Antibodies against loxosceles pld, their combinations, uses, methods and associated diagnostic kits

Abstract

The invention relates to a method for obtaining monoclonal antibodies, a method using said monoclonal antibodies, either individually or in combination, or their fragments, for the detection of venom from spiders of the genus Loxosceles, and a diagnostic kit for the rapid and accurate detection of venom from spiders of the genus Loxosceles, in particular the species Loxosceles laeta, Loxosceles intermedia, Loxosceles reclusa and Loxosceles gaucho. The invention also relates to monoclonal antibodies capable of detecting antigenic epitopes common among the various isoforms of the phospholipase D protein family present in the venom of spiders of the genus Loxosceles, for use in a two-zone immunochromatographic kit.

Description

[0001] ANTIBODIES AGAINST LOXOSCELES PLD, THEIR COMBINATIONS, USES, METHODS AND ASSOCIATED DIAGNOSTIC KITS.

[0002] TECHNICAL FIELD

[0003] The invention falls within the field of in vitro diagnostics. In particular, it relates to a diagnostic test for the rapid and accurate detection of the presence of venom from spiders of the genus Loxosceles in patients with suspected skin lesions attributable to spider bites, and can also specifically differentiate the species Loxosceles laeta.

[0004] BACKGROUND OF THE INVENTION

[0005] Bites from synanthropic spiders (those that live in close proximity to humans) can be common in countries with a wide variety of arachnid genera. However, these primarily cutaneous lesions often go unnoticed, or it is not possible to confirm whether the sting (caused by an insect) or bite (caused by an arachnid) was inflicted by them. Furthermore, it can be difficult to determine the specific genus of the spider that caused the envenomation.

[0006] The arachnids of the genus Loxosceles are spiders that possess a venom capable of causing necrotic ulcers and dermal inflammation, a clinical picture called loxoscelism, which can progress with different degrees of complexity, from a mild local or cutaneous condition, to a moderate or severe one with systemic involvement.

[0007] Local or cutaneous loxoscelism, whether mild or moderate, is the most common form, which develops rapidly within the first 24 hours, and the injury caused by the spider bite can cause serious vascular alterations, with areas of vasoconstriction and others of hemorrhages that immediately lead to local ischemia and sometimes to gangrenous plaque in the skin.

[0008] Systemic loxoscelism is less common and considered more serious than cutaneous loxoscelism. It is characterized by hemolysis and disseminated intravascular coagulation, with symptoms including fever, nausea, vomiting, chills, headache, dark urine (hemoglobinuria and hematuria), and decreased hematocrit. Patients may develop acute renal failure, which is frequently fatal.

[0009] Currently, the diagnosis of the clinical picture of Loxoscelism is essentially presumptive, based on three aspects: the characteristics of the lesion, the epidemiological history of the presence of the spider in the home (endemic area and contact with the arachnid) and the capture and identification of the specimen as a member of the Loxosceles spider genus.

[0010] Despite the wide distribution of Loxosceles species across the Americas, loxoscelism is usually caused by or attributed to a predominant species. For example, in Chile, loxoscelism is caused by Loxosceles laeta, while in the United States it is primarily caused by Loxosceles reclusa, and in Mediterranean and Middle Eastern countries by Loxosceles rufescens. In Brazil, Argentina, and Peru, loxoscelism is mainly caused by Loxosceles intermedia, Loxosceles gaucho, and Loxosceles laeta.

[0011] Regardless of the species causing loxoscelism in Latin America, one of the main problems in diagnosing the condition is the delay in seeking medical attention, which varies from 4 to 24 hours after the bite. This delay can contribute to the spread of the local lesion, since the skin necrosis and systemic symptoms caused by Loxosceles venom are irreversible and begin very early, within a few hours of the bite. Diagnosis is primarily clinical, and currently there is no complementary laboratory test to confirm it. Given these diagnostic challenges, the treating physician must be able to establish an early presumptive diagnosis of loxoscelism and differentiate it from other causes of skin necrosis.This differential diagnosis should be made in comparison with bites from other insects and arachnids, herpes simplex, herpes zoster, erysipelas, carbuncle, cutaneous lupus erythematosus, angioneurotic edema, and vasculitis.

[0012] Therefore, early patient attendance at healthcare centers and timely diagnosis are critical aspects to avoid serious consequences in patients with Loxoscelism.

[0013] For example, the Chilean Ministry of Health publishes the "Guide for the Management of Brown Recluse Spider Bites," which details a strategy for confirming Loxoscelism. This strategy considers only the clinical history of the injury and the identification of the spider for diagnostic purposes, and then suggests observation or hospitalization of the patient to rule out a systemic condition. To this end, serial hemoglobinuria tests are performed over a period of 6 to 8 hours, with follow-up for 48 hours if the result is negative. In the case of a positive result, hospitalization in the intensive care unit (ICU) is indicated due to the risk of systemic damage.

[0014] State of the Art

[0015] The spider genus Loxosceles comprises 146 species and is commonly known for its highly toxic venom, which consists of a complex mixture of proteins and peptide toxins. Loxosceles venom toxins include toxins expressed in large quantities, such as phospholipases-D (PLD), metalloproteases (Astacin-type), and insecticidal toxins (ICK peptide-type), as well as toxins expressed in low quantities, such as hyaluronidases, serine proteases, serine protease inhibitors (serpins), allergenic factors, and translation-controlled tumor protein (TCTP) (Gremski et al., 2021). The Phospholipase-D (PLD) family of toxins has been considered the most toxic component of the venom, and its effects on humans include a severe inflammatory response, hemolysis, dermonecrosis, and cytotoxicity in kidney, liver, and heart tissues (Gremski et al., 2022).This type of toxin has been found expressed in 20% of the total venom transcripts of L intermedia and between 16% and 69.3% of the total venom transcripts of L laeta (Gremski et al., 2010; Fernandes-Pedrosa et al., 2008; Medina-Santos et al., 2022). In this latter species, the diversity of possible isoforms expressed according to intra-species variations is demonstrated, which can occur in response to the diet and geographic location of L laeta.

[0016] The intra- and inter-species diversity of phospholipase D (PLD) in spiders of the genus Loxosceles is an important topic for venom research for biotechnological purposes. PLDs are the most relevant component of the venom of these spiders, contributing to their cytotoxic effects. Studies have revealed a remarkable diversity of PLDs in Loxosceles spiders, both in terms of sequence variation and enzymatic activity. For example, the venom of Loxosceles intermedia contains several PLD isoforms with different enzymatic and biological characteristics (Binford et al., 2009). Furthermore, compared to other Loxosceles species, the venom of L. laeta contains PLDs with unique sequence variations and enzymatic characteristics (Gremski et al., 2020; Catalán et al., 2011).The different recombinant PLD isoforms or vahant forms of Loxosceles venom are designated with a wide variety of nomenclatures, such as dermonecrotic toxin, sphingomyelinase-D (SMasaD), Loxnecrogin, LoxTox, and SicTox. These names often include combinations of species initials (e.g., LI = L. laeta) followed by one of the aforementioned terms and finally a number or letter to denote their sequential isolation or purification (Gremski et al., 2020). This intricate diversity underscores the complexity of spider venom composition and the difficulty of conducting consistent comparative studies of Loxosceles spider phospholipase D, focusing on the potential for diverse pharmacological effects.

[0017] Recombinant PLDs from Loxosceles spider venom have been considered an important tool for biotechnological applications focused on immunological responses, immunotherapy, and the diagnosis of loxoscelism. This is because recombinant PLD proteins can be used as antigens to produce monoclonal or even monospecific polyclonal antibodies, which would be useful for ELISA and lateral flow immunochromatography (LFI) assays. In immunotherapy, they would be useful for overcoming the low availability of venom antigens for antivenom production, since recombinant toxins can provide sufficient antigen material, and the resulting antibodies can produce molecules with high affinity, avidity, and greater interaction strength with the antigens, as well as neutralizing capacity, making them valuable tools for diagnosis and treatment.

[0018] In order to have a diagnostic method that allows the detection of Loxosceles spider venom and in turn provide a timely treatment response for the envenomation, some sandwich-type enzyme-linked immunoassay (ELISA) methods have been developed, in the experimental testing stage for the detection of L intermedia venom from rabbit polyclonal antibodies, detection of L reclusa venom from horse polyclonal antibodies, and recently a competitive sandwich-type ELISA test for the detection of L intermedia venom, based on monoclonal antibodies.These tests are aimed at detecting the venom of certain species of the genus Loxosceles that are important in their respective geographical areas; however, they have deficiencies in detecting the venom of other species, such as the species L laeta, and also show cross-reactions with the venom of other spider genera.

[0019] Thus, the article “ELISA for the detection of venom antigens in experimental and clinical envenoming by Loxosceles intermedia spiders” describes a sandwich ELISA test based on the use of the F(ab')2 region of equine polyclonal IgG antibodies produced for the detection of L. intermedia venom. This test was able to specifically detect L. intermedia venom inoculated into mice, but to a lesser extent the venoms of L. gaucho and L. laeta. Furthermore, it was specific in not detecting the venom of other venomous animals, such as R. nigreventer, scorpion venom, and snake venom. In addition, the assay was able to detect 0.8 ng of venom per test and was able to detect L. intermedia antigens in clinical serum samples from patients with Loxoscelism (Chaves-Olortegui et al., 1998).

[0020] The article titled “A new assay for the detection of Loxosceles species (brown recluse) spider venom” describes a sandwich ELISA assay for the detection of L. reclusa venom, using the F(ab')2 region of rabbit-produced polyclonal IgG antibodies; this assay was able to detect 40 ng of L. reclusa venom. However, cross-reactivity was observed at concentrations greater than 40 ng with the venom of other arachnid genera, such as Scytodes fusca and Kukulcania hibernalis, and the assay was also unable to detect the venom of other Loxosceles species (Gomez et al., 2002).

[0021] US patent document 7927828, by the same authors and entitled “Immunoassay for venom detection including noninvasive sample collection,” describes a method and immunoassays in ELISA format for diagnosing the bite of venomous organisms. It specifies a method for collecting the sample from the site of the lesion using a swab, which is then placed in contact with a polyclonal antibody against venom antigens present in the sample. This method is also capable of detecting 20 pg in samples stored for three weeks. This assay differs from the present invention in its diagnostic format; the former is an ELISA assay, unlike the IFL format of the present invention.In turn, it uses polyclonal antibodies against the venom of the L. reclusa spider, while the present invention considers the use of monoclonal antibodies produced against the venom component phospholipase D, and which are also capable of detecting the four main species of Loxosceles, including L. reclusa. This characteristic is not present in the polyclonal antibodies used by the patent's authors.

[0022] Furthermore, the aforementioned test has been reported for the confirmation of cutaneous loxoscelism in patients taking a non-invasive sample and in cases of systemic loxoscelism (Stoecker et al., 2006; Akdeniz et al., 2007; Stoecker et al., 2009).

