MX2010012363A - Point of care test for the detection of exposure or immunity to dengue virus. - Google Patents

Abstract

The present invention provides a "point of care test" (POCT) for detecting a subject who has had exposure to a dengue virus or immunological relative thereof or who has achieved some level of immunity against a dengue virus antigen. The method is predicted in part on the detection of a binding partner which is specific for a dengue virus or an immunological relative thereof. The POCT comprises contacting a biological sample from the subject with a mixture of dengue virus specific immunogenic components; and determining the presence of a complex that forms between a binding partner in the biological sample and a dengue virus specific immunogenic component; and characterizing the binding partner in the complex with an anti-binding partner antibody conjugated with gold colloid using reverse flow technology.

Description

PROOF OF IMMEDIATE DIAGNOSIS FOR THE DETECTION OF EXPOSURE OR IMMUNITY AGAINST DENGUE VIRUS The present invention relates generally to the field of diagnostic tests. More specifically, the present invention provides an immediate diagnostic test (POCT) for detecting the recent exposure of a subject to a dengue virus or an immunologically related virus or a vaccine against the dengue virus. Immediate diagnosis and equipment for evaluation of serum for the dengue virus or antibodies specific for the dengue virus are also part of the present invention.

BACKGROUND The bibliographic details of the publications mentioned by the author in this specification are compiled alphabetically at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as an acknowledgment or any other form of suggestion that the prior art forms part of the general knowledge common in any country.

Dengue fever is one of the most common diseases transmitted by mosquitoes in humans. The disease is best controlled by reducing the regular activities of the mosquito routine, Aedes aegypti. The control of the dengue virus requires a rapid response to viral infection clusters and the early and reliable diagnosis of dengue infection or exposure is essential for this effort, which aims to stop the transmission of the dengue virus. There has been good progress in recent years in the development of diagnostic tools for dengue, which results in the availability of adequate tests for each stage of the disease. In addition to the IgM / IgG antibodies of the dengue virus and real-time RT-PCR, the recent development of antigen detection of NS1 in ELISA and the rapid lateral flow platform has offered clinical laboratories a promising tool for early diagnosis (Kumarsamy et al., J Methods Virol 40: 75-79, 2007).

Unfortunately, all these techniques require more than one platform and are not sensitive enough to detect most cases of dengue. This is because none of these techniques covers two diagnostic windows [antigen / virus (first stage) and antivirus signature molecules (last stage)]. This deficiency results in the misdiagnosis of many patients with dengue. In addition, this type of approach is expensive and not available at all levels of health care systems.

Dengue virus contains a positive-strand RNA approximately 11kb in length and is composed of three structural protein genes that encode nucleocapsid or core protein (C), a membrane-associated protein (M), a protein of cover (E) and seven non-structural protein (NS) genes.

The order of the genes for the dengue virus, as well as other Flavi viruses, is 5'C-prM (M) -E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3 '. There are four different serotypes, serotypes 1 and 4 (called DEN1, DEN2, DEN3 and DEN4). The infection induces a long-life protective immunity with the homologous serotype but only confers partial and transient protection against subsequent infections by the other three serotypes. In contrast, it has been generally accepted that secondary infection or infection with secondary or multiple infections that have different serotypes of the dengue virus is an important risk factor for dengue hemorrhagic fever (DHF) and dengue shock syndrome (SSD). ) due to the antibody-dependent increase. Other factors that have been postulated as important in the pathogenesis of DHF include virulence of the virus, genetic background of the host, activation of immune cells, viral load and autoantibodies. As an attempt is made to eradicate Aedes aegypti, the most efficient mosquito vector of the dengue virus, it has not been successful in countries where dengue is endemic, dengue control will be possible only after an effective vaccine has been developed . At present, there is no effective vaccine against dengue has achieved widespread use.

The laboratory diagnosis of dengue virus infection can be made by the detection of specific virus, viral antigen, genomic sequence and / or antibodies. Currently, the three basic methods used by most laboratories for the diagnosis of dengue virus infection are viral isolation and characterization, detection of genomic sequence of a nucleic acid amplification technology assay and detection of specific antibodies against the dengue virus. After the onset of the disease, the virus is found in serum or plasma, circulating blood cells and selected tissues, especially those of the immune system, for about 2 to 7 days, which corresponds more or less to the period of fever.

Two patterns of serological response can be observed in patients with dengue virus infection: the primary and secondary antibody response, depending on the immunological status of the infected individuals. A primary antibody response is; observe in individuals that are not immune to dengue or other members of dengue viruses. A secondary antibody response is seen in individuals who have previously had dengue or infection with the dengue virus. For serums in the acute and convalescent phase, serological detection of antibodies based on the capture of immunoglobulin M (IgM) IgG enzyme linked immunosorbent assay (ELISA) has become the new standard for the detection and differentiation of primary and secondary infections due to dengue virus. This is important, since a sensitive and reliable analysis for the detection and differentiation of primary versus secondary or multiple infection by the dengue virus is fundamental for the analysis of data for epidemiological, pathological, clinical and immunological studies.

Progress in the detection of antigens in acute phase serum samples by serology has been slow due to the low sensitivity of the test for patients with secondary infections, since these patients have pre-existing IgG antibody immunocomplexes. virus. However, recent studies using the ELISA and Dot-Blot technique directed at the envelope membrane antigen (E /) (the denKEY team, Globio Co., Beverly, MA) and the NS1 antigen showed that high antigen concentrations E / M and NS1 in the form of an immune complex can be detected in the acute-phase sera of patients with primary infections of the dengue virus and patients with secondary infections of the dengue virus up to 9 days after the onset of the disease . Koraka et al., J.

Clin. Microbiol. 47: 154-4159, 2003, recently reported on the detection of a Dot-Blot antigen immunoassay NS1 dissociated-immune complex in patients with dengue virus infections and concluded that the detection of NS1 antigens by Dot-Blot immunoassay in serum non-dissociated and dissociated and plasma samples from patients with primary and secondary dengue virus infections produce the largest number of patients with dengue-positive antigen compared to the numbers obtained by RT-PCR and with the denKEY team. The recent development of the NS1 protein based on ELISA showed a high level of detection of dengue cases in particular during the early stage of infection, but remained sensitive during early convalescence (Blacksell et al., 2007).

The serological diagnosis of dengue virus infection is quite complicated for the following reasons: (i) patients may have multiple and sequential infections with the four serotypes of the dengue virus due to the lack of protective cross-neutralization antibodies, (ii) multiple and sequential infections of the dengue virus make a differential diagnosis difficult due to the presence of pre-existing antibodies and the original antigenic sin (many B cell clones in response to dengue virus infection are first re- they stimulate to synthesize antibodies early and with greater affinity for the first virus infection than for the infectious virus present in all the infections of the dengue virus later) in the regions where two or more dengue tactics circulate jointly, (iii) IgG antibodies have a high degree of cross-reactivity with homologous and heterologous antigens of dengue virus, and (iv) the serological diagnosis of past, recent and current dengue virus infections is difficult due to the persistence of IgG antibodies (6 months, as measured by ELISA IgM capture specific for E / M, or long-lived, as measured by indirect IgG ELISA covered with E / M antigen) in many patients with dengue with secondary infections. Therefore, among the viral infections that can be diagnosed by serology, infection by the dengue virus is more difficult.

Several methods have been described for the serological detection of specific antibodies against the dengue virus, including haemagglutination (HI) inhibition, neutralization test, indirect immunofluorescence antibody test, ELISA, complement fixation, Dot Blot, Western Blot and the rapid immunochromatography test. Among them, the capture of ELISA IgM and / or IgG, ELISA IgM and / or indirect IgG coated with antigen and the HI test are the most used techniques for routine serological diagnosis of dengue virus infections. Traditionally, the HI test was used to detect and differentiate primary and secondary dengue virus infections due to its simplicity, sensitivity and reproducibility. Patients are classified as having secondary dengue virus infections, when the titration of the HI test in the serum is greater than or equal to 1: 2560 and classified as having primary infection of the dengue virus if the titration of the HI test is lower than 1: 2560. Recently the HI test has become less popular and has been gradually replaced by ELISA IgM and IgG capture specific to the E / M and due to the inherent disadvantages of the HI test.

Many rapid diagnostic test kits that use the principle of immunochromatography are commercially available. Most of these devices can simultaneously detect IgM and IgG antibodies against dengue virus in human whole blood, serum or plasma within 15 and 30 minutes. Some of these teams say that it is possible to differentiate the infections of the dengue virus, primary and secondary, although this is not always reliable. The results showed that these equipments tend to have greater sensitivity for the detection of IgG, but less sensitivity for the detection of Ig and different specificities in comparison with the results of ELISA IgM and capture IgG specific for E / M. Although rapid diagnostic test equipment (RDT) has the advantage of easy performance and rapid transmission of results, it should serve better as a screening test for doctors in hospitals. The accuracy of RDT is highly variable, particularly during the acute phase of infection with sensitivities ranging from 6.4% to 65.3% and specificities from 69.1% to 100% (Blackwell and others 2006).

