WO2014195534A1

WO2014195534A1 - Method for selecting specific antibodies of european hake (merluccius merluccius) for assigning individuals to the populations thereof according to whether they are of atlantic origin or mediterranean origin

Info

Publication number: WO2014195534A1
Application number: PCT/ES2014/000038
Authority: WO
Inventors: Elena Guacimara GONZÁLEZ JIMÉNEZ; José Manuel BAUTISTA SANTA CRUZ
Original assignee: Universidad Complutense De Madrid
Priority date: 2013-06-07
Filing date: 2014-03-14
Publication date: 2014-12-11
Also published as: ES2526468B2; ES2526468A1

Abstract

The invention relates to a method for selecting peptides from proteins of tissues or organs of European hake in order to produce specific antibodies for identifying the origin of individual European hakes, assigning them to the Mediterranean basin or the Atlantic basin. Starting from the brain, peptides located in the following proteins are selected: ATP synthetase, subunit alpha; 14-3-3; and those of the voltage-dependent anion channel, respectively. The invention also relates to the combination of antibodies given the selected peptides, with which an immunodetection method has been designed in order to identify the populations of origin of the European hake. As well as the antibodies produced, the invention also relates to a kit for identifying the origin of the hakes.

Description

Title

Method of selection of specific European hake antibodies (Merluccius merluccius) to assign individuals to their populations according to whether they are of Atlantic origin or of Mediterranean origin

Technical Sector

The present invention relates to specific antibodies against hake {Merluccius merluccius) as discrimination tools for their populations according to their origin (Mediterranean or Atlantic basin). The evaluation of said discriminant capacity is based on the quantitative analysis of the levels of expression of the antigens of samples of hake individuals captured in both basins against said antibodies. The invention can be used in diagnostic analysis format based on immunodetection techniques.

State of the art

Currently, there is an increase in the need to develop traceability tools, both for fish and processed fish products, for the protection of the final consumer and as a legal framework for the application of regulations and laws that reduce illegal fishing , uncontrolled and unregulated (IUU). These traceability tools focus not only on determining the fish species but also the origin of the catch. Thus, fish or processed fish products that are mislabeled, in which the origin of fishing is incorrectly indicated, have an indirect negative effect on the management of fishing stocks or stocks, whose final consequence may constitute depletion of its resources (Stokstad, E. 2010, Science, 330: 1468-1469). In this context, the fight against IUU fishing is a priority for the European Union (EU), due to the serious threat it represents globally to marine ecosystems and the sustainability of global fisheries. European hake (Merluccius meríuccius, Linnaeus, 1758) constitutes a species of high fishing interest within the EU and is exploited for fishing throughout its distribution. European hake is geographically distributed from the western Atlantic Ocean (approximately 62 ° N, near the Shetland Islands, including the Norwegian fjords, up to 23 ° N in Mauritania) to the Mediterranean Sea.

The management of the exploitation of European hake stocks is carried out by the International Council for the Exploration of the Sea (ICES; in English, the International Council for the Exploration of the Seas, ICES) and the General Fisheries Commission for the Mediterranean ( CGPM; in English, General Fisheries Commission for the Mediterranean, GFCM). Within the EU, Spain is the largest producer of hake, with more than 23,000 tonnes of catches (FAO, 2012, FAO yearboo / r. Fisheries and Aquaculture Statistics 2010). Hake populations in the Northwest Atlantic are considered to be overexploited (Murua, 2010, Advances in Marine Biology, 58: 97-154). That is why these organizations have expressed the need to maintain the biomass of these populations at a level that allows their sustainable exploitation in accordance with the maximum sustainable yield (ICES, www.ices.dk). For this, a correct determination of the origin of the catches is necessary to control the illegal traffic of the populations. The microsatellites constitute the most used molecules as traceability tools and for the analysis of genetic diversity and determination of the structuring of hake populations. Studies based on the use of these markers have been able to differentiate two genetically different stocks or populations of hake, one corresponding to the Atlantic Ocean and another to the Mediterranean Sea, with the contact area located on the coast near Almeria and Oran (Lundy et al., 1999, Molecular Ecology 8: 1889-1898). But nevertheless, microsatellites have calibration and reproducibility limitations between laboratories and are not usually used as a control instrument.