[0023] Additionally, the production and use of different monoclonal antibodies against the recombinant sphingomyelinase D (phospholipase D) rLiD1 protein of the L. intermedia species has been reported for assembling a sandwich ELISA assay. This assay used the L1 mAb (7) antibody as the capture antibody and the F(ab')2 region of peroxidase-conjugated horse IgG polyclonal antibodies as the detection antibody, enabling the detection of less than 0.2 ng of L. intermedia venom (Alvarenga et al., 2003). However, this assay was not able to cross-detect the venoms of the spiders L. laeta (Brazil), L. laeta (Peru), and L. gaucho.Despite the above, this same monoclonal antibody, LimAb7, has been used to design a competitive ELISA and a sandwich ELISA for the detection of L intermedia venom, by being modified as a bifunctional protein formed by scFv fused to alkaline phosphatase (scFv-LimAb7 / AP), which was able to detect up to 39 ng / ml of L intermedia venom, however again without showing cross-detection with the venom of other Loxosceles species (Jiacomini et al., 2016).

[0024] The scientific article published by Dias-Lopes et al., 2014, describes another monoclonal antibody (L¡D1 mAb16) produced from the recombinant sphingomyelinase D (phospholipase D) antigen rLiD1 of the species L. intermedia, which was able to cross-detect the venom of L. intermedia, L. gaucho, and L. laeta, indicating its ability to detect the linear epitope TYHGIP. However, the detection capacity of the L¡D1 mAb16 antibody does not appear to depend on the presence of the highly conserved histidine residue in Loxosceles PLDs; rather, its antigenic capacity is determined by the preceding amino acid residues of the peptide FDDNANPEYTYHGIP, since the TYHG peptide shows reduced detection by the monoclonal antibody.In the present invention it is shown that part of said peptide would correspond to an antigenic epitope exclusive to the amino acid sequences of PLD of the Loxosceles species belonging to phylogenetic cluster 1, in which the species L laeta is not present, which would explain the low cross-reactivity of the antibody L¡D1 mAb16 at dilutions greater than 1 : 100 against the venom of L laeta compared to the species L intermedia and L gaucho.

[0025] The invention patent BR102012033561, by the same authors, entitled “Monoclonal Antibody, Production Process and Use in the Diagnosis and Treatment of Loxoscelism,” describes the procedure for obtaining the monoclonal antibody L¡D1 mAb16, its use in the diagnosis and treatment of loxoscelism, and its potential use in treatment and immunodiagnostic assays. However, it does not specify or exemplify any diagnostic assay demonstrating its use in detecting the three venoms of L. intermedia, L. gaucho, and L. laeta. The monoclonal antibodies of the present invention, on the other hand, are notable for their cross-detection capacity for the species L. laeta, L. intermedia, L. gaucho, and L. reclusa at dilutions greater than 1:1000, and their use in an IFL-type immunodiagnostic assay.

[0026] Patent BR102019016826A2, entitled “kit and method for diagnosing Loxosceles bite,” describes a diagnostic method and kit for detecting Loxosceles spider venom in urine samples using antibodies conjugated to gold nanoparticles. The antibodies are rabbit polyclonal IgG antibodies against the total venom of L. similis. However, the cross-detection capability of these antibodies against the venoms of L. intermedia, L. gaucho, and L. laeta is not demonstrated. Furthermore, detecting Loxosceles venom from a urine sample would only be applicable to patients with kidney damage or systemic envenomation, thus limiting its potential for early diagnosis. Although BR102019016826A2 anticipates conjugation with gold nanoparticles, the present invention differs from this document in terms of the specificity of the monoclonal antibodies obtained.

[0027] While the aforementioned ELISA assays exhibit significant sensitivity, their main disadvantages for diagnostic use in Loxoscelism are related to: 1) their inability to detect the venom of different Loxosceles spider species (since they only detect the venom of the species for which they were designed); 2) the limited or nonexistent number of clinical validation trials; and 3) the average time required to perform the assay (between 3 and 4 hours). Furthermore, these solutions lack early diagnostic applicability, a critical aspect for the management of patients with Loxoscelism, and could only be used for subsequent confirmation of the presence of venom or for epidemiological purposes.Currently, both the diagnosis and research in other clinical areas of loxoscelism are limited by the absence of a gold standard assay or a methodology that demonstrates its sensitivity and specificity for detecting the venom of spiders of the genus Loxosceles. Without a confirmatory diagnostic test, the risk of developing loxoscelism persists due to delays in seeking medical attention, the difficulty in differentiating the skin lesion from other pathologies, and the low percentage of patients who manage to capture the spider for morphological identification of the causative agent. Thus, the lack of a rapid diagnostic test for the accurate diagnosis of loxoscelism presents a clear opportunity for study and technological development. BRIEF DESCRIPTION OF THE INVENTION.

[0028] Although illustrative embodiments of one or more aspects of the present invention are provided herein, it may be implemented by various technical means without departing from the scope defined in the appended claims. The experimental examples and configurations described below are for illustrative purposes only and are not intended to limit the invention to the specific embodiments shown.

[0029] The present invention relates to monoclonal antibodies and fragments thereof directed against phospholipase D (PLD) in the venom of spiders of the genus Loxosceles, obtained from partial or complete consensus amino acid sequences. The invention comprises the process for producing said antibodies, individual antibodies, antibody combinations, in vitro detection methods using said antibodies, a diagnostic kit based on lateral flow immunochromatography and said antibodies, and uses of these antibodies and their combinations in venom detection.

[0030] In one aspect, the invention provides a process for obtaining monoclonal antibodies and / or fragments thereof, from partial or complete consensus amino acid sequences of Loxosceles PLD, where said sequences correspond to SEQ ID NO: 1 to 4. The process comprises cloning the sequences into a genetic vector, introducing said vector into an expression system capable of producing recombinant PLDs, expressing and purifying the resulting proteins, and using them as immunogens in an animal immunization protocol for the generation of monoclonal antibodies or fragments thereof.

[0031] In another aspect, the invention includes individual monoclonal antibodies or fragments thereof obtained according to the process described above. These antibodies are selected from those produced by hibdomes 6E5, 1H11, 10A11, 12D4, 19C8, 34H3, and 35H4, and have affinity for phospholipase D of Loxosceles venom, thus enabling its detection.

[0032] In an additional aspect, individual monoclonal antibodies or their fragments exhibit cross-reactivity against PLD antigens from two or more species of the genus.

[0033] In another aspect, the individual monoclonal antibodies belong to the IgG1 antibody subclass. In one aspect, the invention provides a combination of monoclonal antibodies or fragments thereof comprising at least two distinct antibodies obtained according to the described process. These combinations include antibodies selected from those produced by the aforementioned hibdomes and exhibit affinity for phospholipase D of the venom of spiders of the genus Loxosceles.

[0034] In an additional aspect, the combination of monoclonal antibodies or their fragments allows the detection of venom from Loxosceles laeta, L intermedia, L gaucho and L reclusa, and may include cross-reactive antibodies towards multiple species.

[0035] In another additional aspect, the monoclonal antibodies of the combination belong to the lgG1 .k subclass.

[0036] In one aspect, the invention relates to an in vitro method for detecting Loxosceles venom in biological samples. The method comprises contacting the sample with a combination of monoclonal antibodies selected from the hibdomes 6E5, 1H11, 10A11, 12D4, 19C8, 34H3, and 35H4, and detecting the antigen-antibody binding by lateral flow immunochromatography. The sample may consist of skin swabs, biological fluids, exudates, tissue from the affected site, or other suitable sample types.

[0037] In one aspect, the invention provides an in vitro detection kit for the venom of spiders of the genus Loxosceles. This kit comprises a plastic device with sample deposition, detection, and control zones, and a membrane incorporating antibodies produced by the 6E5 hibdom and, optionally, one or more antibodies selected from 1H11, 10A11, 12D4, 19C8, 34H3, and 35H4. The kit can be configured in various ways.

[0038] In one modality, the kit comprises a general detection zone that incorporates as a capture antibody one selected from those produced by the hibhdomes 34H3, 35H4, 10A11, 12D4 or 19C8, in combination with a conjugated antibody produced by the hibhdome 6E5.

[0039] In an additional configuration, the kit includes one or more specific detection zones targeting particular Loxosceles species. These specific zones may incorporate the following capture antibodies:

[0040] • antibodies produced by 1 H11 , 10A11 , 12D4 or 19C8 for the detection of Loxosceles laeta, • antibodies produced by 34H3, 10A11 , 12D4 or 19C8 for the detection of Loxosceles intermedia',

[0041] • antibodies produced by 35H4 or 6E5 for the detection of Loxosceles gaucho or Loxosceles red usa.

[0042] In another configuration, the kit can be set up as a dual format, comprising two detection zones selected from the specific zones described. Some configurations include combinations for L laeta + L intermedia or L laeta + L gaucho / L reclusa.

[0043] In another mode, the kit can be configured as a triple format, comprising three detection zones selected from the defined specific zones, including configurations capable of simultaneously detecting L laeta, L intermedia and L gaucho / L reclusa.

[0044] In another modality, the kit is presented as a general format comprising at least one general detection zone as set out in the claims, usable when a broad identification of the genus is required without distinguishing the species involved.

[0045] In one aspect, the invention contemplates the use of individual monoclonal antibodies, obtained according to the described process, for the in vitro detection of the venom of spiders of the genus Loxosceles.

[0046] In another aspect, the invention contemplates the use of combinations of monoclonal antibodies or fragments thereof, for the in vitro detection of the venom of spiders of the genus Loxosceles.

[0047] Other variants, applications and modes of embodiment will be evident from the following description and experimental examples, without limiting the scope of the present invention, which is defined exclusively by the attached claims.

[0048] BRIEF DESCRIPTION OF THE FIGURES

[0049] Figure 1: A) Representative diagram of a sandwich-format lateral flow immunochromatography (LFL) device for the detection of Loxosceles spider venom. The three zones are shown: i) conjugated with colloidal gold, ii) common detection zone, and iii) specific detection zone. B) Sample adsorption stage in each zone. C) Top view of a lateral flow immunochromatography (LFL) device. Figure 2: Phylogenetic analysis of consensus phospholipase D from spiders of the genus Loxosceles. A) Maximum likelihood phylogenetic tree of the amino acid sequences of phospholipase D from L. laeta. B) Maximum likelihood phylogenetic tree of the consensus amino acid sequences of phospholipase D from Loxosceles spiders.

[0050] Figure 3: Heat map of the amino acid sequence identity percentage of continuous antigenic epitopes of phospholipase D from Loxosceles species. The nine continuous antigenic epitopes identified from Loxosceles species in phylogenetic group 1 (a) were color-coded on the heat map to indicate higher (green) or lower (red) identity percentages. These epitope sequences were then compared with Loxosceles species from phylogenetic group 2 (b). Similarly, the nine continuous antigenic epitopes identified from Loxosceles species in phylogenetic group 2 (c) were also compared with Loxosceles species from either phylogenetic group 1 (c) or phylogenetic group 2 (d).

