BR102017006706A2 - flow cytometry multiplex serological test - Google Patents

Abstract

The official serological methods available for the diagnosis of canine visceral leishmaniasis (LVC) have performance limitations. Among the deficiencies, we highlight the low sensitivity in detecting asymptomatic cases of the disease, and the cross reactivity with antibodies from other canine parasitic infections, or with antibodies produced after vaccination against lvc. Flow cytometry methodologies have been proven effective in increasing the detection sensitivity of asymptomatic dogs, as well as minimizing the possibility of cross reactions. To achieve a gain in diagnostic performance, the present patent proposal seeks to establish a methodological breakthrough in the diagnosis of LVC by flow cytometry by creating antigenic systems characterized by using functional polystyrene microspheres conjugated to recombinant proteins rlci1a and rlci2b, produced and developed. by researchers from our group. Thus, this patent application seeks to present a flow cytometric diagnostic tool that allows a higher accuracy assay in the diagnosis of lvc, performed through a single serological assay by simultaneous analysis with two recombinant antigens.

Description

(54) Title: MULTIPLEX SERUM TEST BY FLOW CYTOMETRY (51) Int. Cl .: G01N 33/569; G01N 33/533; C07K 14/44 (73) Holder (s): FEDERAL UNIVERSITY OF OURO PRETO (72) Inventor (s): ALEXANDRE BARBOSA REIS; HENRIQUE GAMA KER (85) National Phase Start Date:

03/31/2017 (57) Abstract: The official serological methods available for the diagnosis of canine visceral leishmaniasis (CVL) have performance limitations. Among the deficiencies, we highlight the low sensitivity in detecting asymptomatic cases of the disease, and cross-reactivity with antibodies from other canine parasitic infections, or even with antibodies produced after vaccination against LVC. Flow cytometry methodologies have proven to be effective in increasing the detection sensitivity of asymptomatic dogs, as well as minimizing the possibility of cross reactions. To achieve a gain in diagnostic performance, the present patent proposal seeks to establish a methodological innovation in the diagnosis of LVC by flow cytometry through the creation of antigenic systems characterized by using functional polystyrene microspheres conjugated to the recombinant proteins rLcilA and rLci2B, produced and developed by researchers in our group. Thus, this patent application seeks to present a diagnostic tool by flow cytometry that allows a test of superior accuracy in the diagnosis (...)

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MULTIPLEX SERUM TEST BY FLOW CYTOMETRY

1. Field of the invention

01) Flow cytometry consists of laser technology that analyzes particles suspended in liquid medium and hydrodynamically focused continuous flow. These particles can be cells, cellular components or non-cellular material such as polymeric microspheres. The use of polymeric microspheres is a worldwide trend in the development of diagnostic approaches by flow cytometry, considering the possibility of creating multiple assays, named in the literature as multiplex assays. Some specialized companies already provide microsphere systems with an open and configurable menu of reagents designed to create multiplex serological assays by flow cytometry. The Flex Set Cytometric Bead Array (CBA) technology (Becton & Dickinson - BD ™) provides the creation of tests to evaluate antibodies present in a serum sample against more than one recombinant antigen by serological reaction. Diagnostic tests based on this technology are synthetically characterized by using functional fluorescent microspheres of polystyrene coupled with different recombinant antigens, and has been gaining prominence in the scope of diagnosis of infectious and parasitic diseases (Ondingo et al., 2012; Fujii et al., 2014 ). In infections by trypanosomatids of the genus Leishmania, serological assays by flow cytometry have been explored in recent years both in American cutaneous leishmaniasis (Rocha et al., 2002, Rocha et al., 2006, Pissinate et al., 2008), and for human and canine visceral leishmaniasis (Andrade et al., 2007; Andrade et al., 2009, Ker et al., 2013a, Ker et al., 2013b). Despite guaranteeing better performance than conventional methods, flow cytometry methodologies developed to date have little potential for developing tests capable of analyzing multiple antigens simultaneously. In the context of multiplex serological evaluations, and in the quest to improve the diagnostic performance of serological methods by flow cytometry, a technological innovation was applied in the investigation of canine IgG antibodies through a serological reaction using an innovative antigenic system characterized by the use of microspheres fluorescent functionalities of polystyrene CBA coupled to recombinant Leishmania infantum proteins with high diagnostic potential.

