ES2209592A1 - Method is for production of antigens excretory-secretory of human or animal parasites and comprises incubation of parasite larvas in appropriate medium - Google Patents

Abstract

The method is for the production of antigens excretory/secretory of human or animal parasites and comprises the incubation of parasite larvas in an appropriate medium with a pH equal to or lower than 5 and at a temperature between 15 and 60 degrees Celsius.

Description

Method for antigen production excretors / secretors of human or animal parasites.

Field of the Invention

The invention relates, in general, to the serological diagnosis of human and animal parasitosis and, in particular, with the production of allergens useful for Serological diagnosis of animal parasites.

Background of the invention

Infections caused by parasites They constitute a serious global problem. The diagnosis of this type of diseases is limited in many cases by the difficulty visualizing the parasite The diagnostic alternative  serological is in turn limited by the existence of reactions crusades that often lead to positive results fake

The parasitic fish nematode Anisakis simplex produces in humans a very diverse pathology known as human anisakidosis. The larvae of A. simplex are usually found in the muscles or viscera of fish in a state of lethargy or developmental arrest and infect humans after ingestion of raw or undercooked parasitized fish. This implies that these larvae interrupt a period of their development, recover mobility and resist digestion with pepsin at low pH before crossing the mucosal barrier. A recent multicenter study carried out by the Spanish Society of Allergology and Clinical Immunology in Spain, concluded that sensitization to this nematode had been found in up to 60% of patients suffering from acute urticaria in those regions where it is consumed raw fish, but not in other parts of the country where fish is eaten frequently, although raw is not consumed (Moneo et al., Alergol. Immunol. Clin. 2000; 15: 255-261). In addition, specific IgE antibodies against Ani s 1, the main allergen of A. simplex , can be found in about 12% of the normal (healthy) population of Madrid, a region where raw anchovies in vinegar are consumed with frequency (Moneo et al., J. Allergy Clin. Immunol. 2000; 106: 177-182). Therefore, the correct and reliable diagnosis of human anisakidosis is of great socio-health interest.

The usual diagnosis of this pathology is currently carried out either by visualizing the larvae by endoscopy or by serological methods, both in vitro and in vivo , through the use of somatic crude extracts, with the consequent non-specificity, which leads to frequent diagnostic errors. . The use of excretory / secretory antigens obtained by prolonged culture (days or weeks) of larvae in stage 3 (L3) infectives has also been proposed. In this case, bacterial and fungal contamination are frequent.

However, as in other parasitosis, the Serological diagnostic tests are not enough specific, probably because the current serological methods use total parasite extracts, that is, raw extracts somatic In general, somatic raw extracts are obtained by homogenization of the complete larvae and contain all soluble parasite proteins. In order to solve the problem previously mentioned a very specific ELISA has been developed and sensitive antigen capture using a monoclonal antibody (Lorenzo et al., Allergy 2000; 55: 627-633) as well as other serological tests using antigens excretors / secretaries. Excretory / secretory antigens result much more specific than somatic antigens because they avoid cross reactions produced by said somatic antigens.

The larval excretory / secretory antigens of A. simplex are obtained, in general, by incubation in culture medium for prolonged periods of time, up to 21 days, and contain only the antigens released by the larvae to the culture medium (Sugane et al., J. Helminthol. 1982; 66: 305-309; Raybourne et al., Exp. Parasitol. 1986; 62: 92-97; Sakanari et al., J. Parasitol. 1990; 76: 625-630; Kennedy et al., Mol. Biochem. Parasitol. 1988; 31: 35-46; Akao et al., J. Helminthol. 1990; 64: 310-318). Some authors consider that the antigens secreted by A. simplex are optimal for performing serological tests due to their low cross-reactivity (Kennedy, Allergy 2000; 55: Suppl. 59: 7-13). However, frequent bacterial and fungal contamination prevent the widespread use of this method.

There is, therefore, the need to develop a precise and reliable method for the diagnosis of parasitosis animals, for example, human anisakidosis, which exceeds previously mentioned inconveniences.

Compendium of the invention

The invention generally faces the problem of providing a method for serological diagnosis of animal parasitosis, for example, human anisakidosis, which overcome all or part of the inconveniences of the methods currently used in the serological diagnosis of Animal parasitosis, in particular, human anisakidosis.