[0051] Figure 4: SDS-PAGE of amino acid sequences of consensus PLD antigens for L. laeta, L. intermedia, L. gaucho and L. reclusa species. A) L. laeta PLD1 consensus antigen (LIPLD1 ). Arrow indicates 33,222 kDa protein. B) L. laeta PLD G2 consensus antigen. Arrow indicates 32,358 kDa protein. C) PLD consensus antigen L.reclusalL.gaucho. Arrow indicates 32,142 kDa protein. D) Consensus antigen PLD L. intermedia. Arrow indicates 32,909 kDa protein.

[0052] Figure 5: Indirect ELISA for titration of Loxosceles anti-PLD consensus mouse sera. MaxiSorp 96-well microplates were adsorbed with 1 pg / ml of antigen in 100 µl per well of carbonate / sodium carbonate buffer, pH 9.6. Each value represents the average of two replicates. Absorbance values ​​at 450 nm were normalized to the blank (PBS). The negative control was IgG antibody from a non-immunized mouse. Sera were diluted 2:1 from a 1:1000 dilution to 1:512,000. A) ELISA for titration of anti-LIPLD1 mouse sera. B) ELISA for titration of anti-LIPLD G2 consensus mouse sera. C) ELISA for titration of anti-PLD Lg / Lr consensus mouse sera. D) ELISA for titration of anti-PLD Li consensus mouse sera.

[0053] Figure 6: Indirect ELISA for the detection of Loxosceles consensus PLD antigens using supernatants from parental hybridoma clones. 96-well microplates were adsorbed with 1 pg / ml of each consensus PLD antigen at 100 pg per well. Results were performed in duplicate. Absorbance values ​​at 450 nm were normalized by subtracting the blank value with PBS. Supernatants were added in duplicate and undiluted. Negative control: Anti-N / A: Unrelated hybridoma supernatant. A) Indirect ELISA for the detection of LIPLD1 antigen using supernatants from 20 parental hybridoma clones. Positive control (PC): M1622 mouse serum used for cell fusion. B) Indirect ELISA for the detection of Llaeta consensus PLD G2 antigen using supernatants from 20 parental hybridoma clones.Positive Control (PC): M1864 mouse serum used for cell fusion C) Indirect ELISA for the detection of PLD consensus Lg / Lr Ag by supernatants from 20 parental hibhdoma clones. Positive Control (PC): M1626 mouse serum used for cell fusion D) Indirect ELISA for the detection of PLD consensus L1 Ag by supernatants from 20 parental hibhdoma clones. Positive Control (PC): M1870 mouse serum used for cell fusion.

[0054] Figure 7: Indirect ELISA for the detection of Loxosceles consensus PLD antigen by hibridoma subclone supernatants. Microplates were adsorbed with 1 pg / ml of antigen in 100 pl per well. Results were performed in duplicate. Absorbance values ​​at 450 nm were normalized by subtracting the blank value with PBS. Supernatants were added in duplicate and undiluted. Negative control: Anti-N / A: Unrelated hibridoma supernatant. A) Indirect ELISA for the detection of Llaeta PLD1 antigen by hibridoma subclone supernatants. B) Indirect ELISA for the detection of Llaeta PLD G2 consensus antigen by hibridoma subclone supernatants. C) Indirect ELISA for the detection of Lg / Lr consensus PLD antigen by hibridoma subclone supernatants. D) Indirect ELISA for the detection of consensus PLD Ag L1 by supernatants of hibhdoma subclones.

[0055] Figure 8: Western blot for the detection of Loxosceles PLD consensus recombinant antigens by hibridoma subclone supernatants. 3 pg of each PLD consensus recombinant antigen were separated by SDS-PAGE on a 12% gel and transferred to a nitrocellulose membrane. The membrane was then incubated with undiluted hibridoma subculture supernatants containing the respective anti-PLD mAbs. Detection was revealed using a Goat anti-mouse IgG / HRP conjugate antibody diluted 1:40,000 followed by ECL reagent. Anti-N / A supernatant: unrelated hybridoma supernatant and PBS-T were used as a negative control. A) WB of LIPLD1 consensus recombinant antigen by anti-rLIPLDI hibridoma subclones. B) WB recombinant LIPLD G2 consensus antigen by anti-rLIPLD G2 hibdoma subclones. C) WB recombinant PLD consensus Lg / Lr antigen by anti-PLD consensus Lg / Lr hibdoma subclones.D) WB recombinant PLD consensus L1 antigen by anti-PLD consensus L1 hibdoma subclones. Figure 9: Indirect ELISA for the detection of Llaeta venom by the 40 different mAbs produced against PLD antigens of Loxosceles species. Microplates were adsorbed with 1 pg / ml of L. laeta venom in 100 ml of carbonate / bicarbonate buffer per well. Results were performed in duplicate. Absorbance values ​​at 450 nm were normalized by subtracting the blank value with PBS. Supernatants from hibdoma clones containing the different mAbs were added in duplicate and undiluted. Negative control: Anti-N / A: Unrelated hibdoma supernatant. A) anti-rLIPLD1 mAb. B) anti-rLIPLD consensus G2 mAb. C) anti-rPLD consensus Lg / Lr mAb. D) anti-rPLD MAb consensus L¡.

[0056] Figure 10: WB for the detection of Llaeta venom by the 40 different mAbs produced against PLD antigens of Loxosceles species. A) mAbs from anti-rLIPLD1 hibdoma subclones. B) mAbs from anti-rLIPLD G2 consensus hibdoma subclones. C) mAbs from anti-rPLD Lg / Lr consensus hibdoma subclones. D) mAbs from anti-rPLD Li consensus hibdoma subclones. E) Negative control: PBS; Anti-N / A antibody (against unrelated Ag).

[0057] Figure 11: Indirect ELISA titration of culture supernatants from Loxosceles consensus anti-PLD subclone lines. Microplates were adsorbed with 1 pg / ml of PLD antigen per well. Results were performed in duplicate. Absorbance values ​​at 450 nm were normalized by subtracting the blank value with PBS. Supernatants were added in duplicate and in serial dilutions from 1:10, 1:30, 1:90, 1:270, 1:810, and 1:2430. Anti-N / A antibody was used as a negative control; unrelated hibdomidase supernatant. A) Titration of anti-LIPLD1 subclones 1H11, 10A11, 12D4, and 19C8 supernatants. B) Titration of supernatants of 6E5 anti-PLD consensus Lg / Lr subclones and supernatants of 34H3 and 35H4 anti-PLD consensus L1 subcultures.

[0058] Figure 12: Evaluation of the conjugation of anti-PLD monoclonal antibodies against Loxosceles with colloidal gold for use in an IFL assay setup. Conjugation of the selected anti-PLD monoclonal antibodies against Loxosceles was performed using the Abeam Gold Conjugation Kit (40 nm, 20 OD) (#ab154873). A) Adsorption of BSA protein (negative conjugation control) and anti-IgG antibody (positive conjugation control) to a nitrocellulose membrane. Three pg of each protein was used. Protein presence was observed by staining with Ponceau Red. B) Detection of anti-PLD monoclonal antibodies against Loxosceles, conjugated with colloidal gold diluted 1:5. PBS was used as a negative control instead of the conjugated antibodies. A positive reaction is visualized by a red coloration imparted by the colloidal gold nanoparticle. Figure 13: Cross-detection of Loxosceles anti-PLD mAbs conjugated with colloidal gold.For this purpose, a concentration between 500 pg / ml and 1 mg / ml (depending on the availability of each antibody) was conjugated with colloidal gold using the Abeam Gold Conjugation Kit (40 nm, 20 OD) (#ab154873). Mouse anti-IgG antibody (1 mg / ml) adsorbed to a nitrocellulose membrane in a dot-blot system was used as a positive conjugation control. BSA (3 pg) adsorbed to a nitrocellulose membrane was used as a negative control. Conjugated monoclonal antibodies (1:5) were applied to a nitrocellulose membrane in a dot-blot system containing 3 pg of the different PLD antigens (LIPLD1, LIPLD consensus G2, Lg / Lr PLD consensus, L1 PLD consensus) and including L. laeta venom. Detection of the mAb- conjugated with their respective antigens was visualized by the appearance of a red mark in the area where the antigen was located, and after 15 minutes of incubation at room temperature.

[0059] Figure 14: Proof of concept lateral flow immunochromatographic assay “Loxo-test+”. The IFL strip was assembled using glass fiber strips impregnated with a 1:5 dilution of monoclonal antibody (mAb) conjugated with colloidal gold (6E5 / 34H3) in conjugation buffer. The sample pad was prepared using glass fiber strips soaked in sample pad wash buffer (NaCl, Tween20, sodium azide), mixed with agitation, and allowed to dry at 37°C for 2 hours. The detection (T) and control (C) zones were prepared from a nitrocellulose membrane by depositing 1 mg / ml of the mAb pair (1H11 / 35H4) to form a linear slot using a Bio-dot / Slot filtration system (Bio-Rad). Mouse anti-IgG goat antibody was also deposited in the control zone. The final strip was assembled by attaching the nitrocellulose membrane to a transparent plastic sheet using double-sided tape.Finally, a cellulose fiber strip (absorbent pad) was placed at the opposite end. 100 µl of PBS was placed on the sample pad for the negative control, while 100 µl of PBS containing 25 pg of Llaeta venom was placed on the sample pad for the assay strip. Both strips were incubated for 15 minutes at room temperature in a horizontal position, allowing sample flow. After the incubation period, the strips were read for the presence of red colored bands in both the detection zone (T) and the control zone (C). A positive result was considered to be the presence of both colored bands, while a negative result was considered to be the presence of only one band in the control zone.

[0060] Figure 15. IFL assay for the detection of L. laeta venom. A) Sensitivity of the assay for the detection of L. laeta venom dilutions (50–0.37 pg / ml). B) Sensitivity of the IFL assay for the detection of recombinant protein rLIPLDI (5–0.1 pg / ml). Negative control: PBS pH 7.4. Green line: corresponds to the control zone with mouse anti-IgG antibody (C). Red line: detection zone (T).

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention provides a combination of recombinant anti-phospholipase D consensus proteins (PLD) monoclonal antibodies from the venom of spiders of the genus Loxosceles, specifically the species Loxosceles laeta, Loxosceles intermedia, Loxosceles reclusa, and Loxosceles gaucho, capable of being applied in a lateral flow immunochromatography (LFI) diagnostic method for cross-detection between Loxosceles species. The monoclonal antibodies used in the invention do not exhibit cross-immunoreactivity against the venom of other arachnids unrelated to Loxosceles spiders.

