Total IgE ELISA Kit

Quantitative Assay for Total IgE Antibodies

Product code GD09
96 tests

Reagents included in the kit are for in vitro diagnostic use only

1. Intended use 5. Other equipment required

The total IgE kit is a sensitive ELISA method for the detection of IgE 1. Test tubes for dilution  graduated cylinder for preparing wash
class antibodies. It is intended as an aid to diagnosis of atopy in buffer  precision pipettes and disposable tips to deliver 10l,
children and adults. The components of the kit are for in vitro 100l, 1ml  EIA microplate washer or multi-channel pipette or
diagnostic use only. wash bottle  distilled or de-ionised water  absorbent paper 
EIA microplate reader with 450nm and optional 620nm
2. Introduction. reference filter. Alternatively, a suitable automated system may
be used.
Immunoglobulin E has a molecular structure similar to that of other 2. Instrumentation, whether manual or automated, should meet the
classes, having two specific antigen binding sites attached to a following criteria: pipettes with better than 3% imprecision with
constant (Fc) region. IgE in serum has no known biological no carry over between pipetting steps; microplate washers
relevance and exerts its effect only when bound to blood basophils or should remove 99% of fluid; automated machines should
tissue mast cells. Mediators are released from these cells are minimise time between washing and adding the next reagent.
responsible for the immediate hypersensitivity reactions.
6. Precautions
The mechanisms regulating IgE synthesis differs from those of the
6.1 Safety Precautions
other classes. Serum levels are generally low in normal individuals.
Levels rise in childhood to reach adult levels by the age of 15 to 20 1. All reagents in this kit are for in vitro diagnostic use only.
years. Because IgE levels are usually low, a specific IgE response 2. Only experienced laboratory personnel should use this
to an allergen will often cause a significant rise in total IgE. test. The test protocol must be followed strictly.
3. All human source material used in the preparation of
The assay is calibrated against the Second International IgE
standards and control for this product have been tested
Reference Serum: 75/502 (IU/ml). An elevated total IgE level is
and found negative for antibodies to HIV, HbsAg and
strongly suggestive of an atopic predisposition, but gives no clue to
HCV. No test method, however, can offer complete
the causative agent. Raised levels are found in patients with
assurance that infectious agents are absent. Therefore,
parasitic disease, tropical eosinophilia and during certain drug
all reagents containing human material should be handled
reactions.
as if potentially infectious. Operators should wear gloves
The measurement of total IgE is most helpful in diagnosis of atopic and protective clothing when handling any patient sera or
disease in young children. In adults there is an overlap between serum based products.
atopic and normal subjects. Values up to 120 IU/ml are found in the 4. Reagents of this kit contain antimicrobial agents and the
normal population and most patients with atopic disease have levels Substrate solution contains 3,3’,5,5’-tetramethylbenzidine.
above 120 IU/ml. There is no gender related difference in normal Avoid contact with the skin and eyes. Rinse immediately
IgE concentrations. with plenty of water if any contact occurs.
5. The Stop Solution contains 0.25M sulphuric acid. Avoid
3. Principle of the test contact with skin and eyes. Rinse immediately with plenty
of water if contact occurs.
Diluted serum samples are incubated with monoclonal anti human
6. Any liquid that has been brought into contact with
IgE immobilised on microtitre wells. After washing away unbound
potentially infectious material has to be discarded in a
serum components, monoclonal mouse anti-human IgE conjugated
container with a disinfectant. Disposal must be performed
to horseradish peroxidase is added to the wells, and this binds to
in accordance with local legislation.
surface-bound IgE during the second incubation. Unbound conjugate
is removed by washing, and a solution containing 3,3’,5,5’- 6.2 Technical Precautions
tetramethylbenzidine (TMB) and enzyme substrate is added to trace