There is a need for an effective and sensitive immediate diagnostic test (POCT) for the detection of exposure of subjects to the dengue virus at an acceptable level of performance during the two acute and convalescent phases of infection and to monitor the efficacy of immunity of the dengue virus.

SUMMARY The present invention provides an "immediate diagnostic test" (POCT) for the detection of a subject who has had exposure to a dengue virus or an immunological relative thereof or who has reached a certain level of immunity against an antigen of the dengue virus. dengue. The method is envisaged, in part, in the detection of a binding ligand (in general, IgA antibodies) that is specific for the dengue virus or an immunological relative thereof. The POCT comprises contacting a biological sample of the subject with a mixture of specific components of the immunogenic dengue virus and determining the presence of a complex that is formed between a binding ligand (eg, IgA antibodies) in the biological sample and a immunogenic component of the specific dengue virus; and characterizing the binding ligand in the complex with an anti-ligand binding antibody (eg, anti-human IgA) conjugated to colloidal gold by reverse flow technology.

In one modality, the POCT is based on the identification of IgA antibodies specific for the dengue virus or its immunological relationship in the biological sample.

The POCT is a low-cost, quick and simple test to determine the current or previous recent exposure of dengue virus. In a modality, the POCT uses the nitrocellulose membrane to maintain the dengue virus immunogen, which subsequently captures binding ligands identified as anti-dengue IgA from a biological sample, such as blood, serum, plasma, saliva, urine or tissue fluids of the subjects that will be treated for exposure to the dengue virus and are last visualized by the anti-IgA antibody conjugated with gold particles.

Accordingly, the POCT of the present invention shows greater specificity and sensitivity compared to another conventional used dengue capture IgM ELISA and more currently providing a fast reverse luxury platform that can specifically identify the antibody (e.g., IgA) produced against the dengue virus or immunological parts thereof in an early stage of dengue infection. In a particular embodiment, the POCT identifies infection with the current (acute) dengue virus.

In another aspect of the present invention, a solid support is provided for use in a POCT to detect the exposure of a subject to a dengue virus or immunological part thereof, the assistance encompassing dengue virus-specific immunogenic components immobilized in the support. In use, the solid support is used in a method comprising contacting a biological sample of the subject with a mixture of specific immunogenic components of the dengue virus or an immunogenic equivalent thereof, to determine the presence of a complex that is form between a binding ligand present in the biological sample and the specific component of the immunogenic dengue virus, and, optionally, characterizing the binding ligand in the complex to correlate the binding ligand with exposure to the dengue virus.

In one embodiment, the biological sample is applied to a nitrocellulose membrane previously loaded with the dengue virus. Conveniently, the antigen or immunogenic component is derived from cells infected by the dengue virus. The complex formed by an immunogenic component of the dengue virus antigen and the binding ligand can then be detected with a detection agent containing a reporter group, wherein the detection agent binds specifically to the component / ligand complex of Union.

More particularly, the binding ligand is IgA and the detection agent is an anti-IgA antibody.

Yet another aspect of the present invention provides a POCT device in the form of a roll for the detection of a binding ligand in a subject that is specific for the dengue virus or an immunological part thereof, or for the detection of the exposure to the dengue virus. Dengue virus, the equipment comprising a solid aid that covers a specific component of the immunogenic dengue virus or immunological part; or, a solid support comprising a specific component of the immunogenic dengue virus or immunological part thereof bound to a second support; at least one detection agent conjugated to a reporter group for the detection of a binding ligand in a biological sample that forms a complex with the specific immunogenic dengue virus component; and, optionally, instructions for using the equipment to further identify the complex binding ligand. In one embodiment, the binding ligand is IgA. In this aspect, the reporter group generally covers anti-IgA antibodies conjugated with gold particles and the platform is the reverse flow technology.

The present invention also provides the individual components of the POCT equipment for use in the method of the invention. The present invention also serves as a method for evaluating the relative risk of one or more subjects exposed to the dengue virus or immunological part thereof at a given location (eg, geographic area, urbanization, means of transportation or a medical treatment center). or evaluation), which comprises; obtaining samples from a representative population in a specific place and assessing the exposure test of the individual members of a population sample to a dengue virus or immunological part thereof by the method comprising the steps of contacting a sample of the subject with a mixture of specific immunogenic component of the dengue virus and determine the presence of a complex that is formed between a binding ligand in the biological sample and an immunogenic component of the specific dengue virus and where the presence of the complex indicates the exposure of the subject to a dengue virus or its equivalent and evaluate the relative risk of exposure at the site defined by the characterization of the binding ligand in the complex. In one embodiment, the binding ligand is IgA.

The risk analysis can be carried out using the software in a computer readable format. Accordingly, the present invention further relates to a computer-readable program and equipment suitable for analysis that covers the exposure of subjects or groups of subjects or a risk of exposure of the subject or group of subjects to a dengue virus or relative immunological of the same.

The present invention further relates to the use of IgA antibodies specific for the dengue virus or an immunological part thereof or an antigen of the dengue virus or an immunogenic component thereof in the formation of a POCT to detect infection or current exposure or recent previous to the dengue virus or an immunological part thereof.

BRIEF DESCRIPTION OF THE FIGURES Some figures contain representations or entities of color. Color photographs are available from the patent holder by request or from an appropriate Patent Office. A right may be imposed if it is obtained from a Patent Office.

Figure 1 shows the optimization of dengue antigens in the reverse flow platform (nitrocellulose membrane) in different dilutions that varied between 0.223 mg / ml to 0.175 mg / ml. Antigen dilution at 0.185 mg / ml showed a higher intensity level (1.35) and was considered as the optimal concentration. (Optimization of dengue antigen using the reverse flow technique).

Figure 2 shows the optimization of the L-protein as control line in the nitrocellulose membrane with different concentrations ranging from 0.02 mg / ml to 0.04 mg / ml. The protein concentration 0.03 mg / ml-L is considered as the optimal dilution used for the development of POCT Den-IgA. (Optimization of Protein-L (line C) against the conjugate of gold against human IgA).

Figure 3 shows optimization of the optimal dilution (OD) of anti-human IgA gold conjugate against L-protein. Gold conjugate of various dilutions: OD that vary from OD-6 to OD-10 were used against protein L (0.03 mg / ml) and OD-9-was found as an optimal dilution and used for the development of POCT Den-IgA. (Optimization of the OD of the human anti-human IgA conjugate against the L-protein (line C)).

Figure 4 shows the optimization of the L-protein against the viral antigen (0.185 mg / ml) and 0.03 mg / ml of the L-protein was found optimal in terms of intensity of the T line (1.35) and used as a dilution optimal for the development of POCT Den-IgA. (Optimization of the OD of human anti-human IgA conjugate against line C (protein L)).

Figure 5 shows the comparative evolution of POCT Den-IgA against Den-IgA, Den NSl Ag and ELISA Den-Cap-IgM. POCT Den-IgA detected more positive cases of dengue compared to other dengue tests. (Comparative performance of POCT Den IgA against Den-IgA, Cap-Ig and ELISA NSl antigen).

Figure 6 shows the performance of POCT Den-IgA using enriched blood (W / B), saliva and serum minced with 25 positive IgA serum samples for dengue. All the added samples even diluted at 1: 3 dilution showed positive reactions (> 0.5) in POCT Den-IgA. (Performance of POCT Den-IgA with whole blood and saliva pitted from a person with negative antibodies for dengue).

Figure 7 shows the comparative evolution of Den-IgA POCT versus other rapid dengue diagnostic tests (dengue IgM and IgG combo) currently available on the market and Den-IgA POCT showed a higher level of detection of positive cases of dengue (80.59%). (Comparison of Den-IgA performance against other dengue diagnoses (ELISA IgM Den-Cap, ELISA antigen NS1, Single rod, Panbio Roll and Roll of a single revision)).

Figure 8 shows the kinetics of IgA anti-dengue antibodies produced in different collection points of samples after the onset of fever. The comparison of results of POCT Den-IgA with Den-NSl Ag and ELISA IgM Den-cap based on the day of onset of the disease. POCT Den-IgA showed a higher level of detection of positive dengue cases than ELISA IgM Den-Cap but lower than ELISA 1 Den-NSl Ag. It was considered that ELISA Den-NSl Ag will be tested for the diagnosis of early dengue.

DETAILED DESCRIPTION All scientific citations, patents, patent applications and the technical specifications of the manufacturer are hereby incorporated by reference in their entirety.

Through this specification, unless the context requires otherwise, the word "comprises", or variations such as "understand" or "understanding", shall be understood to imply the inclusion of an established element or whole number or group of elements or whole numbers but not the exclusion of any other element or a whole number or a group of elements or whole numbers.

It should be understood that unless otherwise indicated, the invention is not limited to the components of the specific analysis, manufacturing methods, diagnostic protocols, or the like, therefore, it may vary. It should also be understood that the terminology used in this document is for the purpose of describing particular modalities and is not intended to be limiting.

