HYDRAGEL 7 ß1-ß2

Ref. 4101

HYDRAGEL 15 ß1-ß2
Ref. 4121

HYDRAGEL 30 ß1-ß2
Ref. 4141

2012/03
 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

INTENDED USE

The HYDRAGEL 7, 15 and 30 ß1-ß2 kits are intended for separation of proteins in human serum and urine by electrophoresis on alkaline buffered
(pH 8.6) agarose gels. By design, the normal serum proteins separate into six major fractions. The kits are used in conjunction with the semi-automated
HYDRASYS instrument. The separated proteins are stained with amidoblack. The electrophoregrams are interpreted visually for pattern abnormalities.
Densitometry provides accurate, relative quantification of individual zones.

Each agarose gel is intended to run:

• 7 samples in the HYDRAGEL 7 ß1-ß2 kit,
• 15 samples in the HYDRAGEL 15 ß1-ß2 kit,
• 30 samples in the HYDRAGEL 30 ß1-ß2 kit.

For In Vitro Diagnostic Use.

NOTE : In this instruction sheet, the name "HYDRASYS" is used for both semi-automated HYDRASYS and HYDRASYS 2 instruments.

PRINCIPLE OF THE TEST1-15

Protein electrophoresis is a well established technique routinely used in clinical laboratories for screening of serum and other body fluids for protein
abnormalities. It is based on the principles of zone electrophoresis performed on a suitable support medium. Agarose has been developed into a versatile
and effective support medium. In routine diagnostic applications, serum proteins are separated into five major fractions (e.g., HYDRAGEL PROTEIN(E)
kits). When a greater resolution is required, then the proteins can be separated into six major fractions using the HYDRAGEL ß1-ß2 kits : albumin,
alpha-1 globulins, alpha-2 globulins, beta-1 globulins, beta-2 globulins and gamma globulins. Each zone contains one or more serum proteins. The urine
protein patterns resemble those of serum. However, the relative intensities of the fractions or their presence may vary greatly depending on the filtration
capability of the kidney.

REAGENTS AND MATERIALS SUPPLIED IN THE HYDRAGEL 7, 15 AND 30 ß1-ß2 KITS

WARNING : See the safety data sheets.

| ITEMS | | | |
| Agarose | | | |
PN 4101 PN 4121 PN 4141

| Buffered | | | |
Gels (ready to use) 10 gels 10 gels 10 gels

| Staining | | | |
Strips (ready to use) 10 packs of 2 each 10 packs of 2 each 10 packs of 2 each

| Amidoblack | | | |
solution diluent (stock solution) 1 vial, 60 mL 1 vial, 60 mL 1 vial, 60 mL

| Applicators | | | |
Stain (stock solution) 1 vial, 20 mL 1 vial, 20 mL 1 vial, 20 mL

|FORFilterOPTIMAL | | | |
(ready to use) 1 pack of 10 (7 teeth) 1 pack of 10 (15 teeth) 2 packs of 10 (15 teeth)
Papers 1 pack of 10 1 pack of 10 1 pack of 10
RESULTS :
All reagents from the same kit must be always used together and according to the package insert instructions.
PLEASE READ THE PACKAGE INSERT CAREFULLY.

1. AGAROSE GELS
Preparation
Agarose gels are ready to use. Each gel contains : agarose ; buffer solution pH 8.6 ± 0.5 ; additives, nonhazardous at concentrations used, necessary
for optimum performance.
Use
Support medium for protein electrophoresis.
Storage, stability and signs of deterioration
Store the gels horizontally in the original protective packaging at room temperature (15 to 30 °C) or refrigerated (2 to 8 °C). They are stable until the
expiration date indicated on the kit package and the gel package labels. (The arrow on the front of the kit box must be pointing upwards). Avoid storage
close to a window or to a heat source. Avoid important variation of temperature during storage.
DO NOT FREEZE.
Discard when:
(i) crystals or precipitate form on the gel surface or the gel texture becomes very soft (all these result from freezing the gel),
(ii) bacterial or mold growth is indicated,
(iii) abnormal quantity of liquid is present in the gel box (as a result of buffer exudation from the gel due to improper storage conditions).

