STSM ICTAN FIBHULP, Madrid 2016

Maciej Kochanowski STSM was carried out in the Madrid (Spain) in Instituto de Ciencia y Tecnología de Alimentos y Nutrición  (ICTAN) and La Fundación para la Investigación Biomédica del Hospital Universitario La Paz (FIBHULP) laboratories. The STSM was supervised by Mercedes Careche (ICTAN) and Miguel Gonzalez-Muñoz (FIBHULP)
The main aim of STSM was training on Anisakidae allergens detection in fish and fishery products. The training consisted of several stages.
1. Inactivation of Anisakis larvae
The first stage of investigation was inactivation of Anisakis larvae in fishery products. For this purpose hake (Merluccius merluccius) fillets were obtained from a local market. Two portions of fillets were prepared in a sandwich form, and they were infected with a known number of L3 Anisakis simplex. Samples prepared that manner were left in 4˚C for 12 hours to allow migration of the larvae into the fish flesh. After that time, sandwiches were put in the thermoresistant plastic bags, and heat treated in an oven (200°C, 15 min). Internal temperature of the fillets during cooking and the temperature of the oven was monitored and recorded by a portable temperature acquisition sensor. Then, after cooling, samples were observed under UV light to detect the heat treated larvae, and all previously added parasites were collected.
2. Extraction of parasite allergens
The next step was extraction of allergens from artificially parasitized fish. From each sample 10 g of prepared fish muscle with 30 ml Tris-buffered saline was mechanically homogenised and the mixture was sonicated. Subsequently, samples were incubated on the rotator. In the next step, mixture was centrifuged. An aliquot of the supernatant (5 ml) was acidified to pH around 1 with HCl solution, and incubated for 15 min. Mixture was neutralized to pH around 7 with NaOH, and centrifuged. The pellets were discarded and supernatant was analysed.
3. Quantification of Anisakis allergens
Anisakis allergens in fishery products were quantified by dot-blotting. The samples were the waste fractions obtained from washing steps performed in the production of surimi, extracts of naturally infected fish and extracts from Anisakis L3 removed from commercial canned cod liver. Three microliter of samples was dotted in duplicate to nitrocellulose membranes and dried. Membranes were hydrated with PBS and subsequently blocked. After blocking membranes were washed three times with Tris-buffered saline. Membranes were incubated with anti-Anis 1 antibody, anti-Anis 4 antibody or anti-A. simplex crude extract antibody. After incubation membranes were washed three times with Tris-buffered saline for 5 min. each time. After that membranes were incubated with anti-rabbit IgG antibody conjugated with alkaline phosphatase. Washed membranes were incubated with substrate BCIP-NBT. Reaction was stopped by washing membranes with the PBS. Reference curve was calculated in duplicate dotting 3 µl of Anis-1, Anis-4, Anisakis crude extract with known concentration of protein. Membranes were scanned and measured by densitometry using Quantity One software (Biorad).
Also, examination of human serum for detection of IgE anti-Anisakis antibody was performed. SDS-PAGE electrophoresis of recombinant Ani s 1, Ani s 4, Ani s 5 and Ani s 11-like protein and Anisakis crude extract was performed. SDS-PAGE electrophoresis was conducted in standard conditions. After electrophoresis proteins were transferred to nitrocellulose by Trans-blot Turbo (Biorad). After blocking, membrane was washed three times with Tris-buffered saline for 5 min. each time. Membrane was incubated with an Anisakis allergic patient serum overnight. Next, membrane was incubated with solution of monoclonal anti-human IgE for 2 h. After incubation, membrane was washed and incubated with solution of anti-mouse IgG antibody conjugated with alkaline phosphatase for 1h. After incubation, membrane was washed and colour developing with substrate BCIP-NBT was performed for 30 min. Reaction was stopped by washing membrane with the PBS.
4. LF NMR relaxometry to differentiate between fresh and frozen/thawed hake
Moreover, low field Nuclear Magnetic Resonance relaxometry was used for hake samples differentiation fresh from frozen/thawed fish. Fillets were cut to small pieces in 1x1x2 cm, and accurately weighted, placed into NMR tubes and T2 relaxation times analysed in the LF NMR Minispec.
In conclusion, the training was successful in terms of established goals. The STSM has successfully contributed to know-how detection of Anisakidae allergens and improving collaboration between NVRI, ICTAN and FIBHULP on Anisakis research. It has to be underlined commitment of the management of ICTAN and FIBHULP. The friendly atmosphere leads to good cooperation that will turns into the fruitful future cooperation.

Maciej Kochanowski.