It should be noted that, as used in the specification of subjects, the singular form "a", "one" and "the" include the plural aspects unless the context clearly indicates otherwise. Thus, for example, the reference to "a dengue virus" includes a single dengue virus, as well as two or more dengue viruses, the reference to "an antibody" includes a single antibody, as well as two or more antibodies, the reference to "the invention" includes a series of individual or multiple aspects of an invention; and so on.

The present invention provides a method for detecting dengue virus infection or exposure. The "test" can also be referred to as a "method" or "protocol" and is an immediate diagnostic test or "POCT".

The reference to "dengue virus" includes an immunological relationship thereof. The latter term includes the variants of one of the serotypes of the dengue virus. An immunological part includes another genus within the Flaviviridae family, or a species of Flavivirus. Potential parties include members of the group of viruses by ticks in mammals, the group of viruses by ticks in seabirds, group of viruses by mosquitoes, the group of viruses by Kokobera, the group of viruses by Ntaya, the group of viruses by Spondiverni and the group of viruses due to yellow fever. The reference to "dengue virus" includes any or all of the serotypes Denl2, Den2, Den3 and Den4 as well as variants thereof. The reference to "exposure" includes a current or recent previous infection, as well as an infection that does not lead to a disease condition. In one embodiment, the POCT screens for a binding ligand such as IgA in a subject that is specific for the dengue or immunological virus thereof and the test comprises contacting a biological sample of the subject with a mixture of dengue virus and Specific immunogenic components and determine the presence of a complex that is formed between a binding ligand in the biological specimen and the specific component immunogenic dengue virus, and that characterize the binding ligand in the complex with an anti-IgA antibody conjugated with gold colloidal This method is specific for the identification of specific binding ligand molecules against the dengue virus. In a particular embodiment, the binding ligand is IgA. This method can be further improved by the use of immunogenic components that have been isolated through an IgA specific for the dengue virus. Accordingly, the immunogenic components can be IgA immunogenic components specific for dengue. The use of the reactive IgA components makes POCT immunogenic specific for IgA antibodies in the biological sample that are specific for the dengue virus or its immunological relationship. However, other binding ligands may also be employed.

Accordingly, an aspect of the present invention provides a method for the detection of a binding ligand in a subject that is specific for the dengue virus, the method comprising contacting a biological sample of the subject with a mixture of reactive immunogenic components. of the dengue virus; and determine the presence of a complex that is formed between the binding ligand in the biological sample and an immunogenic component specific for dengue; and that characterize the binding ligand in the complex with an antibody specific for the binding ligand conjugated with colloidal gold.

In another embodiment, the present invention provides a method for the detection of IgA in a subject that is specific for the dengue virus, said method comprising contacting a biological sample of the subject with a mixture of immunogenic components of the dengue virus reactants; and the presence of a complex that is formed between a binding ligand in the biological sample and a specific component of the immunogenic dengue is determined; and that characterize the binding ligand in the complex with an anti-IgA antibody conjugated with colloidal gold.

In another aspect, the present invention provides a POCT using the reverse flow technique for the detection of a subject's exposure to a dengue or immunological relative virus of its method comprises contacting a biological sample of the subject with a mixture of components immunogénicos specific for dengue virus, the determination of the presence of a complex that is formed between a binding ligand in the biological sample and immunogenic component specific for the dengue virus, which characterize the binding ligand in the complex, and the correlation of the binding ligand with exposure to the dengue virus.

In a particular embodiment, the binding ligand is IgA.

Therefore, in a particular aspect, the present invention provides a rapid POCT reverse flow test for dengue reactive IgA, which in a particular embodiment, uses the serum in order to use the saliva of patients infected by dengue as the biological sample. Saliva contains high levels of IgA (19.9 mg / 100 ml) compared to IgG and IgM (1.4 mg / 100 ml and 0.2 mg / 100 ml respectively). The technique shows a high level of performance in terms of anti-dengue IgA antibody detection compared to the detection of specific antibodies against dengue by means of ELISA techniques. The POCT of the present invention is compatible with the diagnosis in primary health care where dengue diagnostic tests with molecular bases and ELISA are not feasible.

The POCT is fast (approximately 10-20 minutes, the average time of 15 minutes), low cost and simple without the need to use laboratory facilities and the special technical knowledge to determine the recent or current exposure to the dengue virus. In accordance with the present invention, the subjects including animals such as mammals and, in particular, humans, are examined for the presence of binding ligands and, in particular, IgA for dengue or an immunological part, their use of the technology of reverse flow. Binding ligands are generally binding ligands derived from subjects such as, but not limited to, immunointeractive molecules. The most useful immuno-interactive molecules are IgA antibodies in particular. The identification of such binding ligands is used as evidence of recent or current exposure before the subject of the dengue virus or an immunological part thereof.

The present invention specifically uses reverse flow technique, where the antigen of the dengue virus is sprayed or loaded in nitrocellulose for the capture of IgA reactive with the dengue from the biological samples of the subjects exposed to the dengue infection. The resulting complex is then detected by anti-IgA antibodies conjugated with gold particles and the technique can be useful at any level of the health system which is called immediate diagnostic test (POC).

Accordingly, the present invention shows greater sensitivity and specificity in comparison with other conventional antibodies and more currently used and the IgA capture antigen (AAC and AAC-ELISA), providing a platform that can identify antibodies produced against the virus of the Dengue or an immunological part of it at an early stage of infection using the reverse flow technique.

Therefore, the present invention provides a specific, rapid and economic POCT using dengue virus antigen, such as immunogenic components of proteins that cover the dengue of the virus, which allow the specific detection of the dengue virus binding ligands. , such as IgA that may be present in the test samples (blood, serum, plasma, saliva, urine or tissue fluids). The test is performed at room temperature (RT). It is a convenient technique for the detection of specific antibodies. The POCT uses the reverse flow platform in a roll format and anti-IgA antibodies conjugated with gold for the detection of IgA of anti-dengue virus of saliva, blood, serum, plasma, urine or phyloid of i tissues. On the other hand, the POCT is used to test a single sample with high sensitivity.

As indicated above, the term "dengue virus" as used herein, refers to all dengue serotypes (Den-1, Den-2, Den-3 and Den-4) associated with a dengue infection or symptoms of dengue infection. The invention is applicable to the detection of infection by the dengue virus or the exposure of any subject, including humans, non-human animals and laboratory animals. Human subjects, however, are the most useful subjects according to the present invention. However, the present invention includes any object that can respond to an infection or vaccination by the dengue virus or an immunological part thereof.

However, the term "infection" includes exposure to the dengue virus, but without necessarily causing the symptoms of the disease. The exposure may also be in the form of an experimental vaccine.

A dengue virus is defined as a group of human RNA viruses consisting of covered particles around 40-50nm in diameter. The viral genome is approximately 11 kb (Stollar et al., Am J Trop ed Hyg. 47 (6): 709-720, 1966). The mature virion consists of the positive RNA genome in a positive sense surrounded by an isometric nucleocapsid. The genome encodes a single open reading frame of around 11,000 nucleotides, which codes for all three structural (C-capsid, M-membrane and E-coat) and seven proteins (NS1, NS2a and NS2b, NS3, NS4a and NS4b, NS5) non-structural.

The dengue virus is transmitted to humans by the bite of infected female Aedes mosquitoes, mainly Aedes aegypti. This is a small, black and white, highly domesticated tropical mosquito that prefers to lay its eggs in artificial containers that are found in and around houses where water collects, such as buckets, vases and other water containers. Adult mosquitoes are rarely found outside; Instead, they rest in dark interior areas, are discreet and prefer to feed on humans or animals during daylight hours, with the highest bite activity occurring in the early morning or at dusk. The female mosquitoes feed nervously, altering the feeding process at the least movement of the host, thus returning to the same or different host to continue feeding. Due to this behavior the mosquito often feeds several people during a blood meal and if it is infectious it can transmit the virus to several people (Platt et al., Am J Trop Med Hyg 57 (2): 119-25, 1997 Scott and others, am J Trop Med. Hyg 57 (2): 235-239, 1997). This behavior has been used to explain the epidemiological observation that dengue diseases occur mainly in children, although in certain places, such as Singapore, this may have changed due to adaptation to vector control measures (Ooi et al., Lancet 357 (9257): 655-686, 2001).

After the bite of an infective female mosquito, the virus goes through an intrinsic incubation period of 3 to 12 days (average of 4 to 7 days) after the person can experience sudden onset of fever accompanied by other signs and symptoms not specific. During this period of viremia (which can be between 2 and 7 days), the virus circulates in the blood of infected people. If an infected Aedes mosquito feeds on the host during this viraemia period, this mosquito becomes infected after a mandatory extrinsic incubation period of 10 to 12 days. Subsequently, it would be able to transmit the virus to other uninfected hosts. In this cycle of transmission, humans are the host amplifiers of the virus, although studies show that monkeys can become infected and perhaps serve as a means of amplifying the virus.