The solution provided by this invention is based on the fact that the inventors have observed that the incubation of larvae of A. simplex under conditions of acidic pH and at a suitable temperature causes the rapid release of excretory / secretory antigens to the incubation medium. The amount of secreted protein accounts for a small part of the total protein content, is free of somatic antigens and has a high value as a diagnostic reagent.

Brief description of the figures

Figure 1 shows the influence of temperature and pH on the release of allergens by the L3 larvae of A. simplex . The result of Coomassie blue staining (Figure 1A) and that of a specific IgE immunoblot using a set of positive sera (Figure 1B) is shown. The larvae were incubated in PBS (lanes 2, 4, 6, 8) or in 50 mmol / L HCl (lanes 1, 3, 5, 7) for 1 hour. Incubation was performed at room temperature (lanes 1, 2, 5, 6) or at 37 ° C (lanes 3, 4, 7, 8). Released: supernatant obtained by centrifugation after incubation. Somatic: soluble proteins obtained after centrifuging the larvae to obtain the secreted product, milling the pellet in a mortar and resuspend it in 1 mL of PBS.

Figure 2 shows the result of an immunoblot specific IgE in which the influence of the acid concentration in the incubation medium. The larvae are incubated for 2 hours in PBS (lane 1) or in different HCl concentrations: 200 mmol / L (lane 2), 100 mmol / L (lane 3), 50 mmol / L (lane 4) and 25 mmol / L (lane 5).

Figure 3 shows the influence of time on Allergen release The larvae were incubated in PBS for 3 hours (lane 1) or in 50 mmol / L HCl for 30 minutes (lane 2), 1 hour (street 3), 2 hours (street 4) or 3 hours (street 5). After centrifuge, the supernatants were electrophoresed and transferred by blotting.

Figure 4 shows the influence of a change in the medium after a first incubation for 1 hour in RPMI 1640 (lane 1) or in HCl 50 mm / L (lane 3). The larvae washed 3 times in PBS, they were resuspended again in RPMI 1640 and were incubated for 1 hour.

\hbox{HCl  50 mmol/L}

con NaCl 500 mmol/L (calle 2) o bien con KCl 500 mmol/L (calle 3).Figure 5 shows the influence of ions on the release of allergens. The larvae were incubated for 3 hours in 50 mmol / L HCl (lane 1) or in

 [HCl 50 mmol / L} 

with 500 mmol / L NaCl (lane 2) or with 500 mmol / L KCl (lane 3).

Figure 6 shows pepsin sensitivity of the released allergens. Specific IgE detection using allergens released without pepsin in the incubation medium (lane 1) or in the presence of 1 mg / mL swine pepsin in the middle (street two).

Figure 7 shows the detection profiles of Specific IgE of 18 positive sera per blot using extract Total somatic crude (Figure 7A) or released allergens (Figure 7B).

Detailed description of the invention

The invention provides a method for production of excretory / secretory antigens from a parasite animal, hereinafter method of the invention, comprising incubating L3 larvae of said animal parasite in an incubation medium that It contains the necessary nutrients for the development of larvae, at a pH equal to or less than 5, and at a temperature between 15 ° C and 60 ° C.

The method of the invention is particularly useful. for the production of excretory / secretory antigens from a animal parasite, useful as allergens for diagnosis serological of said animal parasite.

As used in this description, the term "animal parasite" refers to any parasite capable of infesting a vertebrate animal, including man. In a particular embodiment, said parasite is a parasite that causes infections in humans, which crosses the digestive tract, such as a parasite that causes human anisakidosis, for example, A. simplex .

L3 larvae are larvae in stage 3 of their development and can be extracted from the host, specifically from the tissues where they are, by conventional methods, by example, by manual extraction. Then the larvae L3 they are grown in an incubation medium comprising, in addition to the nutrients necessary for the development of larvae, an acid in sufficient quantity to provide the incubation medium with a pH equal to or less than 5, preferably between about 1 and about 4, for example, between about 1.5 and approximately 3. The acid present in the incubation medium it can be any acid, organic or inorganic, non-toxic to  larvae, capable of providing the incubation medium with said pH, by example, hydrochloric acid or acetic acid. In a particular embodiment said acid is hydrochloric acid that is added to the medium of incubation at concentrations between 25 mmol / L and 200 mmol / L, preferably between 50 and 100 mmol / L.