[0063] The invention comprises the sequence of the four consensus antigens of the phospholipase D protein (PLD) from the venom of Loxosceles spiders used for the production of monoclonal antibodies (mAbs), the process of producing the mAbs, their characteristics and selection process, as well as their application by means of a combination or set of monoclonal antibodies (mAbs) produced against conserved antigenic determinants of the consensus phospholipase D antigens and which can be used as capture and / or conjugate antibodies in diagnostic assays for loxoscelism.The monoclonal antibodies in question have differentiating characteristics from other available monoclonal antibodies (mAbs) because they are produced for the detection of common antigenic epitopes present in consensus sequences of the phospholipase D toxin family from different spider species of the genus Loxosceles. This allows them to detect between two and four of the main Loxosceles species in the Americas (North and South America), such as L. laeta, L. intermedia, L. gaucho, and L. reclusa. This characteristic distinguishes them from other monoclonal antibodies produced against a single recombinant isoform of PLD from a single species, whose cross-detection capacity with other Loxosceles species is reduced, especially against the venom of L. laeta. Furthermore, the mAbs of the present invention belong to the IgG1 class and exhibit high detection titers (>1:2).430), avidity indices (antibody affinity for its antigen) equal to 1 (highest value for the index), and the ability to cross-detect the venom of at least two of the four species: L. laeta, L. intermedia, L. gaucho, and L. reclusa. Specific detection by Loxosceles venoms, as well as cross-detection among spider species of the genus Loxosceles, is maintained after conjugation with colloidal gold. The monoclonal antibodies produced against consensus PLD antigens of the species L. intermedia, L. reclusa, and L. gaucho are capable of detecting the venom of L. laeta, which exhibits significant antigenic differences compared to all known Loxosceles spider species. Additionally, the invention considers the use of monoclonal antibodies with exclusive PLD detection capability for the venom of the L. laeta spider, allowing for the specific identification of the venom causing the envenomation.

[0064] The antibodies according to the invention have application as capture and / or conjugate antibodies in the assembly of diagnostic assays of lateral flow immunochromatography (IFL), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), western blot, and flow cytometry (FC).

[0065] Ten monoclonal antibody (mAb)-producing hibdomes were obtained for each of the four different recombinant consensus PLD antigens used. Each of the 10 subclones produced mAbs, which were purified from culture supernatant, and were capable of strongly recognizing their respective antigens. Of the 40 mAbs analyzed, 3 mAbs were selected, produced against the recombinant consensus PLD antigens of the species L. gaucho, L. reclusa, and L. intermedia, which were also capable of recognizing L. laeta venom. In addition, 4 different mAbs were selected that were capable of specifically detecting L. laeta venom and L. laeta consensus PLDs. A set of 7 mAbs was obtained for use in assembling immunodiagnostic assays for Loxosceles spider venom.Three monoclonal antibodies (mAbs) show cross-detection capacity among the four Loxosceles species; therefore, they can be considered for inclusion in the common detection zone mAb capture / conjugate pair of the assay, which allows for the detection of venom from any of the Loxosceles species. All mAbs exhibit high avidity indices (close to 1) and high detection titers for their antigens. Furthermore, the monoclonal antibodies (mAbs) are characterized by the absence of cross-detection with venoms from other arachnids such as Sicarius thomisoides, Grammostola rosea, and Paraphysa sp.

[0066] Combination of monoclonal antibodies or fragments thereof

[0067] The invention comprises a combination of monoclonal antibodies (mAbs) that enables the detection of venom from spiders of the genus Loxosceles based on their affinity for consensus sequences of phospholipase D in the venom of spiders of the genus Loxosceles. In particular, the invention comprises monoclonal antibodies corresponding to the antibodies produced by clones 6E5 (obtained from consensus PLD antigen Lg / Lr), 19C8 (obtained from PLD1 antigen of L. laeta), 34H3, and 35H4 (both obtained from consensus PLD antigen L1), wherein the monoclonal antibodies or fragments thereof are capable of detecting the four Loxosceles species of interest: Loxosceles intermedia, Loxosceles gaucho, Loxosceles reclusa, and Loxosceles laeta.

[0068] Optionally, the invention further comprises at least one antibody capable of detecting the venom of PLD of the species L laeta, obtained from clones 1 H11, and 10A11.

[0069] 6E5 clones produce consensus MACs against PLD of the Lgaucho / Lreclusa species with affinity also for Loxosceles intermedia and Loxosceles laeta.

[0070] The 19C8 clones produce AcM against PLD1 consensus of the Loxosceles laeta species with affinity also for the other Loxosceles species.

[0071] The 34H3 and 35H4 clones produce consensus PLD mAbs of the Loxosceles intermedia species with affinity also for the other Loxosceles species.

[0072] Methodology and device

[0073] The present invention provides a selection of monoclonal antibodies for performing a lateral flow immunochromatography assay [or other assays such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), western blot, and flow cytometry (FC)] that delivers optimal results. This includes a common detection zone for Loxosceles venoms from different species in an array format, which allows for increased assay sensitivity through the use of mixtures of the aforementioned monoclonal antibodies. This type of array refers to the arrangement of two or more monoclonal antibodies capable of detecting all four Loxosceles spider species, thus enabling the simultaneous detection of venom from multiple species of Loxosceles spiders in a single test.The main advantage of the invention lies in its ability to detect venoms from different Loxosceles species in a single test, increasing efficiency and reducing costs compared to performing individual tests for each species. Other available formats include a device for the specific identification of each of the species L. laeta, L. intermedia, L. gaucho, and L.independently, in addition to a device that includes the simultaneous detection of the venom of any of the four species in a common detection zone followed by a second specific detection zone for the relevant Loxosceles species according to the geographical region where it is triply in device, this combination being (common detection zone of Loxosceles venom + specific detection zone of L laeta venom', common detection zone of Loxosceles venom + specific detection zone of L intermedia venom', common detection zone of Loxosceles venom + specific detection zone of L gaucho venom', common detection zone of Loxosceles venom + specific detection zone of L reclusa venom).

[0074] For this purpose, six cell lines of subclones capable of specifically detecting the D-phospholipases in L. laeta venom are available. These can be used in the specific detection zone for L. laeta, allowing for the development of an analytical kit for epidemiological purposes. Optionally, the specific detection zone could be replaced to detect L. intermedia, L. gaucho, or L. reclusa, using any of the monoclonal antibodies (mAbs) capable of recognizing their respective consensus antigens.

[0075] The in vitro detection method for the presence of venom from spiders of the genus Loxosceles in a sample comprises the following steps:

[0076] - contact the sample with the combination of monoclonal antibodies or fragments thereof produced by clones 6E5, 34H3, or 35H4 + anti-PLD mAb from L laeta clones 1H11, 10A11, or 19C8.

[0077] - detect the binding of antibodies to the antigen by immunochromatography.

[0078] The sample corresponds to a skin swab recovered from the injury area using a flocked-tip swab covered with sterile, disposable short nylon fiber, which is rubbed over the affected area and deposited in pH 7.4 phosphate-buffered saline solution, or any other type of sample that is considered appropriate.

[0079] The device or kit consists of an analysis with two stages or detection zones:

[0080] 1) Stage / Zone 1: able to recognize venom of the genus Loxosceles, through the combination of conjugated and capture mAbs directed against conserved antigenic epitopes of the phospholipase D family (recombinant proteins and / or conserved antigenic peptides).

[0081] 2) Stage / Zone 2: Specific detection of the venom of one species, preferably L. laeta, using conjugated and capture monoclonal antibodies specific to this venom. Alternatively, a specific antibody set for any of the other species of epidemiological interest can be used.

[0082] The analyzed sample corresponds to samples suspected of being caused by a spider bite. In a preferred embodiment, the sample is a swab or torula, where a swab or torula is rubbed onto the suspected skin lesion and then placed in a tube containing phosphate-buffered saline. The buffer is allowed to emulsify, and then the sample, in a volume of 100 to 200 ml, is deposited in the sampling area of ​​the device and subjected to analysis according to the present invention. Other types of samples, such as serum, are excluded because they could generate false positives.

[0083] In one embodiment of the invention, a lateral flow immunochromatographic (IFL) kit is developed for the rapid detection of Loxosceles spider venom, comprising a plastic device that includes:

[0084] • sample deposit areas

[0085] • Detection zone, preferably with a first zone for detecting the venom of spiders of the genus Loxosceles and a second optional zone for the specific detection of a species. Each zone contains the respective antibodies.

[0086] • test confirmation or control zone

[0087] • membrane in which the antibodies produced by subclones 6E5 are incorporated and optionally, at least one of the antibodies produced by subclones 34H3 and 35H4, plus at least one of the antibodies produced by the clones against PLD of the species L laeta ^ m , 10A11 , or 19C8)

[0088] The kit can be used at room temperature, with reading times for a positive result of between 10-15 minutes.

[0089] In the event of a positive detection, a colored band will form. The confirmation or control zone must also be colored whenever the sample successfully migrates from the deposition zone to the control zone of the membrane inside the device.

[0090] Advantages of the invention

[0091] • The monoclonal antibodies used in the present invention are characterized by being specific for the detection of the venom of spiders of the genus Loxosceles, without cross-detection with venoms of other arachnids such as Sicarius thomisoides, Grammostola rosea and Paraphysa sp.

[0092] • The immunochromatographic analysis according to the invention is performed in less than 15 minutes, i.e., it is a rapid test, compared to comparable analyses that take between 3 and 4 hours.

[0093] • The analytical capability of the monoclonal antibodies used for the present invention was verified by performing parallel tests using ELISA and Western blot. In ELISA, absorbance values ​​were obtained above the established cutoff point, and in Western blot, a detection band appeared at the molecular mass corresponding to the different recombinant proteins (antigens) representative of each species.

[0094] • The immunochromatographic assay is able to detect Loxosceles venom in bite site samples in the range of ng to mg of protein, and at skin injury times between 30 min to 48 hours post-bite or inoculation.

[0095] The binding capacity of the mAb to its antigen was verified by an ELISA assay in the presence of urea by comparing the absorbance values ​​between the assay in the presence and absence of urea, calculating the avidity index.

[0096] EXAMPLES

[0097] Example 1:

[0098] Figure 1 shows the setup scheme of a sandwich-format lateral flow immunochromatography (IFL) assay for the detection of Loxosceles spider venom in skin lesion samples.

[0099] This assay includes the monoclonal antibodies in question as capture in their common detection zone (any species of Loxosceles spider) or specific (species specific for the detection of L laeta), or as conjugated with colloidal gold.

[0100] Thanks to the detection capacity of the monoclonal antibodies used, it is possible to detect the main venoms of the species present in Latin America, such as L laeta, L intermedia, L gaucho and L reclusa, present in the dermonecrotic lesion sample of patients under study.

[0101] Example 2: Selection of consensus sequences of phospholipase D from Loxosceles spiders.

