TITAN GEL

IMMUNOFIX-PLUS PROCEDURE
Cat. No. 3067, 3068, 3069 Helena Laboratories

TITAN GEL ImmunoFix-Plus is intended for the identification of materials completely dissolved.
monoclonal gammopathies using protein electrophoresis and Storage and Stability: The packaged buffer should be stored at 15 to
immunofixation. 30°C and is stable until the expiration date indicated on the package.
Diluted buffer is stable two months stored at 15 to 30°C.
SUMMARY Signs of Deterioration: Discard packaged buffer if the material shows
Immunofixation electrophoresis (IFE) is a two stage procedure using signs of dampness or discoloration. Discard diluted buffer if it becomes
agarose gel high resolution protein electrophoresis in the first stage and turbid.
immunoprecipitation in the second. The specimen may be serum, urine or 3. Acid Blue Stain
cerebrospinal fluid. There are numerous applications for IFE in research, Ingredients: The stain is comprised of acid blue stain.
forensic medicine, genetic studies and clinical laboratory procedures. The WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO NOT
greatest demand for IFE is in the clinical laboratory where it is primarily INGEST.
used for the detection and identification of monoclonal gammopathies. A Preparation for Use: Dissolve the dry stain in 1000 mL 5% acetic acid.
monoclonal gammopathy is a primary disease state in which a single Storage and Stability: The dry stain should be stored at 15 to 30°C and
clone of plasma cells produces elevated levels of an immunoglobulin of a is stable until the expiration date indicated on the package. The stain
single class and type. Such immunoglobulins are referred to as solution is stable for six months when stored at 15 to 30°C in a closed
monoclonal proteins, M-proteins, or paraproteins. In most cases they are container. Used stain may be returned to the container and reused.
indicative of a malignancy such as multiple myeloma or Waldenstrom’s Signs of Deterioration: The diluted stain should be homogeneous
macroglobulinemia. Differentiation must be made between polyclonal and mixture free of precipitate.
monoclonal gammopathies because polyclonal gammopathies are only a
4. TITAN GEL IFE Protein Fixative
secondary disease state due to clinical disorders such as chronic liver
Ingredients: The fixative contains 10% sulfosalicylic acid and 10%
diseases, collagen disorders, rheumatoid arthritis, and chronic infections.
acetic acid.
Alfonso first described immunofixation in the literature in 1964.1 Alper and WARNING: FOR IN-VITRO DIAGNOSTIC USE. CORROSIVE.
Johnson published a more practical procedure in 1969 as a result of their NEVER PIPETTE BY MOUTH. DO NOT INGEST.
work devoted to the detection of genetic polymorphisms of ceruloplasmin Preparation for Use: The fixative is ready for use as packaged.
and Gc-globulin and the conversion of C3 during activation.2 They later Storage and Stability: The fixative should be stored at 2 to 6°C and is
extended their studies to genetic polymorphisms of complement stable until the expiration date indicated on the vial.
components and the identification of alpha1 antitrypsin.3, 4 Immunofixation Signs of Deterioration: The fixative should be a clear, yellow solution.
has been used as a procedure for the study of immunoglobulins since
5. Antisera to Human IgG, IgA, IgM, Kappa Light Chain and Lambda
1976.5, 6 The TITAN GEL ImmunoFix methods offer many advantages.
Light Chain
These include ease of interpretation, excellent resolution, reagent
Ingredients: Antisera vials in the kit contain monospecific antisera to
conservation, enhanced sensitivity, within-run quality control and rapid
human immunoglobulin heavy chains, IgG, IgM, IgA and to human light
turnaround.