Dengue virus infection produces a spectrum of diseases in humans according to the infecting virus, age and immunological conditions of the host. It can result in an asymptomatic disease or varies from a disease similar to undifferentiated flu (viral syndrome) to dengue fever (DF), dengue hemorrhagic fever (DHF) and severe and fatal dengue shock syndrome (DSS) (Organization World Health Organization, Dengue haemorragic fever: diagnostics, treatment, prevention and control, 2nd ed., 1997).

The World Health Organization has established standards for the classification of the severity of hemorrhagic dengue. There are four degrees of severity of which grade III and grade IV are considered DSS.

Grade I: Fever with non-specific constitutional symptoms and hemorrhagic manifestation is only a positive tourniquet test and / or easy bruising.

Grade II: In addition to the manifestation in grade I, a spontaneous bleeding in the forms of the skin or other hemorrhages.

Grade III: circulatory insufficiency manifested by a weak and rapid pulse, narrowing of the pulse pressure or hypotension with the presence of cold, wet skin and agitation.

Grade IV: deep shock with blood pressure or imperceptible pulse (WHO, 1997).

Classic DF is more common in older children, adolescents, and adults, and is less likely to be asymptomatic. The fever is abrupt in its onset with high fever, headache, myalgia and disabling arthralgia, nausea, vomiting and macular or maculopapular rash. The fever usually lasts 5-7 days and sometimes it can follow a biphasic course (appearance of a saddle).

DHF is mainly a disease of younger children under the age of 15, although it can also occur in adults and is mainly associated with secondary dengue infections. The critical stage of hemorrhagic dengue is at the time of the disappearance of fever, when the temperature is normal. The main factors that determine the severity of the disease at the moment are plasma loss due to increased vascular permeability and abnormal homeostasis and other common hemorrhagic manifestations such as petechiae, purpuric lesions and ecchymosis. These symptoms and a positive tourniquet test are useful for the diagnosis of DHF (Gubler, Clin Microbiol Rev. 77: 480-496, 1998).

DSS is the terminal phase of hemorrhagic dengue and is manifested by hypovolemic shock due to plasma loss (World Health Organization, 1997 supra). There are four warning signs of DSS: constant abdominal pain, persistent vomiting restlessness or lethargy, and a sudden change of fever to hypothermia with sweating and prostration. Early recognition and appropriate treatment by experienced health personnel can lower the DSS lethality rate to 0.2% but once the shock is established, the mortality rate can be higher than 40%.

Protein E, the largest and only structural protein exposed on the surface of the virus, is the main protein involved in immunological reactions, such as receptor binding, hemagglutination and neutralization. Infection in humans by one of the serotypes provides a long life immunity for said serotype but only temporary protection against other serotypes.

The nucleoplasm is in turn surrounded by the lipids that contain the membrane and cover proteins. In addition to the coat and capsid proteins, the dengue virus has seven non-structural proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5.

As indicated above, the term "immunological relationship" as used herein and applied to dengue viruses is intended to include similar molecules that may elicit the same or similar response as the dengue virus or a structural or non-structural protein that I could get from the dengue virus. For example, several antigens expressed by the dengue virus in the various stages of the virus infection particles or several fragments or can cause similar effects that causes complete virus. The answer may be an immune response. { non-clinical response) or it may be an infectious response (clinical response) or due to vaccination.

The present invention is applicable to the detection of exposure to the dengue virus or a relative immunological thereof. The exposure may be current or be a previous exposure to the dengue virus or an immunological part of it. In general, exposure is sufficient to elicit a reaction or immune response in the body in order to induce a binding ligand in response to the dengue or immunological virus thereof. Once the subject is exposed, the method of the invention can be applied at any stage of the exposure as described above. In general, the method is used to detect exposure, where there are no signs and symptoms that are evident from an infection with the dengue virus. The POCT here detects the subject's exposure at a stage of dengue virus infection at an early stage of acute secondary infection or at a late convalescence stage of exposure to the dengue virus or its equivalent at primary infection or vaccination . Exposure may not always manifest in a dengue virus infection or noticeable signs or symptoms, but will elicit a response in order to induce a binding ligand (eg, IgA). The answer is particularly an immunological response.

The subject may have been exposed to the dengue virus, but does not need to show visual symptoms of the infection. The current method detects exposure that may lead to infection or may indicate prior exposure that does not exhibit overt symptoms.

An "immune response" or "immune response" is understood as a selective response mounted by the immune system of vertebrates in which specific antibodies or fragments of antibodies and / or cytotoxic cells are produced against the invading pathogens and the antigens that are They recognize as strangers in the body.

The binding ligand is any molecule or cell that is produced against the foreign or immunological dengue virus in connection therewith. The binding ligand is generally an antibody or immunologically active fragment thereof, or a cytotoxic cell. The binding ligand includes an immuno-interactive molecule that can interact with the antigen of the dengue virus or equivalent and more particularly an IgA molecule.

As indicated herein, the binding ligand is generally an immuno-interactive molecule, which refers to any molecule that comprises an antigen binding portion or a derivative thereof. In particular, the immuno-interactive molecule is an antibody against any part of the dengue virus proteins or epitopes produced during the humoral response in the subject of an infection or exposure of the dengue virus.

As indicated herein, a particular binding ligand is an antibody produced in the subject for a dengue virus or virus-related components. However, a targeting antibody binding ligand can abe used. An example of a binding ligand is an anti-idiotypic antibody or a specific and discriminatory antibody of an antibody specific to a subject for a dengue virus or virus-related components.

As used herein, an "anti-idiotypic antibody" is an antibody that binds to the specific antigen of the binding site of another antibody generated in response to exposure to a component derived from dengue virus or immunological parts thereof.

As used herein, the terms "antibody" or "antibodies" include the entire antibody and fragments of antibodies that contain functional portions thereof. The term "antibody" includes any monospecific or bispecific compound formed from a sufficient portion of the variable region of a single strand and / or the variable region of the thick strand of binding effect for the epitope for which the whole antibody has binding specificity . The fragments may include the variable region of at least one immunoglobulin or light-coarse polypeptide, and include, but are not limited to, Fab fragments, 2 F (ab ') fragments, and Fe fragments.

In a particular embodiment, the binding ligand is an antibody and more particularly is an IgA molecule specific for dengue or an antigen-specific IgA molecule for the dengue virus.

The method of the present invention detects exposure to the dengue virus or immunological part thereof by using a biological sample obtained from a subject as it has been potentially exposed to the virus. The biological sample may be a sample of the body that may contain a binding ligand. These biological samples may be selected from the group including blood, saliva, marrow fluid, B cells, T cells, plasma, serum, urine and amniotic fluid. Conveniently, the biological sample is serum or plasma. More conveniently, the biological sample is serum or saliva.

It is also useful that the biological sample is obtained from subjects suspected of exposure to a dengue virus. A biological sample can also be modified before use, such as centrifugation, dilution, etc. Accordingly, a biological sample may refer to a homogenate, lysate or extract prepared from a complete organism or a subset of its tissues, cells or components, or a fraction or part thereof.

It should be noted that a biological sample may also be devoid of a binding ligand that may interact with the dengue virus or an immunological part thereof. This occurs when the subject has not been exposed to the dengue virus or an immunological part of it. Therefore "the determination of the presence of a complex that is formed between a binding ligand in the biological sample and an immunogenic component of the specific dengue virus" can give a result of zero, since a complex can not be formed in the absence of binding ligands. A control can be performed with the immunological agent specific for the dengue virus, such as a monoclonal antibody designed to compete with the binding ligands in the biological sample.

Reference is made to a biological sample that is contacted with a component, such as an immunogenic component or immunological part thereof should be understood as a reference to any method for facilitating the interaction of one or more immuno-interactive molecules of biological samples with an antigen component of dengue derived from the infection of cells with the dengue virus or an immunological part thereof. The interaction must be such that the coupling or binding or other association between the immuno-interacative molecule and a specific immunogenic component of the dengue virus antigen derived from the cells infected with the dengue virus or an immunological part may occur. of the same.

The biological sample is contacted with a mixture of specific immunogenic components of the dengue virus, preferably derived from the cells infected with the dengue virus or an immunological part thereof. The dengue virus provides the immunogenic viral components that can be provided by the dengue virus at any stage of its development. In the early stages of convalescence of dengue virus infection, antibodies, especially IgA developed or derived from the infection of the previous dengue is one of the signs of infection by the dengue virus either primary or secondary, which it can be detected by the formation of a complex between it and a specific component of the immunogenic dengue virus, especially an immunological component of the virus.

Vero cells infected with the dengue virus can be cultured for a period, usually for a period that allows the dengue virus to establish itself and infect the cell. More specifically, the cells are cultured until a cytopathic effect can be verified in the cell culture indicating an active infection of the virus in the cells.

At this point, the dengue virus is collected from the infected cells, either by lysis of infected cells or the supernatant of the infected cells by any other available method. Later, the viral antigen was inactivated and purified by the standard protocol and concentrated to 1 mg / ml.

Conveniently, the antigen of the dengue virus is coat protein.

In particular, the specific immunogenic component of dengue is a protein of the dengue virus particle. More specifically, the structural protein is the E-Coated protein, which may be more immunogenic for IgA anti-dengue antibodies, which are exposed in the coat of the virus.

