IgG, Cat. No. 9232 

Antiserum to Human IgA, Cat. No. 9231 

Antiserum to Human IgM, Cat. No. 9234 

Antiserum to Human IgD, Cat. No. 9249 

Antiserum to Human IgE, Cat. No. 9250 

Antiserum to Human Kappa Light Chain, Cat. No. 9262 

Antiserum to Human Lambda Light Chain, Cat. No. 9257 

Trivalent Antiserum to Human Immunoglobulins (Heavy Chain 

Specific for IgG, IgA, IgM), Cat. No. 9236 

Antiserum to Human Serum, Cat. No. 9233 

Pentavalent antiserum to Human Immunoglobulins 

(Specific for IgG, IgA, IgM, IgD, IgE), Cat. No. 9251 

Ingredients: Each vial of antiserum contains the specificity as 

indicated on the vial. The antisera are prepared in horse, 

sheep, goat, donkey or rabbit. Each vial contains 0.1% 

sodium azide as a preservative. 

WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO 

NOT INGEST. Refer to the sodium azide warning. 

Preparation for Use: The antisera are in liquid form and are 

ready for use as packaged. 

Storage and Stability: The antisera should be stored at 2°C 

to 8°C and are stable until the expiration date indicated on 

the vial. 

Signs of Deterioration: The antisera should be colorless to 

light yellow. 

3. IEP Normal Human Serum Control, Cat. No. 9010 

Ingredients: The control contains pooled normal human 

serum with 0.1% sodium azide as a preservative. 


NOT INGEST. Refer to the sodium azide warning. This 

material has been determined negative for Hepatitis B Antigen 

(HBsAg), HIV I/II and HCV antibodies; however, it should be 

handled with the same precautions as those observed when 

handling any human serum. 

Preparation for Use: The control is in liquid form and is ready 

for use as packaged. Before using, add two drops of albumin 

marker to the control vial. 

Storage and Stability: The control should be stored at 2°C to 

8°C and is stable until the expiration date indicated on the vial. 

Stability is not affected by the addition of albumin marker. 

Signs of Deterioration: The control should be light yellow 

and slightly hazy before the addition of marker.

4. Electra ® B 1 Buffer, Cat. No. 5016 

Ingredients: Contains barbital-sodium barbital, pH 8.3-8.7. 


NOT INGEST. The buffer contains barbital which, in sufficient 

quantity, can be toxic. 

Preparation for Use: Dissolve one package of buffer in one 

liter of deionized water. The buffer is ready for use when it is 

completely dissolved. 

Storage and Stability: The packaged, dry buffer should be 

stored at room temperature (15 to 30°C) and is stable until the 

expiration date on the package. Buffer solution is stable for 

two months at 15 to 30°C. 

Signs of Deterioration: Discard packaged buffer if the 

material shows signs of dampness or discoloration. Discard 

buffer solution if it becomes turbid. 

5. Albumin Marker, Cat. No. 9011 

Ingredients: The albumin marker is 0.5% bromophenol blue 

in aqueous solution. 


NOT INGEST. 

Preparation for Use: Add two drops of Albumin Marker to 

each vial of IEP Normal Human Serum Control. The 

bromophenol blue binds with the albumin in the control. The 

tagged albumin allows verification of protein mobility during 

electrophoresis. 

Storage and Stability: The marker should be stored at 15 

to 30°C and is stable until the expiration date indicated on 

the vial. 

Signs of Deterioration: Discard if the solution color changes 

from the yellow-brown color. 

SODIUM AZIDE WARNING 

To prevent the formation of toxic vapors, do not mix with acidic 

solutions. When discarding, always flush sink with copious 

amounts of water. This will prevent the formation of metallic 

azides which, when highly concentrated in metal plumbing, are 

potentially explosive. In addition to purging pipes with water, 

plumbing should occasionally be decontaminated with 10% 

NaOH. 

SPECIMEN COLLECTION AND HANDLING 

Specimen: Fresh human serum or urine are the specimens of 

choice. Urine samples should be tested unconcentrated as well 

as concentrated (10X to 50X) due to the wide range of light 

chain concentrations. 

Patient Preparation: No special patient preparation is 

necessary. 

Interfering Factors: 

1. Samples showing evidence of hemolysis should not be used. 

2. Inaccurate results may be obtained on specimens left 

uncovered, due to evaporation. 

3. Microbial contamination of samples will cause protein 

denaturation affecting results. 

4. Patient age, sex, history and clinical presentation will affect 

immunoglobulin levels and must be considered. 