The method of the invention can be carried out at a temperature between 15 ° C and 60 °, typically between 20 ° C and 40 ° C. In a particular embodiment, various tests (see Example) have shown that the optimum release temperature of excretory / secretory antigens from A. simplex is 37 ° C (see Figure 1).

The incubation of the larvae according to the method of invention can be carried out under the conditions indicated during a variable time period, generally between 30 minutes and 96 hours, preferably between 1 and 2 hours.

Excretory / secretory antigens released to the incubation medium according to the method of the invention, in particular, those useful as allergens (their usefulness can be readily demonstrated by immunoblotting) can, if desired, be extracted from the incubation medium or said antigens. Excretors / secretors containing allergens can be used in the serological diagnosis, both in vitro and in vivo , of animal parasitosis, including human parasitosis. In a particular embodiment, the diagnosis can be made by a skin test comprising inoculating said excretory / secretory antigens containing one or more allergens or, alternatively, one or more of the separate allergens of said excretory / secretory antigens, between the layers of the skin of an animal or a human being, usually in the forearm; optionally, inoculating a control substance at another point of the human or animal body, said control substance containing an element to which animals or humans react, for example, a yeast or fungus, and observing the inoculated areas after a certain period of time, to determine the presence of swelling or redness symptoms, symptoms that would be indicative of a positive result.

As described in more detail in the Example accompanying this description, the invention is illustrated in relation to the production of excretory / secretory antigens from A. simplex . For this, L3 larvae of A. simplex were incubated for different periods of time in varying concentrations of HCl and under various conditions of temperature and phonic concentration. The presence of excretory / secretory antigens of A. simplex useful as allergens in the incubation medium was detected by immunoblotting with IgE using sera from allergic patients.

The results obtained showed that the larvae released a considerable amount of protein (excretory / secretory antigens) to the medium. This phenomenon is detected after 30 minutes, and almost completed after of 120 minutes of incubation. The optimal release was obtained at 37 ° C in 50 mmol / L up to 100 mmol / L hydrochloric acid, and it was inhibited by the addition of 500 mmol / L NaCl.

The results obtained showed that the L3 larvae of A. simplex released large amounts of protein when incubated in dilute hydrochloric acid. The optimal acid concentration was similar to that of a simulated gastric fluid, which could explain the acute allergic symptoms that appear in some patients several hours after ingestion of fish. The released allergens could be used for serological diagnosis of A. simplex showing the same sensitivity as the total crude larval extract.

Example 1. Materials and Methods Human sera

Serums from sensitized patients were used to the parasite and previously characterized by immunoblot. These Serums were stored at -20 ° C until use. In most of the assays a set ("pool") of serum obtained was used mixing said sera.

Larval incubation method 1. Larval extraction

The A. simplex larvae were manually extracted from the bacaladilla viscera ( Micromesistius poutassou ) carrying such larvae. The larvae were washed 5 times with distilled water and resuspended in the different media at a rate of 50 larvae / mL.

2. Study of the effect of acid concentration and temperature

To study the effect of the concentration of acid present in the incubation medium and the influence of the temperature, the larvae were incubated for 2 hours at different temperatures (room temperature and 37 ° C) and concentrations of hydrochloric acid (25-200 mmol / L). Then, the larvae were centrifuged at 1,000 g for 5 minutes and They collected the supernatants. When necessary, the larvae centrifuges were resuspended in phosphate buffered saline (PBS) and they ground in a mortar as described by Moneo et al. (Moneo et al., J. Allergy Clin. Immunol 2000; 106: 177-82). After centrifuging again at 4,500 g for 5 minutes, the Supernatant was stored at -20 ° C. Said supernatant is the considered "somatic extract". The protein content is measured with the BCA protein titration reagent kit (BCA Protein Assay Reagent kit, Pierce, Rockford, IL) using as bovine serum albumin pattern.

