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Titration of the concentration HDM challenge in allergy model

In a recent experiment sensitized and non-sensitized mice (Female, 6-8 week old BALB/c mice) were compared in their response to 3 intranasal challenges with concentrations of 1µg, 5 µg and 25 µg protein. This experiment was performed with Citeq D. pteronyssinus extract (batch 15G10). The results can differ per batch but it gives a reference to determine the concentration of HDM extract to be used as an inhalational challenge.

Sensitization of mice

Three groups of mice (3 groups of n=6) will be immunized by intraperitoneal (i.p.) administration with 5 µg HDM (Citeq) /2.25 mg alum (Pierce) and three groups will receive nothing on days 0, and 14.

HDM challenge

At day 21, 23 and 25 the mice are challenged intranasal (i.n) in 30 µl PBS containing:

  1. 25 µg HDM extract
  2. 5 µg HDM extract
  3. 1 µg HDM extract
Group Day 0/ 14 Day 39/ 42/ 44
1. 5 µg HDM + 2.25 mg alum i.p. 25 µg HDM i.n.
2. 5 µg HDM + 2.25 mg alum i.p. 5 µg HDM i.n.
3. 5 µg HDM + 2.25 mg alum i.p. 1 µg HDM i.n.
4. No i.p. 25 µg HDM i.n.
5. No i.p. 5 µg HDM i.n.
6. No i.p. 1 µg HDM i.n.

In the most optimal scenario, a Der p 1/ Der p 2 therapy should suppress the response similar to the level observed in non-sensitized mice. To determine which concentration would not induce a Th2 like pathology independent of a previous HDM sensitization, these concentrations were tested. All the parameters are summarized in the table below. 

Overview of effect of HDM challenge in sensitized mice vs non sensitized mice: 

  S25 S5 S1 NS25 NS5 NS1
Eosinophils ++ ++ +
% Eosinophils +++ +++ ++ +
Total BALF +++ ++ +
IL-33 +++ ++ + + +
IL-4 +++ +++ ++ + +
IL-5 +++ +++ +++ ++ +
IL-13 +++ +++ +++ + +
IFNg + + + ++ +
IL-10 +++ +++ +++ ? ? ? –
IL-17A ++ +
HDM IgE ++ ++ -/+
HDM IgG1 ++ ++ ++
HDM IgG2a
Airway hyperreactivity ++ ++ + ++
Mucus ++ ++ + +/-
Inflammation ++ ++ + + + +

 

The challenge with 1 µg HDM in non-sensitized mice did not induce any parameter tested (except for recruitment of inflammatory cells based on histology, not reflected in BALF), while 25 µg HDM induced most of the parameters except the immunoglobulin response. The concentration of 5 µg HDM induced some parameters, but often less profound as seen with 25 µg HDM. In conclusion, the concentration of 5 µg HDM as an inhalational challenge is sufficient to induce a clear Th2 driven immune response while 1 µg HDM induces a mild inflammation. 5 µg HDM in non-sensitized mice induced a slightly increased innate immune response compared with 1 µg HDM challenge. A concentration of 25 µg HDM induced a significant innate immune response, as seen in eosinophil recruitment, besides the Th2 driven immune response and is therefore less suitable to use in a vaccine model. The choice has to be made between a mild HDM inflammation (1 µg) with apparently no innate immune response and a strong HDM inflammation (5 µg) with a modest innate immune response.

We should consider the following points:

  1. S1 showed an increase in eosinophils, IL-33, Th2 cytokine production and even in HDM sp IgE (in ELISA with Citeq HDM bi) albeit much stronger than seen in S 5 (S 1 and S 5 both potentially suitable)
  2. a strong inflammation might be more difficult to suppress (S1 preferred choice)
  3. a less strong inflammation provides “less” amelioration (S 5 preferred choice; with S 1 only in case of a complete inhibition it will give a significant effect of therapy)
  4. the difference between S 5 and S 1 (secondary immune response) is greater than between NS 5 and NS 1 (innate response). (S5 preferred choice).

Our HDM products

Read more about: HDM induced asthma models

Often we receive questions about house dust mite (HDM) induced asthma models. Which models are researchers using, which model do we recommend, what are the differences? To answer these questions we have contacted multiple research institutes all over the world and collected their models and background information. On this page a selection of these models.

Quantification of antigen specific serum antibodies

To determine whether immunizations of mice with house dust mite (HDM) have been successful you can look at the amount of specific antibodies against HDM proteins in the serum of mice.

Together with our biotinylated HDM allergen products, antibodies can be used to quantify HDM allergen specific IgE/ IgG1/ IgG2a antibodies in mouse serum using an ELISA method. The antibody can be coated on an ELISA plate and act as a capture antibody to immobilize serum IgE/ IgG1/ IgG2a antibodies. In the next step biotinylated HDM allergens are added to facilitate the detection of HDM allergen specific antibodies of the IgE/ IgG1/ IgG2a isotype. Using a reference serum or by comparing multiple sera the HDM allergen specific antibodies can be (relatively) quantified.