However, the present invention is that it is not limited solely to the use of protein E antigens. A mixture of other dengue virus molecules / immunogens (present in cells infected with dengue virus, which may contain dengue virus particles and other immunological components, usually structural and nonstructural proteins) against which antibodies may also be used develop during the course of exposure / infection of dengue virus.

The specific immunogenic component for dengue virus is generally from the lysate and culture supernatant. The biological sample is contacted so that a complex can be formed between the immunogenic viral components of the lysate and the binding ligand contained in the biological sample. In particular, the immunogens of the dengue virus particles, including but not limited to those of the structural and non-structural proteins captured by anti-dengue IgA, are complexed with the binding ligands. Preferably, the specific binding ligands are antibodies or fragments thereof derived from the biological sample. These will only be present when the subject has been exposed / infected or immunized against the dengue virus.

A complex is formed between an antibody, in particular an IgA reactive for the dengue virus or for immunological thereof.

The methods and methods of the present invention seek to detect the components and binding ligands that form complexes and indicate an infection by the dengue virus. These components and binding ligands are generated in the course of an infection / immunization by the dengue virus.

The complex may comprise one or more binding ligands linked to one or more components derived from the dengue virus or an equivalent thereof. However, not all dengue viruses are specific for IgA. Other immunological molecules such as IgG and IgM can also bind.

A second binding molecule is also loaded onto the nitrocellulose membrane that binds all immunoglobulins (IgA, IgG and IgM) called L-protein and the protein is used as a control line to indicate the validity of the assay.

The biological sample is contacted with the component derived from the dengue virus or an equivalent thereof for a sufficient time and conditions that allow the stable formation of the complex.

The specific immunogenic components of the dengue virus and the biological sample are contacted so that a complex can be formed between the components and a binding ligand of presence in the biological sample. Conveniently, the immunogens of the dengue virus particles, including but not limited to those of the structural and non-structural proteins, especially captured by the anti-dengue virus IgA have a specific binding site to the antigen of the dengue virus, are form complexes with any of the binding ligands or a specific immunological agent for the competing dengue virus such as specific IgA.

In particular, the specific binding ligands are antibodies or fragments thereof present in the biological sample. These will only be present when the subject has been exposed / immunized with the dengue virus.

In particular, the complex forms between an antibody, in particular an IgA specific for the dengue virus antigen member or its equivalent and a viral component of dengue captured from the anti-dengue virus IgA. This then indicates specific IgA for the dengue virus in the sample and therefore recent or previous exposure.

In one embodiment, the present invention is based on the detection of specific binding ligands for the dengue virus, particularly IgA present in the biological sample that are specific for a component of the antigen of the dengue virus present in the cellular lysate derived from a cell. infected with the dengue virus or an immunological part of it that has been captured using anti-dengue virus IgA. The complex may comprise one or more binding ligands linked to one or more components derived from the dengue virus or an equivalent thereof. However, the identification of an IgA bound to the complex is what indicates the previous exposure in this present invention.

A non-immunological complex is formed with the biological samples (saliva, serum) together with the charge line with the L-protein that contains the three types of immunoglobulins (IgA, IgG and IgM). This complex does not indicate the exposure of dengue viruses to the subject, but gives the validity of the analysis.

Another aspect of the present invention provides a solid support for use in a method for detecting exposure of a subject to a dengue virus or its equivalent, the method comprising contacting a biological sample of the subject with a mixture of specific immunogenic components. for dengue virus or an immunological part thereof, determine the presence of a complex that is formed between a binding ligand present in the biological sample and a component of the specific immunogenic dengue virus, and, optionally, that characterize the binding ligand in the complex that correlate the binding ligand with exposure to the dengue virus; the support comprising specific immunogenic dengue virus components immobilized on the support.

The solid support can be of any material known to those skilled in the art to which a binding ligand or immunogenic component specific for the dengue virus can be attached. For example, the solid support can be a test well in a microtiter plate or nitrocellulose or a suitable membrane. On the other hand, the support can be a bead or a disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinyl chloride.

The binding ligand or immunogenic component specific for dengue virus can be immobilized on the solid support with a variety of techniques known to those skilled in the art, which are widely described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to adsorption of the antigen on the nitrocellulose membrane after spraying by the BioDot machine. Immobilization by adsorption of the antigen on the membrane (prima-85). In such cases, the adsorption can be achieved by spraying antigen in 7"of the bottom of the membrane using the BioDot machine in a suitable buffer solution.Two more lines are sprayed on the same membrane in 14" and 12"with glass violet and protein-L After spraying the membrane it is allowed to dry in a dryer and finally blocked by the blocking buffer.

The specific immunogenic component of the dengue virus derived from a dengue virus or a charged / sprayed equivalent on the membrane is allowed contact with the biological samples that are passed over the membrane, once added to the sample pad, which they allow the stable formation of a complex. Once the biological sample matches the indicator line (crystal violet) a detection system that is anti-IgA antibody conjugated with gold particles is allowed to flow over the membrane on the other side of the ribbon which in turn detects the immunocomplex specific proteins by specific binding to IgA molecule.

The detection of the complex between the components derived from the dengue virus or an equivalent thereof and a binding ligand derived from the subject such as specific recognition molecules, can be based on any convenient method, which will be known to those skilled in the art. technique.

It is contemplated that methods useful for the detection of complex binding components and ligands that are indicative of dengue virus infection in a biological sample include, but are not limited to, immunological assays, such as immunoblotting, immunocytochemistry. , immunohistochemistry or affinity chromatography of antibodies, Western blot analysis, lateral flow or variations or combinations of these techniques or otherwise, as known in the art.

In general, the binding components and ligands, which form complexes and indicate an infection by the dengue virus, can be detected in a biological sample obtained from a subject by all means available to the skilled addressee. In one embodiment, the detection method employs a more specific detection agent, such as anti-body conjugated with the enzyme, gold / colloid particles that allows the detection of the complexes with the binding ligands.

In one embodiment, methods as described herein involve the use of a dengue virus antigen on the nitrocellulose membrane to which a binding ligand of a biological sample can be absorbed / bound. The complex formed by a component of the antigen of the dengue virus and the binding ligand can then be detected with a detection agent containing a reporter group and specifically binds to the component / binding ligand complex. The detection agent may comprise, for example, an anti-antibody or other agent that specifically binds to the binding ligand, such as an anti-immunoglobulin (ie, antibodies), G protein, protein A, protein-L or a lectin .

In one embodiment, the detection reagent is an antibody or secondary antibody or an antigen-binding fragment thereof, capable of binding to the binding ligand of the biological sample. The antibodies can be prepared by any of a variety of techniques known to those skilled in the art (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, from Cold Spring Harbor Laboratory, 1988).

The secondary antibody that can be conjugated to a tag can be added to the complex to facilitate detection. A series of labels that provides a detectable signal can be used. The label can be selected from a group including chromogen, an enzyme, a catalyst, a fluorophore, colloidal gold and a direct visual label. In the case of a direct visual label, a metal or non-metallic colloidal particle, a dye particle, an enzyme or a substrate, an organic polymer, or a latex particle can be used. A large number of enzymes suitable for use as labels are disclosed in U.S. Patent Nos. 4,366,241, 4,843,000 and 4,849,338. Enzyme labels suitable in the present invention include alkaline phosphates, horseradish peroxidase, preferably horseradish peroxidase. The enzyme label can be used alone or in combination with a second enzyme, which is in the solution. In the present invention, a secondary antibody connected with horseradish peroxidase, which in turn reacts with the DAB substrate and produces a change in color perceptible to the naked eye, preferably achieves detection of the complex.

In general, the antibody is an anti-IgA antibody conjugated to colloidal gold and detects both the IgA binding ligands that have bound to specific immunogenic components of the dengue virus.

This analysis can be performed first by contacting a binding ligand of a biological sample that has been immobilized on a solid support, the nitrocellulose membrane, with specific immunogenic components of the dengue virus as described herein, so that a component that is allowed to bind to the immobilized binding ligand such as an antibody. Alternatively, the specific immunogenic components of the dengue virus can be bound to the solid support so that the binding ligands are allowed to bind to the immobilized component and then the detection reagent which can be the specific reporter group is added. The unbound sample is then removed from the immobilized complex by the reverse flow technique.

More specifically, once the binding ligand or the specific immunogenic component of a dengue virus is immobilized on the support as described above, the rest of the binding sites for the support are usually blocked. Any suitable blocking agent known to those skilled in the art, such as bovine serum albumin or milk casein, with either Triton X 100 or Tween 20 (Trademarks) [Sigma Chemical Co., St. Louis, Missouri] . The binding component or ligand is used undiluted during contact with the immunogenic component of the dengue virus. In general, an adequate contact time of 5-10 minutes is sufficient to allow the presence of an immunological component of the specific dengue virus in the biological samples that bind to the immobilized binding ligand, or vice versa. Preferably, the contact time is sufficient to achieve a level of binding to the target epitope on the specific immunogenic component of the adjuvant dengue virus which is at least 95% achieved in equilibrium between bound or unbound ligand binding or component immunogenic specific for the dengue virus. Those with common knowledge in the field recognize that the time necessary to reach equilibrium can be easily determined by analyzing the level of union that occurs over time.