chains, Kappa and Lambda. The antisera have been prepared in sheep
In addition, the ImmunoFix-Plus method offers a larger 12-lane gel which and goat. Each vial of antiserum contains sodium azide as a
allows greater diversity and efficiency in testing protocols. Some of the preservative and a stabilizer.
various uses include simultaneously testing a patient’s serum and urine, WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. Refer to the
batching urines to run kappa and lambda antisera with protein Sodium Azide Warning.
separations or adding IgD and IgE to the standard serum profile. Preparation for Use: The antisera are ready for use as packaged.
Storage and Stability: The antisera should be stored at 2 to 6°C and
PRINCIPLE are stable until the expiration date indicated on the vial.
Proteins are first resolved by electrophoresis. In the second stage, the Signs of Deterioration: Extremely cloudy antisera may be indicative of
soluble antigen and its antibody are allowed to react. The resultant bacterial contamination.
antigen-antibody complex(es) may become insoluble (as long as the
antibody is in slight excess or near equivalency) and precipitate. The SODIUM AZIDE WARNING
precipitation rate depends on the proportions of the reactants, To prevent the formation of toxic vapors, do not mix with acidic solutions.
temperature, salt concentration and the pH of the solution. The unreacted When discarding, always flush sink with copious amounts of water. This
proteins are removed by a washing step and the antigen-antibody will prevent the formation of metallic azides which, when highly
complex (which might be visible as a white cloudy band in unstained gel concentrated in metal plumbing, are potentially explosive. In addition to
against a dark background), is visualized by staining. The bands in the purging pipes with water, plumbing should occasionally be
protein separation are compared with the precipitin bands obtained with decontaminated with 10% NaOH.
immunofixation.
SPECIMEN COLLECTION AND HANDLING
REAGENTS Specimen: The specimen may be serum, cerebrospinal fluid or urine.
1. TITAN GEL IFE-Plus Gel Serum Specimen Preparation:
Ingredients: Each gel contains agarose in tris-barbital/aspartate buffer 1. Dilute all serum samples with 0.85% saline. Sample dilutions should be
with 0.1% sodium azide and thimerosal added as preservatives. freshly prepared on day of use.
WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. CAUTION: DO a. Dilute serum 1:2 for the SPE reference pattern.
NOT INGEST. The gel contains barbital which, in sufficient quantity, can b. Dilute serum 1:10 for the immunofixation electrophoresis patterns.
be toxic. Refer to the Sodium Azide Warning. c. When typing minimonoclonal specimens, if the sample IgG level
Preparation for Use: The gels are ready for use as packaged. exceeds 1500 mg/dL, the sample should be diluted 1:20 for the IgG
Storage and Stability: The gels should be stored at room temperature position only.
(15 to 30°C) and are stable until the expiration date indicated on the d. When typing IgM or Lambda proteins in specimens containing
package. The gels must be stored in the protective packaging in which minimonoclonal bands, a sample dilution of 1:5 is recommended for
they are shipped. DO NOT REFRIGERATE OR FREEZE. the IgM and Lambda patterns.
Signs of Deterioration: Any of the following conditions may indicate Urine Specimen Preparation:
deterioration of the gel: (1) crystalline appearance indicating the Detection of Bence Jones proteins (free kappa and lambda light chains):
agarose has been frozen, (2) cracking and peeling indicating drying of If necessary, concentrate urine sample to 100 mg/dL of total protein for
the agarose, (3) bacterial growth indicating contamination. testing for all patterns.
