Total IgE Conjugate 100 and 400

Fluoroenzymeimmunoassay

Directions for Use 52-5238-01/09

INTENDED USE

ImmunoCAP Total IgE is an in vitro test system for the quantitative measurement of circulating total IgE in human serum or plasma samples. It is intended for in vitro diagnostic use as an aid in the clinical diagnosis of IgE-mediated allergic disorders in conjunction with other clinical findings, and is to be used in clinical laboratories.

ImmunoCAP Total IgE Conjugate 100 and 400 is intended to be used with the instrument

ImmunoCAP 250 and ImmunoCAP 1000.

SUMMARY AND EXPLANATION OF THE TEST

Since 1967, when the first assays for serum immunoglobulin E (1-3) were described, these measurements have become well established components of the investigation of allergic patients. The serum concentration of IgE is significantly elevated in patients suffering from extrinsic asthma, hayfever or atopic eczema. The increase during childhood is slow.

Adult values are not stabilized until 15-20 years of age (6-7).

PRINCIPLE OF THE PROCEDURE

Anti-IgE, covalently coupled to ImmunoCAP, reacts with the total IgE in the patient serum specimen. After washing, enzyme-labeled antibodies against IgE are added to form a complex. After incubation, unbound enzyme-anti-IgE is washed away and the bound complex is then incubated with a developing agent.

After stopping the reaction, the fluorescence of the eluate is measured. The fluorescence is directly proportional to the concentration of IgE in the serum sample. To evaluate the test results, the response for the patient samples are transformed to concentrations with the use of a calibration curve.

REAGENTS FOR ImmunoCAP 250 AND ImmunoCAP 1000

Reagents are packaged in separate units, each purchased separately. All units are required to perform an assay, though; calibrators are not required for additional assays while the stored curve is valid. The expiration date and storage temperature for each of the units are stated on the outer label. However, each component is stable until the date stated on each individual component's label.

Note!

It is not recommended to pool any reagents.

ImmunoCAP Total IgE Conjugate 100 (Art No 10-9319-01 )

(Fluoroenzymeimmunoassay for 6 x 100 det.)

Ready for use

Store at 2 − 8 °C until expiration date

Do not freeze!

Total IgE Conjugate 100

ß-Galactosidase-anti-IgE (mouse monoclonal antibodies)

Approximately 2 μ g/ml

Sodium Azide 0.06%

Color coded blue; 5.3 ml

6 vials

ImmunoCAP Total IgE Conjugate 400 (Art No 10-9480-01 )

(Fluoroenzymeimmunoassay for 6 x 400 det.)

Total IgE Conjugate 400

ß-Galactosidase-anti-IgE (mouse monoclonal antibodies)

Approximately 2 μ g/ml

Sodium Azide 0.06%

Color coded blue; 20.5 ml

6 vials Ready for use

Store at 2 − 8 °C until expiration date

Do not freeze!

ImmunoCAP Total IgE Calibrator Strip (Art No 10-9387-01)

(Reagents for 5 calibration curves)

Total IgE Calibrator Strip

(human IgE in buffer)

Conc. 2, 10, 50, 200, 1000 and 5000 kU/l

Kathon CG 0.15%

Color coded yellow; 0.2 ml

5 strips

Each Strip contain a calibration curve

ImmunoCAP Total IgE Curve Control Strip (Art No 10-9325-01)

(Reagents for 5 x 3 sets of Curve Control)

Ready for use

Store at 2 − 8 °C until expiration date

Total IgE Curve Control Strip

(human IgE in buffer)

Kathon CG 0.15%; Color coded yellow

0.1 ml

5 strips

Each Strip contains

3 CC-1 and 3 CC-2

Ready for use

Store at 2 − 8 °C until expiration date

08-04-03 12:26

ImmunoCAP Total IgE Anti-IgE (Art No 14-4509-01)

ImmunoCAP Total IgE Anti-IgE

Mouse monoclonal antibodies

Kathon CG 0.15%

1carrier of 16

ImmunoCAP

ImmunoCAP Stop Solution (Art No 10-9442-01)

(Reagents for 6 x 194 determinations)

Stop Solution

Sodium carbonate 4%, 120 ml

6 bottles

Ready for use

Store at 2 − 8 °C until expiration date

ImmunoCAP Washing Solution (Art No 10-9202-01)

For information see separate Washing Solution Directions for Use.