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1.1.Foundations of the invention and state of the art

02) Research has been carried out to obtain serological diagnostic tests using different approaches. In part of the cases, the innovation in serological tests, especially in canine visceral leishmaniasis, lies in the purification and obtaining of new antigens from different processes that employ multidisciplinary biotechnological tools. We can verify this fact in searches carried out on different bases (INPI, ESPACENET, USPTO, LATIPAT, and THOMSON INNOVATION), in which it is possible to track several patented methods, both nationally and internationally: BR8304073A; BRPI0603872A (2); ES2373262A1; US7780969 (B2), among others. Even with the significant advance in the identification and production of new antigens, little is still done in the sense of developing new methodologies to improve serological assays using these new antigens. The ELISA method is conventionally adopted for the evaluation of antigens reported in the literature. However, the ELISA assay methodology has a low potential for serological evaluation of a biological sample by multiple antigens simultaneously. This is a limitation that must be overcome, since a simultaneous evaluation of several antigenic matrices has the advantage of reducing costs and working time, providing a result supplemented by more than one antigenic matrix. Thus, in view of a large panel of proteins with diagnostic potential, the lack of a serological assay methodology that allows for a multiparametic evaluation becomes even more evident. Seeking to overcome these challenges, the present patent application aims to propose a new methodology for serological diagnosis by flow cytometry using recombinant Leishmania infantum proteins coupled to CBA microspheres. Two recombinant Leishmania infantum proteins called rLcilA and rLci2B were used as antigen, which were previously expressed, isolated and purified by Souza et al., (2012) and the patent application registered by the National Institute of Industrial Property (INPI) under the terms of PI0900961-2, with the title: “The use of Leishmania antigens in methods for diagnosis, treatment and vaccine for leishmaniasis”. Recombinant antigens

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3/13 rLcilA and rLci2B proved to be promising candidates for use in serodiagnostic tests (Fraga et al., 2014). The flow cytometric diagnostic test proposed here is capable of evaluating both antigens simultaneously in a single serological assay. The rLcilA and rLci2B antigens are used to build two distinct recombinant microsphere-protein systems that, when combined, form the antigenic matrix of a diagnostic test by flow cytometry that is based on an immunosorological reaction applied in the investigation of canine IgG antibodies . Its advantages are the high levels of sensitivity and specificity, directly enabling an innovative methodology for the diagnosis of infectious and parasitic diseases.

2.0. Summary of the Invention

03) The official serological methods available for the diagnosis of canine visceral leishmaniasis (CVL) have limited performance. These tests have deficiencies to be overcome such as low sensitivity in the detection of asymptomatic cases of the disease, and cross-reactivity with antibodies from other canine parasitic infections, or with antibodies produced after vaccination against LVC. The flow cytometry methodologies studied to date have proven to be effective in increasing the detection sensitivity of asymptomatic dogs as well as minimizing the possibility of cross reactions. To achieve a gain in diagnostic performance, the present work seeks to establish a technological innovation in the diagnosis of CVL through the coupling of recombinant antigens (rLcilA and rLci2B) to functional fluorescent polystyrene microspheres of the configurable kit BD ™ CBA Flex Set. In this context, the The present patent presents a new diagnostic methodology for flow cytometry that allows a simultaneous serological analysis of two recombinant antigens of remarkable applicability in the diagnosis.