specific antibody binding. Addition of stop solution terminates the 1. Strips and solutions should not be used if the foil bag is
reaction and provides the appropriate pH for colour development. damaged or liquids have leaked.
The optical densities of the standards, positive control and samples 2. Allow all reagents and the microplate to reach room
are measured using a microplate reader at 450nm. temperature before use. Ensure that the microplate foil
bag containing any unused strips is well sealed and
4. Materials included in the kit contains the desiccant to avoid moisture. Store at 2 – 8oC
after use.
 Microplate: 96 wells in 12 X 8 break-apart strips, pre-coated with 3. Include the positive control in every test run to monitor for
monoclonal anti-IgE, with holder in a foil bag with desiccant reagent stability and correct assay performance.
 Reagent 1: Sample Diluent 10mM Buffered protein solution, pH 4. Strictly observe the indicated incubation times and
7.2 with antimicrobial agent, 50 ml, (blue), ready to use temperature.
 Reagent 2: Wash Buffer Concentrate (X 10), 100mM Tris- 5. When automating, consider excess volumes required for setting
up the instrument and dead volume of robot pipette
buffered saline with detergent, pH 7.2, 100ml
6. Ensure that no cross-contamination occurs between wells.
 Reagent 3: Conjugate mouse anti-human IgE conjugate Keep all pipettes and other equipment used for enzyme
(horseradish peroxidase) in protein stabilising solution and conjugate completely separate from the substrate reagent.
antimicrobial agent, 12 ml, ready to use 7. When pipetting Conjugate or TMB Substrate, aliquots for
 Reagent 4: TMB Substrate aqueous solution of TMB and the required numbers of wells should be taken to avoid
hydrogen peroxide, 12 ml, ready to use multiple entry of pipette tips into the reagent bottles.
Never pour unused reagents back into the original bottles.
 Reagent 5: Stop Solution 0.25M sulphuric acid, 12 ml, ready to
8. Do not allow microwells to dry between incubation steps.
use
9. Strictly follow the described wash procedure. Insufficient
 Standards: 0, 50, 150, 375, 1250 IU/ml, 1ml of 10mM Tris- washing may cause high background signal.
buffered saline containing human serum IgE, ready to use 10. Avoid direct sunlight and exposure to heat sources during
 Positive Control: 1ml of 10mM Tris-buffered saline containing all incubation steps.
human serum IgE, ready to use 11. Replace colour-coded caps on their correct vials to avoid
cross-contamination
 Instructions for use
12. It is important to dispense all samples and the positive
control into the wells without delay. Therefore ensure that
all samples are ready to dispense.
7. Shelf life and storage conditions Reference Ranges
The following normal ranges (mean + 2 Standard deviations) are
On arrival, store the kit at 2 - 8C. Once opened the kit is stable for given for guidance only as laboratories should always establish their
three months (or until its expiry date if less than three months). Do own reference range. Geographical factors may influence the results.
not use kits beyond their expiry date. Do not freeze any kit
component. The diluted Wash Buffer has a shelf life of 3 months if Age Values (IU/ml 75/502)
o
stored in a closed bottle at 2 – 8 C. Newborn < 11
Up to 3 months < 25
8. Specimen collection and storage 3 to 12 months < 37
1 to 5 years < 135
Serum or plasma samples may be used and should be stored at - 5 to10 years < 144
20C for long-term storage. Frozen samples must be mixed well
after thawing and prior to testing. Repeated freezing and thawing can Adult < 188
affect results. Addition of preservatives to the serum sample may
adversely affect the results. Microbially contaminated, heat-treated Patients with IgE values below the above age related levels have a
or specimens containing particulate matter should not be used. low probability of atopic disease. Results must be interpreted in
Grossly haemolysed, icteric or lipaemic specimens should be conjunction with other clinical information relating to each patient.
avoided. Assay sensitivity
The minimum detectable concentration of Total IgE is 0.9 IU/ml
9. Preparation of reagents
13. Limitations of the Procedure
1. Dilute the Wash Buffer (Reagent 2) 1: 9 in distilled water