With the characterization of specific European hake antibodies capable of allocating hake samples with sufficient precision by immunoassay analysis, a possible legal framework for the application of regulations and laws that reduce IUU fishing is opened. However, to date the existence of these antibodies or their discriminatory capacity has not been described by means of an immunoassay technique.

Detailed description of the invention

Method of selection of specific European hake antibodies (Meríuccius meríuccius) to assign individuals to their populations according to whether they are of Atlantic origin or of Mediterranean origin

The present invention relates to a method of selection and subsequent application of specific European hake antibodies (Meríuccius meríuccius) as tools of discrimination of their populations according to their origin (Mediterranean or Atlantic basin), by immunodetection techniques. In crude extracts of European hake tissues or organs, proteins with different levels of expression are identified, using specific polyclonal antibodies against peptides present in the proteins in question of this species. Subsequently, based on the statistical analysis of expression levels, it is possible to classify a hake sample as coming from the Atlantic region or the Mediterranean region.

In a first aspect of the invention a method is presented in which different criteria for peptide selection are applied, which are subsequently used for the production of specific antibodies against Meríuccius meríuccius. The use of several criteria in the selection of the peptides that have served as antigens in this invention has led to obtaining results with a power of discrimination far superior to the use of these same criteria separately.

First, the method includes the analysis of the protein expression of European hake tissues or organs in individuals from the Atlantic Ocean and in individuals from the Mediterranean Sea. Next, those proteins whose levels of expression are different in individuals from the Atlantic Ocean with respect to individuals from the Mediterranean Sea have been selected and these proteins have been identified. In a preferred embodiment of the invention, the organ used has been the brain and the selected proteins have been: the ATP synthetase protein, a 60 kDa alpha subunit, characterized by SEQ ID NO: 1; 14-3-3 28 kDa protein, characterized by SEQ ID NO: 2; and the 30 kDa voltage-dependent anion channel protein, characterized by SEQ ID NO: 3. From these proteins, the amino acid sequences that are predicted as constituents of transmembrane a-helix segments exposed to the matrix have been identified. extracellular and specific antibodies have been produced against the amino acid sequences of said extracellular segments.

In this way, three peptides have been determined that have been used for the production of antibodies capable of discriminating against European hake populations. These peptides are characterized by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

Likewise, the method may include the addition of 1 or 2 amino acids and / or molecules to the selected peptides, in order to increase their immunogenic capacity. The invention also includes combinations of specific European hake antibodies to assign individuals to their populations according to whether they are of Atlantic origin or of Mediterranean origin that include one or more antibodies generated according to the method just described. These combinations may include one or more of the specific antibodies generated against the peptides characterized by SEQ ID NO: 4, SEQ ID NO: 5 and / or SEQ ID NO: 6 which, in addition, may include 1 or 2 amino acids and / or molecules, with the aim of increasing their immunogenic capacity.

In another aspect of the invention, a method of classifying hake protein extract samples in Atlantic or Mediterranean populations is presented, which comprises the detection and quantification of antigens corresponding to proteins of the Merluccius merluccius species, by immunodetection techniques from of the antibodies generated in the present invention, and the quantification of the expression levels of the corresponding proteins. The analysis of the expression levels for each protein turns out to be significantly different and characteristic of Mediterranean or Atlantic populations.

By applying immunodetection techniques with antibodies produced against peptides characterized by SEQ ID NO: 4, SEQ ID NO: 5 and / or SEQ ID NO: 6 against antigens present in the brains of European hake individuals , these individuals can be assigned to their populations of origin as they come from the Atlantic or the Mediterranean.