3.0. State of art

3.1. The biotechnological system Cytometric Bead Array (CBA).

04) The BD ™ CBA Flex Set system provides an open and configurable menu composed of functional fluorescent polystyrene microspheres and reagents

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4/13 designed to create multiparametric tests called multiplex. Each microsphere in this system has a unique fluorescence intensity, determined by a discrete level of fluorescent labeling, so that it can be distinguished by its fluorescence intensity measured in the fluorescence channel 3 (FL3) of the flow cytometer. In this sense, microspheres with different fluorescence intensities can be conjugated with different recombinant antigens, and combined to create new serodiagnostic methodologies. The microspheres used in this patent application correspond to the Functional Bead CBA A4, and Functional Bead CBA E4 models. Of the other items commercially available in the BD ™ CBA Flex Set system and which are used in the patent application, are the BD ™ CBA Functional Bead Conjugation Buffer Set caps, composed of two items, the coupling buffer and the storage buffer.

3.2. Coupling of recombinant proteins to CBA microspheres.

05) The coupling of proteins to the CBA microspheres occurs from a stable covalent bond between the amine groups of the proteins and the sulfhydryl groups of the microspheres. The chemical mechanism of binding of recombinant proteins to the microspheres can be divided into 3 stages: (i) preparation of the microspheres; (ii) preparation of the protein; (iii) conjugation of microspheres to proteins. The first stage of the reaction (i) is characterized by the preparation of the microsphere with the reagent Ditioteitrol (DTT), responsible for the reduction of the disulfide group present on the surface of the microspheres and formation of a sulfhydryl group. The second step (ii), is characterized by the reaction of the primary amine present in the terminal portion of the protein with a hydroxysuccinamide of the Succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SulfoSMCC) molecule, forming a maleimide group characterized by be highly reactive with sulfhydryl groups. The third step (iii) consists of joining the products prepared in the two previous steps, and comprises the formation of the sulfhydryl-maleimide bond. This bond is effectively stabilized in the presence of N-Ethylmaleimide (NEM).

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3.3. The serological reaction for canine IgG antibodies

06) A serological reaction based on the search for canine IgG antibodies was standardized using an antigenic matrix composed of two recombinant microsphere-protein systems, namely, the AA-rLcilA and E4 — rLci2B system. First, different concentrations of canine serum samples (negative control [CN] and positive control [CP]), are incubated in the presence of this antigenic matrix. After an incubation period of 90 minutes at room temperature and protected from light, the matrix is washed with PBS buffer supplemented with casein and lecithin in the proportion of 2.0% w / v. The consecutive step deals with an incubation of the antigenic matrix with an anti-canine IgG antibody fluorinated with fluorescein isothiocyanate (FITC). This incubation takes place under the same conditions as described above (90 minutes at room temperature and protected from light). After another washing procedure, the antigenic matrix is read on a flow cytometer. With this processing, it is expected that the antigenic matrix incubated in the presence of a CN sample, does not present an increase in the number of events with a significant increase in green fluorescence - related to FITC marking. For incubation with PC samples, the antigenic matrix should exhibit an increase in the number of events with a significant increase in green fluorescence detection resulting from the FITC marking.

4.0. Detailed description of the invention

07) The microspheres of the BD ™ CBA Flex Set kit are polystyrene spheres of uniform size of 7.5pm with color codes that can be classified using a flow cytometer. These microspheres can be treated with reagents and buffers designed to provide covalent binding of proteins soluble in aqueous medium to the surface of the microspheres. Within this context, the development of the serological test for the diagnosis of Canine Visceral Leishmaniasis is characterized by the use of CBA fungi fluorescent polystyrene microspheres conjugated to recombinant proteins with diagnostic potential. In this way, an innovative flow cytometry methodology based on technology-based principle is available

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6/13 multiplex diagnostics. In the assay proposed here, two different models of microspheres were conjugated to two recombinant proteins with diagnostic potential for canine visceral leishmaniasis, forming two different microsphere-recombinant protein systems. The combination of these two recombinant microsphere-protein systems forms the antigenic matrix for application in a serological reaction to investigate canine IgG antibodies by flow cytometry on a single platform.

