WO2021184135A1

WO2021184135A1 - Portable device for the direct detection of pathogenic leptospira in animals using blood and urine samples simultaneously

Info

Publication number: WO2021184135A1
Application number: PCT/CL2020/050026
Authority: WO
Inventors: Camilo Abraham TOMCKOWIACK JELDRES; Miguel Ángel SALGADO ALFARO; Claudio Alejandro HENRÍQUEZ SCHIRMER; Hugo Folch Vilches
Original assignee: Universidad Austral De Chile
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2021-09-23

Abstract

In general terms, the present invention relates to a portable device for the direct detection of pathogenic leptospira in animals with an active infection, said device allowing simultaneous detection of the bacteria in blood and urine samples from the animals at any stage of the infectious cycle thereof.

Description

PORTABLE DEVICE FOR THE DIRECT DETECTION OF PATHOGENIC LEPTOSPIRAS IN ANIMALS FROM BLOOD AND URINE SAMPLES SIMULTANEOUSLY SCOPE OF THE INVENTION

The present invention, in general terms, refers to a portable device for the direct detection of pathogenic leptospires in animals that allows the detection of the bacteria simultaneously from blood and urine samples of animals.

BACKGROUND OF THE INVENTION

Leptospirosis is a bacterial disease that affects various animal species, including humans. This disease induces abortion in livestock industry animals, such as cows and pigs, where it can also cause death in humans. Leptospirosis is currently difficult to detect because specialized laboratories are required to diagnose it, which delays and impairs early treatment of this disease. Leptospirosis is classified as an under-diagnosed disease, the WHO indicates that only 1 in 8 cases of human Leptospirosis is correctly diagnosed, with more than 1 million cases annually worldwide with a mortality rate close to 10%. i Currently, the technique for the diagnosis of human or animal Leptospirosis is the microscopic agglutination technique (MAT), which does not have a commercial format to perform it, and where pure cultures of various Leptospira serovars are performed, investment in equipment is also required. special for its realization, in addition to specialized personnel. However, this technique has several disadvantages, such as, for example, it uses a limited number of serovars, and may present false negatives if antibodies against another serovar are present. Also, animals that are vaccinated will present antibodies that can show false positives to this technique (without infection). In addition to the MAT technique, in recent years rapid Leptospirosis detection tests focused on the detection of antibodies have been generated, however, they maintain the same disadvantages of serological diagnosis and have not proven to be a confirmatory diagnosis to be able to displace the MAT technique.

For example, patent application BR 10201602061 7 A2 describes methods to obtain a conjugate of LipL32 - colloidal gold and an anti-lipl32 antibody, where in addition an in vitro method for the diagnosis of leptospirosis in serum is disclosed, an immunochromatographic device of the type dipstck (tape) and a kit for the in vitro detection of Leptospira spp in serum comprising the protein LipL32 conjugated with recombinant colloidal gold, applied to a glass fiber membrane that is superimposed on cellulose, an anti-LipL32 antibody produced in rabbit, and a dipstick immunochromatographic device.

Patent application US 2007/0197432 A1 describes the design of a peptide with a high immunogenic effect and obtaining mono- or polyclonal antibodies for the diagnosis of leptospirosis in biological samples or contaminated water. The document points out the use of Seq ID No. 1 (KAKPVQKLDDDDDGDDTYKEERHNK), which is part of the PPL protein sequence (LipL32).

Additionally, patent application CN101363868 discloses the cloning of a LipL32 sequence in an expression vector; obtaining the cloned protein; obtaining mono and polyclonal antibodies; the conjugation of the antibodies with colloidal gold to develop a Leptospira antigen detection kit. The kit is effective for studying biological samples such as urine, blood, plasma and serum. Patent application US 2003/0124567 A1 provides immunogenic peptides and a detection system for Leptospira through specific antibodies. This document describes the isolation and characterization of a protein present in pathogenic and highly immunogenic strains of Leptospira, specifically mentioning SEQ ID No 1 (TFLPYGSVINYYGYVK) corresponding to epitopes of the LipL32 protein, by obtaining expression vectors that express immunogenic peptides of Leptospira. The obtaining of antibodies against peptides according to SEQ ID No corresponding to epitopes of the LipL32 protein is also described, and that said antibodies are used to detect pathogenic Leptospira strains in urine and blood samples. Patent application US 2002/0127239 A1 provides a method to detect

Leptospira in a sample by contacting the sample containing the spirochete with a binding agent, which can be an antibody. The method also provides an antibody that detects LipL32 polypeptide or fragments thereof in a sample, and in addition a kit is provided for the detection of LipL32, nucleic acids that encode it and antibodies that bind to LipL32.