2. BUFFERED STRIPS
Preparation
Buffered sponge strips are ready to use. Each contains : buffer solution pH 8.5 ± 0.5 ; additives, nonhazardous at concentrations used, necessary for
optimum performance.
Use
Buffered strips function as electrophoresis buffer reservoir and ensure contact between the gel and electrodes.
Storage, stability and signs of deterioration
Store the buffered strips horizontally in the original protective packaging at room temperature or refrigerated. (The arrow on the front of the kit box
must be pointing upwards).
They are stable until the expiration date indicated on the kit package or buffered strips package label.
DO NOT FREEZE.
Discard buffered strips if the package is opened and the strips dry out.

-8- SEBIA INSTRUCTIONS - English

 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

3. STAINING SOLUTION DILUENT

Preparation
The stock staining solution diluent must be used as described in paragraph "AMIDOBLACK STAIN".
It contains an acidic solution pH ≈ 2.
Use
For the preparation of the amidoblack staining solution.
Storage, stability and signs of deterioration
Store the stock staining solution diluent at room temperature or refrigerated. It is stable until the expiration date indicated on the kit package or staining
solution diluent vial labels. DO NOT FREEZE.
Do not add any sodium azide.

4. AMIDOBLACK STAIN
Preparation
The amidoblack concentrated stain is a visquous solution which may gelify. The integrity of the stock staining solution is not altered by the increase in
viscosity or solidification.
In all cases, to obtain a perfect reconstitution of the stain, we advise you to respect the following procedure:
1. Add 15 mL of stain diluent to the concentrated amidoblack vial.
2. Close carefully the vial.
3. Shake very vigorously the vial during approximately 5 seconds.
4. Pour this solution in the container for staining solution processing.
5. Repeat this step twice, three times if necessary.
6. Pour the remaining diluent in the container and complete the volume to 300 mL with distilled or deionized water.
7. Mix contents of stain cubitainer well for 5 to 10 minutes.
The staining solution is ready to use.
NOTE : An incomplete reconstitution of the stain will lead to an under-evaluation of albumin fraction (low percentage or white hole inside the fraction).
After dilution, the working staining solution contains : acidic solution pH ≈ 2 ; amidoblack ; ethylene-glycol ; additives, nonhazardous at concentrations
used, necessary for optimum performance.
Use
For staining gels with electrophoretic protein separations.
IMPORTANT : The staining solution is designed to stain only 10 gels. Change the solution after 10 staining steps.
Storage, stability and signs of deterioration
Store both stock and working staining solutions at room temperature or refrigerated in closed containers to prevent evaporation. Stock staining solution
is stable until the expiration date indicated on the kit package or staining vial labels.
Working staining solution is stable for 1 month. Its stability may be extended for 3 months if the working solution is refrigerated. The closed container
must be stored refrigerated immediately after each use.
Do not store the working staining solution close to a heat source.

5. APPLICATORS
Use
Precut, single use applicators for sample application.
Storage
Store the applicators in a dry place at room temperature or refrigerated.

6. FILTER PAPERS
Use
Precut, single use, thin absorbent paper pads for blotting excessive moisture off the gel surface before sample application.
Storage
Store the thin filter papers in a dry place at room temperature or refrigerated.

REAGENTS REQUIRED BUT NOT SUPPLIED

WARNING : See the safety data sheets.

1. DESTAINING SOLUTION
Preparation
Each vial of stock Destaining Solution (SEBIA, PN 4540, 10 vials, 100 mL each) to be diluted up to 100 liters with distilled or deionized water. It is
convenient to dilute only 5 mL of the stock solution to 5 liters, the volume of the destaining solution container.
After dilution, the working destaining solution contains an acidic solution pH ≈ 2.
Use
For destaining, that is removal of excess and background stain from the gels.
For rinsing of the staining compartment after wash step.
To neutralize the acidity of the destaining solution, pour 15 mL of a 50 % solution of Sodium Hydroxide, into the empty waste container.
Storage, stability and signs of deterioration
Store the stock destaining solution at room temperature or refrigerated. It is stable until the expiration date indicated on the kit package or destaining
solution vial labels. Working destaining solution is stable for one week at room temperature in a closed bottle. Do not add any sodium azide.
Discard working destaining solution if it changes its appearance, e.g., becomes cloudy due to microbial contamination.
To prevent microbial proliferation in the diluted destaining solution to be stored more than one week, add 5 µL/dL of ProClin 300.
Working destaining solution added with ProClin is stable in a closed bottle at room temperature or refrigerated until the expiration date indicated on the
kit package or destaining solution vial labels.