The immunodetection techniques comprising the invention may be immunoenzymatic techniques (such as the ELISA technique), immunofluorescence (such as the FPIA technique), immunoblot techniques (such as the Westernblot technique) and agglutination techniques (such as the DAT or FAST technique ) or any other method based on the detection of antigens.

On the other hand, the invention includes a kit for assigning European hake individuals {Merluccius merluccius) to their populations according to whether they are of Atlantic origin or of Mediterranean origin by means of an immunodetection method in which at least one of the antibodies produced against the peptides characterized by SEQ ID NO: 4, 5 and / or 6 or against these peptides which, in addition, include 1 or 2 amino acids and / or molecules, in order to increase their immunogenic capacity. Brief description of the figures

Figure 1: 10% SDS polyacrylamide gel corresponding to the purified antibodies obtained against SEQ ID NO: 4, 5 and 6 (lanes 2-4, respectively) and to the unpurified antibodies obtained against SEQ ID NO: 4, 5 and 6 (lanes 6-8, respectively). Lane 1 corresponds to the standard protein size (Bio-Rad. No. Ref 161-0318). Approximately 40 μg of each antibody was loaded into each lane.

Figure 2: Separation of protein extracts by 10% polyacrylamide-SDS gel and immunodetection by generated antisera. Representative bands are shown that correspond to the migration size of the protein from which the peptide used as an antigen has been selected. All bands were analyzed, but only some representative ones are shown for each of the five sampled populations. ATL: individuals from the Atlantic Ocean populations. MED: individuals from the Mediterranean Sea populations. Lines 1, 2 and 3 correspond to the three different antibodies tested.

Figure 3: "Box plot" graph of the statistical analysis of the quantitative values of proteins detected in "Western" immunoblotting. The average statistical value of the analytical signal (represented by a black line), the upper and lower quartiles and the outliers are also indicated. A, C and E correspond to individuals from the Atlantic Ocean populations. B, D and F correspond to individuals from the Mediterranean Sea populations. Lines 1, 2 and 3 correspond to the three types of antibodies tested. Embodiment of the invention

The present invention is further illustrated by the following examples, which are not intended to be limiting in scope. Example 1

Selection of amino acid sequences

Two different criteria for selecting the amino acid sequences used for the production of the antibodies were followed. On the one hand, the proteins that presented the greatest capacity for discrimination among populations were selected (that is, those with the highest levels of differential expression at the statistical level). Subsequently, from these proteins, those peptides or amino acid sequences were selected which, by predictive analysis, exhibited exposure in the extracellular matrix. Thus, 35 bands or spots ("spots") of proteins from hake brain were selected by analysis of the variation in their expression comparing samples from different geographic populations and analyzing them using 2D / DIGE and mass spectrometry, from a total of 145 proteins (González et al. 2010, Journal or Proteome Research, 9: 6392-6404). Once these 35 proteins were selected, they were identified by mass spectrometry analysis (MALDI-TOF MS). MALDI-TOF MS analyzes were performed on a 4800 Proteomics Analyzer MALDI-TOF / TOF (Applied Biosystems, Framingham, MA) mass spectrometer, operating in positive ion reflector mode with an acceleration voltage of 20,000 V. MALDI- TOF / TOF generated peptides or peptide fingerprint ("mass fingerprint"), and peptides or amino acid sequences (with a +1 charge) with a signal-to-noise ratio greater than 10 were collected and grouped into a list of monoisotopic molecular weights , filtering the rest of the peaks using the GPS Explorer v3.6 program (Applied Biosystems, Framingham, MA). In order to identify the proteins, the resulting peptide footprint was compared with the expected peptide masses for each available entry in the NCBI database specific to fish protein sequences (consisting of 257,377 sequences and 83,362,140 residues as of September 25, 2009), using the MASCOT v2.1 (Matrix Science) program. Of the 35 proteins with significant levels of expression, those three proteins with discriminant capacity of more than 90% were statistically selected using Fisher's Discriminant Function. Thus, the following were selected: ATP synthetase protein, 60 kDa alpha subunit (NCBI accession number: gi | 223648354; SEQ ID NO: 1, amino acid sequence corresponding to the ATP synthetase protein alpha subunit of Salmo salar species ); 14-3-3 28 kDa protein (NCBI accession number: gi | 50844461; SEQ ID NO: 2, amino acid sequence corresponding to protein 14-3-3 of the Oreochromis mossambicus species) and channel protein 30 kDa voltage-dependent anions (NCBI accession number: gi | 169642540; SEQ ID NO: 3, amino acid sequence corresponding to the voltage-dependent anion channel protein of the Salmo sa ^ species.