The detection agent is generally an anti-IgA antibody. Preferred reporter groups include the groups mentioned here. The detection agent is released from the gold pad on the membrane that forms the immunocomplexes with the antigen of dengue virus and IgA abti-dengue from biological samples. An adequate amount of time can usually be determined by analyzing the level of the joint that occurs over time. The unbound detection agent is removed and the bound agent is detected by detection of the reporter group.

The method used to detect reporter group 1 depends on the nature of the reporter group. For radioactive groups, or autoradiographic scintillation counting methods are generally appropriate. The spectroscopic methods can be used to detect dyes, light groups, chromogenic enzymes and fluorescent groups. Chromogenic enzymes include, but are not limited to, peroxidase and alkaline phosphatase. Fluorescent groups include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) 1 rhodamine, Texas Red, colloidal gold and phycoerythrin. Biotin can be detected by avidin, along with a different reporter group (commonly a radioactive or fluorescent group or an enzyme).

As used herein, "binding" refers to a non-covalent association between two separate molecules such that a complex is formed. The ability to bind can be evaluated, for example, the determination of a binding constant for complex formation. The binding constant is the value that is obtained when the concentration of the complex is divided by the product of the concentrations of the components. In general, the two compounds are said to "bind", in the context of the present invention, when the binding constant for complex formation exceeds 10 3 L / mol. The constant bond can be determined using methods well known in the art.

The membranes provided by the method and kits of the present invention include any membrane to which the binding ligand or components derived from the flaviviruses, or their equivalent, can bind. Examples of the membranes are, without being limited to, nitrocellulose membranes, polytetrafluoroethylene membrane filters, cellulose acetate filters of the membrane and cellulose nitrate membrane filters with paper filter vehicles. Preferably, the membrane is a nitrocellulose membrane.

On the other hand, the diagnostic methods of the present invention can adopt an automatic method of analysis using a biological microcircuit. For example, a diagnostic kit can be structured to perform immunoblotting using a glass slide covered with the cell lysate component. This diagnostic equipment may include a biological microcircuit on the surface of which a flavivirus-specific immunogenic component is immobilized, an appropriate regulatory solution, a standardized sample comprising a detectable level of binding agent and a secondary detection reagent, as described at the moment.

The method and equipment of the present invention can detect the specific exposure of humans or animals to a dengue virus or any specific member of the family or equivalent thereof either during acute infection or in convalescent phase. As used herein, "acute infection" refers to the time when a virus has infected a host and actively replicates and / or causes symptoms associated with the infection such as fever, rash, joint pain or abdominal pain. The "convalescent phase" refers to the stage of the infection cycle of the dengue virus when the dengue virus does not multiply or remain in the blood of the host and has developed binding ligands such as, but not limited to, antibodies. Using the method and equipment of the present invention, exposure can be detected at any time after the generation of a binding ligand in the infected patient or patient derived from their previous infection / infections.

In another aspect of the present invention, POCT equipment is provided for detecting IgA in a subject that is specific for a dengue virus or mime equivalent or for detecting exposure to dengue virus comprising: a solid membrane support including an immunogenic component specific for the dengue virus or equivalent thereof; or a solid support that includes a specific immunogenic component for the dengue virus or equivalent thereof bound to a second support: at least one detection agent conjugated to a reporter group to detect a binding ligand in a biological sample that forms a complex with the immunogenic component specific for the dengue virus; and optionally instructs the use of the equipment to further identify the binding ligand of the complex.

Optionally, the equipment will also include additional parts such as plastic dropper, search solution, roll and instructions as necessary to carry out the method.

Accordingly, the present invention provides a device for detecting the exposure of a subject for the dengue virus or any member of the family or its equivalent. The equipment can be any convenient form of testing that allows a binding ligand in a biological sample to interact with a viral component of the dengue virus captured by IgA antidengue. The result is an indication, due to the presence of specific binding ligands of dengue virus, such as IgA in the biological sample, from the previous exposure to the dengue virus. Preferably the kit includes a solid support as described herein adapted to receive or comprise viral component of dengue virus captured from anti-dengue IgA. The equipment may also be constituted by reagents, a reporter of molecules capable of providing detectable signals and, optionally, instructions for its use.

The equipment can be a modular equipment comprised of one or more members in which at least one member is a support that includes the virus component of the dengue loaded / sprayed virus particle or equivalent or cell lysate that includes a data component derived from a dengue virus or its equivalent.

The present invention also provides individual components of the equipment for use in the method of the present invention. The invention provides solid support including anti-dengue virus IgA reagent components of dengue virus for use in detecting exposure to dengue virus. In an incarnation, a nitrocellulose membrane is used to attach the viral antigen, either for use as a viral component of the dengue virus captured by IgA antidengue reactive or as a Line Blot technique. Preferably the membranes include components selected from the group of structural dengue viral antigens and non-structural proteins, flavivirus particles and fragments, glycoproteins, lipids and carbohydrates derived from flaviviruses or any mixture thereof.

The present invention also provides individual components of the equipment for use in the method of the present invention. The invention provides solid supports that include reactive IgA components of the anti-dengue virus. In one embodiment, a nitrocellulose membrane is used to bind the viral host, either as a viral component of dengue reagent IgA antidengue virus or as a Line Blot technique. Preferably the membranes include components selected from the framework of structural and non-structural proteins of viral antigens of dengue, flavivirus particles and fragments thereof, glycoproteins, lipids and carbohydrates derived from flaviviruses or any mixture thereof.

The solid support can also be a microtiter plate, glass slide or biological microcircuit wherein the components of the cellular lysate are immobilized. These solid supports can be subjected to the biological sample to detect exposure to flaviviruses. Preferably, the nitrocellulase membrane is used to bind the antigen of the dengue virus by spraying it on the membrane of the cells infected with the dengue virus.

In yet another aspect, the present invention also provides a method for assessing the relative risk of one or more subjects who have been exposed to the dengue virus or an equivalent thereof within a defined location (e.g., geographic area, state of accommodation, means of transport or center for medical treatment or evaluation), comprising: obtaining samples from a representative population within a defined location; and evaluating evidence of exposure of individual members of a sample population to a dengue virus or equivalent thereof or a method comprising the steps of contacting a biological sample of the subject with a mixture of immunogenic components specific to the virus of the dengue; and determining the presence of a complex that is formed between a binding ligand in the biological sample and an immunogenic component specific for the dengue virus and wherein the presence of the complex indicates the subject's exposure to a dengue virus or equivalent thereof; and evaluate the relative risk of exposure within the defined location characterizing the binding ligand in the complex.

Risk analysis can be performed using software in a form that can be read on a computer. Consequently, the present invention also relates to a program that can be read on computer and computer suitable to analyze the exposure of subjects and group of subjects or a risk of exposure of subject or group of subjects to a dengue virus or equivalent thereof.

The method or technique of the present invention allows the epidemiological study or sero-supervision of inconveniences of the infection caused by dengue virus or any member of the family or its equivalent. These studies provide valuable information that advances multiple facets of research in the area of dengue virus disease. For example, epidemiological studies help to identify the rate of an infection. This information allows the identification of a defined location from which the virus source responsible for viral dysfunction originates.

Additionally, the technique / method of the present invention allows rapid identification or isolation of subjects who are infected with a dengue virus or equivalent thereof without primary laboratory equipment or even in field conditions.

Such information assistants to identify subjects, which require medical treatment as well as define locations that require additional research or disease control approaches such as identification of breeding sites and their control. In addition, the technique of the present invention allows the monitoring of an infected patient to determine the presence of specific IgA for anti-dengue virus. The relief of IgA titration or its presence in an early stage of infection may be the indication of secondary infection and therefore, help monitor subsequent phases of dengue virus infection such as CHEF or DSS.

In addition, the technique of the present invention provides a means to identify subjects who are infected with any specific member of the dengue virus genotype and serotype involved that allows for rapid detection, additional infection risk, which point to the location of a control strategy of infection and disease.

The present invention is further directed to the use of IgA antibodies specific for the dengue virus or an immunological part thereof or an antigen of the dengue virus or an immunogenic component thereof in the manufacture of a POCT to detect current or recent previous infection or exposure to the dengue virus or an immunological part thereof.

The present invention is now described with reference to the following non-limiting Examples. However, it should be understood that the following description is only illustrative and should not be taken as a restriction of the generality of the invention described above.