Reagents for ImmunoCAP 250

ImmunoCAP Development Solution (Art No 10-9441-01)/ (Art No 10-9440-01)

(Reagents for 6 x 200 determinations)/ (Reagents for 6 x 315 determinations)

Development Solution

4-Methylumbelliferyl-ß-D-galactoside 0.01%

Kathon CG 0.05%; 11 ml / 17 ml

6 bottles Ready for use

Store at 2 − 8 °C until expiration date

Do not freeze!

Ready for use

Store at 2 − 8 °C until expiration date

Reagents for ImmunoCAP 1000

ImmunoCAP Development Solution (Art No 10-9439-01)/ (Art No 10-9314-01)

(Reagents for 6 x 1200 determinations)/ (Reagents for 6 x 2000 determinations)

Development Solution

4-Methylumbelliferyl-ß-D-galactoside 0.01%

Kathon CG 0.05%; 65 ml / 112ml

6 bottles Ready for use

Store at 2 − 8 °C until expiration date

Do not freeze!

ImmunoCAP Stop Solution (Art No 34-2271-51) (Reagents for 1200 determinations)

Stop Solution

Sodium carbonate 4%; 850 ml

1 bottle Ready for use

Store at 2 − 8 °C until expiration date

Precautions

• For diagnostic use. Not for internal or external use in humans or animals.

• Do not use reagents beyond their expiration dates.

• This kit contains reagents manufactured from human blood components. The source materials have been tested by immunoassay for hepatitis B surface antigen, for antibodies to HIV1, HIV2 and hepatitis C virus and found to be negative.

Nevertheless, all recommended precautions for the handling of blood derivatives should be observed. Please refer to Human Health Service (HHS) Publication No.

(CDC) 93-8395 or other local/national guidelines on laboratory safety procedures.

• The reagents and their residues must not be allowed to come into contact with ruminating animals or swine.

• Reagents that contain sodium azide as a preservative must be handled with care.

• Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide buildup. Please refer to decontamination procedures as outlined by Centers for Disease

Control and Prevention (CDC) or other local/national guidelines.

Handling of ImmunoCAP

Keep the carrier closed to avoid evaporation of buffer. Do not leave the carrier open for more than 1 day at room temperature, otherwise, discard the first ImmunoCAP.

Indication of instability

ImmunoCAP 250 and ImmunoCAP 1000 software has built-in acceptance limits for the calibration curve and the curve controls. For more information see ImmunoCAP 250 User

Manual and ImmunoCAP 1000 User Manual.

INSTRUMENTS

ImmunoCAP 250 and ImmunoCAP 1000 are continuous random access systems that perform all steps of the assay. For further information regarding handling of the systems see ImmunoCAP 250 User Manual and ImmunoCAP 1000 User Manual.

SPECIMEN COLLECTION AND PREPARATION

Serum and plasma (EDTA or heparin) samples from venous or capillary blood can be used. Collect blood samples using standard procedures. Keep specimens at room temperature (RT) for shipping purposes only. Store at 2 − 8 °C up to one week, or else at –20 °C. Avoid repeated freezing and thawing.

For information about interfering substances see references (8,9).

Preparation of Samples

No dilution of sample is usually required, however, for determination of values higher than 5000 kU/l IgE, samples can be diluted with:

ImmunoCAP IgE/ECP/Tryptase Sample Diluent (10-9360-01)

PROCEDURES

See ImmunoCAP 250 User Manual and ImmunoCAP 1000 User Manual for detailed description.

Parameters of the procedure

Volumes per determination:

Sample

Stop Solution

40 l

μ l

Development Solution 50 μ l

600 μ l

The process time from entering the first sample is 1 hour and 45 minutes.

Incubations are performed at 37 °C by ImmunoCAP 250 and ImmunoCAP 1000.

Procedural steps

See ImmunoCAP 250 User Manual and ImmunoCAP 1000 User Manual for detailed description.

Material

Materials provided by Phadia AB:

See under REAGENTS.

Materials required but not provided by Phadia AB:

• Measuring cylinder 1000 ml

Calibration

ImmunoCAP Total IgE Calibrator Strip is run in duplicate to obtain a calibration curve.

The curve can be stored. Use two Curve Controls, in single determinations to evaluate subsequent runs against the stored curve. For more information see ImmunoCAP 250

User Manual and ImmunoCAP 1000 User Manual.