Storage and Stability: Serum and urine samples should be 

assayed fresh if possible. Samples may be stored at 2 to 8°C for 

up to 5 days after collection. Serum and urine refrigerated 

samples should be warmed to room temperature and mixed well 

prior to testing. 

PROCEDURE 

Materials Provided by Helena: 

Cat. No. 

Titan IV IEPlates, 8 wells, 7 troughs 9050 

Titan IV IEPlates, 7 wells, 6 troughs 9061 

Electra ® B 1 Buffer 5016 

IEP Normal Human Serum Control 

Albumin Marker 

9010 

9011 

Antiserum to Human IgG 9232 

Antiserum to Human IgA 9231 

Antiserum to Human IgM 9234 

Antiserum to Human IgD 9249 

Antiserum to Human IgE 9250 

Antiserum to Human Kappa Chain 9262 

Antiserum to Human Lambda Chain 9257 

Trivalent Antiserum to Human 

Immunoglobulins (IgG, IgA, IgM) 9236 



Antiserum to Human Serum 9233 

Microdispenser and Tubes 6210 

TITAN GEL Chamber (Zip Zone ® Chamber, 

Cat. No. 1283, may be used) 4063 

IEP Sponge Wicks, Long 9015 

Materials Needed but Not Supplied: 

Saline Solution (0.85%) 

5% acetic acid 

SUMMARY OF CONDITIONS 

Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Titan IV IEPlate 

Buffer . . . . . . . . B 1 Buffer dissolved in 1.0 L deionized water 

Sample Volume. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 µL 

Migration Distance . . . . . . . . . . . . . . . Cat. No. 9061, 20 mm 

Cat. No. 9050, 35-40 mm 

Electrophoresis Time . . . . . . . . . . . . . . . . . . . 40-50 minutes 

Voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 V 

Antisera Volume . . . . . . . . . . . . . . Cat. No. 9050, 75-100 µL 

Cat. No. 9061, 150 µL 

Incubation Time . . . . . . . . . . . . . . . . . . . . . . . . . 18-24 hours 

Incubation Temperature . . . . . . . . . . . . . . . . . . . . 15 to 30°C 

Wash Solution . . . . . . . . . . . . . . . . . . . . . . . . . 0.85% Saline 

Saline Wash Time. . . . . . . . . . . . . . . . . . . . . . . . 48-72 hours 

Staining Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 min. 

Destaining Time . . . . . . . . . . . . . . . . . . . . . . 90-180 minutes 

STEP-BY-STEP METHOD 

A.Preparation of TITAN GEL Chamber 

1. Dissolve 1 package of Electra B 1 Buffer in one liter of 

deionized water. 

2. Pour 100 mL of buffer into 

each outer section of the 

chamber (total of 200 mL of 

buffer used). The buffer can 

be reused one time by 

reversing the polarity of the 

chamber. The IEPlates must then be placed with the 

wells on the left side (formerly the anodic side). 

3. Place a long IEP Sponge Wick in each buffer-filled 

compartment. Allow the sponges to become saturated 

with buffer. Place the sponges against the chamber 

walls as shown. 

4. Cover the chamber until ready to use. 

B.Sample Application 

1. Remove the Titan IV IEPlate(s) from the refrigerator. 

Allow the plate(s) to come 

to room temperature prior 

to use (requires 20-30 

minutes). 

2. Add two drops of Albumin 

Marker to a vial of Normal 

Human Serum Control. 

3. Remove the lid from the 

plate. 

4. Apply 4 µL of the control to 

every other well in the plate

using a Microdispenser. Take care not to damage the 

wells during sample application. 

5. Apply 4 µL of the patient sample to every other well. 

C.Electrophoresis of the IEPlate(s) 

1. Quickly put the plate(s) in the electrophoresis chamber, 

agarose side down, with the wells toward the cathode (- 

) side of the chamber. 

Make sure that the agarose makes good contact with 

the sponge wicks. Two plates may be electrophoresed 

in one chamber. 

2. Put the cover on the electrophoresis chamber and wait 

30 to 60 seconds before applying current. This allows 

the plate(s) to equilibrate with the buffer. 

3. Electrophorese the plate(s) at 100 volts for the 

appropriate migration distance. This requires approximately 

40 to 50 minutes. Migration distance can be 

verified visually by observing the position of the Albumin 

Marker. 

The migration distances for the plates are as follows: 

Cat. No. 9061: 20 mm 

Cat. No. 9050: 35-40 mm 

D.Antisera Application 

1. Remove the plate(s) from the chamber and put them on 

a flat surface, agarose side up. 