2. TITAN GEL IFE Buffer Cerobrospinal Fluid Specimen Preparation:
Ingredients: The buffer contains barbital and sodium barbital with 0.1% Concentrate CSF to an IgG level of 100-200 mg/dL for typing oligoclonal
sodium azide and thimerosal as preservatives. bands in CSF. Use concentrated specimen for all patterns.
WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. TOXIC- Interfering Factors:
CAUTION: DO NOT INGEST. The buffer contains barbital which, in 1. Evaporation of uncovered specimens may cause inaccurate results.
sufficient quantity, can be toxic. Refer to the Sodium Azide Warning. 2. Plasma should not be used because the fibrinogen may adhere to
Preparation for Use: Dissolve one package of buffer in 1500 mL the gel matrix resulting in a band in all patterns across the gel.
deionized or distilled water. The buffer is ready for use when all Storage and Stability: Fresh serum, CSF or urine is the specimen of
choice. If storage is necessary, samples may be stored covered at 2 to Refer to Summary of Conditions for all other testing parameters.
6°C for up to 72 hours.
STEP-BY-STEP METHOD
PROCEDURE PART I: PROTEIN ELECTROPHORESIS
Materials Provided: The following materials are contained in the kits A. Patient Sample Preparation
associated with TITAN GEL ImmunoFix-Plus. 1. Dilute the patient serum samples with 0.85% saline as follows:
Cat. No. 1:2 (1 part serum + 1 part saline) for the serum protein pattern
TITAN GEL ImmunoFix-Plus Kit 3067 1:10 (1 part serum + 9 parts saline) for identification of all
TITAN GEL IFE-Plus Gels (10) immunoglobulins.
TITAN GEL IFE Buffer (1 pkg) Identification Dilution for Serum
Acid Blue Stain (1 vial) SP 1:2
TITAN GEL IFE Sample Templates (10) IgG, IgA, IgM, 1:10
TITAN GEL IFE Antisera Templates (10) Kappa, Lambda
TITAN GEL Blotter A-Plus (30) 2. If necessary, concentrate urine and spinal fluids according to
TITAN GEL Blotter C-Plus (30) instructions provided in SPECIMEN COLLECTION AND HANDLING.
TITAN GEL Blotter D-Plus (10) B. Preparation of TITAN GEL Chamber
TITAN GEL Blotter X-Plus (10) 1. Dissolve one package of TITAN GEL IFE
TITAN GEL IFE Antisera Kit 3068 Buffer in 1500 mL distilled or deionized
TITAN GEL IFE Protein Fixative (1 x 0.75 mL) water. Mix well for complete dissolution.
TITAN GEL IFE Antiserum to Human IgG (1 x 0.75 mL) 2. Pour 50 mL buffer into each inner section
TITAN GEL IFE Antiserum to Human IgA (1 x 0.75 mL) of the TITAN GEL Chamber. Total buffer
TITAN GEL IFE Antiserum to Human IgM (1 x 0.75 mL) volume = 100 mL. (Alternately, pour 25 mL
TITAN GEL IFE Antiserum to Human Kappa (1 x 0.75 mL) into each of the sections of the EWC
TITAN GEL IFE Antiserum to Human Lambda (1 x 0.75 mL) Electrophoresis Chamber. Total buffer
TITAN GEL IFE Light Chain Antisera Kit 3069 volume for EWC = 50 mL.)
TITAN GEL IFE Protein Fixative (2 x 0.75 mL) 3. Cover the chamber until ready to use to prevent evaporation.
TITAN GEL IFE Antiserum to Human Kappa (2 x 0.75 mL) C. Sample Application
TITAN GEL IFE Antiserum to Human Lambda (2 x 0.75 mL) 1. Remove the TITAN GEL IFE-Plus Gel from the protective package.
Materials provided by Helena Laboratories but not contained in the 2. Gently blot the surface of the gel with TITAN GEL Blotter C-Plus.
kits above: 3. Place the TITAN GEL IFE Sample Template on the gel so that the
Cat. No. small hole, in the end of the template, is positioned at the lower left,
ImmunoFix Controls (3 x 0.5 mL) 9400 and the application slits align with the arrows on the gel edges.
TITAN GEL IgD (1 x 1.0 mL) 9409 Proper placement of the template
TITAN GEL IgE (1 x 1.0 mL) 9410 is with the slightly rough side of
κ κ λ λ
IFE Trivalent Antisera (1 x 1.0 mL) 9411 the slits away from the gel SP G A M SP G A M