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 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

2. HYDRASYS WASH SOLUTION

Preparation
Each vial of the stock HYDRASYS Wash Solution (SEBIA, PN 4541, 10 vials, 80 mL each) to be diluted up to 5 liters with distilled or deionized water.
After dilution, the working wash solution contains : buffer solution pH 8.7 ± 0.5.
Use
It serves for cleaning of the HYDRASYS Staining Compartment. Use periodically, e.g., if the instrument is used daily, wash the staining compartment
weekly.
See the package insert for directions to use.
Storage, stability and signs of deterioration
Store the stock and working wash solutions in closed containers at room temperature or refrigerated. They are stable until the expiration date indicated
on the wash solution vial label.
Discard working wash solution if it changes its appearance, e.g., becomes cloudy due to microbial contamination.

3. FLUIDIL
Preparation
Fluidil (SEBIA, PN 4587, 5 mL) is ready to use.
Use
To dilute samples with hindered diffusion through the sample applicator teeth (for example, viscous or turbid samples, e.g., sera containing cryoglobulin
or cryogel, samples with polymerized Ig M, …) or giving low intensity electrophoretic pattern.
Storage, stability and signs of deterioration
Store at room temperature. It is stable until the expiration date indicated on the Fluidil vial label.
Fluidil must be free of precipitate.

NOTES :
The assays that were performed for the validation of reagents demonstrated that, for the different solutions and using an adapted equipment for the
reconstitution volume, a variation of ± 5 % on the final volume has no adverse effect on the analysis.
The distilled or deionized water used to reconstitute solutions, must be free of bacterial proliferation and mold (use a 0.22 µm filter) and have a
resistivity higher than 10 Megohms x cm.

EQUIPMENT AND ACCESSORIES REQUIRED

1. HYDRASYS System SEBIA: HYDRASYS 2 SCAN PN 1200, HYDRASYS 2 PN 1201, HYDRASYS 2 SCAN FOCUSING PN 1202, HYDRASYS 2
FOCUSING PN 1203, HYDRASYS PN 1210 or PN 1211 or HYDRASYS FOCUSING PN 1212.
2. Micropipettor, either manual or automated, such as HYDRAPLUS SEBIA, PN 1216, HYDRAPLUS 2 SEBIA, PN 1217 or ASSIST SEBIA, PN 1218,
for an alternative way of loading the sample applicators.
3. Wet Storage Chamber, PN 1270, supplied with HYDRASYS.
4. Container Kit supplied with HYDRASYS.
5. Pipettes: 10 µL and 200 µL.
6. Densitometer / scanner capable of scanning 82 x 51 mm or 82 x 102 mm gel plates : HYRYS SEBIA, GELSCAN SEBIA, DVSE SEBIA or
PHORESIS software for flat-bed scanner. Refer to manufacturer’s instructions for operation and calibration procedures.
7. Gel holder for half-gels SEBIA, PN 1278.

SAMPLES FOR ANALYSIS

Sample collection and storage

Fresh serum samples are recommended for analysis. Sera and urine must be collected according to established procedures used in clinical laboratory
testing. Refrigerate samples (2 to 8 °C) as soon as possible after collection for up to one week. (NOTE : the beta-2 fraction, C3 complement,
disappears after 3 days). For longer storage periods, keep samples frozen (stable at least one month).
Freezing serum samples with sodium azide, 0,02 g/dL improves the storage stability.
Freezing urine samples with HEPES 0.1 M (pH 6.75) and sodium azide, 0,02 g/dL improve the storage stability.
IMPORTANT: Avoid boric acid as preservative.
Thawed samples can give slight application marks due to protein or lipoprotein denaturation. Storage at 2 to 8 °C and freezing cause anodic shift
of beta-lipoproteins from beta-zone to alpha-2 or alpha-1 zones ; the older the serum, the greater the shift.
Sample preparation
1. Sera
Use undiluted serum samples.
Treatment of serum samples with Fluidil :
The treatment of serum samples with Fluidil must be applied in the following cases :
- Serum samples with hindered diffusion through the sample applicator teeth, for example, viscous or turbid sera after storage at 2 to 8 °C or
after freezing (particularly those containing cryoglobulin or cryogel).
- Serum samples with polymerized Ig M.
- Serum samples giving low intensity electrophoretic pattern.
In such cases, add 25 µL Fluidil to 75 µL serum and vortex for 15 seconds. Then follow the standard procedure.
2. Concentrated urines
Analysis is performed on samples previously concentrated to a total protein concentration of about 1.5 - 2.0 g/dL (with an adapted device).
IMPORTANT: Some urines have a salt content. This can cause a gel deformation during migration and consequently, distortion of the migration
profiles. If such a distortion makes interpretation inacurate, the urine should be dialyzed to remove the salts.
Diffusion of urine samples into the applicator tips may be hindered when the urine (neat or concentrated) is turbid. It is recommended to remove
the particulates by centrifugation (e.g., 10 minutes at 3,000 rpm) or filtration (e.g., 0.45 µm syringe filter).

