TWI491880B - A method for improving antibody detection and applications in detecting flavivirus infection - Google Patents

Description

Method for enhancing antibody detection and application of the method for detecting yellow virus infection use

The invention relates to a method for enhancing antibody detection, in particular to a method for detecting an antibody against non-structural protein 1 of flavivirus.

Flaviviruses, such as Japanese encephalitis virus, West Nile virus, Yellow fever virus, St. Louis encephalitis virus (SLEV), And Dengue virus, etc., will cause a variety of major human diseases. The diseases caused by these viruses are recorded in most countries and continue to spread. Therefore, there is a need for a method for accurately detecting a viral infection in clinical care.

Viral isolation assays or viral RNA assays currently used in the field provide reliable detection results, but viral or viral RNA is usually only present in patients for a short period of time, limiting the benefit of such assays. Therefore, antibody detection is a more practical alternative to practice. Antibody assays typically detect a virus-specific immunoglobulin M/G (IgM and IgG) to diagnose or identify a subject's infection status. Studies have shown that the flavivirus-specific immunoglobulin M is produced within five days of the onset of infection and lasts for several months, after which it gradually decreases. Too little to detect. The World Health Organization (WHO) recommends a hemagglutination inhibition (HI) test for testing. Hemagglutination inhibition tests are also commonly used for serological classification of flavivirus infections. However, hemagglutination inhibition tests are too time consuming, and cross-reactions among Flavivirus are another important point that affects the utility of the trial.

On the other hand, the plaque reduction neutralization test (PRNT) is considered to be the gold-standard method in the test, but this test method is difficult for most laboratories. And it often takes several days to get the test results, which limits its clinical application.

In summary, in order to prevent epidemics and accelerate clinical testing, there is a need in the art for an accurate, time-saving, and cost-effective assay for detecting flavivirus infection.

It is an object of the present invention to provide a method for detecting a flavivirus infection which is more accurate in detecting the sensitivity of the antibody detection.

Another object of the present invention is to provide a method for detecting a flavivirus infection which is sufficient to determine whether the source of the antibody is a natural infection or is produced by a vaccine by increasing the sensitivity of the antibody detection.

It is still another object of the present invention to provide a kit for detecting a flavivirus infection which has superior antibody detection sensitivity and thus has the advantages of time saving and cost consideration.

In order to achieve the above object, the present invention provides a method for enhancing antibody detection, wherein the antibody is an antibody against a non-structural protein 1 of flavivirus; and the aforementioned method comprises removing an antibody against a fluffy protein of the anti-flavivirus.

Preferably, the step of removing the antibody of the anti-flavivirus envelope protein of the present invention comprises using the envelope protein of a flavivirus as an antigen to capture the aforementioned antibody against the envelope protein of the flavivirus.

Preferably, the foregoing method is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof.

Preferably, the aforementioned immunoassay is an antigen capture type enzyme immunoassay.

The invention further provides a method for detecting a flavivirus infection, comprising the steps of: (i) removing an antibody against a flavivirus envelope protein; and (ii) detecting a non-structural protein 1 against the flavivirus in the aforementioned sample Antibodies.

Preferably, the aforementioned step (i) comprises using an envelope protein of a flavivirus as an antigen to capture an antibody against a flavivirus envelope protein in the aforementioned sample.

Preferably, the aforementioned step (i) is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof.

Preferably, the aforementioned immunoassay is an antigen capture type enzyme immunoassay.

Preferably, the aforementioned step (ii) is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof.

Preferably, the aforementioned immunoassay is an antibody-collecting enzyme immunological fraction. Analysis method.

Preferably, the antibody-trapping enzyme immunoassay is: an IgM antibody-capturing enzyme immunoassay for the non-structural protein 1 of the flavivirus, and an IgG antibody-capturing enzyme immunoassay for the non-structural protein 1 of the flavivirus. Law, or a combination thereof.

Preferably, the aforementioned step (ii) is performed by means of a peroxidase color reaction or an alkaline phosphatase color reaction to interpret the results of the foregoing detection.

Preferably, the aforementioned sample is the blood of an individual to be determined.