As the second selection criteria, from the proteins identified above, the amino acid sequences that had the greatest exposure to the extracellular matrix were chosen. For this, the HMMTOP v2.0 program was used, which is able to predict the transmembrane topology of proteins from their complete amino acid sequence (Tusnády and Simón, 2001, Bioinformatics, 17 (9): 849-850).

As a result, three amino acid or peptide sequence regions were selected:

1) EAYPGDVFYLHSR, characterized by SEQ ID NO: 4, internal fragment (334-347) of SEQ ID NO: 1,

2) AVTEQGAELSNEER, characterized by SEQ ID NO: 5, internal fragment (27-41) of SEQ ID NO: 2,

3) VNNSSLVGLGYTQTLKPGIK, characterized by SEQ ID NO: 6, internal fragment (236-256) of SEQ ID NO: 3. The numbers in parentheses refer to the residues of the sequence deduced from the cDNA, as they appear in the corresponding sequences. Example 2

Production and purification of polyclonal antibodies

The peptides selected according to example 1 were commissioned to synthesize the company Mimotopes (Mimotopes Pty. Ltd., Clayton, Victoria, Australia). The resulting peptides were purified by HPLC. To obtain a stronger immune response, a cysteine residue (C) was added during the synthesis at the N-terminal end of the peptides, which was used to conjugate them with a barnacle hemocyanin molecule (KLH) by binding with maleimide . These peptides thus prepared were used as antigens in the process of immunization and production of polyclonal antibodies described below.

The entire experiment was carried out at the facilities of the Complutense University of Madrid (UCM), and the guidelines of the International Scientific Council of Laboratory Animals were followed. Six female rabbits of the New Zeland White (NZW) breed were purchased with an average weight of 2.5-3.5 kilograms from the farm San Bernardo (Navarra, Spain), disease free. They were kept at the UCM's animarians with free and continued access to food and water. For the first immunization, a mixture (1: 1 dilution) was made with the peptide (dissolved in phosphate buffered saline -PBS- at a concentration of 1 mg) and the complete Freund's adjuvant (Sigma). Each peptide was inoculated into two animals to obtain two replicates of the same antibody. The complete Freund's adjuvant (AFC) was used only in the first immunization, where 300 ng of the emulsion prepared above was administered intradermally to each female rabbit. In the following immunizations, the AFC was replaced by Freund's incomplete adjuvant (AIF). Thus, three weeks after the first immunization, three other administrations were performed every two weeks at a lower concentration (200 ng). Blood samples were collected before and after immunizations. The extracted blood was left at room temperature for 3-4 hours, after which it was centrifuged at 3000 rpm for 15 minutes at 4 ° C to separate the sera. In this way, six heterologous sera (containing the antibodies) were isolated from the rest of the blood fractions, and subsequently stored at -20 ° C.

In order to avoid nonspecific interactions in subsequent immunochemical analyzes, the antibodies thus obtained were purified using the Spin Nab kit (Pierce Biotechnology, Inc.), following the supplier's protocol. The purified fraction of immunoglobilins-G (IgG) was analyzed by electrophoresis separation in gels from SDS-PAGE. The results indicated strong antibody production at the end of the immunization process.

This purification process can be summarized in Figure 1.