4.1. Preparation of microspheres

08) The procedure described here was carried out in the microsphere CBA A4 and in the microsphere CBA E4 to enable coupling with recombinant proteins of diagnostic potential. The preparation of the microspheres started with sonication in an ultrasonic bath for 1 minute at a frequency of 40 Hertz. The microspheres were homogenized by vortexing for 1 minute and 30 seconds, and 75pL of the microspheres were collected in a 1.5mL eppendorf tube. Then, 1.9 µL of DTT at 1 M concentration was added. The reaction to form the sulfhydryl group on the surface of the microspheres occurred under continuous orbital agitation for 1 hour at room temperature and protected from light. At the end of this step, 3 washes were performed with 700pL of coupling buffer by centrifugation at 900 x g for 3 minutes. At the end of the wash, the supernatant was carefully aspirated, the microsphere pellet suspended in 50pL of the coupling buffer, and storage at 4 ° C until the moment of use.

4.2. Protein preparation

09) The procedure described here was conducted with the recombinant proteins rLcilA and rLci2B to enable coupling to the CBA microspheres. Prior to chemical treatment, the recombinant proteins underwent a dialysis process to replace the residual elution buffer from the purification process. Protein aliquots were dialyzed on a 12-14 KDa Molecular weight cut-off (MWCO) semipermeable membrane in PBS buffer pH 7.9, for 24 hours, at 4 ° C under agitation. After this process, aliquots of the proteins at 1mg / mL were prepared and stored in -20 ° C fieezer until the time of use. For each protein, 90pL of stock solutions were used together with 2pL of the Sulfo-SMCC solution (2mg / mL)

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7/13 in an incubation under orbital shaking for 1 hour at room temperature and protected from light. After completing this process, the proteins treated with Sulfo-SMCC are ready to begin the process of conjugation to the microspheres.

4.3. Conjugation of proteins to microspheres

10) The tubes containing the microspheres prepared after reaction with DTT were quickly homogenized by vortexing. Then, the entire contents of proteins treated with Sulfo-SMCC were transferred to the tubes containing the CBA microspheres. This mixture was incubated on an orbital shaker for 1 hour at room temperature and protected from light. At the end of this process, 2pL of NEM (2mg / ml) was added and the tube was again incubated in an orbital shaker for 15 minutes at room temperature and protected from light. The material was washed 3 times with 700pL storage buffer by centrifugation at 1000 xg for 3 minutes. After washing, the supernatant was carefully aspirated and the microsphere pellet suspended in 500pL of storage buffer. The microspheres remained for a minimum period of 18 hours at 4 ° C to stabilize the bond. After the sequence of steps in this protocol the final concentration of microspheres is approximately 6 x 10 ⁶ particles / ml. It is worth mentioning that the recombinant microspheres-protein systems that composed the antigenic matrix of this work correspond to the CBA A4 microsphere conjugated with the recombinant protein rLcilA (AA-rLcilA system) and by the CBA E4 microsphere combined with the recombinant protein rLci2B (system E4-rLci2B) .

4.4. Analysis of data from the multiplex serological assay by flow cytometry for canine IgG antibodies applied to the diagnosis of canine visceral leishmaniasis

11) In this patent application, the innovative antigenic matrix composed of the A4-rLcilA and E4-rLci2B systems was initially evaluated under conditions that were based on the experience of carrying out serological assays by flow cytometry. Didactically, to confirm the viability of the serological reaction of the antigenic matrix, it is important to analyze three elements: (i) the reaction behavior in the control of the conjugate; (ii) the behavior of the reaction with the incubation of a serum sample