[0102] Consensus sequences of phospholipase D (PLD) from the main Loxosceles spider species that cause arachnidism in the Americas were used as antigens for the production of monoclonal antibodies. These included Loxosceles laeta, Loxosceles intermedia, Loxosceles gaucho, and Loxosceles reclusa. From the amino acid sequences available in the NCBI database, identified as “phospholipase D,” “dermonecrotic toxins,” “LoxTox toxins,” and “SicTox toxins” (the latter considered alternative nomenclature for phospholipase D), it was possible to obtain the different sequences for each Loxosceles spider species. Multiple sequence alignment was then performed using the Clustal Omega platform. and then visualized using Jalview software version 2.11.3.3. From the multiple sequence alignments, one or more consensus amino acid sequences were identified for each Loxosceles spider species, which were subsequently grouped into phylogenetic clusters using a phylogenetic tree constructed by the Maximum Likelihood method with Mega X software version 10.2.4. Due to the high variability of amino acid sequences in the species L. laeta, with three phylogenetic groups, two consensus PLD sequences were chosen from the two main phylogenetic groups as potential antigens (Figure 2a). These were named LIPLD1 (representing phylogenetic cluster 1 of L. laeta) and L1 consensus PLD group 2 (LIPLDcG2) (from phylogenetic cluster 2 of L. laeta). The species L. laeta belongs to a different phylogenetic cluster than the species L. intermedia, L. gaucho, and L. reclusa (Figure 2b).In addition, a consensus PLD sequence was used for the species L intermedia (consensus PLD L1) belonging to phylogenetic cluster 1 of Loxosceles, and a single consensus sequence for the species L reclusa and L gaucho, both belonging to phylogenetic cluster 1 of Loxosceles. The PLD sequences of both species showed a high sequence identity (between 80 and 100%), and the consensus sequence was designated (consensus PLD Lg / Lr). The consensus PLD sequences identified for each Loxosceles spider species were compared, revealing high conservation among some species grouped in phylogenetic cluster 1, while others, such as L. laeta, showed high divergence compared to the rest of the Loxosceles species and belong to a different phylogenetic cluster (phylogenetic cluster 2 Loxosceles) (Figure 2b).

[0103] The amino acid sequences of the different consensus PLDs used as antigens were as follows:

[0104] L. laeta PLD1 (SEQ. ID. NO.1)

[0105] AETDVGERADNRRPIWNLAHMVNAVKQIPTFLDLGANALEADVTFKGSVPTYTYHGTPCD

[0106] FGRDCIRWEYFNVFLKTLREYTTPGNAKYRDGFILFVLDLKTGSLSNDQVRPAGENVAKE LLQNYWNNGNNGGRAYVVLSLPDIGHYEFVRGFKEVLKKEGHEDLLEKVGYDFSGPYLP SLPTLDATHEAYKKAGVDGHIWLSDGLTNFSPLGDMARLKEAIKSRDSANGFINKIYYWSV DKYSTTRTALDVGVDGIMTNYPNVLIDVLNEDGYKDNYRLATYDDNPWETYKK

[0107] L. laeta PLD consensus G2 (SEQ. ID. NO.2)

[0108] ADKRRPIWIMGHMVNKIEQIDEFLDLGANSIETDITFDELAYPEYTYHGVPCDCKRWCTKW EYVNDFLEALSRATTPGNSKYRKELTLWFDLKTGGLDASRAYKSGKAFAEKLAFHYWNG SNDAGRAYIVLSLPDLDHYEFIKGFREHFKNSTHKDLLEKVGYDFSGNDDLGLTRVALNKA GVNDREHVWQSDGITNCILRGLGRVKAAVANRDSSNGYINKVYVWTIQKYSSVRDALDAE VDGIMTNEPDVIANVLKEDAFKDRFRLATYEDNPWETFKR

[0109] PLD Consensus L. intermedia (SEQ. ID. NO.3)

[0110] MLXXIXLIXGCWSVLSEGAETDVEERADKRRPIWNMGHMVNAIYQIDEFVDLGANSIETDV SFDDNANPEYTYHGVPCDCGRSCLKWEYFNDFLKGLRSATTPGDAKYQEKLILVVFDLKT GSLYDNQAYDAGKKFAKNLLQHYWNNGNNGGRAYIILSIPDLNHYPLIKGFKEQLTKDGHP ELMDKVGYDFSGNDDIGDVGKAYKKAGVTDKEHVWQSDGITNCILRGLSRVREAVANRD SANGFINKVYYWTVDKRASTRDALDAGVDGIMTNYPDVIVDVLNEGAYKKKFRVATYEDN PWVTFKK

[0111] Consensus PLD L. gaucho / L. Reclusa (SEQ. ID. NO.4)

[0112] MLLYVTLILGCWAFSESAETDVAEAANKRPIWIMGHMVNAIAQIDEFVNLGANSIETDVSFD KNANPEYTYHGIPCDCGRSCLKWEYFNDFLKGLRKATTPGDSKYHEKLVLWFDLKTGSL YDNQAYDAGKKLAKNLLQHYWNNGNNGRAYIVLSIPNLNHYKLITGFKETLKSEGHPEL MDKVGYDFSGNDAIGDVGNAYKKAGVTGHVWQSDGITNCLLRGLSRVKAAVKNRDSSN GFINKVYYWTVDKRATTRDALDAGVDGIMTNYPDVITDVLNESAYKAKFRIATYDDNPWET FKN

[0113] In general, at least nine common regions for the presence of linear sequences of antigenic epitopes were identified for all consensus PLD sequences of Loxosceles species, both for the consensus PLD sequences of species belonging to phylogenetic cluster 1 of Loxosceles and for the consensus PLD sequences of species belonging to phylogenetic cluster 2 of Loxosceles (Figure 3). Variations were observed in the percentage of identity for the epitopes identified in species of phylogenetic cluster 1 versus the epitope sequences identified in species of phylogenetic cluster 2, to which L. laeta belongs, demonstrating significant antigenic variation at the intra- and inter-species levels (Table 1 and Table 2). The presence of antigenic epitopes specific to L. laeta was not found in sequences of L. intermedia, L. gaucho, and L. reclusa.Thus, in order to generate a diagnostic assay with broad detection capacity, it was necessary to produce monoclonal antibodies against the PLDs of L laeta from both intra-species phylogenetic groups, as well as to produce monoclonal antibodies against the other PLDs of different species of Loxosceles (L intermedia, L gaucho or L reclusa) in order to achieve detection of interspecies antigenic epitopes.

[0114] Table 1. Summary of linear antigenic epitopes present in amino acid sequences of phospholipase D from phylogenetic group 1 of Loxosceles spiders.

[0115] 1 Length of the antigenic epitope according to the number of amino acid residues that compose it.

[0116] Table 2. Summary of linear antigenic epitopes present in amino acid sequences of phospholipase D from phylogenetic group 2 of Loxosceles spiders.

[0117] 1 Length of the antigenic epitope according to the number of amino acid residues that compose it.

[0118] Example 3: Expression and purification of recombinant consensus phospholipase D (PLD) proteins from different Loxosceles species.

[0119] Each consensus phospholipase D antigen from the species L. laeta, L. intermedia, L. gaucho, and L. reclusa, to be used in the production of monoclonal antibodies, was expressed and purified as a recombinant protein. The amino acid sequences of the four PLD consensus antigens were cloned into a pET-30a(+) vector, transformed into E. coli BL21 (DE3) bacteria, and expressed as recombinant fusion proteins with a 6-His tag in the N-terminal region, and then purified using a Ni-NTA column + Sepharose Q. The final recombinant protein used for immunization does not contain the 6-His tag, as it was removed by enzymatic digestion with TEV (Tobacco Etch Virus) protease. The molecular masses of the different recombinant consensus PLD antigens were as follows: Consensus Antigen 2 PLD Group 1 L. laeta (L. laeta PLD1), 33,222 kDa. L laeta PLD consensus G2 (LIPLD consensus G2) Antigen, 32,358 kDa.Consensus antigen PLD LreclusalLgaucho (consensus PLD Lg / Lr), 32,142 kDa. Consensus antigen PLD L intermedia (consensus PLD Li), 32,909 kDa (Figure 4). A minimum of 4 mg of purified protein was required for the production of monoclonal antibodies.

[0120] Example 4: Production of monoclonal antibodies (mAbs) against consensus PLD antigens.

[0121] Using four different recombinant consensus PLD proteins, monoclonal antibodies were produced using a total of 4 mg of purified recombinant protein. These proteins were used as immunogens following a four-phase monoclonal antibody production scheme, prepared by GenScript (860 Centennial Ave. Piscataway, NJ, USA) through its mAb production service. Phase 1: Immunization of mice with the different immunogens. For this, five BALB / c mice per immunogen were inoculated intraperitoneally with each of the four immunogens at a dose of 10 to 25 pg of protein in phosphate-buffered saline (PBS; 500 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 10% Glycerol, pH 7.4) per mouse, following a three-boost procedure.Followed by evaluation of sera prior to performing the fourth and final immunization boost by indirect ELISA using 1 pg / ml of protein per well of a 96 well Maxisorp plate (Nunc, Thermo Fisher) in a volume of 100 pl of 0.05 M Sodium Carbonate / Sodium Bicarbonate buffer pH 9.6 per well and incubated for 1 hour at 37 °C and then overnight at 4 °C. The wells were then blocked with 200 pl of blocking buffer (PBS 1x pH 7.4 / Tween20 0.05% in the presence of 5% skimmed milk; PBS-T-milk) for 1 hour, washed once with 200 pl of wash solution (PBS 1x pH 7.4 / Tween20 0.05%; PBS-T), and subsequently incubated for 1 hour at 37°C with 100 pl of the different sera in duplicate and in serial dilution from 1:1000 to 1,512,000. Subsequently, each well was washed 3 times with PBS-T wash solution, and then incubated for 1 hour with 100 pl of the HRP-labeled Goat anti-mouse IgG (H+L) conjugate antibody (Invitrogen, Cat.626250) dilution 1:5000. The wells were then washed three times with 200 µl of PBS-T wash solution and incubated with 100 µl of Tetramethylbenzidine (TMB) substrate (SIGMAALDRICH, Cat. T0440) for 30 min in the dark. The reaction was then stopped with 100 µl of 3N sulfuric acid. Absorbance readings were performed at 450 nm using a Tecan Infinite M200 Pro plate reader spectrofluorometer. The presence of antibodies was considered based on the absorbance values ​​of the supernatants relative to the absorbance value for the negative control with PBS (cutoff point) and the absorbance value of wells in the presence of an unrelated recombinant protein fused with a 6-His tag. The absorbance values ​​at 450 nm were normalized by subtracting the PBS blank value. An unrelated monoclonal hybridoma supernatant was used as a negative control (Neg: Anti-N / A).Figure 5 shows that all immunized mice generated a response to the immunogens with detection titers equal to or greater than 1:512,000. The titer considered for the selection of mice that continue to the cell fusion phase was that they had absorbance values ​​greater than 1 for the 1:8000 serum dilution.