Patent application BR102014011648 discloses obtaining anti-Leptospira interrogans IgG antibodies by inoculating rabbits with L. interrogans cultures. The method comprises purifying the anti-L. interrogans IgG antibodies. An objective of this patent is to use the antibodies obtained in the diagnosis of L. interrogans from biological samples and to provide an IgG-DMAE-NHS diagnostic kit.

By virtue of the problems found in the prior state of the art, the present invention is capable of detecting the presence of the Leptospira bacteria in blood and urine from humans or animals, on the other hand, existing developments detect Leptospirosis through the antibodies. Since the symptoms of leptospirosis are not specific in the first stage of the disease, its early detection is often problematic. Currently, commonly used microagglutination tests (MAT) rely on the development and presence of the antibody, which has a high false negative rate during early stages. On the other hand, the detection of antibodies is classified as a diagnosis of the indirect type, which requires confirmation.

In contrast, the present invention when detecting the presence of the bacteria, indicates an active infection in the animal or human, thus seeking to be a confirmatory detection device. Finally, the present invention differs from the state of the prior art in several points: first, it detects an active infection state; • it is additionally compatible with vaccination; identifies the disease in both early and late stages; and

• is capable of simultaneously detecting the presence of Leptospira bacteria in urine and blood samples.

SUMMARY OF THE INVENTION

The present invention relates to a portable device for the direct detection of pathogenic leptospires in animals that allows the detection of the bacteria simultaneously from blood and urine samples of animals, more particularly in a mammal. The device of the present invention comprises:

• A first container for receiving a first sample, and a second container for receiving a second sample, where each of said containers includes: o a component for receiving the sample; or a component containing gold-conjugated antibodies; or a component containing line dried antibodies; and o a component for adsorption of the one-sided liquid flow;

Furthermore, an antibody is described that is included in the first container and second container of the device described in the present invention, which allows the direct and early detection of infection by pathogenic leptospires from urine and blood samples in the form Simultaneously, said antibody can be monoclonal or polyclonal and comprises immunogenic epitopes of the protein of LipL32 membrane of the genus Leptospira, according to the amino acid sequences SEQ ID N ° 1, SEQ ID N ° 2 and SEQ ID N ° 3.

BRIEF DESCRIPTION OF THE FIGURES Figure N ° 1 is (A) a front view of the double device described in the present invention containing a first container (1) for receiving blood samples and a second container (2) for receiving of urine samples. In (B) a front isometric view of a container for receiving blood and urine samples is shown, according to the device described in the present invention, including: receiving strip (3) for the sample (4); support (5); conjugated antibody strip (6); nitrocellulose membrane (9) with line-dried antibodies for sample assays (7) and control assays (8); and absorbent strip (10).

Figure No. 2 is a front view of the device described in the present invention, including: reception strip (3); conjugated antibody strip (6); nitrocellulose membrane (9) with line-dried antibodies for sample assays (7) and control assays (8); and absorbent strip (10).

Figure N ° 3 shows assays using SDS-PAGE (AC), SDS-PAGE and Western blot techniques (B and C), and Indirect Immunofluorescent Detection (DE). Figure N ° 4 shows preliminary assembly tests under the lateral flow technique. DETAILED DESCRIPTION OF THE INVENTION

• A first container (1) for receiving a first sample that includes: o a component for receiving the sample (3); or a component containing gold-conjugated antibodies (6); or a component containing line dried antibodies (9); and o a component for adsorption of the one-sided liquid flow (10);

• A second container (2) for receiving a second sample including: o a component for receiving the sample coupled to a second whole blood separator paper; or a component containing gold-conjugated antibodies (6); or a component containing line dried antibodies (9); and o a component for adsorption of the one-sided liquid flow (10);

The first container (1) and the second container (2) include antibodies that make it possible to detect infection by pathogenic leptospires. Particularly, the first container (1) comprises a component containing gold-conjugated antibodies (6) and a component containing line-dried antibodies (9); and the second Container (2) comprises a gold-conjugated antibody-containing component (6) and a line-dried antibody-containing component (9).

The antibody included in the first container (1) and second container (2) of the device described in the present invention, allows the direct and early detection of infection by pathogenic leptospires from urine and blood samples simultaneously, said The antibody can be monoclonal or polyclonal and comprises immunogenic epitopes of the LipL32 membrane protein of the genus Leptospira, in accordance with the amino acid sequences SEQ ID N ° 1, SEQ ID N ° 2 and SEQ ID N ° 3, as described in the Table N ° 1:

Table N ° 1: List of sequences of the present invention.