➞
IFE Antisera to Free Kappa Light Chains (1 x 1.0 mL) 9412 ensuring uniform absorption of the ➞

IFE Antisera to Free Lambda Light Chains (1 x 1.0 mL) 9413 sample. Apply slight fingertip
Dialamatic Microdispenser and Tubes (1-10 mL) 6210 pressure to the template making TITAN GEL IFE-PLUS
+
Dialamatic Microdispenser and Tubes (10-100 mL) 6275 sure there are no air bubbles
TITAN GEL Chamber 4063 between it and the gel.
I.O.D. (Incubator, Oven, Dryer) 5116 Note: When wearing rubber gloves to perform this step, place a
EWS Digital Power Supply 1520 Blotter A-Plus over the template and then apply fingertip pressure.
Titan Plus Power Supply 1504 The powder from the gloves can produce artifacts on the gel.
TITAN GEL Isoenzyme Incubation Chamber 4062 4. Apply 2.0 µL of the appropriate serum sample dilution or
TITAN GEL Multi-Staining Set 1558 concentrated urine or cerebral fluid onto the template slit. If urine
EWC Staining Rack for TITAN GEL Iso•Dot 1556 and CSF have been concentrated, use the concentrated sample in
Immuno SuperPress® 9035 every position across the gel.
EWC (Electrophoresis Work Center) 1551 5. Wait five (5) minutes after the last sample has been applied to allow
(Includes electrophoresis chambers, incubation the samples to diffuse into the agarose.
chamber, dryer and staining racks). 6. After allowing the samples to absorb into the agarose, gently blot
Materials and Supplies Needed but not Supplied:
Glacial Acetic Acid
Destaining Solution: 5% acetic acid. Store at 15 to 30°C ➞
SP G A M κ λ SP G A M κ λ

Saline (0.85%) TITAN GEL IFE-PLUS

+

Laboratory Rotator ➞
SP G A M κ λ SP G A M κ λ

TITAN GEL IFE-PLUS

+

SUMMARY OF CONDITIONS the template with a new TITAN GEL Blotter A-Plus and then
Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TITAN GEL IFE-Plus Gel carefully remove the template.
Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dissolve in 1500 mL
Buffer Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mL each side D. Electrophoresis of the Sample Gel
Serum Dilution . . . . . . . . . . . . . . . . 1:2 (.85% Saline) for SP Pattern 1. Place the TITAN GEL IFE-Plus Gel in the inner
. . . . . . . . . . . . . . 1:10 (.85% Saline) Immunoglobulin identification sections of the electrophoresis chamber,
Blotter A-Plus . . . . . . . . . . . . . . . . . . . . . . . . . . Blot application area agarose side up, with the edges of the gel in
Sample Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 µL the buffer. The application point should be on
Sample Absorption Time . . . . . . . . . . . . . . . . . . . . . . . . . 5 minutes the cathodic (-) side. Only 1 gel can be run per
Application Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cathode chamber. A maximum of 2 chambers can be run on the power
Electrophoresis Time/Voltage . . . . . . . . . . . . . . 20 minutes/120 volts supply.
Fixative Volume . . . . . . . . . . . . . . . . . . . . 1 drop (approx. 35-50 mL) 2. Place the cover on the TITAN GEL Chamber.
Antisera Volume . . . . . . . . . . . . . . . . . . . 1 drop (approx. 35-50 mL) 3. Electrophorese the gel:
Incubation Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 minutes TITAN GEL Chamber: 120 volts/20 minutes
Press Conditions . . . . . Press 1- 1 Blotter C-Plus & 1 Blotter X-Plus EWC Chamber: 120 volts/25 minutes
Press 2- 1 Blotter C-Plus & 1 Blotter D-Plus ALTERNATE ELECTROPHORESIS PROCEDURE
Press Time . . . . . . . . . . . . . . . . . . . . . . . . . . 5 minutes and 1 minute 1. While the sample is absorbing into the gel, pour 125 mL buffer into
Wash Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 minutes each of the outer sections of the TITAN GEL Chamber. Total volume
Drying Time/Temp . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 minutes/56°C = 250 mL. Remove the TITAN GEL Chamber Cooling Device from
Staining Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 minutes the refrigerator and place it in the center of the TITAN GEL
Destaining Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 x 2 minutes Chamber. Wet the entire surface of the cooling device with a few
Drying Time/Temp . . . . . . . . . . . . . . . . . . . . . . . . . 2-4 minutes/56°C drops of buffer.
2. Quickly place the TITAN GEL IFE Gel in the electrophoresis
Recommended EWC Parameters: chamber, agarose side up, on top of the TITAN GEL Chamber
Buffer Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 mL each side Cooling Device. The application point should be on the cathodic (-)
Electrophoresis Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 minutes side. Avoid trapping air bubbles between the agarose gel and glass
Electrophoresis Voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 V of the Chamber Cooling Device. Run one gel per chamber.
 3. Prepare a wick for each side of the gel by placing three TITAN GEL gel in a drying oven at 56-60°C for 1 minute or until the agarose is
IFE Wicks together in two sets making two thick wicks. Evenly align completely dry.
the edge of each set of wicks, and dip them into the chamber buffer. 7. Stain the gel 4 minutes in Acid Blue Stain. Again a single gel can be
Then attach the wicks to each side of the gel parallel to the edge of laid in a shallow staining dish or 2 gels can be stained together in a
the TITAN GEL Chamber Cooling Device. Gently rub one finger TITAN GEL Multi-Staining Dish and Rack.
across the gel at the wick contact area to insure good contact and to 8. Place the gels in 2 washes of Destain Solution for 1 minute each or
displace trapped bubbles. until the background is clear. (See Materials and Supplies Needed
4. Place the cover on the TITAN GEL Chamber. but not Supplied for destain formulation.)
5. Electrophorese the gel at 250 volts for 15 to 18 minutes. 9. Place the gels in the I.O.D. at 56-60°C until destain has evaporated
PART II: IMMUNOFIXATION and gels are completely dry (about 2-4 minutes).
1. Remove the electrophoresed gel from the chamber. 10. Observe the control wells for the presence of precipitin rings
2. Place the gel in the TITAN GEL Isoenzyme Incubation Chamber indicating appropriate reactivity in the antisera, and interpret results.
which has been lined with a damp blotter or filter paper. Be sure the Stability of End Product: The completed, stained and dried
gel is laying flat against the wet blotter. Should the gel maintain a immunofixation gel is stable for an indefinite period of time.
bowed shape after removal from the electrophoresis chamber, Quality Control: The ImmunoFix Controls (Cat. No. 9400) are
moisten the blotter in the incubation chamber sufficiently to hold it recommended for use as qualitative controls for verification of the
flat. appropriate reactivity of the antisera. The set contains three monoclonal
3. Apply the IFE Controls proteins; IgG Kappa, IgA Lambda and IgM.
a. Since the control wells are very small and maybe filled with buffer
after electrophoresis, blot them very carefully with Blotter A-Plus
to ensure that the wells will hold all control material applied.
b. Align the Antisera Template on the
gel so that the slits in the template
are aligned over the antisera G A M κ λ SP G A M κ