Example 3

Western blot antibody detection

A total of 72 hake brain samples were analyzed to determine the ability of the method to significantly assign individuals to each basin of origin (Atlantic or Mediterranean), using the antibodies generated according to example 2. Extraction of tissue proteins from Brain was carried out following the procedures previously described (González et al. 2010, Journal or Proteome Research, 9: 6392-6404). Brain samples of European hake from geographically remote regions were used, specifically from two regions in the Atlantic Ocean: the Bay of Biscay, (corresponding FAO area: 27.VIII.C) and the coast of Portugal, (FAO area: 27. IX.a); and three others in the Mediterranean Sea: Aegean Sea, (FAO area: 37.1.1), Adriatic Sea, (FAO area: 37.2.1) and Sardinia coast, (FAO area: 37.1.3). All samples were captured in the same period of 2008.

80 μg of total protein extracted from each of the 72 individuals that were mixed with loading buffer for SDS-PAGE gels were used and heated at 100 ° C for 5 minutes. The composition of the loading buffer used was: 50 mM Tris-HCI, 5% SDS, 10% glycerol, 0.042% Bromophenol Blue and 5% 2'-mercaptoethanol, pH 6.8. The samples were loaded on SDS-PAGE gels (at a concentration of 10%) and separated first at 70 mA for 30 minutes and then at 90 mA for one hour and twenty minutes, using a Bio-Rad transfer apparatus ( 1 mA / cm ² for each gel). Subsequently, the proteins thus separated were transferred to polyvinyl difluoride membranes (PVDF-Amersham Biosciences). For this, the polyacrylamide gel and the membrane were previously equilibrated in transfer buffer, with orbital agitation for 15 minutes. The composition of the transfer buffer used was: 50 mM NaCI, 2 mM Tris-HCI, pH 7.4.

The transfer of the proteins from the gel to the membrane was carried out at 150 mA for one hour and twenty minutes with a Bio-Rad transfer apparatus (1 mA / cm ² for each gel). This process was carried out refrigerated (at 4 ° C) and using transfer buffer. Subsequently, the membranes (with the transferred proteins) were blocked by incubation with orbital shaking for one hour at room temperature in phosphate buffered saline (PBS) containing 5% skimmed milk powder (Central Lechera Asturiana).

The antibodies used in Western blots, obtained according to example 2, were prepared at a dilution of 1: 1000, while the secondary antibody (anti-rabbit IgG, Amersham Biosciences) was used at a dilution of 1: 5000. Thus, the blocked membranes were first incubated with each antibody of example 2 in PBS buffer containing 10% skimmed milk powder, overnight, with orbital shaking to 8 ° C The membranes were then washed by three serial incubations in PBS buffer containing 0.05% Tween, with orbital shaking, at room temperature for five minutes, and one last incubation in PBS buffer containing 0.05% Tween and 5% of skimmed milk powder, with orbital stirring at room temperature, for five minutes. After washing, the secondary antibody (anti-rabbit IgG) was added and the membranes were incubated therewith for one hour at room temperature. Finally, the membranes were washed twice with orbital shaking at room temperature, for five minutes, with each of the following buffers consecutively: 1 ^o ) PBS buffer containing 0.05% Tween and 5% skimmed milk powder, 2 ^o ) PBS buffer containing 0.05% Tween; and 3 ^o ) PBS buffer.

The membranes thus prepared were revealed using the SuperSignal West Pico Rabbit IgG Detection (Thermo Scientific) detection kit by exposing sensitive photographic films that were subsequently scanned at high resolution.

The results indicated the existence of antibody reaction bands of size corresponding to the molecular weight of the protein of origin, that is, the antibodies obtained had a specific reactivity to the corresponding antigens. In addition, the expression pattern turned out to be different according to the sampled geographic population. The result of this process can be summarized in Figure 2.