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CN canine 8/13; and (iii) the behavior of the reaction with the incubation of a canine serum sample from the CP. In the data analysis represented in Figure 1, information regarding the size, granularity and relative fluorescence intensity (FL1 - green fluorescence / FL3 - red fluorescence) of 2000 microspheres for each sample was acquired. Initially, a selection of the microsphere population is made on a size chart (Forward Scatter [FSC]) versus granularity (Side Scatter [SSC]) as shown in Figure IA. By using two different microspheres, each microsphere chosen to compose the test has a unique intensity of red fluorescence, determined by a discrete level of fluorescent marking distinguishable by its fluorescence intensity measured in the fluorescence channel FL3 of the flow cytometer. In this sense, a graph of the average intensity of red fluorescence FL3 as a function of SSC (Figure 1B) proves the differentiation of the different populations of microspheres that make up the A4-rLcilA system (blue) and the E4-rLci2B (green). From this differentiation, it becomes possible to determine the intensity of green fluorescence (FL1) obtained by the two microsphere-protein recombinant systems of the antigenic matrix. Thus, histograms of the average green fluorescence intensity FL1 were constructed as a function of the number of events. The sequence outlined by Figures 1C, ID and 1E show the differences in the average intensity of green fluorescence FL1 as a function of the number of events in the antigenic matrix corresponding to the conjugate control of the reaction (Figure 1C), in the incubation with CN sample (Figure ID), and with CP sample (Figure ΙΕ). The analysis of the result was determined by measuring the Percentage of Fluorescent Particles (PPF) independently among the systems of the evaluated antigenic matrix. The set of blue histograms (Figure 1F, Figure 1G and Figure 1H) illustrate the sequence of analysis of the A4-rLcil A system of the antigenic matrix. In the control of the reaction conjugate (Figure 1F), a reactivity threshold (marker M) was drawn, which follows as a criterion a maximum limit of 1% of the PPF. From the establishment of the M marker in this control, it is possible to determine the PPF values related to the A4-rLcilA system incubated in the presence of canine serum from the CN (Figure 1G) and from the CP (Figure 1H). Following the same guidelines, the set of histograms

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9/13 green (Figures II, 1J and 1L) represents the sequence of analysis for the E4-rLci2B system. Likewise, the reactivity threshold (marker M) in the control of the reaction conjugate was determined following the maximum limit criterion of 1% PPF (Figure II), followed by the analysis of the PPF for the E4-rLci2B system incubated in the presence of canine serum from the CN (Figure 1J) and the CP (Figure 1L). It is worth noting by the set of figures that with this sequence of analysis it is possible to differentiate samples from the CN in relation to the CP by the two systems of the antigenic matrix.

4.5. Standardization of multiplex serological assay by flow cytometry for canine IgG antibodies applied to the diagnosis of canine visceral leishmaniasis

12) The microsphere-protein systems were rapidly homogenized by vortexing and then in the ultrasonic bath for 2 minutes. An aliquot of 150pL antigenic matrix formed by an equal proportion of the A4 — rLcilA and E4 — rLci2B systems was first diluted in 5,000pL of PBS containing 0.5% w / v bovine serum albumin. After homogenization, the diluted antigenic matrix was distributed in a volume of 50pL per well of the 96-well U-bottom plate. Then 50pL of the canine serum was added in different titrations (1:50; 1: 100; 1: 200; 1: 400; 1: 800; 1: 1600; 1: 3200; 1: 6400; and 1: 12,800) previously diluted in PBS containing 2% w / v casein and 2% w / v lecithin. 13) The plate was incubated at room temperature for 90 minutes in the dark. After incubation, the antigenic matrix was washed three times with the application of 100pL of PBS containing 0.5% w / v of bovine serum albumin, followed by centrifugation at 2,200 rpm for 10 minutes at 24 ° C, and the supernatant discarded in the end of the process. For the subsequent step of fluorescent labeling, 100pL of anti-IgG antibody, canine labeled with fluorescein isothiocyanate-FITC diluted in different titers (1: 1,000, 1: 2,000, 1: 4,000 and 1; 8,000) in PBS containing 2% p / v casein and 2% w / v lecithin were added to each well of the plate. A second incubation at room temperature for 90 minutes in the dark was performed. The antigenic matrix was again washed three times with 100pL of PBS containing 0.5% w / v bovine serum albumin, centrifuged (2,200rpm, 10 minutes, 24 ° C) and the supernatant discarded. Finally, 180pL of PBS containing 0.5% w / v of

