FAQ

Purified Allergens

Allergens on-Demand Videos

Check out our on-Demand videos about purified allergens. Dr.Sabina Wünschmann, Head of Allergen Manufacturing, R&D discusses advantages of using purified allergens, and provides insight into Indoor Biotechnologies Allergen Manufacturing processes. The 11 minute videos highlights Pros, Cons, and applications of natural and recombinant allergens.

Q1 How should allergens be stored?

A: Allergens should be stored frozen in a -20ºC freezer.

Q2 How are allergens purified?

A: The purification protocol varies for different allergens. We use affinity chromatography, ion exchange, hydrophobic interaction and size-exclusion chromatographic methods for purification of natural, recombinant and LoTox™ allergens.

Q3 In which buffer is the allergen supplied?

A: Our natural and recombinant allergens are supplied in preservative-free and carrier-free phosphate buffered saline, pH 7.4 and are sterile filtered.
LoTox™ allergens are supplied in Preservative and carrier-free sterile, endotoxin-free, phosphate buffered saline, pH 7.4.

Q4 What are LoTox™ allergens?

A: LoTox™ allergens contain very low endotoxin levels below 0.03 EU/µg protein and are ideal for human and murine cellular studies using T-cells, APC’s or dendritic cells.

Q5 Why should I use LoTox™ products for my T-cell studies?

A: The LoTox™ product is ideal because it is almost endotoxin free and avoids the issue of whether the T-cell responses may be driven by endotoxin.

Q6 Why is it important to work with purified allergens rather than allergen extracts?

A: Allergen extracts are heterogeneous and contain non-allergenic proteins and other macromolecules. Investigators often use extracts because they tend to give high T cell responses, but the interpretation of these responses is questionable. The specificity of the response cannot be verified and it is quite possible that the responses could be driven by non-allergenic proteins or other substances. Thus we recommend using purifed allergens to measure, for example, IgE and IgG antibody responses, histamine release experiments and animal models. We strongly recommend LoTox™ products for any cellular studies, where endotoxin may influence the cellular response.

Q7 Should I use natural or recombinant allergens?

A: Recombinant allergens compare structurally very well to their natural counterparts and show comparable binding in IgE antibody assays. Pichia pastoris expressed recombinant proteins tend to be hyperglycosylated which may interfere with IgE binding. We offer recombinant LoTox™ Fel d 1 (LTR-FD1D), rBla g 2 (RP-BG2) and rDer p1 (RP-DP1D) as de-glycosylated products.

Q8 How do I activate the cysteine protease activity of Der p 1?

A: Der p 1 is activated by 5 mM cysteine or 1mM Dithiothreitol to regenerate its thiol group, which becomes oxidized during purification (Gough et al. Journal of Experimental Medicine, 1999).

Q9 Which substrate do I use to measure Der p 1 cysteine protease activity?

A: DTT-dependent cysteine protease activity can be measured in a fluorescent assay using the fluorescent peptide substrate Boc-QAR-MCA.

Q10 Do I need to activate Der p serine protease activity?

A: No, the Der p serine proteases (NA-DPSP) do not need to be activated.

Q11 Which substrate do I use to measure Der p serine protease activity?

A: DTT-independent serine protease activity can be measured in a fluorescent assay using the fluorescent peptide substrate Boc-QGR-MCA.

Q12 Which species of shrimp was used for manufacturing of natural shrimp tropomyosin?

A: The shrimp used for manufacturing of the shrimp tropomyosin are Carolina shrimp bought fresh from a local seafood market. They are boiled before extraction. The seasons for white (Penaeus setiferus) and brown shrimp (Penaeus aztecus) in the Carolina’s overlap and thus the shrimp we buy are likely a mixture of these two species. This is why we do not use the allergen nomenclature for this product.

MARIA®

Statement on use of Bovine Serum Albumin (BSA) 

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

A: 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.

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.

Instructions for MARIA® Data Processing

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 biannual training courses at our facility in Charlottesville, VA. Please check our website for more information. We also offer custom training in MARIA® on a year-round basis.

ELISA

Q1 What is the difference between the Der p 1 ELISA kits, EL-DP1 and EL-DP1A?

A: INDOOR Biotechnologies markets two ELISA kits to measure Der p 1 which differ in the combination of monoclonal antibodies that are used in the assays:

EL-DP1: Uses capture mAb 5H8 and biotinylated mAb 4C1 for detection EL-DP1A: Uses capture mAb 10B9 and biotinylated mAb 5H8

The most widely used ELISA kit is EL-DP1 (5H8/4C1). The biotin 4C1 mAb is also used in the Der f 1 ELISA (EL-DF1). EL-DP1 is recommended for most routine analyses of dust and air samples for Der p 1. However, there is a degree of cross-reactivity for Der f 1 in the EL-DP1 assay, of about 2% (see Luczynska et al, J Immunol Meth, 1989). For routine analyses, this level of cross-reactivity is not significant.