[0122] Phase 2: Cell fusion and screening of parental supernatants. In this stage, spleen cells from selected mice were fused with SP2 / 0 myeloma cells and cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1x hypoxanthine / thymidine (HT). The culture supernatant from the 20 parental hibhdomas was analyzed for each of the four immunogens initially used (total of 80 parental hibhdoma clones). The presence of antibodies capable of detecting their respective immunogen was determined by indirect ELISA in 96-well Maxisorp flat-bottom plates containing 1 pg / ml of recombinant protein in a volume of 100 µl of 0.05 M sodium carbonate / sodium bicarbonate buffer, pH 9.6, per well and incubated for 1 hour at 37 °C and then overnight at 4 °C.The wells were then blocked with 200 µl of blocking buffer (PBS 1x pH 7.4 / Tween20 0.05% in the presence of 5% skim milk; PBS-T-milk) for 1 hour, washed once with 200 µl of wash solution (PBS 1x pH 7.4 / Tween20 0.05%; PBS-T), and subsequently incubated for 1 hour at 37°C with 100 µl of undiluted culture supernatant from each parental hibdom. Detection was performed using 100 µl of HRP-labeled Goat anti-mouse IgG (H+L) conjugate antibody (Invitrogen, Cat. 626250) diluted 1:5000 and incubated for 1 hour. This was followed by washing and incubation with 100 pl of TMB substrate for 30 min in darkness, then stopping the reaction with 100 pl of 3N sulfuric acid. The absorbance reading was performed at 450 nm and the blank absorbance value was subtracted from each absorbance value obtained.

[0123] Five parental clones were selected for each immunogen used for subsequent subcloning, based on the criterion of exhibiting the greatest reactivity against their respective antigens. In addition, the criterion of cross-detection against the other PLD antigens was considered (Figure 6).

[0124] Phase 3: Subcloning, expansion, and cryopreservation of hibhdomas. From each of the five parental clones, expansion was performed, generating two subclones per clone, resulting in a total of ten subclones for each initial immunogen used. Figure 7 shows that both subclones produced from each of the five parental clones selected for each consensus PLD immunogen were able to strongly recognize their respective immunogens. Therefore, the parental hibhdoma lineage designation will be used to identify each monoclonal antibody (mAb) produced by the subclone lines. Detection was confirmed by Western blot, where each cell culture supernatant from the hibhdoma subclones detected their respective immunogens at the corresponding molecular weight (Figure 8).

[0125] Example 5: Cross-detection of monoclonal antibodies against Loxosceles laeta venom.

[0126] The culture supernatants of the hybridoma lines of the subclones previously selected for each of the four PLD consensus immunogens were evaluated for their ability to detect L. laeta venom using an indirect ELISA assay. For this purpose, a 96-well Maxisorp flat-bottom plate was adsorbed with 1 pg / ml of L. laeta venom in 100 µl of carbonate / bicarbonate buffer pH 9.6 and blocked with PBS-T-milk. Subsequently, the microplate wells were incubated in duplicate for 1 hour at 37 °C with 100 µl of each hybridoma culture supernatant containing monoclonal antibodies (mAbs) produced for the four different PLD consensus immunogens (10 hybridoma subclones per PLD immunogen). After 3 washes with 200 pl of PBS-T, 100 pl of conjugated antibody at a 1.5000 dilution was added to each well, which was incubated for 1 hour at 37 °C.After performing three additional washes with 200 µl of PBS-T, 100 µl of TMB substrate was added to each well, and the ELISA microplate was incubated for 30 minutes in the dark at room temperature. The reaction was then stopped by adding 100 µl of stop solution (6N HCl). Absorbance values ​​at 450 nm were read using a Tecan Infinite M200 Pro plate reader and normalized by subtracting the absorbance value of the PBS blank.Figure 9 shows that the 10 subclones produced against the recombinant immunogen LIPLD1 were able to detect L. laeta venom. The subclones with the lowest detection rates were those derived from the 5G10 hybridoma, while the remaining subclones (1H11, 10A11, 12D4, and 19C8 hybridomas) were able to strongly detect Llaeta venom (Figure 9a). However, the subclones produced against the recombinant immunogen Llaeta PLD consensus G2 (a group formed by Llaeta isoforms with less conserved amino acid sequences) were not able to detect Llaeta venom, with the slight exception of clone 9F5 (Figure 9b). This demonstrates the high intra-species variation of PLD isoforms present in L. laeta.Furthermore, of the mAb-producing subclones against the consensus PLD immunogen of L. gauchol-L. reclusa species, only subclones 6E5-1 and 6E5-2 (both from the same parental hybridoma 6E5) were able to cross-detect L. laeta venom (Figure 9c). Finally, of the mAb-producing subclones against the consensus PLD immunogen of L. laeta, only subclones 34H3-1 and 34H3-2 (from the parental clone 34H3) and 35H4-1 and 35H4-2 (from the parental clone 35H4) were able to cross-detect L. laeta venom (Figure 9d). Clones 6E5, 34H3, and 35H4 showed broad detection of PLDs from different species, with cross-detection against all four immunogens, including LIPLD1, LIPLD consensus G2, PLD consensus Lg / Lr, and PLD consensus Li.Furthermore, the cross-detection of L. laeta venom by each of the monoclonal antibodies produced against the four different Loxosceles consensus PLD antigens was analyzed by Western blot. For this purpose, a 5 pg sample of L. laeta venom was isolated.

[0127] The sample was stained by SDS-PAGE on a 12% gel and then transferred to a nitrocellulose membrane. The membrane was then blocked with PBS-T-milk blocking buffer (PBS1x pH 7.4; 0.1% Tween20 and 5% skim milk) for 1 hour with horizontal shaking at room temperature. The membranes were then washed once with PBS-T wash buffer (PBS pH 7.4 / 0.1% Tween20) and incubated with each culture supernatant containing undiluted mAb produced by the antigen (10 mAbs per antigen) for 1 hour with shaking at room temperature. Subsequently, the membranes were washed 6 times with PBS-T wash buffer and then incubated for 1 hour with shaking at room temperature with a Goat anti-mouse IgG / HRP conjugate antibody diluted 1:40,000 in PBS-T. After a further 6-time wash with PBS-T, the presence of specific bands was revealed using the ECL reagent (SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific, Cat.34095) and visualized on a ChemiDoc™ Imaging System (BIORAD). An unrelated hybridoma supernatant (Anti-N / A) and PBS-T were used as negative reaction controls. Figure 10 shows the detection of L. laeta venom, where the mAbs were able to detect the 25-35 kDa region, corresponding to the phospholipase D family. The 34H3 and 35H4 mAbs produced against the consensus PLD protein L1 showed the best detection, apart from the mAbs produced against the L. laeta consensus PLD antigen. The negative controls, unrelated mAb (anti-NA) and PBS, did not show venom detection (Figure 10). Therefore, the aforementioned mAbs are the main candidate mAbs for use in the kit assembly, in their common detection zone for all Loxosceles species.In addition, the 6E5 subclones produced against the consensus PLD antigen Lg / Lr will be included, which also detect the consensus PLD antigen L1 and L laeta venom as a second mAb to be used in the common detection zone.

[0128] Example 6: Characteristics of Loxosceles anti-PLD mAbs for use in diagnostic assay setup.

[0129] In addition to the cross-detection capacity of the different monoclonal antibodies (mAbs) with L. laeta venom, the detection titers and avidity index of the selected mAbs for use in the diagnostic assay were evaluated. For avidity determination, a pre-existing indirect ELISA protocol was used in the presence and absence of 6M urea. For this, each PLD immunogen at a concentration of 1 pg / ml was adsorbed onto 96-well ELISA plates using 100 ml of carbonate / bicarbonate buffer (pH 9.6) and blocked for 1 hour with PBS-T-milk. Then, 100 pl of each cell culture supernatant from the selected clone lines (1 H11 , 10 A11 , 12D4, 19C8, 6E5, 34H3 and 35H4) was added undiluted in duplicate to each well of a previously defined area of ​​the plate for treatment with Urea, and an additional 100 pl in duplicate to the wells of the plate defined without treatment with Urea.As a negative control, PBS and a supernatant of an unrelated monoclonal antibody (Anti-N / A) were used in both the urea-treated and non-urea-treated areas of the plate. The plates were then incubated for 1 hour at 37°C. After removing the contents, the wells were washed three times with 200 µl of PBS-T wash buffer. The wells were then incubated for 10 minutes at room temperature with 100 µl of 6M urea solution, while parallel wells (without urea) were incubated with 100 µl of PBS-T. After incubation, the contents were removed, and the plate wells were washed four times with 200 µl of PBS-T wash buffer. Subsequently, the plates were incubated with 100 pl of the HRP-labeled Goat anti-mouse IgG (H+L) conjugated antibody (Invitrogen, Cat. 626250) dilution 1:5000.The wells were then washed three times with 200 µl of PBS-T wash solution and incubated with 100 µl of TMB substrate for 30 min in the dark. The reaction was then stopped with 100 µl of 3N sulfuric acid. Absorbance readings were taken at 450 nm using a Tecan Infinite M200 Pro plate reader / spectrophotometer. Absorbance values ​​at 450 nm were normalized by subtracting the PBS blank value. The avidity of each IgG monoclonal antibody was expressed as the Avidity Index (AI), which was calculated from the absorbance obtained for each sample in the presence and absence of 6M urea using the following formula: AI = (average OD450 of the urea-treated sample / average OD450 of the non-urea-treated sample).

[0130] For the titration of the culture supernatants of the selected clones (1 H11 , 10A11 , 12D4, 19C8, 6E5, 34H3 and 35H4), an indirect ELISA protocol was used to detect each of the four PLD immunogens adsorbed to a 96-well plate at a concentration of 1 pg / ml, and then blocked with PBS-T-milk blocking buffer. The supernatants were then added in duplicate and in serial dilutions from 1:10, 1:30, 1:90, 1:270, 1:810, and 1:2430. The reaction was detected using HRP-labeled Goat anti-mouse IgG (H+L) conjugate antibody at a 1:5000 dilution, followed by washing and incubation with 100 µL of TMB substrate for 30 min in the dark, then stopping the reaction with 100 µL of 3N sulfuric acid. Absorbance readings were taken at 450 nm. The absorbance values ​​at 450 nm were normalized by subtracting the PBS blank value.The detection titer was assigned as the highest dilution with absorbance / negative values ​​greater than 0.5. For the identification of the isotype of each monoclonal antibody produced against the Loxosceles PLD immunogens, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit Cat No. 114993027001 (Roche Diagnostics GmbH, Sandhofer Strasse 116, Mannheim, Germany) was used according to the manufacturer's instructions. This assay allows for the determination of the antibody isotype (IgG1, IgG2a, IgG2b, IgG3, IgA, or IgM), as well as the identification of the antibody light chains (A or K). For this purpose, each test tube containing the detection strip was placed vertically in a test tube rack and labeled according to the monoclonal antibody to be analyzed. Then the culture supernatant sample from each AcM-producing hibhdoma cell line was diluted 1:10 in a volume of 150 pl of pH 7.4 phosphate buffered saline (PBS) along with 1% Bovine Serum Albumin (BSA).Each mixture of mAb diluted in PBS / BSA was placed inside the test tube, and then an isotype detection strip was inserted, allowing the end of the strip to become saturated with the solution. The assay was incubated at room temperature for 5 minutes, and then each strip was read for the appearance of blue colored bands corresponding to the antibody isotype, and the results were recorded.