Additionally, in one of the embodiments of the present invention, the portable device for the direct detection of pathogenic leptospires in animals, comprises a first container (1) that includes:

• a component for receiving the sample (3), whose material can be selected from cotton strip or fiberglass;

• a component containing antibodies conjugated to gold (6), whose material can be selected from fiberglass or matrix membrane of a layer; • a component containing line-dried antibodies (9), the material of which can be selected from nitrocellulose strip; and

• a component for adsorption of the unilateral liquid flow (10), whose material can be selected from cotton strip. Additionally, in one of the embodiments of the present invention, the portable device for the direct detection of pathogenic leptospires in animals, comprises a second container (2) that includes:

• a component for receiving the sample that is coupled to a second whole blood separator paper, the material of which can be selected from cotton strip or fiberglass;

• a component containing antibodies conjugated to gold (6), whose material can be selected from fiberglass or matrix membrane of a layer;

• a component containing line-dried antibodies (9), the material of which can be selected from nitrocellulose strip; and · a component for adsorption of the one-sided liquid flow (10), whose material can be selected from cotton strip.

In one of the embodiments of the present invention, the portable device for the direct detection of pathogenic leptospires in animals comprises a first container (1) to preferably receive urine samples; and a second (2) container to preferably receive blood samples.

In one of the embodiments of the present invention, the portable device for the direct detection of pathogenic leptospires in animals, comprises a first container (1) and a second container (2), each including line-dried antibodies (9) that allow analysis of urine and blood samples (4) from an animal, and that also include a reaction control to rule out false positives and / or false negatives. In one of the embodiments of the present invention, the device detects the presence of the Leptospira bacteria in blood and urine from animals, more particularly in a mammal, where the animal is a human mammal and / or said animal is a non-human mammal. In one embodiment of the present invention, the non-human mammal can be selected from cattle, goats, horses, sheep, pigs, and / or domestic mammals.

In one of the embodiments of the present invention, the use of the device is described for the direct and early detection of Leptospirosis in an animal, in urine and blood samples simultaneously. In one of the embodiments of the present invention, a process is described to prepare the antibody described in the amino acid sequences SEQ ID N ° 1, SEQ ID N ° 2 and SEQ ID N ° 3, which comprises immunizing an animal with the immunogenic epitopes of the LipL32 membrane protein of the genus Leptospira, in accordance with the amino acid sequences SEQ ID N ° 1, SEQ ID N ° 2 and SEQ ID N ° 3. The production of antibodies, whether polyclonal or monoclonal, is based on inoculation animal of synthetic peptides (amino acid sequences EP3, EP4 and EP6) that simulate the LipL32 protein of the outer membrane zone. Once obtained the antibodies, the marking is carried out by conjugating them with colloidal gold and then added and dried on the conjugate strip.

The visualization of the direct detection reaction of pathogenic leptospires in animals in the device is carried out by the line of antibodies (9) dried on the nitrocellulose strip in high concentration, where a sandwich type complex is created creating a red line visible (Figure N ° 2 and 3).

EXAMPLES

Example No. 1: production of the anti-LipL32 antibody. The production of the polyclonal antibody was carried out based on the inoculation of the peptides EP3, EP4 and EP6 in rabbits. Polyclonal antibodies were obtained from the serum for the detection of the LipL32 protein, a protein found in all pathogenic leptospires. Example No. 2: functionality of the anti-LipL32 antibody.

The functionality of the antibody of the present invention was tested both at the homogenate level of Leptospira and in Leptospira integra. It was tested in 6 Leptospira isolates from different pathogenic serovars L. interrogaos Icterohaemorrhagiae; L. interrogaos Bratislava; L. interrogaos Canicola; L. interrogaos Pomona; L. interrogaos Hardjo; L. interrogate Tarassovi. Figure 3 shows assays using SDS-PAGE (AC), SDS-PAGE and Western blot (B and C) techniques, and Indirect Immunofluorescent Detection (DE). Figures AC show an SDS-PAGE assay of molecular weight standard immunoblot proteins (lines 1 and 9) and lysates of L. interrogans (lines 2-7); buffered saline solution (line 8).

A: proteins stained with Coomassie stain. SDS-PAGE gel for total protein lysate (20 pg) of six strains (lines 2-7) and immunogenic peptide

(line 10).

B: SDS-PAGE and Western blot analysis for 20 pg of total protein using anti-LipL32 serum (Rabbit).

C: SDS-PAGE and Western blot analysis (negative control) of total protein using serum antibodies (anti-LipL32) pre-absorbed overnight with immunogenic peptide. The tips of the black arrows show the strong immunodetection signal (B) and the weak immunodetection signal when using pre-absorbed serum antibodies (C). These are representative images of three independent determinations. DF: indirect immunofluorescent detection of the LipL32 protein in L. interrogans serovar Icterohaemorrhagiae. An absent (D), strong (E), and weak (F) in situ signal is displayed when using only secondary antibodies, primary serum antibodies, and pre-absorbed serum antibodies, respectively. White asterisk in D, shows DNA staining cell with propidium iodine (2ug / ml), white arrows and head arrows in E, shows elongated or curled cells, red asterisk in F, shows displacement of primary binding of antibody-antigen produced when pre-absorbed antibody is used. Scale bars, 20 pm. These are representative images of three independent determinations.