application areas on the gel. Make

sure the template makes good TITAN GEL IFE-PLUS
+
Antisera
contact with the agarose using Template

gentle fingertip pressure along the Damp Paper IFE Plate

edges and over the channel

dividers.
c. Apply 2 µL of the controls to the appropriate wells. The IgG Kappa Figure 1: An example of a TITAN GEL ImmunoFix-Plus gel showing the
control is applied to both the “G” and “Kappa” wells. The IgA protein electrophoresis pattern and immunofixation results and precipitin
Lambda control is applied to the “A” and “Lambda” wells, and the rings obtained with the controls.
IgM control is applied to the “M” well only.
d. Close the incubation chamber and allow the controls to absorb
INTERPRETATION OF RESULTS
The majority of monoclonal proteins migrate in the cathodic (gamma)
into the agarose for two and a half (2.5) minutes.
region of the protein pattern. But, due to their abnormality, they may
e. Open the incubation chamber and blot the control wells with a
migrate anywhere within the globulin region on protein electrophoresis.
Blotter A-Plus to ensure that the excess unabsorbed control
The monoclonal protein band on the immunofixation pattern will occupy
material does not float out of the well during antisera application
the same migration position and shape as the monoclonal band on the
resulting in a poorly defined control ring.
reference protein electrophoresis pattern. The abnormal protein is
4. Apply TITAN GEL Antisera and Protein Fixative
identified by the corresponding antiserum used. Figure 1 illustrates the
The antisera and fixative are packaged in dropper vials and can be
results obtained with immunofixation. When low concentrations of M-
applied directly to the gel from the vial. No pipetting is required.
protein are present, the immunofixation band may appear on the stained
The IFE Protein Fixative is applied to
background of the polyclonal immunoglobulin. A stained background may
the “SP” position of the gel to develop
also appear when the M-protein is present along with a large polyclonal
a complete protein pattern. The
increase.
monospecific antisera are applied to SP G A
For an in-depth discussion of IFE interpretation, call Helena Laboratories
their respective channels. To apply, ➞
toll free and request the free publication “ImmunoFixation for the
squeeze the vial until a drop of
Identification of Monoclonal Gammopathies” Form R5.
antiserum or Fixative is “hanging” on TITAN GEL IFE-PLUS
+