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 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

Sample to avoid
• Do not use hemolysed serum samples. Hemolysis increases alpha-2 and beta-zones.
• Avoid plasma samples. Fibrinogen gives a band close to the application point which might be taken for a monoclonal immunoglobulin and would
offset percentage of corresponding zone.
• Avoid aged, improperly stored urine samples where enzymatic degradation of proteins might occur.

PROCEDURE

The HYDRASYS system is a semi-automated multi-parameter instrument. The automated steps include processing of HYDRAGEL agarose gels in
the following sequence: sample application, electrophoretic migration, drying, staining, destaining and final drying. The manual steps include handling
samples and gels, and setting up the instrument for operation.
READ CAREFULLY HYDRASYS / HYDRASYS 2 INSTRUCTION MANUAL.

I. MIGRATION SET UP
1. Switch on HYDRASYS instrument.
2. Place one applicator for HYDRAGEL 7 ß1-ß2 (7 samples) and HYDRAGEL ß1-ß2 15/30 (15 samples), or two applicators for HYDRAGEL
ß1-ß2 15/30 (30 samples), on a flat surface with the well numbers in the right-side-up position (Fig. 1).
- Apply 10 µL of neat serum or concentrated urine sample in each well. Load each applicator within 2 minutes.
- Place the applicator(s) into the wet storage chamber with the teeth up. [Handle it (them) by the plastic tooth protection frame]. Let the
samples diffuse into the teeth for 5 minutes after the last sample application. For later use (up to 8 hours), keep the entire chamber under
refrigeration.
See wet chamber package insert for further details.
3. Open the lid of the Migration Module and raise the electrode and applicator carriers.
WARNING: Never close the lid while the carriers are raised !
4. Select "7 B1-B2" migration program for HYDRAGEL 7 ß1-ß2 or "15/30 B1-B2" for HYDRAGEL ß1-ß2 15/30 migration program from the
instrument menu (left side of the keyboard).
5. Remove buffered strips from the package ; handle them by the plastic ends. Engage the punched ends of the strip's plastic backing to the pins
on the electrode carrier ; the strip's plastic backing must face the carrier (Fig. 2).
6. Unpack the HYDRAGEL plate.
- Roll quickly and uniformly one thin filter paper onto the gel surface to absorb the excess of liquid. Remove the paper immediately.
WARNING: Do not leave the filter paper for a too long contact with the gel to avoid its dehydration.
- Pool 120 µL of distilled or deionized water for HYDRAGEL 7 ß1-ß2, or 200 µL for HYDRAGEL ß1-ß2 15/30, on the lower third of the frame
printed on the Temperature Control Plate of the migration module.
- Place the gel plate (the gel side up) with its edge against the stop at the bottom of the printed frame (Fig. 3).
- Bend the gel and ease it down onto the water pool (Fig. 3). Ensure that no air bubbles are trapped, water is spread underneath the entire
gel plate and the gel is lined up with the printed frame.
7. Lower both carriers down. In this position the buffered strips do not touch the gel. DO NOT FORCE THE CARRIERS ALL THE WAY DOWN.
8. Remove the applicator(s) from the wet chamber. Handle it (them) by the protection frame.
- Snap off the applicator teeth's protection frame.
- For 7 and 15 samples analysis, place the aplicator into position No 6 on the carrier.
- For 30 samples analysis, place the two applicators each into position No 3 and 9.
IMPORTANT: The numbers printed on the applicator(s) must face the operator (Fig. 4).
9. Close the lid of the migration module.
10. Start the procedure immediately by pressing the green arrow "START" key on the left side of the keyboard.
IMPORTANT: Make sure that the ventilation air inlet on the right side of the instrument is not blocked.