EXAMPLE 1 Dengue antigen Type 2 a) Dengue antigen: Dengue antigen type 2 (Biodesign, International, USA) was used to develop POCT using reverse flow technology. The dengue virus was developed in cell culture and was concentrated from culture supernatants by precipitation and ultracentrifugation, which was inactivated by formaldehyde. After the formaldehyde was neutralized by sodium bisulfite and inactivation was confirmed by Vero cell culture. The resulting inactivated type 2 dengue antigen was tested against antibodies against the four serotypes of dengue virus (1-4) and found positive. a) Protein-L: Protein L (Pierce Biotechnology, USA) is a recombinant immunoglobulin binding protein that was originally derived from the bacterium Peptostreptococcus magnus. Protein L has the ability to bind to the kappa light chain of all classes and subclasses of immunoglobulins without interfering with antigen-antibody reactions and was used to bind the IgA molecule of the biological samples used in this development. c) Anti-human IgA-Gold conjugate (40 nm): The goat anti-human IgA antibodies conjugated with colloidal gold were prepared using our domestic protocol and compared with the commercial product (Code-HIAIO, Millenia Diagnostics, Inc. USA) ). The IgA antihuman gold conjugate was used in this test as secondary antibodies and was used at a dilution of 90D (6520 nm). d) Serum samples: a total of 184 positive serum samples either by antigen NS1 or IgM ELISA of anti-dengue capture was used as positive samples for dengue. A number of 125 serum samples collected from healthy donors were used as a negative control. All these samples were obtained from the serum bank library of MP Biomedicals Asia Pacific Pt Ltd, Singapore. 124 additional serum samples used for field validation of the analysis were obtained from the Virology Department of Bangabandhu Sheikh Mujib Medical Univerisity, Bangladesh. e) Serological Analysis; IgM capture ELISA, a commercial kit (Pan-bio, Australia) was used for the measurement of specific anti-dengue IgM antibodies in the samples used in this study. The test was carried out during the procedures described by the manufacturer. f) Rapid diagnosis of dengue: Three types of rapid dengue diagnostic equipment available on the market either in the form of roll or test strip were used as Competence Teams to validate and evaluate Den-POCT IgA. g) Dengue NS1 antigen ELISA: Dengue NS1 antigen commercial equipment called Dengue Platelia NS1 Ag ELISA (Bio-Rad Corporate HQ, Hercules, USA) was used for detection of NS1 protein specific for dengue samples of serum used in this study. The test was carried out according to the procedures described by the manufacturer. h) Saliva samples: The 25 saliva samples collected from healthy donors were used as negative and chopped samples with positive IgA dengue serum. i) Whole blood: Whole blood was collected from the healthy donor (5 ml) and used to chop the dengue IgA positive serum. These samples, which were chopped with whole blood, were used to study the performance of POCT Den-IgA (Figure 6).

Optimization of Dengue Virus Antigen. The antigen optimization of dengue virus (Biódesign, International, USA) is carried out lx PBS according to Table 1. A minimum volume of 500μ1 was made for each concentration before starting the optimization.

TABLE 1 Optimization of dengue virus antigen Dengue E protein was sprayed in the position established for the separation of the Den-IgA Rapid Test in 4 pieces of membrane (30 cm each). Protein L is used as control line and crystal violet as indicator line. After spraying, the membrane is dried, blocked using blocking buffer solutions and dried again and laminated, cut and assembled for testing. j) Optimization of Protein L. Protein L (Pierce Biotechnology, USA) was diluted in 1 x PBS according to Table 2 mentioned below. A minimum volume of 500 μ? It was formed for each concentration for the optimization process.

TABLE 2 Dilutions of Protein L The assembly roll was used for the optimization of antigen and L protein sprayed / loaded on the panel using the positive, weak and negative anti-dengue IgA positive serum sample membrane. For protein L, the C line with 1.5 to 2.5 of intensity with serum was considered as the optimal reaction level, while with saliva the intensity was 2.5 to 3. For the optimization of viral protein, the T line with positive positive serum 2.5, 1.5 at 1.0 moderate, 0.5 to 0.25 weak positive while for the negative no line was considered as the level of optimal reactions.

After optimization of antigen (Figure 1) and protein L (Figures 2 &4), 500 rolls were prepared for the study of sensitivity, specificity, positive predictive value and negative predictive value.

Colloidal gold optimization of anti-human IgA: Anti-human IgA gold colide was prepared using standard procedure and optimized using different OD level varying from 06 to 10 and 09 OD was found optimal against positive and negative samples of dengue ( Figure 3).

Analysis Procedure Complete Blood Step 1 - 20 μ? were added to square samples of whole blood followed by 1 drop of search solution to same well.

Step 2 - The sample absorbed by the sample pad and then the serum / plasma moves on the membrane (blood cells that are not red blood cells) and once the blue control line (A) is reached, three (3) ) drops of Regulatory Solution Search on the square pad of the sample and with time control started. The reading turned out after 15 minutes.

Serum / Plasma / Saliva samples Step 1 - 15μ1 of sample was added to the sample wells followed by 1 drop of search buffer to the same well.

Step 2 - Sample absorbed by the sample pad and then moved over the membrane and once the blue control line (A) is reached, three (3) drops of Search Regulatory Solution were added to the conjugate pad.

Step 3 - Remove the plastic ear (marked with Den-IgA) until the resistance is felt and add another drop of search buffer to the square pad of the sample and start the time control. read the result after 15 minutes.

INTERPRETATION OF POCT Den-IgA The result of POCT was interpreted using the interpretation card that has a range of color intensities ranging from 025 to 3.0. For the test line (line T) any intensity from 0.25 to 3.0 was considered as a positive sample for den-IgA while 0.0 is considered negative. For the intensity of the control line (line C) between 1.5 and 3.0 was considered as a validity test. 1) ELISA IgA anti-dengue capture antigen (ACA-ELISA): the 96-well plate (Max-absorbed-NINC) was checked with 100 μ? by dengue antigen well (3 μg) using lxPBS and incubated overnight at 4 ° C or 1 hour at 37 ° C. After blocking the plate with blocking buffer for 1 hour at 37 ° C, the plate was washed with 4 washings with washing and used for ACA-ELISA. 100 μ? of test serum at 1: 100 in diluent buffer was added to each well and further incubated for 30 minutes at RT. After incubation, the plate was washed again 6 times and 100 μ? of TMB (Sigma, USA) was added to each well and incubated for 10 minutes at RT. In addition the color revelation was stopped using sulfuric acid (2.75% and the plate was read in an ELISA reading at 450 nm) The results were calculated using an OD value formula of the test sample divided by an average OD of samples negative s and multiplying the value by 10. f) Comparative analytical sensitivity of the immediate diagnosis test (POCT) of Den-IgA: The analytical sensitivity of POCT den-IgA was studied using strong, medium and weak dengue IgA positive serum samples. Serum samples were diluted 1: 5 and 1: 160 in whole blood, serum or saliva from a healthy person who is negative for dengue antibodies (IgG, IgM and IgA). Fifteen microliters (15 μ?) Of each diluted sample were added to the roll sample pad and the test was performed as described above.

EXAMPLE 3 Comparative analysis of POCT Den-IgA against antigen ELISA IgM, ACA and NS1 capture of Den.

The sensitivity level of POCT Den-IgA carried out using positive dengue serum obtained from the R & amp; D compared to other ELISA. The results showed that Den-IgA POCT detected 77.52% of cases of: dengue compared to 59.55% of NS1 protein, 60.67% of capture IgM den and 58.42% of anti-Den IgA ELISA (Figure 5).

EXAMPLE 3 POCT Den-IgA The performance of POCT Den-IgA was further evaluated using commercially available POCT using 67 dengue-positive sera (the sera were positive either for ELISA of NS1 antigen or ELISA IgM den-capture). The sensitivity of POCT Den-IgA was found to be 80.59% compared to 77.61%, 70.15% and 77.61% by Imi-check immersion IgM / IgG, an IgM / IgG roll and Pan-bio IgM / IgG roll respectively (Figure 7). ).

EXAMPLE 4 Specificity and Sensitivity of POCT Den-IgA The specificity of Den-IgA POCT using sera collected from healthy subjects and with febrile disease that is not dengue.

Two-by-two table showing sensitivity and specificity of POCT Den-IgA.

TABLE 3 EXAMPLE 5 POCT Den-IgA The performance of POCT Den-IgA against saliva and whole blood minced with positive and negative serum samples showed a high level of consistency at dilution of 1: 3 Figure 6).

EXAMPLE 6 The kinetics of Den-IgA production by POCT Den-IgA Of 67 samples of positive dengue serum (ELISA) AG Den-NSl and IgM Den-Cap) collected during the acute phase of infection 1-8 days), 57.14% (12/21) between 1-3 days ,. 90.48% (38/42) between day 4-6 and 100% (4/49 between day 7-8 showed the presence of anti-dengue IgA (Figure 8).

Newly developed POCT Den-IgA showed high performance compared to another POCT and early dengue ELISA (ELISA capture of IgM and NS1 antigen). This is due to the elimination of non-specific IgA interference for dengue particularly in the early phase of dengue disease. This performance is even better to ACA-ELISA due to the use of a high amount of serum (15 μ? Compared to 1: 100 in ACA-ELISA), in addition the POCT Den-IgA platform can be used against any type of samples collected from patients such as whole blood, serum / plasma and saliva.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It will be understood that the invention includes variations and modifications. The invention also includes all steps, aspects, compositions and compounds referred to or indicated in this specification, individually or collectively and any of all combinations of two or more of the steps or aspects.