Preferably, the aforementioned flavivirus is Japanese encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus, or dengue virus.

Preferably, the flavivirus is a dengue virus, and the envelope protein of the aforementioned flavivirus as an antigen is a sleeve protein of a serotype type 2 dengue virus, and a set of a serotype type 3 dengue virus. Membrane proteins, or combinations thereof, or combinations thereof with envelope proteins of dengue viruses of other serotypes.

The present invention further provides a kit for detecting a flavivirus infection, comprising: an antibody removing unit for removing an antibody against a flavivirus envelope protein; and a non-structural protein 1 against flavivirus Detection unit for antibodies.

Preferably, the aforementioned antibody removal unit comprises a membrane protein using a flavivirus as an antigen.

Preferably, the antibody removal unit is a first immunoassay device, a first affinity chromatography column, a first antigen magnetic bead device, or a combination thereof.

Preferably, the first immunoassay device is an antigen capture type enzyme immunoassay device.

Preferably, the detection unit of the non-structural protein 1 antibody against flavivirus is a second immunoassay device, a second affinity chromatography column, a second antigen magnetic bead device, or a combination thereof.

Preferably, the second immunoassay device is a pair of flavivirus non-structural protein 1 specific IgM antibody capture type enzyme immunoassay device, and a pair of flavivirus non-structural protein 1 specific IgG antibody capture type enzyme immunoassay Device, or a combination thereof.

Preferably, the detection unit of the aforementioned non-structural protein 1 antibody against flavivirus is judged by a color reaction of a peroxidase or a color reaction of an alkaline phosphatase.

Preferably, the aforementioned flavivirus is Japanese encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus, or dengue virus.

Preferably, the flavivirus is a dengue virus, and the envelope protein of the aforementioned flavivirus as an antigen is a sleeve protein of a serotype type 2 dengue virus, and a set of a serotype type 3 dengue virus. Membrane proteins, or combinations thereof, or combinations thereof with envelope proteins of dengue viruses of other serotypes.

In summary, the present invention provides a method for enhancing antibody detection and a method and a kit for detecting a flavivirus infection using the spirit of the method. By enhancing the sensitivity of antibody detection, the detection method of the method and the set of the invention is accurate, and has the advantages of time saving and low cost.

10‧‧‧ bottom of ELISA test tray

11‧‧‧Multiple antibodies against rabbit anti-JEV (class) virus particles

12‧‧‧(class) virus particles

13‧‧‧anti-E antibody

[abbreviation description]

E‧‧‧Flavor envelope protein (envelop)

Nonstructural protein of NS1‧‧‧ flavivirus

Anti-E antibody ‧‧‧anti-envelop antibody

Anti-NS1 antibody ‧‧‧Anti-structural protein 1 antibody

VLP‧‧‧ virus-like protein

IgM‧‧‧ immunoglobulin M (Immunoglobulin M)

IgG‧‧‧ immunoglobulin G (Immunoglobulin M)

MHIAF‧‧‧murine hyper-immune ascitic fluid

JEV‧‧‧Japanese encephalitis virus

WNV‧‧·West Nile virus

YFV‧‧‧Yellow fever virus

SLEV‧‧‧St.Louis encephalitis virus

DENV‧‧ Dengue virus

HRP‧‧‧Mountain Peroxidase (horseradish peroxidase)

NHS‧‧‧ Blood samples from healthy individuals (Normal Human Serum)

MAC-ELISA ‧ ‧ IgM antibody capture enzyme immunoassay (IgM antibody capture ELISA)

GAC-ELISA ‧ ‧ IgG antibody capture ELISA

The first figure illustrates the pre-adsorption step of the present invention.

The second graph shows the experimental results confirming the effect of the pre-adsorption step of the present invention; wherein (+) indicates that the infection has occurred.

The third figure shows the pre-adsorption step of the present invention for detecting anti-NS1 Effect of antibody; (A) sample of JEV infection; (B) sample of WNV infection; (C) sample of DENV infection; wherein (+) indicates that a pre-adsorption step is performed, and (-) indicates that no pre-adsorption step is performed.