MIGRATION - DESCRIPTION OF THE AUTOMATED STEPS

• The two carriers are lowered so that buffered strips and applicator(s) contact the gel surface.
• Sample applicator carrier rises up.
• Migration is carried out under 10 W constant for HYDRAGEL 7 ß1-ß2 or 20 W constant for HYDRAGEL ß1-ß2 15/30, at 20 °C controlled by Peltier
effect, until 36 Vh have accumulated, for about 7 minutes.
• The electrode carrier rises to disconnect the electrodes.
• The temperature of the control plate rises to 65 °C for 10 minutes to dry the gel.
• The control plate is cooled down ; when it reaches 50 °C, an audible beep signals that the migration module lid unlocks. The plate temperature
remains at 50 °C until the lid is opened. Then, the temperature keeps decreasing until it reaches 20 °C (in less than 5 minutes) after which a new
migration run may start.
NOTE: The migration module lid remains closed during all migration steps.

II. GEL PROCESSING SET-UP

1. Open the lid.
2. Remove the applicator(s) and discard.
3. Raise both carriers, remove the buffered strips by their plastic ends and discard.
4. Remove the dried gel film for further processing.
5. After each use, wipe the electrodes and the temperature control plate with a soft wet tissue.
6. Open the Gel Holder. Lay it flat and position the dried gel (with gel side facing up) into the grooves of the two rods and close the holder. Make
sure that the film is correctly positioned inside the holder (Fig. 5).

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 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03
7. Place the gel holder into the Gel Processing / Staining Module.
IMPORTANT: Before starting the gel processing / staining program, check the following:
- the staining container is filled with 300 mL of staining solution ;
- the destaining container contains at least 1 liter of destaining solution ;
- the waste container is empty.
For reagent line connection: refer to the information displayed on the screen of the instrument (select key: REAGENT LINES).
IMPORTANT: Do not forget to block up the unused lines.
8. Select "PROTEIN(E)/ß1-ß2" staining program from the instrument menu and start the run by pressing the "START" key (green arrow on the
right side of the keyboard).

During staining, destaining and drying steps, the compartment remains locked.
After cooling step, an audible beep signals that the compartment unlocks (the ventilation is maintained until the gel holder is removed).

III. GEL PROCESSING COMPLETION

1. Remove the gel holder from the compartment, open it and remove the dried gel.
NOTE : After gel staining / destaining and before densitometry / scanning, a gel may be put through an additional wash step, if needed, to
further clarify the gel background and to remove any residual stain that may appear as blue spots. Wash the gel using the "WASH
ISOENZ/GEL" program.
2. If needed, clean the back side (the plastic support side) of the dry film with a damp soft paper.
3. Scan using a densitometer / scanner by selecting the appropriate scanning program.
NOTE : The lengths of electrophoretic migrations may be slightly different with gels containing 2 or 3 analysis rows, without any adverse effects
on performance.

RESULTS

Quality control
It is advised to include an assayed control serum (Control Serum, SEBIA PN 4785) into each run of samples.

* US customers : Follow federal, state and local guidelines for quality control.

Values
Densitometer scanning of stained electrophoregrams yields relative concentrations (percentages) of individual protein zones.

Normal values (mean ± 2 SD) for individual major electrophoretic serum protein zones on HYDRAGEL ß1-ß2 15/30 gels in the HYDRASYS system
have been established from a healthy population of 136 adults (men and women).
The protein quantification in UV on CAPILLARYS gives similar values to nephelometric procedure (especially for albumin). SEBIA proposes a
HYDRAGEL - CAPILLARYS/NEPHELOMETRIC Equivalency of values obtained on HYDRAGEL after calibration of scanning systems.

