Technology & Assay

  1. How many components are there on ImmunoCAP ISAC?

    112.

  2. Do we have any experience when it comes to automation of the ISAC assay procedure (pipetting, washing or incubation)? Is the device „Beeline 220s“ suitable for this purpose?

    We did some experiments in-house with automation – but not with the device mentionned. It can more or less be concluded that transferring the ISAC assay procedure to automatic devices is not an easy project so we have no recommendation to give at this moment.

  3. ISAC is a semi-quantitative technology. What does it mean?

    The definition of a semi-quantitative test according to CLSI IL/A20-A2 is not related to the accuracy or quality of a test results, it is related to the calibration methodology. ImmunoCAP ISAC® sIgE 112 calibration is made against an in-house reference preparation (called control sample) and measured IgE antibody concentrations are expressed as arbitrary units; ISAC Standardized Units for IgE (ISU-E). The ImmunoCAP ISAC® sIgE 112 in-house reference preparation is calibrated against ImmunoCAP Specific IgE, which is standardized against the WHO reference preparation 75/502 for IgE. In other words: ImmunoCAP ISAC is formally, according to CLSI IL/A20-A2, considered as "semi-quantitative" because the dose response curve used to estimate the levels of IgE antibodies is not directly calibrated to the WHO IgE reference preparation for IgE. The results for IgE antibodies, expressed in arbitrary ISAC Standardized Units for IgE (ISU-E), are proportional to the concentration of specific antibodies in test samples. The results are continuous and range from 0 to 100 ISU-E. This range approximately corresponds to a concentration range of 0 -100 kU/l IgE.

  4. A customer is looking at the IgE intensity variation. Is there any possibility of interference from, for example, IgG?

    a) If a patient is having SIT treatment, thus having elevated IgG levels in the blood – it is possible that IgG might cause some interference. b) The intensity variation could also be sample dependent due to sample stickiness. It has been noted that this results in higher CVs than for other samples if tested repeatedly.

  5. What is the difference between ImmunoCAP ISAC and ImmunoCAP component tests?

    ImmunoCAP ISAC is a test where you can test a patient for several antigens at the same time. The chip contains 112 components and you get information about these plus many related proteins. This useful information can be of help when diagnosing multi-sensitized patients or patients that haven’t responded satisfactory to treatment. Using ImmunoCAP ISAC you will get an “overall picture” of your patient. With ImmunoCAP components tests you get detailed information on single component tests, which is useful when you have one or a few components in focus. ISAC is a microarray technique where the allergens are coupled to a glass slide surface while ImmunoCAP component tests have a three dimensional surface with a larger amount of antigens bound, allowing more antibodies to bind. ISAC is a manual assay while ImmunoCAP component tests are automated.

  6. How much sample is required?

    30μl sample for each run and patient.

  7. What is the reason for the change from 2 to 3 guide dots (GD) in the left bottom corner?

    The 2 GDs date back to the time when customers also used other scanners. Now all customers use scanners with barcode readers – thus the orientation is given by the barcode (If customers put the slide into the reader the other way around the barcode will not be scanned).

  8. In the ISAC DFU on page 5 under the Washing Solution section it says: “For information see separate Washing Solution Directions for Use.” but when I refer to the Washing Solution DFU (52-5216-EN/15) it mentioned that one should mix 17.2 ml Washing Solution additive and one bottle of washing solution concentrate (80 ml) to 1 L of purified water. Is this correct for the new ISAC 112 assay as well?

    Yes, that is correct.

  9. The fridge broke down for a week and during that time the ISAC kits and reagents were not refrigerated. Will this have caused any problems or is the product still ok to use?

    Most probably the product is still ok. We made a stability test, where the product was shown not to be negatively affected. The transport simulation protocol: Storage at +32°C for one week, during that week the test material was moved to +2°C to +8°C for 18-24 hours at two different occasions, but not in the beginning or in the end of the transport simulation.

  10. Why are there positive IgG spots in one of the corners even though they are running an IgE assay.

    These spots are not positive IgG spots, in reality they will always shine either you run an IgE or IgG assay as they are so called guide spots and are needed for the fit of the array over the spots on the chip. The spots called IgG in the array are labeled in the same dye as IgE detection antibody. That’s why they shine in an IgE assay. The corner spots named IgE are labeled with the same dye as for the IgG detection antibody whereas they will shine in an IgG assay.