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10/13 bovine serum albumin and the reading performed on the flow cytometer. The reactivity profile of the antigenic matrix with canine IgG antibodies related to the A4-rLcilA system (Figure 2) and the E4-rLci2B system (Figure 3) show the variations of the serological reaction according to the canine serum title and the title of the FITC-labeled canine anti-IgG antibody. Noteworthy is the presence of intervals with excellent differentiation between a serum sample from CN compared to CP in both microsphere-recombinant protein systems, and the gray rectangular bands outlined in Figure 2 and Figure 3 indicate the regions curve selected to carry out the other steps of this patent proposal.

4.6. Construction of the ROC (Receiver Operating Characteristic) Curve to define the cutoff point.

14) In this object of the patent application, the receiver operating characteristic curve analysis strategy, known as the ROC curve, was used to help define the best cutoff point for the serological diagnostic test by flow cytometry. The study of the ROC curve demonstrated in this patent application was carried out on samples of animals from the CN (n = 30) and CP (n = 35) groups and which presented, respectively, negative and positive results, in parasitological diagnostic tests (gold standard ) that included direct demonstration of the parasite by microscopy and / or isolation of the parasite in culture. The construction of the ROC curve was performed from the determination of arbitrary cut-off values within a range of the PPF. Cutoff points of PPF> 20, PPF> 30, PPF> 40, PPF> 50 and PPF> 60 were evaluated.

15) The ROC curve consists of the graphical representation of sensitivity (true positive) on the vertical axis, and the complement of specificity (false positive rate) on the horizontal axis, for different cutoff points of the evaluated diagnostic test. The higher the AUC, which means values closer to 1, the better the diagnostic performance of the evaluated method. The ROC curves and their respective AUC for different cutoff points evaluated in the work as a function of the PPF values are shown in Figure 4. The A4-rLcilA system (Figure 4A), the E4-rLci2B (Figure 4B). and the combination of the two systems in a single serological test (Figure 4C) showed excellent performance under the cutoff conditions evaluated. You can

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11/13 classify a diagnostic test according to the value of the AUC in: no value (AUC = 0.5), low accuracy (0.5 <AUC <0.7), moderate accuracy (0.7 <AUC < 0.9), highly accurate (0.9 <AUC <l) and as a perfect test (AUC = 1). In the patent application proposed here, all the cutoff points evaluated showed values greater than 0.9 and indicate that the studied antigenic systems may constitute a highly accurate serological test in the diagnosis of canine visceral leishmaniasis.

4.7. Field of use

16) The multiplex serological test by flow cytometry can be used as a diagnostic test for canine visceral leishmaniasis. To evaluate the potential of this test, standard samples from the seroteca of the Laboratory of Immunopathology of the Nucleus for Research in Biological Sciences at UFOP were used. Seroteca make up: samples from uninfected control dogs; (ii) dogs naturally infected with other pathogens important in canine infections (Leishmania braziliensis, Ehrlichia canis and Babesia canis); (iii) dogs immunized with different anti-LVC vaccines (Leishmune®, Leish-Tec® and LBSap); (iv) dogs naturally infected with Leishmaia infantum with different clinical forms of the disease (asymptomatic, oligosymptomatic and symptomatic).

17) The development of multiplex tests is a worldwide trend in high performance diagnostic approaches. The standardized immunosorological test applied to the diagnosis of canine visceral leishmaniasis composed of the antigenic matrix from the association of the recombinant microsphere-protein systems A4-rLcilA and E4-rLci2B ensured an outstanding performance in tests with the samples of the seroteca (Figure 5). In this way, the consolidation of the development of a new methodology for the diagnosis of LVC provides a relevant conquest of interest in public health worldwide, which leads to the provision of new and effective tools applied in the diagnosis and control of neglected diseases. Thus, by reversing the knowledge produced in products or processes that can improve the quality of services, a direct contribution is made in expanding the reach of new technologies to improve

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12/13 health actions. The development of the new method proposed here, increases the repertoire of associated antigens in a single diagnostic test, providing a synergistic additive effect on the performance of serology in LVC.