2. Apply the appropriate antiserum to each trough in the 

plate using the appropriate volume of antisera as 

follows: 

Cat. No. 9061, 150 µL/trough 

Cat. No. 9050, 75-100 µL/trough 

Fill the troughs by placing the tip of a pipette in the end 

of the trough farthest from 

the sample well. Holding 

the pipette in place, slowly 

depress the plunger and 

dispense the antiserum 

into the trough. The 

antiserum will flow down 

the trough by capillary 

action. Severe overfilling 

may cause antisera to contaminate other troughs, 

yielding erroneous results. 

3. Before moving the plates, allow the antisera to absorb 

for approximately 3 to 5 

minutes. 

4. Stack the plates in an 

enclosed container with a 

moist paper wick. 

5. Incubate the plate(s) for 

18 to 24 hours at room 

temperature (15 to 30°C). 

The minimum incubation 

time is 18 hours. After 

incubation, place the 

plates in a 0.85% saline wash to stop the precipitin 

reaction. 

E. Interpretation of Unstained Plates 

1. Place the plate on a light box for viewing. The precipitin 

arcs will appear as dense opaque white arcs in the 

agarose layer. 

2. Compare the patient arcs with the Normal Control in 

order to determine the presence or absence of an 

abnormal immunoglobulin. 

3. For a permanent record, photograph the IEP plate. 

Stability of End Product: The dried Titan IV IEPlate is stable 

for an indefinite period of time. 

Quality Control: IEP Normal Human Serum Control with 

(-) 

C 

P 

C 

P 

C 

P 

C 

P 

C = Control 

P = Patient Sample 

TITAN GEL 

40mm 

IEPlate 

1 

2 

3 

4 

5 

6 

7 

(+) 

IEPlate (Cat. No. 9050) 

ANTISERA 

IgG 

IgA 

IgM 

Human Serum 

Trivalent 

Kappa 

Lambda 

Albumin Marker added should be used as a control for each 

antiserum specificity used. 

RESULTS 

The formation of a precipitin arc between a well containing test 

specimen and a trough containing antiserum indicates the 

presence of the protein specific to the antiserum. The lack of a 

precipitin arc indicates that a detectable amount of the protein is 

not present in the test specimen or that prozoning has occurred. 

The size, location, and shape of the precipitin arc, as compared 

to the control, are indications of the amount of protein in the test 

sample. IEP is a semiquantitative technique. 

In general, when protein concentrations are below normal, 

precipitin arcs are shortened and located farther from the 

antiserum trough compared to the corresponding arc in the 

control. When protein concentrations are above normal, 

precipitin arcs are thicker and located closer to the antiserum 

trough compared to the control. 

Further Testing Required: 

A. IgM Typing (IgA) 

Interpretation of Light Chain reactions associated with 

monoclonal IgM is often difficult due to the umbrella effect of 

IgG. The following method may be used to depolymerize IgM 

(19S) into single molecular units (7S), which diffuse through 

the agarose more rapidly. In some instances, this may be 

necessary for IgA typing. 

Perform this procedure under a fume hood. The vapor 

from 2-mercaptoethanol (2-ME) may cause skin irritation. 

Avoid contact with skin, eyes and clothing. 

1. Materials: 

2-mercaptoethanol 

10 µL disposable pipettes 

100 µL disposable pipettes 

Fume hood 

2. Make 1:10 dilution, 2-ME to deionized or distilled water. 

3. Add 10 µL of diluted 2-MI to 100 µL of patient sample. 

4. Assay immediately, running the plate(s) with treated and 

untreated patient sample in alternate wells. 

B. Correction for Antigen Excess 

Antigen excess (prozoning) is an incomplete precipitin 

reaction caused by antigen excess (too high an antigen-toantibody 

ratio). Prozoning should be suspected if a 

precipitin arc appears to “run” into a trough or if a light chain 

appears fuzzy when a heavy chain is increased or if an arc 

appears to be incomplete. To correct for antigen excess, 

use one of the following three methods. 

1. After incubation, but before washing in saline, add an 

additional 75-100 µL of antisera to the troughs in 

question and incubate an additional 18 to 24 hours. 

2. Retest the sample using twice the volume of originally 

specified antiserum in the trough(s) in question. Add half 

the volume and allow the antiserum to diffuse into the 

plate, then add the additional half volume. 

3. Make a 1:2 dilution of the patient sample with saline and 

repeat the entire procedure. 

INTERPRETATION OF RESULTS 

A sample immunoglobulin profile is illustrated below. The 

pattern of precipitin arcs is interpreted comparing the patient 

sample to the control. In the figure, the patient serum forms a 

dense, bowed arc against IgG antiserum. There appears to be 

a diminished IgA level and virtually no IgM in the patient serum 

when compared to the IgA and IgM in the control. The 

abnormal IgG band is also visible against both Human Serum 

and Trivalent antisera. The patient sample reveals a bowed, 

abnormal kappa arc and a decreased lambda arc. The

composite is indicative of an “IgG Monoclonal Gammopathy 

Kappa type”. 

