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Use of biotinylated allergens

Biotinylation is a process of attaching biotin to proteins and/or other macromolecules. Biotinylated proteins can, due to the high specifity of biotin-avidin interaction, be used for the detection/labelling of specific antibodies against allergens in serum of animal models. With this method it is, for example, possible to verify in an early stage if an animal model is correctly immunized and in a later stage of the experiment verify if the animal responds to the treatment (an increase or decrease in the levels of specific antibodies). The use of biotinylated proteins is to detect specific binding of the different antibodies induced in an animal model, e,g, specific IgG1 IgG2a and IgE. With this, specific antibodies are detected and the amount could be related to different mouse serum standards.

Biotinylated proces explained

A micro well plate is coated with anti-IgE (or IgG1/ IgG2a). The next step is to add serum, in which allergen specific antibody detection has to be determined, to the plate. After plate washing the biotinylated allergen is added, which will be captured by the serum antibodies (if present). Streptavidin conjugate has a high affinity for biotine and is linked to an enzyme which converts e.g. TMB (tetramethyl benzene) substrate into a colour. With the colour the level of specific anti-bodies which are present in the serum can be measured.

Biotinylated house dust mite proteins

Citeq has developed house dust mite related biotinylated proteins. These biotinylated proteins include biotinylated D. pteronyssinus extract FD, biotinylated D. farinae extract FD, and natural purified biotinylated Der p 1 protein and biotinylated Der p 2 protein.  Biotinylated proteins can be applied in many protein assays such as western blot analysis, fluorescence-activated cell sorting (FACS) and flow cytometry and enzyme-linked immunosorbent assay (ELISA).

Our biotinylated products

Results of using biotinylated proteins

Using the biotinylated proteins from Citeq for the detection of HDM specific IgE (or IgG1/ IgG2a) in mouse serum shows a highly recognizable binding. The OD for biotinylated-HDM from Citeq is much higher compared to biotinylated HDM extracts by research groups with a commercially available kit.

As shown in the comparison below, the OD for a commercial kit is 0.05, whereas the OD for the biotinylated HDM of Citeq is 0.5.

comparison biotinylation

Protocol for using biotinylated products

Protocol for using biotinylated products (It is recommended that the instruction sheet be read in its entirety before using this product).

Required materials

Required equipment

  • Microplate reader with software
  • Microplate washer (optional) 

Method

  1. Coat micro wells with 100 µl per well anti-IgE (or IgG1/ IgG2a) capture antibody diluted in coating buffer. Incubate overnight at 4°C.
  2. Aspirate wells and wash 3 times with 300 µl/well wash-buffer.
  3. Block plates with 300 µl blocking-buffer. Incubate at room temperature for (at least) 1 hour.
  4. Aspirate wells and wash 1 time with 300 µl/well wash-buffer.
  5. Dilute serum samples and controls/references in assay diluent. Pipette 100 µl of each in duplointo the wells. Incubate for 2.5 hours at room temperature.
  6. Aspirate wells and wash 5 times with 300 µl/well wash-buffer.
  7. Add 100 μl/well of diluted biotinylated HDM/Der p 1/Der p 2 to each sample and incubate for 1 hour at room temperature.
  8. Aspirate wells and wash 5 times with 300 µl/well wash-buffer.
  9. Add 100 µl of diluted Streptavidin-Peroxidase to each well, incubate for 30 minutes at room temperature.
  10. Aspirate wells and wash 7 times with 300 µl/well wash-buffer.
  11. Add 100 µl of substrate solution to each well.
    Incubate plate at room temperature in the dark until:
    – blanks are staining or
    – until the positive control stains clearly.
  12. Add 50 µl of stop solution to each well.
  13. Read absorbance at 450 nm within 30 minutes of stopping reaction. Use A 650 as a reference filter.