Description of figures:

18) Figure 1: Schematic representation of the sequence of the analysis of the serological reaction data for canine IgG antibodies using an antigenic matrix composed of the recombinant microspheres-protein systems A4-LcilA and E4-Lci2B. (A) Selection of the microsphere population using the parameters of size (Forward Scatter - FSC) and granularity (Side Scatter - SSC). Differentiation of microsphere populations in fluorescence intensity graphs FL3 versus SSC. The fluorescence intensities FL1 of the set A4-rLcilA and E4-rLci2B obtained for the control of the reaction conjugate (C), after incubation with a non-infected dog serum sample - Negative Control - CN (D) and after incubation with sample of dog serum infected with Leishmania infantum - Positive Control - CP (E). The individual histograms for determining the negativity threshold and the Percentage of Positive Fluorescent Particles (PPF) obtained with the control of the reaction conjugate independently in the AA-rLcil A (F) and E4-rLci2B (I) system; after incubation with CN dog serum sample in the A4-rLcilA (G) and E4-rLci2B system (J) and after incubation with CP dog serum sample in the A4-rLcilA (H) system and E4-rLci2B ( L).

19) Figure 2: Canine IgG reactivity titration curves for the serological test by flow cytometry in the microsphere-protein AA-rLcilA system: sera from dogs in the negative control group (CN = - · -) and the positive control group ( CP = -) incubated with different titers of conjugated anti-canine IgG antibody labeled with fluorescein isothiocyanate (FITC). The results were expressed as Percentage of Fluorescent Particles (PPF). The gray shading represents the optimal range of reactivity of the samples and the dotted line highlights the dilution of choice of serum.

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20) Figure 3: Canine IgG reactivity titration curves for the serological test by flow cytometry in the microsphere-protein system E4 — rLci2B: sera from dogs in the negative control group (CN = - · -) and the positive control group ( CP = -) incubated with different titers of conjugated anti-canine IgG antibody labeled with fluorescein isothiocyanate (FITC). The results were expressed as Percentage of Fluorescent Particles (PPF). The gray shading represents the ideal range of reactivity of the samples and the dotted line highlights the dilution of choice of the serum.

21) Figure 4: ROC curves of the serological test by flow cytometry constructed from the sensitivity and 1-specificity indices in samples of dogs from the CN and CP group in the 1: 1,600 dilutions of the serum and 1: 4,000 conjugate. The results are expressed by the area under curve index (AUC). (A) Ideal cutoff point for serological assay with the recombinant microsphere-protein system A4-rLcil A. (B) Ideal cutoff point for serological assay with the recombinant microsphere-protein system E4-rLci2B. (C) Ideal cut-off point for serological testing with the combination of both recombinant microsphere-protein systems (A4-rLcil A and E4-rLci2B).

22) Figure 5: Evaluation of the performance of the antigenic matrix composed of A4-rLcilA and E4-rLci2B recombinant microspheres-protein systems in a multiplex serological assay by flow cytometry for the diagnosis of Canine Visceral Leishmaniasis (LVC). By the individual dispersion profile of each sample, and considering a cut-off of PPF> 40, the results were obtained using standard dilutions of the 1: 1,600 sera and the 1: 4,000 conjugate. (A) Negative control dogs (CN = ·) and positive control dogs (CP = M) with different clinical forms; (B) Dogs from the CP group stratified according to the clinical classification of LVC in asymptomatic dogs (CA = o), oligosymptomatic dogs (CO = □) and symptomatic dogs (CS = Δ); (C) Dogs naturally infected with other canine pathogens: Leishmania braziliensis (O), Ehrlichia canis (□) and Babesia canis (Δ); (D) Dogs vaccinated against CVL with Leish-Tec® (·), Leishmune® () and LBSap (à) vaccines.