the tip of the vial and touching the LIMITATIONS

agarose. Maintain fingertip pressure on the vial, but do not continue 1. Antigen excess will occur if there is not a slight antibody excess or
to squeeze more antiserum from the vial. Pull this drop down the antigen/antibody equivalency at the site of precipitation. Antigen
immunofix channel. The antisera (or Fixative) will be quickly and excess in IFE is usually due to a very high level of immunoglobulin in
evenly applied in this manner. the patient sample. The dissolution of immunoprecipitation is
5. Incubate the gel for 10 minutes at room temperature (15 to 30°C) in manifested by a loss of protein at the point of highest antigen
the closed incubation chamber. concentration resulting in staining in the margins and leaving the
6. Wash and press the gels to clear unprecipitated protein. central area with little demonstrable protein stain. In this case it may
NOTE: The kit contains 3 different types of blotters used in this step. be necessary to adjust the protein content of the sample by dilution.
Note that Blotter X-Plus is used in the first pressing step and Blotter Electrophoresing excessive amounts of antigen decreases resolu-
D-Plus is used in the second pressing step. Blotter C-Plus is used in tion and requires higher concentrations of antibody. For optimum
both pressing steps and is always placed directly on the agarose. separation and sufficient intensity for visual detection, care must be
a. Remove the gel from the incubation chamber and rinse the gel in taken in adjusting antibody content, sample concentration, time and
0.85% saline before removing the antisera template. This can be voltage. The TITAN GEL ImmunoFix methods has been optimally
accomplished by quickly dipping the gel in and out of a small developed to minimize the antigen excess phenomenon.
container of saline. The antisera template will wash off in the 2. Monoclonal proteins may occasionally adhere to the gel matrix,
process. This will wash excess antisera from the surface and especially IgM. These bands will appear in all five antisera reaction
thoroughly wet the surface of the gel. areas of the gel. However, where the band reacts with the specific
b. Remove the antisera template (usually washes off in the saline). antisera for its heavy chain and light chain, there will be a marked
c. Place 1 Blotter C-Plus, dampened in saline, on the surface of the increase in size and staining activity, allowing the band to be
gel followed by 1 Blotter X-Plus. identified.
d. Place the gel with blotters on top in the Immuno SuperPress, 3. The level of monoclonal protein in urine may not correlate well with
tighten it and press the gels for 5 minutes. Up to 15 gels with the total protein quantitation.
blotters can be stacked in the press.
e. Remove the gels from the press, discard the blotters and place Further Testing Required:
the gels in 0.85% saline wash for 4 minutes. Specimens containing a band on serum protein electrophoresis
A single gel can be washed by laying it in a suggestive of a monoclonal protein, but which do not react with IgG, IgA
shallow dish and covering it with 50 mL or IgM antisera, may require further testing as follows:
saline. 1. Serum samples which have a precipitin band with Kappa or Lambda
f. Remove the gel(s) from the saline wash. Light Chain antisera but no corresponding band with IgG, IgA or IgM
Place 1 Blotter C-Plus, dampened in saline, antisera may have a free light chain or they may have no IgD or IgE
followed by 1 Blotter D-Plus on each gel. monoclonal protein. Such sera should be tested with TITAN GEL