to make sufficient buffer for the assay run e.g. add 50ml 1. Results of this assay should be interpreted in conjunction
wash buffer concentrate to 450ml water. with clinical findings and patient history.
2. The IgE response is short lived. Patients who have not
10. Assay Procedure had a recent atopic challenge may show low IgE levels.
3. Results of this assay are not diagnostic proof of the
Assemble the number of strips required for the assay.
presence or absence of atopic disease.
1. Dispense 100 l of each standard and positive control. .
14. Performance Characteristics
Dispense 20l of each sample to be tested. Dispense
80l of sample diluent into each sample well (to make a
Inter-assay precision
1/5 dilution). Tap the plate rapidly to mix the well IgE IU/ml CV%
contents. Sample A 497 3.0
Sample B 614 3.1
2. Incubate for 60 minutes at room temperature. During all Sample C 2013 4.7
incubations, avoid direct sunlight and close proximity of
any heat sources. Intra-assay precision
3. After 60 minutes, decant or aspirate the well contents and CVs typically <12%
wash the wells 3 times using automated washing or the
manual wash procedure (see below). Careful washing is Linearity
the key to good results. Do not allow the wells to dry Serial dilutions of a serum yielded a linear response.
out. Dilution Measured IgE Corrected IgE
Neat 784 784
Manual Wash Procedure: 1:1 387 774
Empty the wells by inversion. Using a multi-channel 1:2 191 764
pipette or wash bottle, fill the wells with wash buffer. 1:4 98 784
Empty by inversion and blot the wells on absorbent paper.
Repeat this wash process 2 more times.
Method Summary
4. Dispense 100l of Conjugate (Reagent 3) into each well.
Incubate the wells for 30 minutes at room temperature.  Dispense 100l standards and the Positive Control
 Dispense 20l of each test sample.
5. After 30 minutes, discard the well contents and carefully
wash the wells 4 times with wash buffer. Ensure that the  Dispense 80l of sample diluent into each sample well
wells are empty but do not allow to dry out. (Reagent 1).
 Incubate for 60 minutes at room temperature.
6. Using a repeating dispenser, rapidly dispense 100l of  Wash the wells three times
TMB Substrate (Reagent 4) into each well. Incubate the  Dispense 100l of Conjugate (Reagent 3) into each well
plate for 10 minutes.  Incubate at room temperature for 30 minutes
 Wash the wells four times
7. Add 100l of Stop Solution (Reagent 5) to each well. To  Add 100l of TMB Substrate (Reagent 4) to each well
allow equal reaction times, the Stop Solution should be  Incubate at room temperature for 10 minutes
added to the wells in the same order as the TMB
 Add 100l Stop Solution (Reagent 5) to each well
Substrate.
 Read the optical density at 450nm (single wavelength) or
8. Read the optical density (OD) of each well at 450nm in a 450/620nm (dual wavelength).
microplate reader within 10 minutes. A 620nm filter may
be used as a reference wavelength.
Further reading
11. Quality control
Pollart SM, Smith TF, Morris EC, et al. Environmental exposure to
Quality control data is supplied on the lot-specific QC certificate cockroach allergens: analysis with monoclonal antibody-based
included in the kit. enzyme immunoassays. J Allergy Clin Immunol 1991;87:50510.
Chapman MD. Allergen specific monoclonal antibodies: new tools for
The positive control is intended to monitor for substantial reagent the management of allergic disease. Allergy 1988;43:714.
failure. Kemeny DM, Urbanek R, Samuel D, et al. Increased sensitivity and
specificity of a sandwich ELISA for measurement of IgE antibodies. J
Any well positive by spectrophotometer but without visible colour
Immunol Methods 1985;78:21726.
should be cleaned on the underside and re-read. If OD-values below
Kemeny DM, Urbanek R, Samuel D,et al. The use of monoclonal and
zero are observed, the wavelengths used should be verified, the
polyspecific antibodies in the IgE Sandwich ELISA. J Immunol
reader re-blanked to air and the measurements repeated.
Methods 1986;87:4550.
12. Interpretation of Results

Using a 4 parameter logistic fit; plot the optical density of each

standard against its concentration. Read the unknowns off this
curve. Values above 1250 IU/ml should be repeated at a higher
dilution, using the sample diluent provided.

263-009-03