The fourth panel presents experimental results of JEV-infected serum samples pre-adsorbed with JEV and WNV (class) virus particles, respectively; (A) is a pre-adsorption step using JEV (class) virus particles, and then anti-antigen detection - PEV ratio of JEV NS1 IgM; (B) pre-adsorption step using JEV (class) virus particles, followed by P/N ratio of anti-JEV NS1 IgG; (C) using WNV (class) virus particles The pre-adsorption step was performed to detect the P/N ratio of anti-JEV NS1 IgM; (D) the pre-adsorption step using WNV (class) virus particles, and then the P/N ratio of anti-JEV NS1 IgG was detected.

The fifth panel presents experimental results of WNV-infected serum samples pre-adsorbed with JEV and WNV (class) virus particles, respectively; (A) is a pre-adsorption step using WNV (class) virus particles, and then anti-antigen detection -PNV NS1 IgM P/N ratio; (B) Pre-adsorption step using WNV (class) virus particles, followed by detection of anti-WNV NS1 IgG P/N ratio; (C) using JEV (class) virus particles The pre-adsorption step was performed to detect the P/N ratio of anti-WNV NS1 IgM; (D) the pre-adsorption step using JEV (viral) virus particles, and then the P/N ratio of anti-WNV NS1 IgG was detected.

The sixth panel examines the effect of the envelope protein of dengue virus using different serotypes on the methods of the invention. (A1) (A2) is sample ID #100, which is a sample infected with type 1 infection; (B1) (B2) is sample ID #56, which is a sample infected with type 2 infection; (C1) ( C2) is sample ID #76, which is a sample infected with type 2 infection.

The seventh panel shows the effect of the pre-adsorption step of the present invention in detecting anti-NS1 antibodies in acute phase samples of dengue infection. (A) not carried out a pre-adsorption step; (B) a pre-adsorption step.

The eighth panel shows the pre-adsorption step of the present invention in detecting the anti-NS1 antibody in a recovery period sample of dengue infection. (A) The pre-adsorption step is not performed; (B) the pre-adsorption step is performed.

Flaviviruses, including Japanese encephalitis virus, West Nile virus, Yellow fever virus, St. Louis encephalitis virus (SLEV), or dengue virus (Dengue virus), both of which are positive-stranded single-stranded RNA viruses, whose positive-stranded single-stranded RNA is translated into a single complex protein in a host cell. The complex single white is then cut into three structural proteins, including capsid (C), premembrane (prM), and envelope (E), and seven non- Structural proteins, including non-structural protein 1 (NS1), non-structural protein 2A (NS2A), non-structural protein 2B (NS2B), non-structural protein 3 (NS3), non-structural protein 4A (NS4A), non-structural protein 4B (NS4B) ), and non-structural protein 5 (NS5).

Most of the antibodies produced by the host after infection produce a specific immune response against the envelope protein of the flavivirus. Therefore, there are many antibodies against the envelope protein in the field (hereinafter referred to as anti-E antibodies, including IgM and IgG). ) Detection of enzyme-linked immunosorbent assay kits. This type of IgM antibody-collecting enzyme immunoassay device (E-MAC-ELISA) or IgG-capture enzyme immunoassay device (E-GAC-ELISA) for antibodies against envelope proteins is considered to be equivalent to the HI assay. Reliability. On the other hand, antibodies against non-structural protein 1 (hereinafter referred to as anti-NS1 antibodies) are also the subject of detection in another study. however, The anti-NS1 antibody is low in the blood of patients and is susceptible to interference from other antibodies in the blood of the patient. Therefore, even if a variety of immunoassay techniques for anti-NS1 antibodies are developed in the field, these immunoassay techniques fail to satisfactorily meet the clinical requirements in the case of a small number of antibodies against non-structural protein 1 and unsatisfactory detection sensitivity. use.

The reason for the poor sensitivity of the anti-NS1 antibody detected by the present invention is mainly due to the presence of too many anti-E antibodies in the blood of the patient. Accordingly, the present invention provides a method of enhancing the detection of an anti-NS1 antibody comprising removing an anti-E antibody as in the present invention.