| | |
| | |
Without HYDRAGEL With HYDRAGEL

| | |
CAPILLARYS/NEPHELOMETRIC CAPILLARYS/NEPHELOMETRIC

| | | ||
Equivalency Values Equivalency Values

| | |
FRACTION HYRYS - GELSCAN - DVSE - PHORESIS HYRYS - GELSCAN - DVSE - PHORESIS

| | | |
Albumin 60.3 - 72.8 % 54.3 - 65.5 %

| | | |
Alpha-1 globulins 1.0 - 2.6 % 1.2 - 3.3 %

| | | |
Alpha-2 globulins 7.2 - 11.8 % 8.3 - 15.0 %

| | | |
Beta-1 globulins 5.6 - 9.1 % 6.5 - 11.5 %

| | | |
Beta-2 globulins 2.2 - 5.7 % 2.5 - 7.2 %
Gamma globulins 6.2 - 15.4 % 7.1 - 19.5 %

It is recommended each laboratory establishes its own normal values.

Interpretation1-15
Some serum samples may show a slight split which depends on the concentration and mobility of the alpha-2 zone protein components, see
MIGRATION PATTERN.
• Some sera have different phenotypes (Haptoglobin, GC Globulin).
• Alpha-1 lipoprotein depends on the concentration and storage of the sample.
Some serum samples may show a small, fairly sharp band corresponding to a component of C3 complement that migrates cathodic to the Beta-2
zone. See MIGRATION PATTERN.
As an aid in interpretation of serum and urine protein electrophoregrams, see BIBLIOGRAPHY.

Interference and Limitations

LDL and HDL lipoproteins are complexed components with a very variable natural electrophoretic mobility moving from the beta zone to the alpha-2
zone. In order to avoid difficulties of integration and interpretation, the HYDRAGEL gels are made of specific chemical composition which generally
positions HDL in alpha-2 zone and LDL in beta zone.
The migration is very sensitive to the following parameters:
- sample storage,
- lipoproteins concentration,
- drug treatment (heparin for instance),
- dehydration level of the gel (storage of the gel),
- raw materials variations, even slight ones.

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 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03
For these reasons, a slight anodic shift of these lipoproteins may be observed, which become more noticeable with a broadening or a slight splitting
of the alpha-2 and/or beta zones.
1) The percentages of alpha-2 and beta fractions remain totally unchanged in spite of the slight splitting due to a variation of the electrophoretic
mobility.
2) The characteristic shape of the beta-lipoprotein fraction (with an important focalisation and an irregular shape) may not lead to a misinterpretation,
only the aspect has been changed.

See SAMPLES FOR ANALYSIS.

Due to the resolution and sensitivity limits of zone electrophoresis, it is possible that some monoclonal components may not be detected with this
method.

Troubleshooting
Call Technical Service of the supplier when the test fails to perform while the instruction for the preparation and storage of materials, and for the
procedure were carefully followed.
Kit reagent Safety Data Sheets and informations on waste products elimination are available from the Technical Service of the supplier.

PERFORMANCE DATA

Reproducibility within gel and run

Three (3) different serum samples each were run in 28 tracks on a HYDRAGEL ß1-ß2 15/30 gel from each of the 2 lots tested. The means, SD and
CV (n = 28) were calculated for each sample, each zone and each gel/lot. The table shows the values for sample A from the two gels/lots tested.
Similar results were obtained for samples B and C.

| | | | |
| | | | |
ZONE MEAN (%) SD CV (%)

| | | | |
Albumin 50.8 ; 51.5 0.8 ; 1.3 1.6 ; 2.5

| | | | |
Alpha 1 2.9 ; 2.9 0.1 ; 0.1 3.2 ; 3.7

| | | | |
Alpha 2 8.8 ; 8.7 0.1 ; 0.2 1.2 ; 2.8

| | | | |
Beta 1 8.6 ; 8.2 0.3 ; 0.2 4.0 ; 2.2

| | | | |
Beta 2 5.3 ; 5.4 0.2 ; 0.5 4.4 ; 8.5
Gamma 24.1 ; 23.3 0.4 ; 0.6 1.7 ; 2.4

Reproducibility between gels and runs

Fifteen (15) different serum samples were run on one gel each from two lots of HYDRAGEL ß1-ß2 15/30 gels for five consecutive days. Each sample
was applied in two tracks on each gel (upper and lower row). The daily and total means, SD and CV were calculated for each sample and each zone.
The results were essentially the same for all samples and both lots. The following example shows the ranges of SD and CV calculated for individual
samples over the period of 5 days and the mean CV calculated from the pooled CV’s for all samples and days and one lot (n = 150).

