Frequently Asked Questions - MARIA™

Q1 Can I store the MARIA™ kit in the freezer or at room temperature?

A No. It is important that the kit and all reagents are stored in the refrigerator at 4°C. Freezing will damage the reagents.

Q2 Is there an expiry date for the MARIA™ kit?

A The kit will be under warranty for 6 months upon receipt, provided that it has been stored correctly.

Q3 Is it necessary to centrifuge the samples before the assay?

A Yes. The MARIA™ is a bead-based assay. Therefore, it is important to minimize the number of foreign particles in the sample.

Q4 Which sample dilutions do you recommend?

A 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.

Q5 How can I prepare the serial dilutions?

A1 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.

A2 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.

Q6 Is it really necessary to sterile-filter the assay buffer?

A Yes! Unfiltered assay buffer has a high particle load that will interfere with measurement in the xMAP sytem. It will cause high bead aggregation ratios and may increase the time it takes to read the plate 3-4-fold.

Q7 Can I use an ELISA plate washer for the MARIA™ assay?

A 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.

Q8 Do I need to shake the plate during incubations?

A No. Before every incubation step of the assay, beads and reagents are mixed thoroughly by vigorous pipetting. Shaking during incubations is not necessary.

Q9 How important is it to perform the incubations in the dark? Can I leave the plate on the bench?

A All incubations have to be performed in the dark, otherwise the microspheres may lose their distinguishable fluorescent label.

Q10 Do I need to cover the plate with aluminum foil during incubations?

A We generally cover the plate with the lid provided and place it in an empty drawer for the incubation periods.

Q11 What instrumentation can I use to read the plate?

A 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).

Q12 How many beads per analyte shall I specify for measurement?

A We recommend counting 100 beads per analyte for maximum reproducibility.

Q13 What curve fit shall I choose?

A We use a 5 parameter logistic (5PL) curve fit. (The fit can be chosen easily in the Bio-Plex Manager software.)

Q14 How can I determine the usable part of the standard curve?

A1 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.

A2 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).

A3 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%.

A4 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.

Q15 I get different results for the different sample dilutions. How do I choose the correct result?

A 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.

Q16 Can I receive training in MARIA™ technology?

A Yes. We offer a training course at our facility in Charlottesville, VA, on September 8-10, 2008. Please check our website for more information.