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Claims (10)

1. MULTIPLEX SERUMOLOGICAL TEST BY FLOW CYTOMETRY characterized by being composed by two distinct microsphere-recombinant protein systems; one composed by the Functional Bead CBA A4 microsphere conjugated to the rLcilA protein (A4-rLcilA), and another formed by the Functional Bead CBA E4 microsphere conjugated to the rLci2B protein (E4-rLci2B). 2. MULTIPLEX SERUMOLOGICAL TEST BY FLOW CYTOMETRY according to claim 1, characterized by using two recombinant microsphere-protein systems associated in equal proportion to form the antigenic matrix of a serological test by flow cytometry. 3. MULTIPLEX SERUMOLOGICAL TEST BY FLOW CYTOMETRY according to claims 1 and 2, characterized by using antibodies from a first incubation of 50pL of the antigenic matrix together with 50pL of the serum sample in concentration ranging from 1:50 to 1: 12,800. 4. MULTIPLEX SERUMOLOGICAL TEST BY FLOW CYTOMETRY according to claims 1, 2, and 3, characterized by using a second incubation for fluorescent labeling of the antibodies bound in the antigenic matrix by the use of an antibody conjugated to the fluorescein isothiocyanate in titers varying from 1: 1,000 to 1: 8,000. 5. MULTIPLEX SERUM TEST PROCESS BY FLOW CYTOMETRY according to claims 01, 02 and 03, and 04, characterized by comprising the following steps: (i) preparing the antigenic matrix; (ii) incubate with a serum sample; (iii) incubate with antibody conjugated to a fluorescent molecule; (iv) perform the reading in a flow cytometer. 6. MULTIPLEX SERUMOLOGICAL TEST PROCESS BY FLOW CYTOMETRY according to claim 5, step (i), characterized by preparing the antigenic matrix by mixing 2.0x10 ⁶ to 4.0x10 ⁶ of the A4- recombinant microsphere-protein system rLcilA with 2.0x10 ⁶ to 4.0x10 ⁶ of the E4-rLciB system that will be diluted by applying a volume of 5 to 10 mL of PBS buffer supplemented with 0.5% w / v bovine serum albumin, followed by Petition 870170038924, of 06/08/2017, p. 17/24 2/2 homogenization in an ultrasonic bath for 1 to 5 minutes at a frequency of 20 to 60 Hertz, and another homogenization in vortex for 30 seconds. 7. MULTIPLEX SERUMOLOGICAL TEST PROCESS BY FLOW CYTOMETRY according to claim 5, step (ii), characterized by incubating in a serological test plate, from 50 to 100 pL of the antigenic matrix together with 50 to 100 pL of the sample of serum previously diluted in PBS buffer supplemented with 2.0% w / v lecithin and casein in titers ranging from 1:50 to 1: 12,800, for an interval of 30 to 120 minutes at room temperature, followed by a plate washing process through centrifugation for 5 to 15 minutes at 1500 to 3000 rpm. 8. MULTIPLEX SERUMOLOGICAL TEST PROCESS BY FLOW CYTOMETRY according to claim 5, step (iii), characterized by incubating from 100 to 200 pL of the antibody conjugated to the fluorescent isothiocyanate molecule in titers ranging from 1: 1,000 to 1: 8,000, for an interval of 30 to 120 minutes at room temperature, followed by a plate washing process through centrifugation for 5 to 15 minutes at 1500 to 3000 rpm. 9. MULTIPLEX SERUM TEST PROCESS BY FLOW CYTOMETRY according to claim 5, step (iv), characterized by applying 100 to 200 pL of PBS buffer supplemented with 0.5% w / v of bovine serum albumin followed by reading in a cytometer flow. 10. APPLICATION according to claims 1 to 9, characterized by comprising the multiplex serological test to be applied in the detection of antibodies. Petition 870170038924, of 06/08/2017, p. 18/24 1/4 FSC - FL3 fluorescence intensity —► - FL1 fluorescence intensity -►