| | | | |
| | | | |
ZONE SD CV (%) MEAN CV (%)

| | | | |
Albumin 0.3 ; 2.1 0.5 ; 3.2 1.4

| | | | |
Alpha 1 0.1 ; 0.2 3.3 ; 10.2 4.8

| | | | |
Alpha 2 0.1 ; 0.3 1.2 ; 6.0 2.8

| | | | |
Beta 1 0.1 ; 0.7 2.7 ; 8.2 4.3

| | | | |
Beta 2 0.2 ; 0.7 6.1 ; 17.8 11.0
Gamma 0.5 ; 1.1 2.9 ; 9.6 5.2

Accuracy
Pathological and normal serum samples (n = 112) were run on HYDRAGEL ß1-ß2 15/30 gels and HYDRAGEL PROTEIN(E) 15/30 gels. The
correlation parameters calculated for individual zones from the pooled data for HYDRAGEL PROTEIN(E) 15/30 gels vs. The comparative gel systems
(y = HYDRAGEL ß1-ß2 15/30) were :

| | | | | Range of %ß1-ß2 |
| | | | |(HYDRAGEL 15/30) |
Zone Correlation coefficient y-Intercept Slope Values

| | | | | |
| | | | | |
Albumin 0.989 0.318 1.010 36.1 - 70.8

| | | | | |
Alpha 1 0.967 0.009 0.952 1.0 - 10.4

| | | | | |
Alpha 2 0.981 0.002 0.985 5.6 - 22.9

| | | | | |
Beta 1 - Beta 2 0.950 0.475 0.950 9.5 - 22.3
Gamma 0.988 0.134 0.988 5.1 - 28.1

Sensitivity
Serial dilutions of two serum samples, each with a monoclonal protein, were electrophoresed on HYDRAGEL ß1-ß2 15/30 gels. The highest dilution
with a discernible monoclonal band corresponded to 44 and 21 mg/dL of the monoclonal protein for the two samples, respectively.
NOTE : According to the position of the monoclonal component and polyclonal background in the gamma zone, the detection limit may vary.

Concentrated urines analysis

Results obtained with HYDRAGEL ß1-ß2 15/30 gels indicate a very good reproducibility within and between runs for concentrated urines samples after
quantitative and qualitative analysis. Twenty eight (28) different samples (pathological and normal urines) were run on HYDRAGEL ß1-ß2 15/30 gels
and another commercially available agarose gel system. There were no visually detectable differences among the two systems. The sensitivity of
detection was determined from the highest serial dilutions of a monoclonal urinary protein giving a discernible band at 0.024 g/dL.

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 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

MIGRATION PATTERN

+
Albumin

Orosomucoid
α1 Antitrypsin

α Lipoprotein
α1 Antichymotrypsin
Ceruloplasmin
GC Globulin
α2 Macroglobulin
Haptoglobin

β Lipoprotein
Transferrin
Hemopexin
Plasminogen
Fibronectin

C3 Complement

γ Globulins

-
G-A-M-D-E

Alpha-2 zone :

2 (± 5) 2 (± 5)
1+2+3+4+5 1 + 2 + 3 + 4 (± 5) 1 + 3 + 4 (± 5)
A B C

1 = α2 Macroglobulin
2 = Haptoglobin
3 = Ceruloplasmin
4 = GC Globulin
5 = α1 Lipoprotein

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 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

