Fish-Check ELISAs

The assay utilises ELISA techniques and detects the presence of cod parvalbumin (the major fish allergen). Imutest’s FISH-CHECK ELISA kits are designed for the detection of a variety of fish species at low levels.

For fresh COD, the assay has a nominal range of 5-125 mg/kg (PPM), in e.g. raw materials and finished food products. The reactivity of other species and of processed (e.g. cooked, canned, pickled) foods may be somewhat different.

Assay originally developed² by the Norwegian Veterinary Institute (NVI); the kits are manufactured by D.I.L. in the U.K. under license from the NVI.

See "How To Use" for an outline of the Sample Preparation and ELISA methodologies.

¹ Kuehn et al, Important variations in parvalbumin content in common fish species; Int. Arch. All. Immunol. 153 (2010) pp359-366.

² Faeste & Plassen, Quantitative sandwich ELISA for the determination of fish in foods; J. Immunol. Meth. 329 (2008) pp45-55.

(Copies available on request)

Click here for the product information sheet.

Food samples are firstly prepared e.g. by milling/grinding/chopping etc to make them homogeneous.

Any parvalbumin protein in the sample are then dissolved using an extraction solution.

This extraction solution is also available stabilised & ready-to-use in the imutest Allergen Extraction kit (Cat. No. A6015), which saves both time and money; the inclusion of yellow dye in pre-filled extraction tubes helps confirm that samples have been diluted prior to testing.

The sample extract is then diluted and added to microwells coated with an antibody to cod parvalbumin; any parvalbumin present binds tightly to these antibodies.

After this first incubation the microwells are washed to remove unwanted proteins/food components.

Cod parvalbumin is then detected by adding anti-parvalbumin to which an enzyme molecule (HRP) has been attached.

After this incubation the microwells are again washed to remove unbound enzyme.

A colourimetric substrate (TMB) is then added which develops a bright blue colour in the presence of HRP enzyme.

The more colour that develops, the more white fish was present in the original sample.

The substrate reaction is then stopped using dilute acid and the colour in the microwells is measured in a plate reader (450nm).

The amount of fish in the original sample is then calculated with reference to a Standard Curve of PPM fish versus OD450nm.

• Laboratory Sample/Kit Controls Preparation
• Prepare                                    Sample by grinding/chopping/blending until homogeneous.
• Add                                                          Test Portion of 0.5g to 4.5ml of Extraction Solution.
• Mix. Extract                                                                                  15 minutes to extract fish.
• Mix. Centrifuge                                               10 minutes or allow to settle for 30 minutes.
• Remove                                                        a portion of the extract above the food pellet.
• Dilute                                                               extracts 1/5 (1:4) in Diluent Solution 2 (1x).

• ELISA Protocol
• Pipette                          100uL Standards and diluted Sample/Control Extracts into wells.
• Mix. Incubate                                                             at room temperature for 40 minutes.
• Wash wells                                                                     three times with wash solution 1X.
• Pipette                                                                                    100uL Anti-Fish HRP into wells.
• Mix. Incubate                                                            at room temperature for 40 minutes.
• Wash wells                                                                       four times with wash solution 1X.
• Pipette                                                                   100uL TMB Substrate reagent into wells.
• Mix. Incubate                                                               at room temperature for 15 minutes.
• Pipette                                                                            100uL Acid Stop Solution 1 into wells.
• Mix. Read                                                                                    wells at 450nm wavelength.
• Calculate                                                  mg/kg (PPM) Fish results for all Controls/Samples.