The "sample" of the present invention, such as the blood of a potential patient; more specifically, the serum of a potential patient. The "antibody" of the present invention is, for example, immunoglobulin M (IgM), immunoglobulin G (IgG), or a combination thereof. The "antibody for removing the envelope protein of the anti-flavivirus in the same embodiment" and "removing the anti-E antibody in the same embodiment" means removing the anti-E antibody in the same manner to a considerable extent, so as to detect the anti The sensitivity of the -NS1 antibody is increased to a level that contributes to clinical use. In principle, the above "substantial degree" need not be specifically defined. The spirit of the present invention is to confirm and teach that when the anti-E antibody in the same embodiment is removed, the sensitivity of detecting the anti-NS1 antibody can be improved, and the general knowledge in the field can be Understand that, under the best conditions, the more complete the removal of anti-E antibodies in the sample, the more helpful the detection of anti-NS1 antibodies.

The above "antibody for removing the envelope protein of the anti-flavor virus in the present embodiment" and the above "removal of the anti-E antibody in the same" can be accomplished by a technical means known in the art. For example, it can be accomplished by immunoassay, affinity chromatography (Affinity chromatography), antigen magnetic beading, or a combination thereof.

In one embodiment of the invention, an immunoassay is employed to remove anti-E antibodies from the sample. Specifically, the envelope protein of a flavivirus is immobilized as an antigen on a substrate (such as an ELISA assay plate), and the anti-E antibody in the sample is removed by the principle of immunoassay. For example, an antigen-capture enzyme immunoassay (Antigen-capture ELISA) can be used to immunologically bind anti-E antibodies in a sample by a flavivirus envelope protein to remove anti-E antibodies from the sample. the goal of. The above "immune binding" refers to the combination of two substances by the specific relationship of antigen antibodies.

In another embodiment of the invention, affinity chromatography is used to remove anti-E antibodies from the sample. Specifically, the membrane protein of a flavivirus is immobilized on the stationary phase of the chromatography column, and when the sample flows through the chromatography column, the anti-E antibody is specifically adsorbed into the chromatography column. Separation from the sample, thereby achieving the purpose of removing the anti-E antibody in the sample.

In yet another embodiment of the invention, the antigen magnetic beads method is utilized to remove anti-E antibodies from the sample. Specifically, the magnetic beads immobilized with a flavivirus envelope protein are reacted with the sample to form a complex of the anti-E antibody in the sample with the magnetic beads of the envelope protein immobilized with the flavivirus to achieve removal. The purpose of the anti-E antibody in the sample.

In the spirit of the enhanced sensitivity of the present invention for detecting an anti-NS1 antibody, the present invention provides a method for detecting a flavivirus infection. One of the keys to the detection method provided by the present invention is to first remove the anti-E antibody in the sample to be tested, thereby increasing the sensitivity of detecting the anti-NS1 antibody. Specifically, the detection method provided by the present invention comprises the steps of: (i) removing the same anti-E antibody; and (ii) detecting the anti-NS1 antibody in the aforementioned sample; wherein step (i) is according to the present invention. Spiritual pre-absorption step, pre-processing the sample to remove the test Anti-E antibody in the sample.

The foregoing step (i) can be carried out by immunoassay, affinity chromatography, antigen magnetic bead method, or a combination thereof; wherein the above immunological analysis method, the aforementioned affinity chromatography method, and the operation details of the aforementioned antigen magnetic bead method are As described in the previous paragraph.

The aforementioned step (ii) can be accomplished by technical means known in the art. For example, immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof can be used.

In one embodiment of the invention, an immunoassay is employed to detect the anti-NS1 antibody in the aforementioned sample. Briefly, an anti-human IgM or IgG antibody is first immobilized on a substrate (such as an ELISA assay plate), and IgM or IgG in the serum of the subject is bound to the substrate by the aforementioned antibody, and then NS1 is added. Antigen to identify IgM or IgG of anti-NS1. Finally, an antibody recognizing anti-NS1 (in this experiment, the immune serum of the mouse is used), and a horseradish peroxidase (HRP) color reaction or alkaline phosphatase (alkaline phosphatase) ; AP) color reaction to interpret the results. For example, an NS1-specific IgM antibody-trapping enzyme immunoassay (NS1-specific IgM-capture ELISA), an NS1-specific IgG-specific IgG-capture ELISA, Or a combination thereof to detect the anti-NS1 antibody in the aforementioned sample.