[0131] Figure 11 shows the titer graphs for the seven selected clones (1H11, 10A11, 12D4, 19C8, 6E5, 34H3, and 35H4), all with titers above 1:2430. Furthermore, all the monoclonal antibodies (mAbs) produced by the selected clones correspond to IgG1 isotypes, and the avidity indices for each of the selected clones are close to 1 (IA range: 0.86–1) (Table 3). The expected values ​​for each parameter were as follows: ELISA detection titer >1:1000, avidity index >0.5, and IgG1 isotype. Thus, all the mAbs from the Loxosceles anti-PLD hibdom clones meet the minimum requirements for use in the diagnostic assay. Example 7: Identification of antigenic peptides specifically detected by anti-PLDs mAbs from Loxosceles and complementarity determining regions (CDR).

[0132] The monoclonal antibodies (mAbs) present in the culture supernatants of hibdomes 6E5, 1H11, 10A11, 19C8, 34H3, and 35H4 were evaluated for the specific detection of different linear antigenic peptides present in the consensus sequences of phospholipase D, belonging to phylogenetic groups 1 and 2 of spiders of the genus Loxosceles. A total of 18 peptides, ranging from 13 to 19 amino acids (Table 4), were synthesized by GenSchpt and then adsorbed onto wells of a Maxisorp Nunc ELISA plate in 100 pL of carbonate / bicarbonate buffer, pH 9.6, at a concentration of 3 pg / ml, and incubated overnight at 4°C. After blocking each well with a solution of PBS 1x pH7.4 + tween20 0.05% (PBS-T) and 3% BSA for one hour at room temperature, 100 pl of each culture supernatant under study were deposited and then incubated at room temperature for 1 hour.Each well of the microplate was washed three times with PBS-T solution and then incubated with 100 µl of HRP-labeled Goat anti-mouse IgG (H+L) antibody conjugate solution (Invitrogen, Cat. 626250) at a 1:5000 dilution. The wells were then washed three times with 200 µl of PBS-T wash solution and incubated with 100 µl of TMB substrate for 30 min at room temperature in the dark. The reaction was then stopped with 100 µl of 3N sulfuric acid. Absorbance readings were taken at 450 nm using a Tecan Infinite M200 Pro plate reader / spectrophotometer. The absorbance values ​​at 450 nm were normalized by subtracting the PBS blank value. As a positive control, adsorbed wells with 1 pg / ml of each antigen were used (LIPLD1, PLD consensus Lg / Lr, and PLD consensus L1).The pores corresponding to each peptide that showed the highest absorbance according to each mAb were considered as the specific antigenic epitopes, as shown in Table 5.

[0133] Example 8: Conjugation of anti-PLD AcM of Loxosceles with colloidal gold.

[0134] The different anti-PLD monoclonal antibodies of Loxosceles were purified from culture supernatants of their respective hibdomes using the Pierce™ Protein A / G Magnetic Agarose Beads kit (Thermo Scientific, Cat. 78609), according to the manufacturer's instructions. A volume of 200 µl of magnetic agarose beads was placed in a 1.5 ml tube and then mounted on a DynaMag™ 2 magnetic support (Thermo Scientific, Cat. 12321 D), allowing for the removal and disposal of the supernatant. The magnetic resin was washed with 500 µl of bonding / wash buffer (1x PBS, pH 7.4), mixed by inversion 3 to 5 times, and then separated from the supernatant by placing the tube on the magnetic support. The supernatant was then removed and discarded. Next, 500 ml of culture supernatant from each hibhdoma was added onto the magnetic resin, and then mixed by inversion 5 times ensuring good homogenization of the resin.Once the tubes were deposited, they were incubated at room temperature for 1 hour with end-to-end mixing using a vertical tube rotator. After the incubation time, each tube was placed on a magnetic stirrer to separate the resin from the supernatant (flow-through), which was collected in a new 1.5 ml tube. The resin was then washed with 500 ml of bonding / wash buffer, thoroughly mixed by vortexing, and the supernatant was removed after the tubes were placed on the magnetic stirrer. The wash was repeated a second time with another 500 ml of PBS, and the supernatant was removed. Finally, a wash was performed with 500 ml of deionized water, mixed, and the supernatant was removed after placing the tubes on the magnetic stirrer. Finally, 100 ml of elution buffer (0.1 M glycine, pH 2.8) was added, mixed, and incubated for 10 minutes in a rotating stirrer.After incubation, the tubes were placed on a magnetic support, and the supernatant containing the purified antibodies was transferred to a new 1.5 mL tube, to which 50 µL of neutralizing solution (Tris-HCl buffer, pH 7.5) was added. Subsequently, the elution buffer was replaced with PBS pH 7.4 using Amicon® Ultra-0.5 mL centrifugation concentration tubes with a 3 kDa cutoff (Cat. UFC500324; Merck Millipore Ltd, Tullagreen, Carrigtwohill, IRL). The eluted volume was added to these tubes along with PBS buffer and centrifuged at 14,000 x g for 15 min at room temperature. The remaining volume was then recovered from the column. The purified monoclonal antibodies were quantified for protein concentration using the Pierce™ 660 nm Protein Assay kit (Thermo Scientific, Cat. 22662), with a bovine serum albumin (BSA) standard curve ranging from 125 to 2000 pg / ml. The different mAbs were stored at 4 °C until use.

[0135] The conjugation of each monoclonal antibody was performed using the Gold Conjugation Kit (40 nm, 20 OD) (Cat. Ab154873; Abeam Limited, Cambridge, UK), according to the manufacturer's instructions. This kit allows for the stable and rapid covalent binding between colloidal gold nanoparticles and purified antibodies. For this purpose, the selected purified monoclonal antibodies (mAbs: 6E5, 1H11, 10A11, 12D4, 19C8, 34H3, and 35H4) were diluted with antibody diluent at a concentration of 0.5 mg / ml to a volume of 12 lp. To each mAb dilution, a volume of 42 lp of Gold 40 nm Reaction Buffer was added and mixed by inversion. Next, a 45 pl volume of the mixture was deposited into a Gold 40 nm vial (which contains 1 pg of colloidal gold nanoparticles), gently mixed by pipetting and incubated for 15 minutes at room temperature.After incubation, 5 pl of Gold 40 nm Quencher was added to complete the conjugation reaction. The conjugate mixture (mAb-colloidal gold) 20 OD was held at room temperature for 5 minutes, then washed with 10 volumes (500 pl) of Gold 40 nm Quencher diluted 1:10, and subsequently centrifuged at 9,000 x g for 10 minutes at room temperature. The supernatant was removed, and the pellet was resuspended in 50 pl of Gold 40 nm Quencher diluted 1:10 + 2% BSA. The conjugated antibody was stored at 4 °C until use. Proper conjugation was assessed by measuring the absorbance at 530 nm for a 1:20 diluted volume of conjugated antibody equivalent to 1 OD at 530 nm.

[0136] The correct conjugation of the monoclonal antibodies (mAbs) with colloidal gold was visually assessed in a dot-blot assay. Three pg of BSA in 100 µl of PBS and 100 µl of mouse Goat Anti-IgG (H+R) antibody diluted 1:100 were applied to a nitrocellulose membrane using the Bio-Dot® / Bio-Dot SF microfiltration system (BioRad, catalog 1703938). The presence of the antigens was confirmed by adding Ponceau Red solution, which was subsequently removed by washing the membrane twice with double-distilled water. The membrane was then blocked for 1 hour at room temperature with a blocking solution containing PBS pH 7.4 / 0.1% Tween / 2% skim milk. The membrane was then dried at room temperature and 5 µl of colloidal gold-conjugated antibody diluted 1:5 in PBS pH 7.4 was deposited and incubated for 15–20 minutes. 5 µl of PBS was deposited as a negative control.Once the incubation period was complete, the membrane was washed with 0.1% PBS-Tween washing solution and visualized for the appearance of a red dot or circular mark on the antigen corresponding to the anti-IgG antibody, while no coloration should be observed on the antigen corresponding to BSA. Additionally, a dot-blot assay was performed using the different antigens and Llaeta venom, which were adsorbed onto a nitrocellulose membrane at a concentration of 3 pg using the Bio-Dot microfiltration system. These were then evaluated as previously described with each of the different monoclonal antibodies conjugated with colloidal gold.

[0137] The successful conjugation of the mAbs with the colloidal gold nanoparticles is shown in Figure 12. All mAbs (1H11 and 10A11, 19C8, 12D4, 6E5, 34H3, and 35H4) were successfully conjugated, as indicated by detection with the mouse anti-IgG antibody (the same one used in the assay control zone). No red coloration of the colloid product was observed when the anti-PLD / conjugated mAbs were incubated against BSA. Furthermore, the conjugated mAbs maintained their cross-detection capacity with other PLDs from different Loxosceles species (Llaeta, Lintermedia, Lgaucho, and Lreclusa), especially mAbs 12D4, 6E5, 34H3, and 35H4 (Figure 13). Example 9: Proof of concept of the prototype lateral flow immunochromatography (IFL) assay for the detection of L laeta venom.

[0138] The reaction strip for assembling a lateral flow immunochromatography (IFL) assay to evaluate the functionality of the selected monoclonal antibodies was prepared using an IFL strip approximately 7 cm long x 0.5 cm wide, consisting of: 1) a detection pad, 2) a sample pad, 3) a conjugate pad, and 4) an absorbent pad on a plastic film. A nitrocellulose membrane was used to prepare the detection pad. A pair of monoclonal antibodies (1H11 / 35H4) were adsorbed onto the membrane at a concentration of 1 mg / ml in pH 7.4 PBS in a slot format to create the detection zone (T) using the slot well tool of the Bio-Dot / Bio-Dot SF microfiltration system (Bio-Rad, Cat. 1703938; Bio-Rad Laboratories, Hercules, CA, USA). Along with this, 0.5 mg / ml of Goat anti-mouse IgG antibody (H+L) was added in a control zone (C).Each detection and control slot was spaced 1 cm apart. The membrane dimensions were 2.5 cm x 0.5 cm. The membrane with the antibodies in each detection zone was subsequently blocked with 1% BSA in PBS for 1 hour with shaking at room temperature, washed once with 0.1% PBS-Tween, and then dried by incubation at 37 °C for 2 hours. The sample pad was prepared using glass fiber (Merck / Millipore; catalog GFCP203000; Millipore Corporation, Burlington, MA, USA), which was cut into 1.7 cm x 0.5 cm strips and then washed for 5 minutes with shaking in a pH 7.3 PBS wash solution containing 0.1 M NaCl, 0.2% Tween 20, and 0.1% sodium azide. Subsequently, the sample pads were dried at 37 °C for 2 hours, until their use in the assembly of the IFL strip.The conjugate pad was prepared using fiberglass (Merck / Millipore; catalog GFCP203000), which was cut into 0.7 cm x 0.5 cm strips. These strips were then incubated with a set of two monoclonal antibodies previously conjugated with colloidal gold (mAb: 6E5 and 34H3) at a 1:5 dilution in 50 ml of conjugation buffer (20 nM sodium borate, pH 8.0; 2% sucrose; 1% BSA; 0.2% Tween20; and 0.1% sodium azide). Once the mAb was deposited onto the conjugate pad, it was dried at 37 °C for 2 hours before being used to assemble the IFL strip. The absorption pad was prepared using cellulose filter paper (Merck / Millipore; catalog FP10102500) which was cut into strips of 2.2 cm x 0.5 cm and stored until its use in the assembly of the IFL strip.