The EL-DP1A kit shows <0.1% cross-reactivity with Der f 1 and is designed for applications that need absolute distinction between Der p 1 and Der f 1. For example, an allergen manufacturer who wants to be sure that they have no cross contamination between species in their dust mite cultures. The EL-DP1A assay has the same level of sensitivity as the EL-DP1 kit (1-2ng/ml).

Q2 We noticed that the assay was very slow in developing (and never reached OD 2.0). Is this a problem?

A1: There is definitely a problem with your results - the control curves should read OD 2.0-2.4 at the highest concentration and have sensitivity down to 1-4ng/ml. If not reaching an OD of 2.0 is the case with all your assays, it suggests that it may have something to do with the substrate system and color development.

A2: Have the biotinylated reagents been frozen at any time during storage? This may reduce the sensitivity of those mAbs.

A3: Are you making up the reagents or using buffer tablets? We have found that some buffer tablets do not work as well as fresh made solutions. We usually keep all solutions for less than a month at 4°C without Thimerosal.

A4: Has sodium azide been used in any of the reagents as preservatives? This would stop the color development since it is an inhibitor of horseradish peroxidase.

A5: When making up Solution B for the ABTS is the correct form of Sodium Phosphate used? The sodium phosphate has to be Dibasic, Heptahydrate (Na2HPO4·7H20).

A6: Are you adding enough hydrogen peroxide to the ABTS solution? Use 1µl of 30% H2O2/ml of ABTS solution.

A7: Has the streptavidin-peroxidase been diluted correctly?

A8: Is the pH of the ABTS citrate buffer correct? The pH of the ABTS solution should be pH 4.2 and should be close to that pH before adjustment. If it is far off, solution A or B or their proportions are probably incorrect.

A9: Plate washer contamination can sometimes hinder color development causing the plate to develop very slowly or never reaching an O.D. of 2.0.  Plate washers should be cleaned on a regular basis.

A10: Direct sunlight exposure via windows will cause both of the above issues.  Leave ELISA plates away from windows during incubation.

Q3 The backgrounds for our assays have been high. What could be the cause of this?

A1: The background on the assay is usually higher in assays where the secondary antibody is a rabbit polyclonal. The background is usually around 0.15 OD. Other causes of the high background may be:

A2: The source of the peroxidase labeled Goat anti Rabbit IgG antibody. We use material from BioSource International and also Jackson Laboratories. Both work well with very low backgrounds. BioSource International Tel: 1-800-242-0607 (Cat # ALI4404) Jackson Laboratories Tel: 1-800-367-5296 (Cat # 111-036-045)

A3: How long is the ABTS substrate kept at 4°C? It is usually stable for about one month in the fridge. It can start to turn green and give high backgrounds if kept for longer periods. The ABTS solution should be more or less colorless for use in the assay. The same goes for the other solutions. We usually keep all solutions for less than a month at 4°C without Thimerosal.

A4: Block the plates for 30 minutes with 1%BSA/PBS-T before adding allergen solutions.

A5: In some cases a cause for high background or random high samples in a sample dilution series  where the first few sample dilutions are low or not detectable can be caused by plate washer contamination.  Plate washers should be cleaned on a regular bases to prevent random well contamination and plate washer blockages.

A6: If washing plates by hand, an increase in washes throughout the assay can help lower the backgroud.  Fill the wells completely without overflow and then dump the wells carefully not to contaminate.  Then gently tap the excess buffer from the plate on a paper towel.

Q4 Our dupilcate standard curves and/or samples have very high CV values.  What could be the cause?

A1: After adding the ABTS shake gently by tapping the plate on the side occasionally and right before reading to ensure the color development is homogenous throughout.

A2: Pipetting is very important when running the ELISA assays.  Be sure that the pipettes being used, especially multichannels, are picking up and dispensing evenly during your doubling dilutions across the plate.  Also be sure that there are no air bubbles during your transfers. 

Q5 Can we keep antibody-coated micro-wells in storage? How long can they be stored and at what conditions?

A: The coated plates can only be stored for a short period e.g. over the weekend or up to a week. We usually leave plates coated with antibody solution and wrapped in plastic film in the lab fridge or cold room. We have not tried longer storage times.

Q6 Can we substitute the ABTS with OPD or TMB? We had difficulties keeping the ABTS under the conditions required by Sigma (in a desiccator, under Argon).

A: You can use OPD or TMB. We do not keep ABTS under Argon (it doesn’t appear to be necessary). We have good results with ABTS.

Q7 Should the dust samples be stored at 4°C or should they be frozen?

A: We routinely keep house dust samples at 4°C prior to assay, but once extracted they should be stored at -20°C.

Q8 How can I stop the enzyme reaction in the ELISA?

A: The reaction can be stopped by adding 0.1ml 0.002M sodium azide to each well. Most ELISA readers scan plates in 5-20 seconds and plates can be read once the OD reaches 2.0-2.4 without stopping the reaction. Azide can be added if a large number (5-10 plates) are set up, or if the plates need to be preserved for any other reason e.g. taking photographs.

Q9 What temperature is used for the ELISA incubations?

A: Coating the ELISA plates with the capture mAb is carried out overnight at 4°C. All other steps are carried out at room temperature, usually 20-25°C.

