T2/HT2 Immunoaffinity Column

$180.00

SKU: BTTE316005 Categories: , ,

T2/HT2 Immunoaffinity Column

3ml, 10 columns

For Research Use Only

Product Developed and Manufactured in Germany by BioTeZ Berlin-Buch GmbH

Additional Information

Assay Principle


This instruction of T2/HT2 determination in food and feed focuses on the enrichment step of extract using immunoaffinity column (IAC) and quantification with HPLC.

Accepted laboratory extraction methods could be maintained. Full performance of the IAC column is given if pronounced criteria regarding organic solvent tolerance, elution process of analyte and working range of column is followed.

Many pretreatment methods for T2/HT2 in food and feed show low sensitivity because of interferrin substances if problematic matrices are applied.

This method of content determination of T2/HT2 combines the high selectivity of an immunoaffinity column (IAC with its potential to concentrate elute and additional step of purification of labelled T2/HT2 by HPLC column.

As said before, this instruction focuses on the handling with the IAC column. For the commodity extraction step a literature method is given. Please see below. The given apparatus (e.g. HPLC system) might serve as example among other possibilities. For your convenience, an example HPLC method for the analysis of T2- toxin is given below.

Sample Preparation (Literature Method Given):
Samples which content of T2/HT2 are to be analyzed, e.g. cereals, are extracted by the method of Visconti et al.1 using methanol-water (90/10 v/v) as extraction solvent. E.g. to 50g of sample are added a volume of 100ml of the extraction solvent and processed as cited.

Enrichment Step IAC:

Samples which content of T2/HT2 are to be analyzed, e.g. cereals, are extracted by the method of Visconti 2ml extract (1g sample equivalent if above mentioned example extraction is followed) is diluted with 18ml 10mM PBS, pH=7.2 resulting a total volume of 20ml and then applied in a reservoir on top of the B-TeZ IAC T2/HT2 3ml column.

If the example is followed the resulting organic solvent concentration of the diluted extract is 9% of methanol which is tolerated by the column. The flow rate is adjusted to lie between 1 to 3ml/min.
According to application and contents expected the applied extract volumes could vary. In case of very low contents even extract volumes of 20ml may be diluted with buffer and applied without significant loss of analyte as long as resulting pH is fairly neutral and alcohol or acetonitrile content lies under 15%.

If latter is not the case the extract must further be diluted with PBS until forementioned maximum allowed organic solvent content during enrichment step using the B-TeZ IAC T2/HT2 3ml column is reached.

Thus, to guarantee maximum efficiency in terms of maximum recovery rates using B-TeZ IAC T2/HT2 3ml column, please ensure that:

organic solvent content in diluted extract which is applied on top of the column is not higher than 15%,
T2/HT2 load per column does not exceed a total of 1μg.

Wash:
After whole sample has passed through the gel the latter is washed with 5ml of 10mM PBS. Remaining liquids in the gel are removed by applying either pressure from top of the column or underpressure from bottom.

Elution:

Sample reservoir on top of the using B-TeZ IAC T2/HT2 3ml column is removed and an appropriate vial is placed below the affinity column. The bound toxins are eluted by using a total of 1.5ml of methanol as elution solvent. The elution process is performed in two steps to ensure complete release of analytes. First, a volume of 0.5ml elution solvent is applied. After that volume has passed through column half a minute is waited before the second portion of 1ml of elutions solvent is eluted through the column. Remaining solvent solutions should be eluted by application of slight under- or overpressure. All methanol fractions are unified to give the column eluate.

The column eluate is concentrated to complete dryness (e.g. using VLM evaporater at 50°C under a permanent stream of nitrogen). The residue is treated with a mixture of 1-Anthroylnitrile and 4- Dimethylaminopyridin as reported1 to give fluorescent labelled derivatives of T2 and HT2 which are then injected to the HPLC apparatus.

Manual

Product Manual