[0139] The strip was assembled by adhering the nitrocellulose membrane to a 7 cm x 0.5 cm transparent plastic sheet using double-sided tape (19 mm thick). The conjugate pad was then placed 0.3 mm thick over the nitrocellulose membrane, followed by the sample pad, which was also adhered and placed 0.3 mm thick over the conjugate pad. At the opposite end of the strip, the absorbent pad was applied, also 0.3 mm thick over the nitrocellulose membrane. A volume of 100 µl of PBS was then placed on the sample pad for the negative control, while 100 µl of PBS containing 25 pg of L. laeta venom was placed on the sample pad of the assay strip. Both strips were incubated for 15 minutes at room temperature in a horizontal position, allowing sample flow.Once the incubation period was complete, the strips were read for the presence of red colored bands in both the detection zone (T) and the control zone (C). A positive result was considered to be the presence of both colored bands, while a negative result was considered to be the presence of only one band in the control zone. Figure 14 shows the results of the proof-of-concept test, where the presence of a red band was observed in both the detection and control zones in the assay incubated with L. laeta venom, while the assay incubated with PBS only showed the presence of the control band.

[0140] Example 10: IFL “Loxo-test” assay for the detection of L laeta venom.

[0141] Using the different mAbs obtained against the consensus PLD immunogens of the species L laeta, L intermedia, L gaucho and L reclusa, it is possible to assemble different options of the IFL detection assay called “Loxo-test”.These options would be the following: Detection devices specific to the prevalent species according to geographical location: 1) IFL specific for the detection of L laeta venom (includes conjugated mAb pair and capture anti-rLIPLD1; 1 H11, 10A11, 12D4, 19C8 or any other mentioned in the present invention produced against the consensus immunogens LIPLD1 and LIPLDcG2); 2) IFL specific for the detection of L intermedia venom (includes conjugated mAb pair and capture anti-PLD consensus LÍ-34H3, and 35H4 or any other mentioned in the present invention produced against the consensus immunogen PLD Li); 3) Specific IFL for the detection of L gaucho / Lreclusa venom (includes conjugated mAb pair and anti-PLD consensus Lg / Lr-6E5 capture or any other mentioned in the present invention produced against the PLD consensus immunogen Lg / Lr).In addition, the following dual detection assay options are considered: 4) IFL detection of L laeta + L intermedia venoms, 5) IFL detection of L laeta + L gaucho / L reclusa venoms, 6) IFL detection of L intermedia + L gaucho / L reclusa venoms, or triple detection assays: 7) IFL detection of L laeta + L intermedia + L gaucho / L reclusa venoms, 8) IFL general detection of Loxosceles spider venom (includes L laeta, L intermedia, L gaucho and L reclusa species (includes conjugated AcM pair set 6E5 / 34H3 and capture AcM pair set 1 H11 / 35H4); 9) IFL for the detection of Loxosceles venom + specific detection of L laeta venom (includes conjugated AcM pair and consensus anti-PLD capture L¡). In the example, an IFL assay was set up according to format No. 1 for the specific detection of the venom of the L laeta spider.In this case, the reaction strip was covered by a plastic device containing a detection window that included the test (Test) and control (C) zones, as well as a sample deposit area. Subsequently, 100 ml of 1x PBS buffer, pH 7.4, containing different concentrations of L. laeta spider venom or recombinant rLIPLDI protein in dilutions ranging from 50 pg / ml (5 pg) to 0.37 pg / ml (37 ng), and from 5 pg / ml (0.5 pg) to 0.1 pg / ml (10 ng), was added. The reaction was incubated for 15 minutes at room temperature. A positive reaction was indicated by the appearance of two colored bands (green: control zone and red: test zones), while a negative reaction was defined as the appearance of a single green band in the control zone. Figure 15 shows the results of L laeta venom detection, through the IFL device in its final format for the specific detection of this spider's venom.In this way, it was possible to detect an amount of 150 ng of L laeta venom, and 10 ng for the recombinant protein rLIPLDI.

Claims

1. CLAIMS 1. A process for obtaining monoclonal antibodies and / or fragments thereof, from partial or complete consensus amino acid sequences of Loxosceles phospholipase D, CHARACTERIZED in that said sequences correspond to the amino acid sequences SEQ. ID. NOs: 1-4, the process comprising the steps of: • clone these amino acid sequences into a genetic vector; • introduce the vector into an expression system capable of producing Loxosceles' phospholipases D, • to express and purify the recombinant phospholipases D obtained from said expression system; and • to use these recombinant phospholipases D as immunogens in an animal immunization protocol for the generation of monoclonal antibodies and / or their fragments.

2. A monoclonal antibody or fragment thereof, obtained in accordance with the method of claim 1, CHARACTERIZED in that said monoclonal antibody or fragment thereof has affinity for phospholipase D sequences of the venom of spiders of the genus Loxosceles, said monoclonal antibody or fragment thereof allows the detection of venom of spiders of the genus Loxosceles; and said monoclonal antibody or fragment thereof is selected from the antibodies produced by the hibdomes clones 6E5, 1H11, 10A11, 12D4, 19C8, 34H3 and 35H4.

3. The monoclonal antibody or fragment thereof, of claim 2, CHARACTERIZED in that said monoclonal antibody or fragment thereof exhibits cross-reactivity against phospholipase D antigens of two or more Loxosceles species.

4. The monoclonal antibody or fragment thereof, of claims 2 and 3, CHARACTERIZED in that said monoclonal antibody belongs to the IgG1 antibody subclass.k.

5. A combination of antibodies or fragments thereof, wherein said antibodies or fragments thereof are produced according to the process of claim 1, CHARACTERIZED in that it comprises at least two distinct monoclonal antibodies or fragments thereof; wherein said monoclonal antibodies or fragments thereof have affinity for phospholipase D sequences of the venom of spiders of the genus Loxosceles, said monoclonal antibodies or fragments thereof allow the detection of venom of spiders of the genus Loxosceles, and said monoclonal antibodies or fragments thereof are selected from the antibodies produced by the hibdomes clones 6E5, 1H11, 10A11, 12D4, 19C8, 34H3 and 35H4.

6. The combination of monoclonal antibodies or fragments thereof, of claim 5, CHARACTERIZED in that said monoclonal antibodies or fragments thereof allow the detection of venom from Loxosceles intermedia, Loxosceles gaucho, Loxosceles reclusa and Loxosceles laeta spiders.

7. The combination of monoclonal antibodies or fragments thereof, of claims 5 and 6, CHARACTERIZED in that said monoclonal antibodies or fragments thereof are capable of cross-detecting different species of Loxosceles.

8. The combination of monoclonal antibodies or fragments thereof, of claims 5 to 7, CHARACTERIZED in that said monoclonal antibodies belong to the IgG1 antibody subclass.k.

9. An in vitro method for detecting the presence of venom from spiders of the genus Loxosceles in a sample, CHARACTERIZED in that it comprises the steps of: • contact the sample with the combination of monoclonal antibodies or fragments thereof produced by clones 6E5, 1H11, 10A11, 12D4, 19C8, 34H3 and 35H4; and • detect the binding of antibodies to the antigen using lateral flow immunochromatography.

10. The method according to claim 9, CHARACTERIZED in that the sample corresponds to a skin swab from a skin lesion, or to a selected biological sample between fluids, exudates or tissues obtained from the affected site, or any other type of sample that is considered appropriate.

11. An in vitro detection kit for the presence of venom from spiders of the genus Loxosceles, CHARACTERIZED in that it comprises: • a plastic device that includes sample deposit zones, a detection zone, and a test confirmation or control zone, wherein, optionally, the detection zone includes a first zone for detecting the venom of spiders of the genus Loxosceles and a second zone for the specific detection of a species; and • a membrane in which the antibodies produced by clones 6E5 are incorporated, and, optionally, at least one of the antibodies produced by clones 1H11, 10A11, 12D4, 19C8, 34H3 and 35H4.

12. The kit of claim 11, CHARACTERIZED in that it comprises configurations with a general detection zone and configurations with two or more specific detection zones, selected from single, double, triple or general formats, according to the number of zones incorporated in the membrane.

13. The kit of claims 11 and 12, CHARACTERIZED in that the general detection zone incorporates as a capture antibody an antibody selected from those produced by the hibdomes 34H3, 35H4, 10A11, 12D4, or 19C8, in combination with a conjugated antibody produced by the hibdome 6E5.

14. The kit of claims 11 to 13, CHARACTERIZED in that it comprises a specific detection zone for Loxosceles gaucho or Loxosceles reclusa, wherein said zone incorporates as a capture antibody an antibody selected from those produced by the 6E5 and 35H4 hibdomes.

15. The kit of claims 11 and 12, CHARACTERIZED in that it comprises a specific detection zone for Loxosceles laeta, wherein said zone incorporates as a capture antibody an antibody selected from those produced by the hibdomes 1 H11 , 10A11 , 12D4 or 19C8.

16. The kit of claims 11 and 12, CHARACTERIZED in that it comprises a specific detection zone for Loxosceles intermedia, wherein said zone incorporates as a capture antibody an antibody selected from those produced by hybridomas 34H3, 10A11, 12D4 or 19C8.

17. The kit of claims 11 and 12, CHARACTERIZED in that it comprises a dual format configuration, comprising two detection zones selected from the detection zones defined in claims 13 to 16.

18. The kit of claim 17, CHARACTERIZED in that it comprises a specific detection zone for Loxosceles laeta and a specific detection zone for Loxosceles intermedia.

19. The kit of claim 17, CHARACTERIZED in that it comprises a specific detection zone for Loxosceles laeta and a specific detection zone for Loxosceles gaucho or Loxosceles red usa.

20. The kit of claims 11 to 16, CHARACTERIZED in that it comprises a triple format configuration, comprising three detection zones selected from the detection zones defined in claims 13 to 16.

21. Use of a monoclonal antibody or fragment thereof, according to claims 2 to 4, CHARACTERIZED in that it serves for the in vitro detection of venom of spiders of the genus Loxosceles.

22. Use of the combination of monoclonal antibodies or fragments thereof, according to claims 5 to 8, CHARACTERIZED in that it serves for the detection of venom of spiders of the genus Loxosceles.

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