Place the gels with blotters in the SuperPress ImmunoFix IgD and IgE antisera.
for 1 minute. 2. Cerebrospinal fluid may contain a non-immunoglobulin band,
g. Remove the gels from the press and discard the blotters. Dry the referred to as gamma-trace, which migrates in the gamma region.
Because gamma-trace is non-immunoglobulin in nature, it will not
 react with antisera against human immunoglobulins. Gamma-trace is 12. Whicher, J.T., et al., Clinical Application of Immunofixation: Detection
often detected in normal cerebrospinal fluid.7, 8 and Quantitation of Complement Activation, J Clin Pathol, 33:781-
3. A CRP band may be detected in patients with acute inflammatory 785, 1980.
response. 9, 10 CRP appears as a narrow band on the most cathodic
end of the high resolution agarose protein electrophoresis pattern.
Elevated alpha1-antitrypsin and haptoglobin (acute phase proteins) TITAN GEL ImmunoFix-Plus System
are supportive evidence for the presence of a CRP band. Patients Cat. No.
with a CRP band will have a positive CRP by latex agglutination or TITAN GEL ImmunoFix-Plus Kit 3067
an elevated quantitative CRP. TITAN GEL IFE-Plus Gels (10)
TITAN GEL IFE Buffer (1 pkg)
PERFORMANCE CHARACTERISTICS Acid Blue Stain (1 vial)
1. Specimens containing monoclonal proteins: Serum samples were TITAN GEL IFE Sample Templates (10)
tested by the Helena TITAN GEL ImmunoFix Procedure, high TITAN GEL IFE Antisera Templates (10)
resolution electrophoresis, immunoelectrophoresis and other TITAN GEL Blotter A-Plus (30)
appropriate procedures to identify abnormals and to classify the M- TITAN GEL Blotter C-Plus (30)
proteins by class and type in serum, with 100% agreement. TITAN GEL Blotter D-Plus (10)
2. Specimens negative for protein abnormalities: In a study, ten patients, TITAN GEL Blotter X-Plus (10)
determined free of protein abnormalities, were tested by immuno-
fixation. All normal individuals’ specimens gave negative results on the TITAN GEL IFE Antisera Kit 3068
TITAN GEL ImmunoFix Method. TITAN GEL IFE Protein Fixative (1 x 0.75 mL)
TITAN GEL IFE Antiserum to Human IgG (1 x 0.75 mL)
BIBLIOGRAPHY TITAN GEL IFE Antiserum to Human IgA (1 x 0.75 mL)
1. Alfonso, E., Quantitation Immunoelectrophoresis of Serum Proteins, TITAN GEL IFE Antiserum to Human IgM (1 x 0.75 mL)
Clin Chem Acta, 10:114-122, 1964. TITAN GEL IFE Antiserum to Human Kappa (1 x 0.75 mL)
2. Alper, C.A. and Johnson, A.M., Immunofixation Electrophoresis: A TITAN GEL IFE Antiserum to Human Lambda (1 x 0.75 mL)
Technique for the Study of Protein Polymorphism. Vo Sang 17:445- TITAN GEL IFE Light Chain Antisera Kit 3069
452, 1969. TITAN GEL IFE Protein Fixative (2 x 0.75 mL)
3. Alper, C.A., Genetic Polymorphism of Complement Components as TITAN GEL IFE Antiserum to Human Kappa (2 x 0.75 mL)
a Probe of Structure and Function. Progress in Immunology, First TITAN GEL IFE Antiserum to Human Lambda (2 x 0.75 mL)
International Congress of Immunology. Edited by Amos B., New
Other Supplies and Equipment
York, Academic Press, 609-624, 1971.
The following items, needed for the performance of the TITAN GEL
4. Johnson, A.M., Genetic Typing of Alpha 1 -Antitrypsin in
Immunofix-Plus Kit, must be ordered individually.
Immunofixation Electrophoresis. Identification of Subtypes of P.M., J
ImmunoFix Controls (3 x 0.5 mL) 9400
Lab Clin Med, 87:152-163, 1976.
TITAN GEL IgD (1 x 1.0 mL) 9409
5. Cawley, L.P. et al., Immunofixation Electrophoretic Technique
TITAN GEL IgE (1 x 1.0 mL) 9410
Applied to Identification of Proteins in Serum and Cerebrospinal