  11. What are the differences between the old (103) and the updated (112) ISAC chip?

    The binding chemistry has been improved on the new chip, which now also includes 112 components instead of 103 plus the calibration concept has been updated The components selection has also been updated and all the information about which components are replaced, updated or added and the rationale behind this, is documented in “New components and the rationale behind” at DiaNet --> Molecular Allergology --> ImmunoCAP ISAC --> Marketing material.

  12. Is the Specific IgE control sample the same for ImmunoCAP as for ISAC?

    No it is not, but anyhow the control sample is used for creating a calibration curve in order to make quantitation possible.

  13. How does the calibration concept for ImmunoCAP ISAC work?

    The two calibration samples (Specific IgE/IgG/IgG4 Control sample) for the different methods are the following: • Specific IgE: CTR02 • Specific IgG/IgG4: KS15 In Phadia MIA 1.1 and 1.2 and earlier versions there is a manual process step to enter what sample on a slide corresponds to a calibration sample. Criteria for calibration curve: - Slope is locked to 1. - Intercept X-axis Specific IgE; 5.5-6.6 (variable) - Intercept Y-axis Specific IgG/IgG4; 7.0-8.2 (variable) - Logaritmic model => All allergens have the same impact.

  14. What QC validation criteria are permitted for the calibration curve?

    The calibration curve must meet the following criteria: a. It should be no older than 30 days b. r² must be > 0.8 c. Not more than 20% of the calibration points are allowed to be bad (red circles) or negative. d. Intercept has defined ranges, which are different for IgE, IgG and IgG4. e. Variation is not considered explicitly for calibration points other than in 2a-e

  15. What components are covered with the control sample CTR02 and what levels can I expect? The new

    The new calibrator CTR02 is a pool and contain ab:s for the following 15 components and ISU levels: 1,0 ISU:, Bet v 1, Der p 2, Ole e 1, Gal d 1. 4,0 ISU: Art v 1, Fel d 1, Phl p 1. 15 ISU: Amb a 1, Can f 1, Der p 1, Gal d 2. 50 ISU: Can f 2, Can f 5, Phl p 5, Pru p 3

  16. What does the IgG4 calibration sample KS15 contain and what values/responses should I expect?

    The expected ISU values for the respective components are the following: Sample Allergen ISU value KS15 Api m 1 9,24 KS15 Bet v 1 7,99 KS15 Bet v 2 0,55 KS15 Gal d 1 10,95 KS15 Gal d 2 9,76 KS15 Phl p 1 2,63 KS15 Phl p 5 12

  17. Sometimes we experience dislocated spots in the analyzing step. Why and what do we do about it?

    Dislocated spots should not occur but the spotting procedure is a complex process. Our device works with piezo-technology not getting in contact with the slide as such. If there is a deflection of a single spot, it might not be detected during our internal quality control. MIA should alert in such cases – e.g., giving red circles indicating that customers should check the spot. If it is not clear to which triplicate group the spot belongs I would suggest to flag it as bad. 2 spots are sufficient for getting a positive result.

  18. This ISAC image has status failed since the guide dots are missing (IgE assay). Is it possible to validate the image anyway?

    <img style="float: left;" src="/FAQ/Allergy/ImmunoCAP%20ISAC/Failed%20assay.jpg" alt="" width="300" height="174" /> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; In this image, the guide dots are negative, which most probably is due to a problem during the detection antibody incubation. There are three possibilities: 1)&nbsp;&nbsp;&nbsp;&nbsp; The detection antibody was not applied sufficiently in the wells 2)&nbsp;&nbsp;&nbsp;&nbsp; The wrong detection antibody was applied (eg. IgG4) 3)&nbsp;&nbsp;&nbsp;&nbsp; The incubation time was not sufficient. The suggestion is to repeat the sample, as you might get false results if the guide dots are missing. If one or two of the guide dots show too weak signal, it might be more possible that the scanner settings are too weak.

  19. Why do we see inhomogenous spot morphology (not perfectly round shaped, but more flowery)

    This is an effect that has been observed when the slides are not adapted to room temperature before opening the vacuum pack. The DfU states to allow the box to reach room temperature before opening the seal (Recommended: 15 minutes).

As in all diagnostic testing, the diagnosis is made by the physican based on both test results and the patient history.