Improper reconstitution of lyophilized (freeze-dried) allergenic extracts can cause variations in allergen content of the same extract after reconstitution and should be avoided. Customers who have used our mould (fungi) extracts had questions about how to dissolve the extract properly. Based on these questions we have tested dissolving Alternaria alternata in different concentrations. On this page we report on the solubility of lyophilized Alternaria alternata medium extract of batch 10M11. This medium extract contains the highest amount of Alt a 1, the major allergen of Alternaria alternata. Batch 10M11 contains about 215 ug/mg (based on BCA determination).
Dissolving the medium extract
Alternaria alternata medium extract of batch 10M11 was dissolved in phosphate buffer solution, pH 6.4, at concentrations of 1, 2, 5, 10, and 20%. The solutions were mixed by vortex mixing for 5 minutes on maximum setting. Then sonication was performed at 4°C for 5 minutes. Solubility was determined by visual observation.
Results of solubility of Alternaria alternata
The solubility of Alternaria alternata medium extract of batch 10M11 differed with the used concentrations. Insoluble particles can be observed in 1 and 2% mixtures, both within the liquid and adhering on the inside of the tubes (see figure 1).
The 5 and 10% concentration mixtures are very opaque, preventing visual observation of insoluble particles within the liquid, however no particles can be seen adhering to the inside of the tubes. The absence of insoluble particles was confirmed in these concentrations by diluting them to less opaque solutions, with visual observation performed directly after dilution and with minimal mixing.
The 20% concentration yielded a very viscous solution that made mixing and correct pipetting difficult.
Figure 1: Tubes with different extract concentrations
Most allergens are soluble in solution. Alternaria alternata medium extract of batch 10M11 can be successfully reconstituted in phosphate buffer, pH 6.4, at concentrations of 5 to 10%. At lower concentrations the extract may not completely dissolve, while concentrations above 10% may result in viscous and impractical solutions. Why the extract does not completely dissolve at low concentrations is not clear.
Extracts from animal, insect or moulds sources contain insoluble parts and preferably should be centrifuged (10 minutes at 14000 rpm) before use.
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