3. Effect of incubation time

To assess the influence of incubation time on the production of excretory / secretory antigens by L3 larvae of A. simplex , 200 larvae were incubated at 37 ° C in 4 mL of 50 mmol / L HCl and aliquots of the supernatant medium were taken at different times (30 minutes, 1 hour, 2 hours and 3 hours).

4. Effect of pH change

The influence of the pH change of the medium of incubation was tested by incubating the larvae in 50 mmol / L HCl for 1 hour at 37 ° C, washing the larvae 3 times in PBS and incubating the larvae for 1 hour in RPMI 1640. Larvae incubated twice in RPMI 1640 they were used as control. The supernatants of different incubation media were collected and tested in Regarding protein content and allergenicity.

5. Influence of the ionic strength of the medium of incubation

To measure the influence of phonic force, the larvae were incubated under different conditions, specifically (i) in 50 mmol / L HCl, (ii) in 50 mmol / L HCl with 500 mmol / L NaCl, and (iii) in 50 mmol / L HCl, with 500 mmol / L KCl. Then the tubes are centrifuged at 4,500 g for 5 minutes and the supernatants were tested by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) and immunoblot.

6. Effect of pepsin on antigens excretors / secretaries

The effect of pepsin on antigens excretors / secretors released to the medium was tested by incubation of the larvae in 50 mmol / L HCl in the presence of 1 mg / mL of swine pepsin (Sigma, St Louis Mo).

SDS-PAGE and immunoblotting

The SDS-PAGE was performed in 16% minigels of acrylamide and stained with Coomassie blue (Coomassie Brilliant Blue). The detection of specific IgE is performed after its transfer to nitrocellulose, during the entire night, using a diffusion method followed by a stage of incubation for 30 minutes in Nonidet P-40 at 3%. United IgE was detected using a monoclonal anti-IgE (Ingenasa, Madrid, Spain) at a dilution of 1/1000 for 3 hours and a goat anti-mouse serum, marked with alkaline phosphatase (Biosource Int., Camarillo, Ca) at a dilution 1/4000 for 1 hour, as described by Moneo et al. (Moneo et al., J. Allergy Clin. Immunol 2000; 106: 177-82). The Most of the trials were carried out using a pool of positive sera, although the response of the different individual sera to the total crude extract and to the secreted proteins To do this, 0.1 mg of the extracts are underwent electrophoresis in a minigel without streets and, after transfer and incubation in Nonidet P - 40, membranes they were placed in a miniblotter and incubated with 0.1 mL of the serum diluted 1/6 in incubation buffer.

2. Results

Figure 1 shows that incubation of the L3 larvae of A. simplex for 1 hour in 50 mmol / L HCl induced the release of several proteins to the incubation medium. The protein concentration found in the incubation medium was twice as high when 50 mmol / L HCl [diluted acid] (0.26 mg / 50 larvae) was used instead of PBS (0.12 mg / 50 larvae). This secretion improved when the incubation was performed at 37 ° C (0.38 mg / 50 larvae). Several of the released proteins bound the IgE as evidenced by immunoblotting (Figure 1B). When somatic extracts obtained after incubation under different conditions were compared by SDS-PAGE, the protein pattern obtained was clearly different after incubation in diluted acid (Figure 1A, lanes 5-8), suggesting that the larvae had suffered some changes. This fact could also be appreciated in the immunoblot (Figure 1B, lanes 5-8).

The tests performed to find out the optimal concentration of HCl so that the release of excretory / secretory antigens showed that HCl concentration was between 50 and 100 mmol / L (Figure 2, streets 3 and 4). A certain reduction in the release of said excretory / secretory antigens when the HCl concentration was 25 mmol / L and 200 mmol / L. Nevertheless, all acid concentrations tested were more effective, in as for the release of excretory / secretory antigens, that the incubation of the same amount of larvae in PBS (Figure 2, street one).

The influence of incubation time on the release of excretory / secretory antigens from L3 larvae of A. simplex was analyzed using the best conditions found, specifically, 37 ° C and 50 mmol / L HCl. As shown in Figure 3, the presence of excretory / secretory antigens in the incubation medium was already detected in the first control, performed after 30 minutes, and slowly increased to reach a plateau after 2 hours with A minimum increase in the next hour.