BIBLIOGRAPHIE / BIBLIOGRAPHY

1. Brouet J.C. Les cryoglobulinémies. La Presse Médicale, 1983, 12, p. 2991 à 2996.
2. Brouet J.C. Orientation diagnostiquée en cas d’anomalies des immunoglobulines plasmatiques (Ig E exclues). La Revue du Praticien, n° 9, 21/03/91,
p. 782 à 785.
3. Garnier J.P., Laurent D., Clauvel J.P., Danon F., Bousquet B., Dreux C. Dosages des protéines sériques : cause d’erreur en cas d’immunoglobuline
monoclonale. Act. Pharm. Biol. Clin., 1987, 4, p. 275 à 278.
4. Guinan J.E.C., Kenny D.F., Gatengy P.A. Detection and typing of paraproteins : comparison of different methods in a routine diagnostic laboratory.
Pathology, 1989, 21, p. 35 à 41.
5. Keren D.F., “High Resolution Electrophoresis and Immunofixation Techniques and Interpretation”, Butterworth-Heinemann, Woburn, MA, USA, 2nd
ed., 1994, 397 pp.
6. Le Carrer D. Gammapathies monoclonales : mise au point sur leur exploration biochimique en 1991 - Première partie : Les techniques de diagnostic
protéinologique, principes et limites. L’Eurobiologiste, 1991, Tome XXV, n° 194, p. 203 à 212.
7. Le Carrer D. Gammapathies monoclonales : mise au point sur leur exploration biochimique en 1991 - Seconde partie : Diagnostic protéinologique du
myélome, de la maladie de Waldenström et des autres gammapathies monoclonales. L’Eurobiologiste, 1991, Tome XXV, n° 195, p. 283 à 285.
8. Le Carrer D. Intérêt du profil protéique, cible immunitaire en biologie clinique. Revue Française des Laboratoires, 1993.
9. Le Carrer D. Électrophorèse et Immunofixation des Protéines Sériques, Interprétations illustrées. Laboratoires SEBIA, 1994, 120 pp.
10. Le Carrer D. L’interprétation de l’électrophorèse des protéines. L’Eurobiologiste, 1989 - Tome XXIII, n° 182, p. 27 à 33.
11. North M.L. Détection et caractérisation d’une immunoglobuline monoclonale. Revue Française des Laboratoires, Mars 1990, n° 203, p. 54 à 58.
12. Peltre G. Électrophorèse, les trois principes de base. Technique et Biologie, 1990, 1, p. 16 à 23.
13. Sicard D. Du bon usage de l’électrophorèse des protéines. Le Concours Médical, 05.05.90, 1990, 112, 16, p. 1513 à 1515.
14. Van Den Abelle. Électrophorèse des protéines sériques. Intérêt, limites, apport du profil protéique. Larc Medical, 1987, n° 7, Vol VI, p. 348 à 351.
15. Wicher J.T., Spence C.E. Serum protein electrophoresis - An out moded test. Ann. Clin. Biochem., 1987, 24, p. 133 à 139.
16. Wendling A. Procédures de diagnostic ou de dépistage : justification et validité d’un test de diagnostic ou de dépistage-sensibilité-spécificité. Impact-
Internat, Septembre 1986.

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 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

SCHÉMAS / FIGURES

Figure 1 Figure 2

15
14
13
12
11
9 10
78
56
34
12

Figure 3 Figure 4

12
34
5 6 sebia
78
9 10
11 12

14 15
12 13
10 11
789
456
123

Figure 5

HYDRASYS
sebia 30
15/30 27 28
29
(E)
EIN 26
PROT
25
24
AGEL
23
22
HYDR
21
20
19
18
17
16

15
14
13
12
11
9 10
8
7
6
5
4
3
2
1

sebia 30
15/30 27 28
29
(E)
EIN 26
PROT
25
24
AGEL
23
22
HYDR
21
20
19
18
17
16

15
14
13
12
11
9 10
8
7
6
5
4
3
2
1

(E)
EIN
7 PROT
AGEL
HYDR

7
6
5

sebia
4
3
2
1

(E)
EIN
7 PROT
AGEL
HYDR

7
6
5
sebia
4
3
2
1

- 167 -
 HYDRAGEL 7, 15 & 30 ß1-ß2 - 2012/03

SCHÉMAS / FIGURES

HYDRASYS 2

sebia 29
30
15/30 26 27
28
(E) 25
EIN 24
PROT 22 23
AGEL 20 21
HYDR 18 19
17
16

15
14
13
12
11
9 10
8
7
6
5
4
3
2
1

30
sebia 29
28
/30 27
) 15 26
(E 25
IN 24
OTE 23
PR 22
EL 21
AG 19 20
DR
HY 17 18
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1

(E)
EIN
OT
7 PR
EL
AG
DR
HY

7
6
5
sebia
4
3
2
1

(E)
EIN
7 PROT
AGEL
HYDR

7
6
5
4
3
1
2
sebia

- 168 -