BIBLIOGRAPHY Blacksell et al., 2007, Diagn Microbiol Infect Dis, 2007 Sep 21 Blackwell et al., 2006, Clin. Infect. Dis, 2006; 42: 1127-34 Gubler, Clin. Microbiol. Rev. 77: 480-496, 1998 Kumarsamy et al., J Virol ethods 40: 15-19, 2007 Ooi et al., Lancet. 357 (9257): 655-686, 2001 Platt et al., Am J Trop Med Hyg. 57 (2): 119-25, 1997 Scott and others, Am J Trop Med Hyg. 57 (2): 235-239, 1997 Stollar et al., Am J Trop Med Hyg. 47 (6): 709-120, 1966 World Health Organization, Dengue hemorrhagic fever: diagnosis, treatment, prevention and control, 2nd ed., 1997

Claims (46)

1. - A method for detecting exposure to a subject to a dengue virus or an immunological part thereof, said method comprising contacting a biological sample of the subject with a mixture of immunogenic components specific to the dengue virus; and determining the presence of a complex that is formed between a binding ligand in the biological sample and a specific immunogenic component of the dengue virus; characterizing the binding ligand in the complex; and correlating the binding ligand to exposure to the dengue virus or its immunological part.

2. - A method of claim 1, wherein the specific inmunglenic components for dengue are sprayed onto the solid cell surface infected with dengue virus or an equivalent thereof.

3. - A method of claim 1, wherein the specific inmunglenic components for the dengue virus are sprayed on the nitrocellulose membrane.

4. - A method of claim 1, wherein the specific inmungénico component for the dengue virus is selected from the list consisting of non-structural proteins, dengue virus particles and fragments thereof, glycoproteins, lipids and carbohydrates derived from dengue virus. dengue.

5. - A method of claim 4, wherein the structural protein is selected from the group including cover proteins, Pr membrane proteins and nucleocapsid proteins.

6. - A method of claim 5, wherein the structural protein is a coat protein.

7. - A method of claim 1, wherein the immunogenic component specific for the dengue virus is selected from the group comprising immunogenic components of dengue virus serotype selected from the group consisting of DENl, DEN2, DEN3 and DEN4.

8. - A method of claim 1, wherein the method detects exposure to a serotype of dengue virus selected from the group consisting of DENl, DEN2, DEN3 and DEN4.

9. - A method of claim 1, wherein the immunogenic component is an antidiotypic antibody to an antigen binding site of a dengue virus antibody generated in response to exposure to a component derived from the dengue virus or an immunological part of the dengue. same.

10. - A method of claim 1, wherein the binding ligand is an antibody specific for the dengue virus or an immunological fragment thereof.

11. - A method of claim 10, wherein the binding ligand is an antibody, expressed at an early stage of a dengue virus infection, during acute or convalescent infection or derived from recent past infection.

12. - A method of claim 1, wherein the binding ligand is an IgA antibody.

13. - A method of claim 1, wherein the relative dengue virus is selected from the group consisting of yellow fever virus, West-Nile and JE virus.

14. - A method of claim 12, wherein the binding ligand antibody is an IgA antibody that is specific for all dengue serotypes (DEN1, DEN2, DEN3 or DEN4).

15. - A method of claim 1, wherein the biological sample is selected from the group consisting of saliva, blood, serum, plasma, spinal fluid, B cells, urine and amniotic fluid.

16. - A method of claim 15, wherein the biological sample is saliva, whole blood, serum, plasma or urine.

17. - A method of claim 12, wherein the binding ligand is characterized using an anti-IgA antibody.

18. - A method of claim 17, wherein the anti-IgA antibody is conjugated with colloidal gold.

19. - A method for detecting IgA in an object that is specific for the dengue virus thereof, said method comprising: contacting a biological sample of the subject with a mixture of reactive immunogenic components of the dengue virus and determining the presence of a complex which forms between a binding ligand in the biological sample and a specific immunogenic component of the dengue virus; and which characterizes the binding ligand in the complex with a human anti-IgA antibody conjugated to colloidal gold.

20. - A method of claim 19, wherein the immunogenic components specific for dengue are sprayed onto a solid surface of the infected cells of the dengue virus or an equivalent thereof.

21. - A method of claim 19, wherein the specific immunogenic components of the dengue virus are sprayed onto the nitrocellulose membrane.

22. - A method of claim 19, wherein the specific immunogenic component for the dengue virus is selected from the list consisting of structural and non-structural proteins, dengue virus particles and fragments thereof, glycoproteins, lipids and carbohydrates derivatives of the dengue virus.

23. - A method of claim 22, wherein the structural protein is selected from the group including cover proteins, Pr membrane proteins and nucleocapsid proteins.

24. - A method of claim 23, wherein the structural protein is a coat protein.

25. - A method of rei indication 19, wherein the specific immunogenic component of the dengue virus is selected from the group comprising immunogenic components of dengue virus serotype selected from the group consisting of DEN1, DEN2, DEN3 and DEN4.

26. - A method of claim 19, wherein the method detects exposure to a serotype of the dengue virus selected from the group consisting of DEN1, DEN2, DEN3 and DEN.

27. - A method of claim 19, wherein the immunogenic component is an anti-idiotypic antibody to an antigen-binding site of a dengue virus antibody generated in response to exposure to a component derived from the dengue virus or an immunological part of the dengue virus. same.

28. - A method of claim 27, wherein the binding ligand is an antibody expressed at an early stage of an infection by dengue virus, during acute or convalescent infection or derived from recent past infection.

29. - A method of claim 19, wherein the relative dengue virus is selected from the group consisting of yellow fever virus, est-Nile and JE virus.

30. - A method of claim 29, wherein the binding ligand antibody is an IgA antibody that is specific for all dengue serotypes (SEN11, DEN2, DEN3 or DE).

31. - A method of claim 19, wherein the biological sample is selected from the group consisting of saliva, blood, serum, plasma, spinal fluid, B cells, urine and amniotic fluid.

32. - A method of claim 31, wherein the biological sample is saliva, whole blood, serum, plasma or urine.

33. - A method of claim 30, wherein the binding ligand is characterized using an anti-IgA antibody.

34. - A method of claim 33, wherein the anti-IgA antibody is conjugated with colloidal gold.

35. - A solid support for use in a method of claim 1 or 19, the support comprising specific immunogenic components for dengue immobilized in the support.

36. - A solid support of claim 35, selected from the group consisting of a bead, a disk, a magnetic particle or an optical fiber sensor, a microtiter plate, glass slide or biological microcircuit or a membrane including nitrocellulose membranes, membrane filters of polytetrafluoroethylene, cellulose acetate membrane filters and cellulose nitrate membrane filters with filter paper vehicles.

37. - A device for detecting IgA in a subject that is specific for a dengue virus or an immunological part thereof or for detecting exposure to the dengue virus comprising a solid support including an immunogenic component specific for the dengue virus or equivalent thereof , or a solid support including a specific immunogenic component for the dengue virus or equivalent thereof bound to a second support; at least one screening test conjugated to a reporter group to detect IgA in a biological sample that forms a complex with the immunogenic component specific for dengue virus; and optionally instructions for using the equipment to further identify the binding ligand of the complex.

38. - A kit of claim 37, wherein the specific immunogenic component of the dengue virus is immobilized on a nitrocellulose membrane.

39. - A kit of claim 38, wherein the immunogenic component specific for the dengue virus is selected from the group consisting of structural and non-structural dengue virus proteins, virus particles and fragments thereof, glycoproteins, lipids and carbohydrates derivatives of the dengue virus.

40. - A rei-indication kit 39, wherein the structural protein is selected from the group including cover proteins, Pr membrane proteins and nucleocapsid proteins.

41. - A kit of claim 40 wherein the structural protein is a coat protein.

42. - A kit of claim 41, wherein the immunogenic component specific for the dengue virus is selected from the group that includes immunogenic components of dengue virus serotype DEN1, DEN2, DEN3, or DE 4.

43. - a method for assessing the relative risk of one or more subjects that are exposed to the dengue virus or an immunological part thereof within a defined location comprising: obtaining samples from a representative population within a defined location; and evaluating evidence of the exposure of individual members of a sample population of a dengue virus or immunological equivalent thereof by the method comprising the steps of contacting a biological sample of the subject with a mixture of immunogenic components specific for the virus of the dengue; and determining the presence of a complex that is formed between a binding ligand in the biological sample and an immunogenic component specific for dengue virus and wherein the presence of the complex indicates the subject's exposure to a dengue virus or equivalent thereof; and evaluates the relative risk of exposure within the defined location characterizing the binding ligand in the complex.

44. The method of claim 43, wherein the binding ligand is IgA.

45. - The method of claim 43 or 44, wherein the defined location is a geographical area, accommodation status, means of transport or center for medical treatment or evaluation.

46. - Use of IgA antibodies specific for dengue virus or an immunological part thereof or a dengue virus antigen or an immunogenic component thereof in the manufacture of a POCT to detect current or recent infection with a dengue virus or a immunological part of it.