Fig. 1: Titan IV IEPlate illustration indicative of an IgG monoclonal 

gammopathy, kappa type. 

BIBLIOGRAPHY 

1. Ritzmann, S.E., Editor, Protein Abnormalities, Volume 1. 

Physiology of Immunoglobulins: Diagnostic and Clinical 

Aspects. Alan R. Liss, Inc., New York, 1982. 

2. Ritzmann, S.E. and Daniels, J.C. “Diagnostic Proteinology: 

Separation and Characterization of Proteins, Qualitative and 

Quantitative Assays”, Chapter 12 in Race, G.J., Laboratory 

Medicine, Harper and Rowe, Hagerstown, Maryland, 1979. 

3. Ritzmann, S.E. and Daniels, J.C., Editors. Serum Protein 

Abnormalities: Diagnostic and Clinical Aspects, 1st Edition, 

Little, Brown and Co., Boston, 1975. 

4. Roitt, I. Essential Immunology, 3rd Edition, Blackwell Scientific 

Pub., London, 1977. 

5. Kyle, R.A. and Greipp, P.R. “The Laboratory Investigation of 

Monoclonal Gammopathies.” Mayo Clinic Proceedings 

53:719-739, 1978. 

6. Solomon, A. “Bence Jones Proteins and Light Chains of 

Immunoglobulins”. Parts 1 and 2, N. Eng. J. Med 29:17-23, 

1976. 

Additional References 

1. Ritzmann, S.E., Editor, Protein Abnormalities, Vol 2. Pathology 

of Immunoglobulins: Diagnostic and Clinical Aspects. Allen R. 

Liss, Inc., New York, 1982. 

2. Kyle, R.A. Bayrd, E.D., The Monoclonal Gammopathies, 

Charles C. Thomas, Springfield, IL, 1976. 

3. Kyle, R.A., Multiple Myeloma: Review of 869 Cases. Mayo 

Clinic Proceedings 50, 29-40, 1975. 

4. Ritzmann, S.E. et al., Bence Jones Proteins: Another Look, 

Lab Management, 17-21, September 1981. 

Titan IV IEPlate System 

Cat. No. 

The following items are available individually: 

Titan IV IEPlates, 8 wells, 7 troughs (5 plates) 9050 

Titan IV IEPlates, 7 wells, 6 troughs (5 plates) 9061 

Electra ® B 1 Buffer (10 pkg/box) 5016 

IEP Normal Human Serum Control (1 x 2.0 mL) 9010 

Albumin Marker (1 x 2.0 mL) 9011 

Antiserum to Human IgG (1 x 2.0 mL) 9232 

Antiserum to Human IgA (1 x 2.0 mL) 9231 

Antiserum to Human IgM (1 x 2.0 mL) 9234 

Antiserum to Human IgD (1 x 2.0 mL) 9249 

Antiserum to Human IgE (1 x 2.0 mL) 9250 

Antiserum to Human Kappa Chain (1 x 2.0 mL) 9262 

Antiserum to Human Lambda Chain (1 x 2.0 mL) 9257 

Trivalent Antiserum to Human 

Immunoglobulins (IgG, IgA, IgM) (1 x 2.0 mL) 9236 



Antiserum to Human Serum (1 x 2.0 mL) 9233 

Microdispenser and Tubes (1 to 10 µL) 6210 

TITAN GEL Chamber (Zip Zone ® Chamber, 

Cat. No. 1283, may be used) 4063 

IEP Sponge Wicks, Long (2/pkg) 9015 

For Sales, Technical and Order Information and Service 

Assistance, call 800-231-5663 toll free. 

Helena Laboratories warrants its products to meet our published specifications and to be free 

from defects in materials and workmanship. Helena’s liability under this contract or otherwise 

shall be limited to replacement or refund of any amount not to exceed the purchase price 

attributable to the goods as to which such claim is made. These alternatives shall be buyer’s 

exclusive remedies. 

In no case will Helena Laboratories be liable for consequential damages even if Helena has 

been advised as to the possibility of such damages. 

The foregoing warranties are in lieu of all warranties expressed or implied including, but not 

limited to, the implied warranties of merchantability and fitness for a particular purpose. 

Shaded areas indicate that the text has been modified, added or deleted. 

Helena 

Laboratories 

Beaumont, Texas 77704-0752 

Pro 22 

1/05(4)

TITAN IV Immunoelectrophoresis Procedure - Helena Laboratories

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