The Mite Group 2 MARIA multiplex assay detects both Der p 2 and Der f 2 allergens. The assay uses a combination of monoclonal antibodies, 1D8 and 7A1, which bind to a common epitope on dust mite Dermatophagoides pteronyssinus allergen, Der p 2 and Dermatophagoides farinae allergen, Der f 2. The Mite Group 2 MARIA immunoassay allergen standard is prepared from purified natural Der p 2.
Note: Der p 2 and Der f 2 cannot be simultaneously quantified in the MARIA assay because they are recognized by the same antibody pair and therefore are not distinguishable. In a sample that contains both Der p 2 and Der f 2 (such as a mixed mite culture), the antibodies will bind both Der p 2 and Der f 2; however, results will be calculated against a Der p 2 standard curve. If separate analysis is needed to quantify both Der p 2 and Der f 2, InBio’s Der p 2 and Der f 2 ELISA 2.0 assays are recommended.
No. Upon receipt of the MARIA® kit it is important that all reagents, except the standards and quality controls (QC-MRA-L and QC-MRA-H), are stored in the refrigerator at 4°C. Freezing will damage the microspheres, BI-MRA and SAP-MRA reagents. The standards and quality controls should be stored frozen at -20°C, unless used immediately.
The kit will be under warranty for 6 months upon receipt, provided that it has been stored correctly.
Yes. The MARIA® is a bead-based assay. Therefore, it is important to minimize the number of foreign particles in the sample.
The recommended sample dilutions depend on the kind of sample you have and the level of sensitivity you require for your study:
- For house dust extracts, we recommend diluting samples at 1/10, 1/100 and 1/10,000. This will give you access to lower limits of detection between 0.1-0.9ng/mL (equivalent to 0.002-0.012µg/g dust).
- If you do not need such high sensitivity, dilute samples only at 1/100 and 10,000 for sensitivity levels of 1-6ng/ml (0.02-0.12µg/g dust). This will allow you to assay 35 samples per plate.
- Air filter extracts contain much lower allergen concentrations. We generally assay these samples undiluted, at 1/10 and 1/50.
For serial dilutions of 1/10, 1/100 and 1/10,000:
- Prepare three microcentrifuge tubes per sample. Add 10µL of sample to 90µL of assay buffer for a 1/10 dilution in tube 1; mix by vortexing. Add 90µL assay buffer into tube 2 and transfer 10µL of the 1/10 dilution (tube 1) into 90µL assay buffer; mix by vortexing. Add 990µL of assay buffer into tube 3 and transfer 10µL of the 1/100 diluted sample from tube 2 into tube 3; mix by vortexing. 50µL of each prepared dilution are used in the MARIA® assay.
For serial dilution of 1/10 and 1/50:
- Prepare two microcentrifuge tubes per sample. Add 90µL of assay buffer into tube 1 and add 10µL undiluted sample; mix by vortexing. Add 80µL of assay buffer to tube 2 and transfer 20µL of diluted sample from tube 1 into tube 2; mix by vortexing.
Yes! Unfiltered assay buffer has a high particle load that will interfere with measurement in the xMAP system. It will cause high bead aggregation ratios and may increase the time it takes to read the plate 3-4-fold.
No. Inverting the plate or rinsing of the wells will wash away the microspheres. All washing steps have to be performed by filtration using a vacuum manifold and the filter plate provided with the kit.
No. Before every incubation step of the assay, beads and reagents are mixed thoroughly by vigorous pipetting. Shaking during incubations is not necessary.
All incubations have to be performed in the dark, otherwise the microspheres may lose their distinguishable fluorescent label.
We generally cover the plate with the lid provided and place it in an empty drawer for the incubation periods.
The kit provided can only be used in conjunction with Luminex Corporation’s laser based fluorescent analytical test instrumentation: Luminex® 100, Luminex 200 and other Luminex Instruments available from Luminex Corporation and from authorized distributors, including Bio-Rad Laboratories (Hercules, CA), Qiagen Corporation (Valencia, CA) and MiraiBio (South San Francisco, CA). We use Bio-Plex instrumentation with Bio-Plex Manager software (Bio-Rad, Hercules, CA).
We recommend counting 100 beads per analyte for maximum reproducibility.
We use a 5 parameter logistic (5PL) curve fit. (The fit can be chosen easily in the Bio-Plex Manager software.)
If the Fluorescent Intensity (FI) of the low end of the curve is close to the FI of the Blank, do not use values that fall within the FI of the Blank plus three times the Standard Deviation of the Blank.
We generally do not use data that fall within the FI of the top flat part of the curve. This means that we normally exclude the top 2-3 points of the curve for Der p 1, Der f 1, Der p 2, Fel d 1, Can f 1, Rat n 1 and Mus m 1. The top part of the Bla g 2 curve is usable, whereas the 3-4 lowest points are too close to the blank (see above).
Some software packets, such as the Bio-Plex Manager® (Bio-Rad, Hercules, CA) automatically show the ratio between expected and observed standard values in the exported Excel file. We use this function as additional quality control and only use values, where the observed/expected ratio is within 15% of 100%.
The coefficient of variance (CV%) between duplicate standards should not exceed 15%. If this is the case, the accuracy of your pipettes and pipetting procedures should be checked.
Depending on the allergen concentration in your sample, you may get different results for different sample dilutions. There are a number of criteria that determine which result to choose:
Only select results that fall within the usable MFI range. If more than one dilution produces similar results: Average results.
For all analytes but Der f 1: If results rise with increasing dilution factors: Choose the result based on the highest dilution factor
- A small percentage of Der f 1 samples show a low-level interference with substances within that particular dust extract that becomes amplified by the calculation of higher dilution factors. This effect is present if you observe results such as 1/10= 7ng/ml, 1/100=74ng/ml, 1/10,000=4500ng/ml. In this case, use the result from the 1/10 dilution, unless this result reaches ~100ng/ml, at which point you can rely on the results from the higher dilution factors. This aberrant effect has only been observed in <5% of our tested samples and only appears to occur in samples with low levels of Der f 1.
A: Yes, contact InBio today for MARIA® training opportunities.
If you don’t find your answer in the questions listed above, please submit a troubleshooting form to support@inbio.com that includes specifics about your issue, kit details, plate data and/or your data file.