Q10 What is the isoform specificity of Indoor Biotechnologies ELISA kits?

A: Isoform Specificity of ELISA or MARIA®

Q11 Could you please tell me how many dust (or air) samples can we analyse with one ELISA kit? How many do you recommend?

A1: Indoor Biotechnologies ELISA kits contain sufficient reagents (capture mAb, biotinylated detector mAb and allergen standard) for 10 plastic 96-well microtiter plates. The number of samples that can be analyzed per plate depends on the number of dilutions used for each sample.

A2: Standard curve in duplicate, with 4 blank (PBS-T) wells, uses 24 microtiter wells, leaving 72 wells for samples on each plate. Typically, we use 4 doubling dilutions for each dust sample (1/10, 1/20, 1/40, 1/80 for mite allergens), which means that 18 samples can be tested on a plate. Under these conditions, one 10 plate ELISA kit could be used to analyze 18 x 10 = 180 samples.

A4: We use four dilutions per sample so that we can in most cases obtain results on >90% of the samples in a single assay. The four dilutions increase the probability of having at least two points on the linear part of the control curve for each sample. It may be possible to use fewer dilutions, if the samples are known to have allergen levels in a particular range. Other samples, e.g. allergenic extracts, may have very high allergen levels and have to be assayed at doubling dilutions starting at 1/100 or 1/1000, and may need to be serially diluted across the ELISA plate.

A5: Air samples usually contain low levels of allergen and are assayed at 1/2, 1/4, 1/8, and 1/16 dilution.

Environmental Testing

Q1 I want to send some samples to Indoor Biotechnologies for allergen analysis. How much sample do you need?

A1: For the analysis lab to analyze for the allergen levels in bulk dust samples, we need 100mg of fine dust. Our recent findings (click link below) indicate that due to increased potential for variability, allergen analysis should not be attempted unless a minimum of 10mg fine dust is available. Samples with less than 10mg available of fine dust will not be analyzed and a handling charge will be applied. (NES - Not Enough Sample)

"Variability introduced into allergen immunoassays during the dust extraction process" 

A2: If using the DUSTREAM™ and sampling for the recommended area and time yields very little or no dust, continue sampling until the filter is 1/4 full.  Our allergen results are not based on area, but on microgram of allergen per gram of dust.

A3: For allergen extracts or extracts of bulk dust or air filters, we need a minimum of 0.5 mL, especially if testing for multiple allergens.

A4: For air sampling cassettes, the whole filter is extracted with 1ml of PBS extraction solution and the results are reported as ng or µg allergen per filter.

Q2 I want to send some samples to Indoor Biotechnologies for endotoxin analysis.  How much sample do you need?

A1: For the analysis lab to analyze for endotoxin levels in bulk dust samples, we need 100mg of fine dust.  Samples of bulk dust will not be processed if there is less than 20mg of fine dust.  If using the DUSTREAM™ and sampling for the recommended area and time yields very little or no dust, continue sampling until the filter is 1/4 full.  Our endotoxin results are not based on area, but on endotoxin Units per gram dust.

A2: It is very important that we receive samples that have not been extracted.  The extraction procedure must be performed in a clean environment with endotoxin free water containing 0.05% Tween.  Samples should be extracted and analyzed on the same day.  These steps are important in preventing endotoxin contamination from other sources. 

A3: Air filter samples can also be sent in for endotoxin analysis.  The whole filter is extracted in 1mL of endotoxin free water with 0.05% Tween.  Results are reported as endotoxin Units per filter.  We recommend sending in a blank filter for testing as well to provide a baseline for the specific filter being used.  

Q3 I am preparing dust samples for analysis in my laboratory. Do I need to sieve the dust?

A: Whether to sieve the dust or not is determined by the quality of the dust. We do not routinely sieve dust collected from beds and soft furnishings. Carpet samples are sieved (e.g. using a No.45 mesh screen, 355um diameter sieve) to separate the fiber and large particles from fine dust. We recommend the use of the DUSTREAM™ collector which greatly reduces the need for sieving dust. After collecting a dust sample using the DUSTREAM™ collector, remove the nylon filter containing the dust sample insert, invert the filter and tap the dust onto a piece of weigh paper. The fine dust falls before the fibers come out of the filter.

Q4 How should dust samples and dust extracts be stored?

A: Dust samples prior to extraction should be stored in a dry place at room temperature. Dust extracts should be stored frozen in a -20C freezer.

Q5 What are the procedures for sampling and submitting samples for INDOOR Allergen Analysis Service?

A1: Please see Collecting Dust Samples and Submitting Samples.

A2: Download Analysis Request Form

Q6 What method do you recommend for preparation of dust extracts for allergen testing?

A1: Please see Sample Extraction Procedure.

A2: Extracts for endotoxin testing need to be performed by Indoor Biotechnologies analysis service lab on the day they are analyzed due to the multiple risks of endotoxin contamination.  Extracts should not be sent in for this service, only dry samples such as bulk dust and air filters.

IgE-QBA™