\hbox{500 mmol/L}

de KCl puso de manifiesto la liberación selectiva de dos antígenos excretores/secretores con pesos moleculares cercanos a 14 kD (véase la Figura 5, calle 3). Los antígenos excretores/secretores liberados resultaron ser muy sensibles al efecto de la pepsina, tal como se muestra en la Figura 6.After ingestion, at least in humans, the A. simplex larvae penetrate the gastric or intestinal mucosa, thus finding completely different conditions in the composition of the surrounding environment. As shown in Figure 4, when the A. simplex larvae were incubated in culture medium (RPMI 1640) after a previous incubation in acid medium (50 mmol / L HCl), the release of excretory / secretory antigens to This culture medium was strongly inhibited, which seems to suggest that a low pH was needed as a stimulus. As is known, the recovery of diapause is controlled in other nematodes by regulated routes, some of them mediated by muscarinic receptors Tissenbaum et al., Proc. Natl. Acad. Sci USA, 2000; 97: 460-465. Due to the fact that these routes may be strongly influenced by the activation of the phonic channels, the larvae were incubated to release the excretory / secretory antigens at the optimal acid concentration, but in the presence of 500 mmol / L of NaCl, similar to the content in salt from seawater, observing a complete inhibition (Figure 5, lane 2). The lowest salt concentrations tested (125 and 250 mmol / L) were not effective as inhibitors (data not shown). On the contrary, the same test performed with

 ? {500 mmol / L} 

of KCl revealed the selective release of two excretory / secretory antigens with molecular weights close to 14 kD (see Figure 5, lane 3). The excretory / secretory antigens released were found to be very sensitive to the effect of pepsin, as shown in Figure 6.

The excretory / secretory antigens released were studied to measure their diagnostic value using 18 different sera from patients with Anisakis allergy. As shown in Figure 7, the total somatic extract and excretory / secretory antigens released by incubation in acidic medium were equally effective in measuring specific IgE in individual sera. These results show the utility as an allergen of excretory / secretory antigens released from the L3 larvae of A. simplex by incubation in an acid medium, and confirm the results obtained by other authors (Kennedy, Allergy 2000; 55: Suppl. 59: 7-13).

3. Discussion

\hbox{HCl 50
mmol/L}

) indujo la secreción al medio de incubación de una gran cantidad de proteínas (antígenos excretores/secretores), entre ellas algunos alergenos. Este efecto se mejoró cuando la temperatura de incubación se elevó a 37°C. La liberación inducida por ácido no fue seguida por una pérdida de movilidad de las larvas, sugerente de una toxicidad inducida por ácido. En agudo contraste, las larvas retuvieron la movilidad en ácido diluido durante más de 96 horas a 37°C. Además, tal como se muestra en la Figura 2, la liberación alcanzó un máximo a 50 a 100 mmol/L de HCl, mostrando una cierta inhibición a concentraciones más altas de HCl. En opinión de los inventores, estos hechos se oponen a un efecto tóxico de las condiciones de la incubación. Sorprendentemente, el pH de las concentraciones óptimas para la liberación del alergeno de las larvas fue próximo al utilizado por Astwood et al. (Astwood et al., Nat. Biotechnol. 1996; 14-1269-73) para la digestión de alergenos in vitro simulando el efecto del pH del jugo gástrico. Por otra parte, la liberación de proteínas fue íntimamente dependiente de la concentración de los iones, un hecho que sugiere que esta liberación era el producto final de sistemas de activación y desactivación finamente regulados.The tests carried out have shown that incubation in dilute acid (

 \ hbox {HCl 50
mmol / L} 

) induced secretion into the incubation medium of a large amount of proteins (excretory / secretory antigens), including some allergens. This effect was improved when the incubation temperature rose to 37 ° C. The acid-induced release was not followed by a loss of larval mobility, suggesting an acid-induced toxicity. In sharp contrast, the larvae retained mobility in diluted acid for more than 96 hours at 37 ° C. In addition, as shown in Figure 2, the release peaked at 50 to 100 mmol / L of HCl, showing some inhibition at higher concentrations of HCl. In the opinion of the inventors, these facts oppose a toxic effect of the conditions of the incubation. Surprisingly, the pH of the optimal concentrations for larval allergen release was close to that used by Astwood et al. (Astwood et al., Nat. Biotechnol. 1996; 14-1269-73) for in vitro allergen digestion simulating the effect of gastric juice pH. On the other hand, protein release was intimately dependent on the concentration of ions, a fact that suggests that this release was the final product of finely regulated activation and deactivation systems.

