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Antibodies and immunoassays for fish antigens

Fish allergy is among most important and dangerous, causing anaphylactic reactions in some patients. The sensitivity of allergic patient towards fish allergens is extremely high; we have checked several patients for their skin sensitivity by skin testing (see typical figure below, showing the titration of skin prick test allergen of Allergopharma).

The task for immunometric determination of fish allergen is to achieve at least the same degree of sensitivity, to detect the common antigen(s) of ALL fishes used in food industry, even after deep processing (canning, cooking, repeated freeze-thaw, extractions).

Antigens of BONE FISH
Two major fish speciei were prepared to obtain heat-resistant antigen of BONE FISH - cod (Gadus) and salmon (Salmon). For mAb screening and antiserum characterization three kinds of extracts were used for each species: raw, cooked, frozen and then thawed. Picture below shows typical SDS-PAGE of the immunogenic extracts.

Antisera and rabbit antibodies against bone fish
The extract of cooked cod was used for rabbit immunization. Affinity adsorbent for purification of antibodies against fish antigens was made of raw cod extract. After the rabbit antiserum (AS363) was ready we purified anti-fish antibodies (AS363 A) and tried them in direct binding in ELISA with extracts of different species of fish and different kinds of meat. The relative reactivity of various fish and seafood extracts is shown on the picture below.

Rabbit anti-fish antibodies: relative reactivity to food extracts (100 % - optical density in wells with raw cod).

Due to high cross-reactivity of raw antisera and even affinity anti-fish antibodies to shrimp, we have adsorbed the antibodies on immobilized shrimp antigen. The resultant antibodies AS363 AA are free from cross-reactivity to shrimp and other Crustaceae (see picture below).

Rabbit anti-fish antibodies AS363 AA (without crossreactivity to shrimp extract; left diagram) and anti-shrimp antibodies AS353 A (right diagram) reactivity to food extracts.

Monoclonal antibodies against bone fish
The extract of cooked cod was used for immunization of mice and obtaining of hybridomas. Most of monoclonal antibodies against fish proteins have shown a considerable preference of binding to raw tissue, or selected fish families. Cartilaginous fishes (sharks, sturgeons) and to certain extent, salmon and related genuses, were most frequently missing from the mAb binding patterns. However, monoclonal antibody X21 has shown full coverage of tested fishes, including cartilaginous fishes and even lamprey. Target antigen of this mAb is a previously unknown protein with apparent MW ca 17 kD, which we designated as Fish Muscle Protein 17 (FMp17) and included into our patent application. This mAb was chosen as PAN FISH capture for our Total Fish version I assay

K363 Total Fish version I EIA (bone fish)
Mono-poly sandwich was constructed from pan-fish specific monoclonal antibody X21 and affinity purified rabbit antibodies AS363 AA labeled with HRP (see IFU). Due to low reactivity of AS363 AA against cartilaginous fishes, the assay has low sensitivity for sharks and sturgeon (see table below)

SpeciesSystematic name (genus)Immunoreactivity in present assay
Sharks (order)Selachimorpha<5%
Lamprey (order)Petromyzontidae<5%
Shrimp (Crustacea)various<5%

Further evaluation of XEMA Total Fish version I EIA was made by the research partner of Calbioreagents Inc, CA (see report)

Anti-fish antibody application: immunoblotting
Immunoblotting studies shown that the target FMp17 antigen is NOT bound by most specific IgE from highly allergic patients, but may serve as the marker of potential presence of fish muscular tissue in foodstuff. Immunoblotting of raw cod extract with (left) or without (right) 2-mercaptoethanol The lanes from left to right: MW markers; Coomassie staining; rabbit anti-Fish AS363 AA visualized by anti-Rabbit-HRP (AS301-HRP); human allergic serum visualized by mouse anti-human epsilon chain (XTE4-HRP), control of second antibodies AS301-HRP, XTE4-HRP

The sensitivity of K363 Total Fish was checked by titration of skin testing allergen preparations of different manufacturers. The necessary degree of sensitivity 10E-6 (comparable to skin prick testing dilution threshold in most allergic patients) was achieved in all tested preparations of codfish.

K363 Total Fish version I EIA application: detection of fish antigen leakage from fish body
The fish antigen test can be used to control a leakage of muscular antigens of fish outside body. This approach, similar to use of cardiac markers for diagnosis of myocardial infarction, may be important to control the intactness of the fish bodies after catch, eg on fish markets.
Trial experiment (see figure below) has shown that the substantial leakage of fish antigen from freshly caught fish body to the storage tray starts as early as after 1 hour of storage even at 4C. The antigen reaches its peak within hours of storage and then undergoes denaturation.
Relatively rapid decrease in antigen concentration after long and improper storage of fish bodies may be circumvented by combination of Total Fish version I EIA with another antibody combinations which ensure the detection of decayed (low MW) fish antigens (data not shown).

X363 Fish: rapid test for fish antigens for kitchen use
The same combination of antibodies as in K363 Total Fish EIA was used to construct a rapid immunochromatographic (lateral flow) test for field or kitchen use. The test shows slightly (5-7 times) less sensitivity comparing to EIA, but is easy to use and robust -please look into instruction for use.

Development of antisera and antibodies to cartilaginous fishes and lamprey
Additional antibodies may be helpful to improve a coverage of Total Fish immunoassays for cartilaginous fishes (sharks, sturgeons) and lamprey. Separately, cartilaginous fishes are prohibited by Jewish religion and their control in fishermans catch can be used for Kosher control. Eel does not belong formally to cartilaginous fishes but is also listed in Kosher prohibition list.
Antisera against sharks (AS363A), sturgeon (AS363Y), eel (AS363U) and lamprey (363M) are available in neat form and partly in affinity purified and adsorbed form.
Additionally, a series of monoclonal antibodies was obtained showing high degree of specificity against sturgeon. A pair of mAbs (eg X252 vs X315-HRP) can form a Sturgeon EIA (K363Y) which can be used for detection of sturgeon antigens, eg in Kosher control. NOTE: This assay does NOT recognize black caviar. Rapid test Sturgeon is in development.
Further assays detecting other non-Kosher fishes (sharks, eel) and lamprey are also in development, both in EIA and rapidformat.

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