Frequently Asked Questions - ELISA

Sensitivity and reproducibiltity of ELISA for indoor allergens

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?

A 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.

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

A 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.

A 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.

A 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).

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

A Has the streptavidin-peroxidase been diluted correctly?

A 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.

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

A 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:

A 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)

A 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.

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

Q4 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.

Q5 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.

Q6 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.

Q7 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.

Q8 What temperature is used for the ELISA incubations?

Q9 I would like Indoor Biotechnologies to check the allergen content of my allergen extracts or solutions. What quantity should I send?

A The INDOOR® Allergen Analysis reference lab needs a minimum quantity of 0.5ml of extract.

Q10 Could you please tell me how many dust (or air) samples can we analyse with one ELISA kit? How many do you recommend?

A Indoor Biotechnologies ELISA kits contain sufficient reagents (capture mAb, biotinylated detector mAb and allergen standard) for either 10 or 20 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 and whether or not a control curve is included on every plate.

If a control curve is included in duplicate on every plate, with 4 control (PBS-T) wells, it will use 24 microtiter wells, leaving 72 wells for samples. 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 20 plate ELISA kit could be used to analyse 18 x 20 = 360 samples. However, the plate to plate variation for most assays is less than 10% and we normally do not need to have a control curve on every plate. For example, we might set up four plates with only one control curve. Under these conditions, 24 samples can be analysed per plate, together with 18 on the same plate as the control curve, or 90 samples in all on 4 plates. This would mean 450 samples could be analysed using the 20 plate ELISA kit.

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.

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