Reference material

The IgE calibrators are traceable (via an unbroken chain of calibrations) to the 2 from World Health Organization (WHO). nd

International Reference Preparation (IRP) 75/502 of Human Serum Immunoglobulin E

Measuring Range

The measuring range for an undiluted sample is 2-5000 kU/l.

Quality control

Record keeping for each assay: It is good laboratory practice to record the lot numbers of the components used, the dates when they were first opened and the remaining volumes.

Control Specimens: Good laboratory practice requires that quality control specimens should be included in every run. Any material used should be assayed repeatedly to establish mean values and acceptable ranges.

Controls available from Phadia AB for day to day quality control:

ImmunoCAP Total IgE Control LMH (10-9447-01)

Proficiency Testing: An external quality assessment program (proficiency testing) is available from Phadia AB for quality assurance purposes (Quality Club):

Quality Club Total IgE (10-9297-01)

CALCULATION OF RESULTS

All calculations are performed by the Operator Software. For complete details, see

ImmunoCAP 250 User Manual and ImmunoCAP 1000 User Manual.

LIMITATIONS OF THE PROCEDURE

A definitive clinical diagnosis should not be based on the results of any single diagnostic method and should only be made by the physician after all clinical and laboratory findings have been evaluated.

EXPECTED VALUES

Good practice recommends that each laboratory establishes its own expected range of values.

Comparison studies

(1) between ImmunoCAP 1000 and ImmunoCAP 100 have been performed with 86 samples. Results for patient samples obtained with ImmunoCAP 1000 and ImmunoCAP 100 show good agreement, see figure below.

Comparison studies

(1) between ImmunoCAP 250 and ImmunoCAP 100 have been performed with 96 samples. Results for patient samples obtained with ImmunoCAP 250 and ImmunoCAP 100 show good agreement, see figure below.

Comparison studies

IgE (run in ImmunoCAP 100) have been performed with 151 samples in two replicates on three occasions. Results show good agreement between the methods, see figure below.

A study (1)

(1) between Pharmacia CAP System IgE FEIA and ImmunoCAP Total was performed with ImmunoCAP Total IgE (run in ImmunoCAP 100) on serum collected from 63 nonatopic blood donors. The results of this study (Geometric Mean =

17.4 kU/l, and Geometric Mean +2SD = 113 kU/l) confirm the applicability of results from previously published expected values studies (6) performed with patient sera from a carefully selected non-atopic patient population using Phadebas IgE PRIST in vitro assay technology.

Since previous studies have shown good agreement between results obtained with

Pharmacia CAP System IgE FEIA and Phadebas IgE PRIST, (see figure below), between

ImmunoCAP 100 vs. Pharmacia CAP System (see figure above), between ImmunoCAP

250 and ImmunoCAP 1000 vs. ImmunoCAP 100 (see data/figures above), the expected values for Phadebas IgE PRIST can also be used for ImmunoCAP Total IgE, run in

ImmunoCAP 100, ImmunoCAP 250 and ImmunoCAP 1000.

Adults: Total IgE levels have been determined using Phadebas IgE PRIST in serum of

412 adult patients with respiratory symptoms, of which 160 were classified non-atopic and 252 had atopic disease, and showed the following distribution pattern between atopic and non-atopic individuals (7): below 25 kU/l - 84% non-atopic, above 100 kU/l - 78% atopic.

When determined using Phadebas IgE PRIST in another study (7), the geometric mean calculated from the total IgE levels in serum of 175 non-atopic adults was 13.2 kU/l, + 2

SD = 114 kU/l.

Children: The data from two independent studies using Phadebas IgE PRIST for the determination of total IgE in serum of 466 carefully selected healthy children (4,5), have been used for calculations leading to the following summary of development of serum total IgE levels during childhood (6).

After the peak at the age of 10 years, serum total IgE levels decline to adult values.

Age Geometric + 1 SD mean (kU

Age Geometric + 1 SD

Weeks Years

2.3

Months

3 1.0

6 1.8

3

4.1

7.3

8.0 32

5 12 48

9 2.6

12 3.2 13 7 16 63

8 18

9 20

10

78

22

PERFORMANCE CHARACTERISTICS

Precision (1)

The following mean coefficients of variation have been obtained, each sample assayed in

2 replicates on 24 different occasions, using the same lot of reagents. Each level contains 3 samples. The results have been obtained with ImmunoCAP 1000. They are expected results for both ImmunoCAP 250 and ImmunoCAP 1000.