Example 4

Signal quantification for each antibody

Each photographic film was scanned (EPSON PERFECTION V750 PRO model scanner) at a resolution of 300 dpi The expression signal of each protein for each immunoassay (measured as the area for each band detected) was quantified using ImageJ software (version 1.44). The absolute value of each measurement was expressed as a percentage of the highest value measurement within each "Western". Expression values for each antibody were statistically analyzed to determine possible correlations between expression levels and geographic regions of sample origin. For the Student's T analyzes, the samples belonging to the Atlantic basins, on the one hand, and the Mediterranean, on the other, were grouped and a significance level ("p-value") of less than 0.05 was established. According to the results, the ATP synthetase alpha subunit protein presented significantly different levels of expression among individuals captured in the Atlantic Ocean or in the Mediterranean Sea (Student's T, t = 5.70, p = 0.023, df = 66), being the populations of the Atlantic those that showed greater overexpression of said protein. On the other hand, protein 14-3-3 turned out to be a unique expression protein for individuals from the Mediterranean populations, which presented expression values, while in those of the Atlantic, said protein was either not expressed or did so at levels not detectable by its corresponding antibody. Finally, the voltage-dependent anion channel protein showed significantly different levels of expression among individuals captured in the Atlantic Ocean or in the Mediterranean Sea (Student's T, t = 7.06, p = 0.000, df = 55), being in this In this case, the Mediterranean populations showed the highest overexpression of said protein. The results indicate that the quantification values obtained with the antibodies make it possible to discriminate hake individuals by their basin of origin in a significant way. Figure 3 graphically summarizes these results.

Claims

one . Method of selection of specific European hake antibodies (Merluccius merluccius) to assign individuals to their populations according to whether they are of Atlantic origin or of Mediterranean origin that includes:

a) analyze the expression of European hake proteins in tissues or organs of individuals from the Atlantic Ocean and individuals from the Mediterranean Sea,

b) select the proteins from step a) whose levels of expression are different in individuals from the Atlantic Ocean with respect to individuals from the Mediterranean Sea,

c) identify the proteins selected in step b),

d) in the proteins identified in c), identify the amino acid sequences that constitute segments in transmembrane α-helix exposed to the extracellular matrix;

e) produce specific antibodies against the amino acid sequences identified in d), using synthetic peptides.

2. Method according to claim 1 wherein the organs selected to perform step a) is the brain.

3. Method according to any of claims 1-2 in which the peptides of step e) include 1 or 2 amino acids and / or molecules that increase their immunogenic capacity.

4. Combination of specific European hake antibodies (Merluccius merluccius) to assign individuals to their populations according to whether they are of Atlantic origin or of Mediterranean origin that includes one or more antibodies selected according to the method defined in any of claims 1-3.

5. Antibody combination according to claim 4 wherein one of the included antibodies specifically binds to a peptide whose amino acid sequence is SEQ ID NO: 4.

6. Antibody combination according to any of claims 4-5 wherein one of the included antibodies specifically binds to a peptide whose amino acid sequence is SEQ ID NO: 5.

7. Antibody combination according to any of claims 4-6 wherein one of the included antibodies specifically binds to a peptide whose amino acid sequence is SEQ ID NO: 6.

8. Antibody combination according to any of claims 4-7 wherein the amino acid sequences include a cysteine at the N-terminal end and a lapa hemocyanin molecule (KLH) conjugated by maleimide binding.

9. Immunodetection method to assign European hake individuals (Merluccius merluccius) to their populations according to whether they are of Atlantic origin or of Mediterranean origin, which is based on a technique selected from the group comprising: immunoenzymatic techniques (such as the ELISA technique), of immunofluorescence (such as the FPIA technique), immunoblot (such as the Westernblot technique) and agglutination (such as the DAT or FAST technique) or any other method based on the detection of antigens and that includes the use of antibody combinations according to They are defined in claims 4-8 against antigens present in the brains of European hake individuals.

10. Kit for assigning European hake individuals (Merluccius merluccius) to their populations as being of Atlantic origin or of Mediterranean origin by a method as defined in claim 9, which includes a combination of antibodies as defined in claims 4- 8.