IFE Trivalent Antisera (1 x 1.0 mL) 9411
Fluid, Clin Chem, 22:1262-1268, 1976.
IFE Antisera to Free Kappa Light Chains (1 x 1.0 mL) 9412
6. Ritchie, R.F., and Smith, R.. Immunofixation III, Application to the
IFE Antisera to Free Lambda Light Chains (1 x 1.0 mL) 9413
Study of Monoclonal Proteins, Clin Chem, 22:1982-1985, 1976.
Dialamatic Microdispenser and Tubes (1-10 µL) 6210
7. Keshgegian, A., Oligoclonal Immunoglobulins in Cerebrospinal Fluid
Dialamatic Microdispenser and Tubes (10-100 µL) 6275
Multiple Sclerosis, Clin Chem, 26(9):1340-1345, 1980.
TITAN GEL Chamber 4063
8. Papadopoulos, N.M., et al., A Unique Protein in Normal Human
I.O.D. (Incubator, Oven, Dryer) 5116
Cerebrospinal Fluid, Clin Chem, 29(10):1842,1844, 1983.
EWS Digital Power Supply 1520
9. Jeppsson, J.E. et al, Agarose Gel Electrophoresis, Clin Chem,
Titan Plus Power Supply 1504
25(4):629-638, 1979.
EWC Adaptor/Chamber 1559
10. Killingsworth, L.M. et al., Protein Analysis, Diagnostic Medicine, 3-
TITAN GEL Isoenzyme Incubation Chamber 4062
15, Jan/Feb,. 1980.
TITAN GEL Multi-Staining Set 1558
ADDITIONAL REFERENCES EWC Staining Rack for TITAN GEL 1556
1. Sun, T., Immunofixation Electrophoresis Procedures in Protein Immuno SuperPress® 9035
Abnormalities, Vol. I., Physiology of Immunoglobulis: Diagnostic and EWC (Electrophoresis Work Center) 1551
Clinical Aspects, Allen, R., Liss Inc., New York, 110-115, 1982. (Includes electrophoresis chambers, incubation
2. Sun, T. et al., Clinical Application of a High-resolution chamber, dryer and staining racks).
Electrophoresis System, Ann of Clin and Lab Sci, 8(3):219-227,
1978.
3. Sun, T. et al., Study of Gammopathies with Immunofixation For Sales, Technical and Order Information and Service Assistance, call
Electrophoresis, AJCP, 72(1):5-11, 1979. 800-231-5663 toll free.
4. Ritchie, R.F., and Smith, R., Immunofixation, I. General Principles
and Application to Agarose Gel Electrophoresis, Clin Chem, 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
22(4):497-499, 1976.
limited to replacement or refund of any amount not to exceed the purchase price attributable to the
5. Ritchie, R.F. and Smith, R., Immunofixation II: Application to Typing
goods as to which such claim is made. These alternatives shall be buyer’s exclusive remedies.
of Alpha1-Antitrypsin at Acid pH, Clin Chem, 22:(10):1735-1737, In no case will Helena Laboratories be liable for consequential damages even if Helena has been
1976. advised as to the possibility of such damages.
6. Pascali, E. et al., Immunofixation: Application to the Identification of The foregoing warranties are in lieu of all warranties expressed or implied including, but not limited
“Difficult” Monoclonal Components, Clin Chem, 28(6):1404-1405, to, the implied warranties of merchantability and fitness for a particular purpose.
1982.
7. Johnson, A.M., Immunofixation Electrophoresis and Electrofocusing.
Clin Chem, 28(8):1797-1800, 1981.
8. Reichert, C.M. et al., High-resolution Zone Electrophoresis,
Combined with Immunofixation, in the Detection of an Occult
Myeloma Paraprotein, Clin Chem, 28(11):2312-2313, 1982.
9. Mestyan, I. and Jobst, K., Improved Immunofixation of Detection of
Complement 3 Conversion, Clin Chem, 28(6):1396-1397, 1982.
10. Koch, F.J. et al., Test for C3 Nephritic Factor Activity by
Immunofixation Electrophoresis, AJCP, 76(1):63-66, 1981.
11. Keshgegian, A.A. and Peiffer, P., Immunofixation as an Adjunct to
Immunoelectrophoresis in Characterization of Serum Monoclonal
Immunoglobulins, Clin Chim Acta, 110:337-340, 1981.

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