These results allow to demonstrate the effective functionality of the antibody of the present invention to detect the LipL32 protein of Leptospira.

Example N ° 3: device mounting tests.

Once the specific binding effectiveness of the antibody to the LipL32 protein of pathogenic leptospires had been demonstrated, preliminary mounting tests were carried out under the lateral flow technique. A homogenate of pathogenic Leptospira was used as a sample, and it was tested on the device mounted under the lateral flow technique. The results shown in Figure N ° 4 allow to demonstrate that the technique is effective for the direct detection of pathogenic leptospires, and that, therefore, the device proposed by the present invention allows solving the problem of the technique and detecting the presence of Leptospira bacteria in blood and urine from animals, simultaneously.

While this invention has been described under the modalities outlined above, it might seem obvious that other alternatives, modifications, or variations would deliver the same results. Consequently, the embodiments of the invention are intended to be illustrative, not limiting. Various changes can be made without departing from the spirit and scope of the invention as defined in the following claims. All patents, patent applications, scientific articles and other public documents that, to the applicant's knowledge, constitute the state of the art, have been adequately cited in this application.

Claims

1. A portable device for the direct detection of pathogenic leptospires, CHARACTERIZED in that it comprises: · A first container (1) for receiving a first sample (4) that includes: o a component for receiving the sample (3); or a component containing gold-conjugated antibodies (6) comprising the amino acid sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; or a component containing line-dried antibodies (9) comprising the amino acid sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; and o a component for adsorption of the one-sided liquid flow (10); · A second container (2) for receiving a second sample (4) that includes: o a component for receiving the sample coupled to a second whole blood separator paper; or a component containing gold-conjugated antibodies (6) comprising the amino acid sequences SEQ ID N ° 1, SEQ ID N °

2 and SEQ ID No. 3; or a component containing line-dried antibodies (9) comprising the amino acid sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; I a component for adsorption of the one-sided liquid flow (10); wherein the detection of pathogenic leptospires is performed simultaneously by the device; and wherein said device detects the presence of Leptospira bacteria in blood and urine from animals.

2. The device according to claim 1, CHARACTERIZED in that the component for receiving the sample (3) from the first container (1) can be selected from a cotton strip and / or fiberglass.

The device according to claim 1, CHARACTERIZED in that the component for receiving the sample (3) of the second container (2) is coupled to a second whole blood separator paper, and wherein said component can be selected made of cotton and / or fiberglass strip.

4. The device according to claim 1, CHARACTERIZED in that the component containing antibodies conjugated with gold (6) of the first (1) and second container (2), can be selected from fiberglass or matrix membrane of a layer, to deposit said antibodies.

5. The device according to claim 1, CHARACTERIZED in that the component containing antibodies dried in line (9) of the first (1) and second container (2), can be selected from nitrocellulose strip.

6. The device according to claim 1, CHARACTERIZED in that the component for adsorption of the one-sided liquid flow (10) of the first (1) and second container (2), can be selected from cotton strip.

7. The device according to claim 1, CHARACTERIZED in that the device detects the presence of the Leptospira bacteria in blood and urine from animals, more particularly in a mammal.

8. The device according to claim 1, CHARACTERIZED in that the animal is a human mammal.

9. The device according to claim 1, CHARACTERIZED in that the animal is a non-human mammal.

10. The device according to claim 1, CHARACTERIZED in that the non-human mammal can be selected from among cattle, goats, equine cattle, sheep, pigs and / or domestic mammals.

11. Use of the device according to claims 1 to 10, CHARACTERIZED in that it serves for the direct detection and throughout its infectious cycle (Leptospiremia and Leptospiruria) of Leptospirosis in an animal.

12. An antibody for the direct detection of pathogenic leptospires, CHARACTERIZED in that it comprises immunogenic epitopes of the LipL32 membrane protein of the genus Leptospira, in accordance with the amino acid sequences SEO ID N ° 1, SEO ID N ° 2 and SEO ID N ° 3 .

13. The antibody according to claim 12, CHARACTERIZED in that it is a polyclonal antibody.

14. The antibody according to claim 12, CHARACTERIZED in that it is a monoclonal antibody.

15. A process for preparing the antibody of claims 12-14, CHARACTERIZED in that it comprises immunizing an animal with the immunogenic epitopes of the LipL32 membrane protein of the genus Leptospira, in accordance with the amino acid sequences SEO ID No. 1, SEO ID No. 2 and SEO ID

N ° 3.