Under adverse conditions, some nematodes suffer reversible arrest in development, for example, dauer larvae, an alternative larval stage adapted for survival (Cherkasova et al., J. Mol. Biol. 2000; 300: 433-448). The larvae of A. simplex are normally obtained from chilled fish viscera for a long period and appear to be in a state of developmental arrest, waiting for their entry into the digestive tract of a new host. Ashton and Shad (Vet. Parasitol. 1999; 20: 487-502) suggest that the resumption of development upon entering a host is likely triggered by signals from the host perceived by amphibious neurons of the parasites. In this case, the same larvae (L3) were found to be sensitive to chemical and thermal signals and have different responsiveness to the presence of ions in the incubation medium.

The fact that the products released were highly sensitive to pepsin digestion becomes controversial any explanation about the biological role of this release, but this sensitivity to pepsin can explain by what allergic patients tolerate the ingestion of large amounts of lyophilized or dead larvae, as it has been recently demonstrated (Satre et al., Allergy 2000; 55: 560-564).

On the other hand, secreted products can contribute to perforate the gastric wall once out of reach of the pepsin, since the excretory pore is located very close from the mouth of the larva. In fact, the best treatment for Gastric anisakidosis is the endoscopic removal of the larvae that have partially penetrated the gastric wall. The symptoms clinical, often severe, experienced by patients sensitized after ingestion of parasitized fish, usually appear hours after ingestion of said parasitized fish and are coincident with this phenomenon of activation / release

In any case, the excretory / secretory antigens released were found to be useful as an allergen and an excellent diagnostic reagent. Being free of the most frequent cross-reactive antigens of a total somatic crude extract, they constitute a possible alternative for the diagnosis of A. simplex . This simple, fast and efficient method of secretion of excretory / secretory antigens (allergens) from A. simplex can contribute to a more accurate diagnosis of said nematode parasite.

Although rapid release of excretory / secretory antigens from A. simplex useful as allergens has been illustrated, the method of the invention can be used to obtain excretory / secretory antigens from other animal parasites, for example, animal parasites that cross the digestive tract, and evaluate its diagnostic value in serological methods.

Claims (9)

1. A method for the production of antigens excretors / secretors of an animal parasite, which includes incubating L3 larvae of said animal parasite in an incubation medium that It contains the necessary nutrients for the development of larvae, at a pH equal to or less than 5, and at a temperature between 15 ° C and 60 ° C. 2. Method according to claim 1, wherein said animal parasite is a parasite that causes infections in animals or in humans, which crosses the digestive tract. 3. Method according to claim 2, wherein said animal parasite is a parasite that causes anisakidosis human 4. Method according to claim 1, wherein said incubation medium also contains an acid, in quantity sufficient to provide the incubation medium with an equal pH or less than 5, preferably between 1 and 4, more preferably between 1.5 and 3. 5. Method according to claim 1 or 4, in the that said incubation medium also contains hydrochloric acid, at a concentration between 25 mmol / L and 200 mmol / L, preferably between 50 and 100 mmol / L. 6. Method according to claim 1, wherein the incubation of said L3 larvae is carried out for a period of time between 30 minutes and

 \ hbox {96 hours} 

, preferably between 1 and 2 hours. 7. A method for the production of excretory / secretory antigens of Anisakis simplex , comprising incubating L3 larvae of A. simplex in an incubation medium containing the nutrients necessary for the development of the larvae, at a pH equal to or less 5, and at a temperature between 15 ° C and 60 ° C. Method according to claim 7, wherein said incubation medium comprises hydrochloric acid, in a concentration between

 \ hbox {25 mmol / L} 

and 200 mmol / L, preferably between 50 and 100 mmol / L. 9. The method according to claim 7, wherein the incubation of said L3 larvae is carried out for a period of time between 30 minutes and

 \ hbox {96 hours} 

, preferably between 1 and 2 hours.