In another embodiment of the invention, affinity chromatography is used to detect anti-NS1 antibodies in the aforementioned samples. Specifically, the non-structural protein 1 of a flavivirus is immobilized on the stationary phase of the chromatography column, and when the sample flows through the chromatography column, the anti-NS1 antibody is specifically adsorbed into the chromatography column. . Afterwards, the anti-NS1 is adsorbed by a suitable buffer. The antibody is shed and the effluent is contacted with NS1 grafted with peroxidase or alkaline phosphatase to interpret the results by means of a peroxidase color reaction or an alkaline phosphatase color reaction.

In still another embodiment of the present invention, the antigen-magnetic beads method is used to detect the anti-NS1 antibody in the aforementioned sample. The basic principle of the antigen magnetic bead method as contained in the academic literature published by Khan et al. in 2006 (Imran H. Khan et al., CLINICAL AND VA CCINE IMMUNOLOGY , Vol. 13, No. 1, 2006) can be utilized. Detection was carried out by capturing the anti-NS1 antibody by the NS1 antigen using a similar design logic as described above for affinity chromatography.

In the spirit of the enhanced sensitivity of the present invention for detecting anti-NS1 antibodies, the present invention provides a kit for detecting flavivirus infection. The kit of the present invention comprises: an antibody removal unit for removing an antibody against a flavivirus envelope protein of the present invention; and a detection unit for a non-structural protein 1 antibody against flavivirus.

The detection unit of the antibody removal unit and the anti-viral protein non-structural protein 1 antibody may be an immunoassay device, an affinity chromatography column, an antigen magnetic bead device, or a combination thereof, respectively, which are subjected to immunoassay respectively. , affinity chromatography, or the principle of antigen magnetic beads. The principles of the aforementioned immunoassay, affinity chromatography, or antigen magnetic bead method are as described in the preceding paragraphs.

For example, the antibody removal unit may be an antigen-trapping enzyme immunoassay device comprising a membrane protein using a flavivirus; the antibody removal unit may be a affinity chromatography column, and the stationary phase comprises a flavivirus The envelope protein; the antibody removal unit may be an antigen magnetic bead device, and the surface of the magnetic beads used is fixed with a yellow virus envelope protein.

For example, the detection unit of the non-structural protein 1 antibody against flavivirus may be a pair of flavivirus non-structural protein 1 specific IgM antibody capture type enzyme immunoassay device, and a pair of flavivirus non-structural protein 1 specific ones. An IgG antibody-trapping enzyme immunoassay device, or a combination thereof; the detection unit of the anti-tumor antibody of the non-structural protein 1 may be a stationary phase of a affinity chromatography column, comprising a non-structural protein 1 of a flavivirus; The detection unit of the non-structural protein 1 antibody against flavivirus may be an antigen magnetic bead device, and the surface of the magnetic beads used is fixed with a non-structural protein 1 of flavivirus.

The results of the present invention and specific embodiments will be described in the following examples in order to facilitate the understanding of the invention. It is to be noted that the following is merely exemplary and should not be construed as limiting the scope of the invention.

Example 1: Comparison of the effect of the pre-adsorption step on the detection of anti-NS1 antibodies. [blood sample]

The serum samples used in this experiment were collected from the Diagnostic and Reference Laboratory of the Department of Arthropod Media Virus Diseases of the US Department of Diseases, Inc. (Arboviral Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers). For Disease Control and Prevention). Serum samples were collected from West China from 1999 to 2003 by 90% plaque reduction neutralization test with West Nile virus (WNV; n=21), St. Louis encephalitis virus (SLEV; n=6), or alpha arterial vector virus (alphaviruses; n=12) neutralizing antibody titers (NT antibody titers). Serum samples confirmed to be infected with DENV (n=24) or JEV (n=16) were collected from national serum samples provided by the Center for Disease Control in Taiwan.