Sample level

(kU/l)

Coefficients of variation (%)

Within assay Between assay

15 - 60

75 - 430

600 - 1840

3

3

3

5

4

7

Sensitivity

(1)

The detection limit is < 2 kU/l.

Specificity

(1)

The cross-reactivity with other human immunoglobulins is non-detectable at physiological concentrations of IgA, IgD, IgM and IgG.

Recovery (1)

Mean recovery is 98%.

WARRANTY

The performance data presented here was obtained using the procedure indicated. Any change or modification in the procedure not recommended by Phadia AB may affect the results, in which event Phadia AB disclaims all warranties expressed, implied or statutory, including the implied warranty of merchantability and fitness for use.

Phadia AB and its authorized distributors, in such event, shall not be liable for damages indirect or consequential.

REFERENCES

1 Ishizaka K, Ishizaka T, Hornbrook MM. J Immunol 1966;97(1);75-85.

2 Johansson SGO, Bennich HH, Wide L. A new class of immunoglobulin in human serum. Immunology 1968;14:265-72.

3 Bennich H, Ishizaka K, Johansson SGO, Rowe DS, Stanworth DR, Terry WD. WHO

Bull 1968;38;151-2.

4 Bhalla RB, Rappaport I, De Filippi I, Schwartz MK. Serum IgE Levels in a northeast

United States causian population. In: Heusghem C, Albert A, Benson E S, eds.

Advanced interpretation of clinical laboratory data. New York:Marcel Dekker Inc,

1982:295-305.

5 Kjellman NI, Johansson SGO, Roth A. Clin Allergy 1976;6:51-9.

6 Björksten B, Weeke B. Allergy 1985;40 (Suppl 4)

7 Zetterström O, Johansson SGO. IgE concentrations measured by PRIST in serum of healthy adults and in patients with respiratory allerqy. Allergy 1981;36:537-47.

8 Friedman RB, Young DS. Effects of Disease on Clinical Laboratory Tests. Second ed. AACC Press 1989;3-133 - 3-134.

9 Tryding N, Hansson P, Tufvesson C, Sjölin T, Sonntag O, editors. Drug Effects in

Clinical Chemistry. Stockholm: Apoteksbolaget, 1992:371.

ImmunoCAP is our brand name and replaces the old name UniCAP

Notes

(1)

Studies performed at Phadia AB, Uppsala, Sweden.

Patents

ImmunoCAP Systems may be covered by the following patents:

US Patent 4,647,655; 4,708,932; 5,822,069 and 5,895,630

European Patent 134 236 and 128 885

Japanese Patent 194 288 1 and 185 589 1

In addition pending patents.

The following designations are trademarks belonging to Phadia AB:

ImmunoCAP, Quality Club.

Kathon is a trademark of Rohm and Haas Company.

Addresses

GREAT BRITAIN Phadia Ltd.

CBX2, West Wing

382-390 Midsummer Boulevard

Central Milton Keynes MK9 2RG

Tel: +44-1 908 84 70 34 Fax: +44-1 908 84 75 54

IRELAND Phadia Ltd. (Irish Branch)

Beaghbeg, Carrigallen

LEITRIM

Tel: +44 1908 84 70 34

Fax: +44 1908 84 75 54

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Rajatorpantie 41 C, FIN-01640 VANTAA

Tel: +358-9 8520 2560 Fax: +358-9 8520 2565

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Shinjuku-ku TOKYO JP-163-1431

Tel: +81-3 5365 8332 Fax: +81-3 5365 8336

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PB. 4814, NO-0422 OSLO

Tel: +47-21 67 32 80 Fax: +47-21 67 32 81

USA Phadia US Inc.

4169 Commercial Avenue

Portage, Michigan 49002

Tel: +1 800-346-4364 (Toll Free) Fax: +1 269 492-7541

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Tel: +886-2 2516 0925 Fax: +886-2 2509 9756

OTHER COUNTRIES

Phadia AB

Distributor Sales

P O Box 6460, SE-751 37 UPPSALA

Tel: +46-18 16 56 16 Fax +46 18 16 63 65

Issued November 2002. Revised February, 2008.

© Phadia AB, Uppsala, Sweden.

) Manufactured by Phadia AB, P O Box 6460, SE-751 37 Uppsala, Sweden Tel:

+46 18 16 50 00 Fax: +46 18 14 03 58