[In vitro production of (type) virus particles]

In this experiment, COS-1 cells (ATCC CRL 1650; Manassas, VA) were used to prepare virus-like particles (VLPs) and water-soluble NS1 proteins (sNS1) required for subsequent experiments in vitro . ). Briefly, a plastid with a gene sequence that can be translated into a flavivirus envelope protein and a pre-membrane protein, or a non-structural protein, is transferred to COS-1 cells (ATCC CRL 1650; Manassas, VA). COS-1 cells are expressed and secreted with the desired (type) viral particles and sNS1 in the culture broth. The culture broth was collected and the (type) virus particles and sNS1 were purified. Since the envelope protein is the outermost part of the structure of the flavivirus, the (viral) virus particles composed of the envelope protein and the front membrane protein expressed by COS-1 cells are immunologically equivalent to the yellow virus particles. The membrane protein.

[Pre-adsorption step]

In this example, anti-E antibodies in serum samples were removed by antigen capture enzyme immunoassay. Take the serum sample of JEV infection as an example. Please refer to the first figure. Fix the rabbit anti-JEV VLP antibody (11) to the bottom of the ELISA test disc (10). ), and then, the JEV will be made in vitro. The (type) virus particles (12) were mixed with the diluted serum sample (dilution factor 1:4000), placed in an ELISA test tray, and placed at 37 ° C for 60 minutes to make anti-E in the serum sample. The antibody (13) sufficiently reacts with the immune complex formed by the antigen of the viroid-like particle (12), and is caught by the polyclonal antibody. Finally, excess serum samples from the test tray are removed for subsequent testing.

The above steps were carried out by using multiple antibodies against rabbit anti-WNV (viral) virus particles and WNV (viral) virus particles prepared in vitro to pre-adsorb anti in WNV-infected serum samples (dilution factor 1:1000). -E antibody for subsequent testing.

[Check the effect of the pre-adsorption step]

This experiment was conducted to confirm whether the aforementioned pre-adsorption step of the present invention did remove the anti-E antibody in the infected serum sample. Taking the JEV-infected serum sample experiment as an example, the ELISA test disc after the pre-adsorption step was obtained, and the JEV-infected mouse high-immune ascites (MHIAF) and anti-grafted with wasabi peroxidase (HRP) were respectively added. Human IgM/IgG antibody was allowed to react at 37 ° C for 60 minutes. Subsequently, the addition of horseradish peroxidase substrate (3,3'5,5'-tetramethylbenzidine) coloring, and the spectrophotometer reading the absorbance at OD _450.

The experimental results are shown in the second figure. The ELISA test plate of the serum sample (Normal Human Serum; NHS) treated with healthy individuals was reacted with the JEV-infected mouse high-immunified ascites and the anti-human IgM/IgG antibody, respectively, and the absorbance values of the obtained OD ₄₅₀ were respectively 1.08, 0.4, and 0.1. The OD ₄₅₀ of the ELISA test disk treated with the serum sample of the infected JEV was 2.64, 1.22, and 0.32, respectively, which showed a significant increase, which means that the pre-adsorption step of the present invention does effectively anti-antigen in the serum sample. E antibody removal.

[Detecting anti-NS1 antibodies in blood samples]

In this experiment, anti-NS1 antibodies were detected using blood samples that were not subjected to the aforementioned pre-adsorption steps and subjected to the aforementioned pre-adsorption steps, and the differences were compared. This experiment will use the NS1-specific IgM antibody-trapping enzyme immunoassay (NS1-specific IgM-capture ELISA) and the non-structural protein-1-specific IgG antibody-capture enzyme immunoassay (NS1-specific IgG-capture ELISA). ), serum samples infected with JEV, WNV, and DENV were separately detected.

Taking a serum sample infected with JEV as an example, first, an antibody against goat anti-human IgM was immobilized at the bottom of the ELISA test disk. Next, a diluted serum sample (dilution factor 1:4000; a serum sample including JEV-infected serum samples as a control group and a healthy individual) was placed, and allowed to react at 37 ° C for 120 minutes. Then, NS1 of JEV prepared in vitro was introduced and allowed to react at 37 ° C for 60 minutes. Thereafter, JEV-infected murine hyperimmune ascites (MHIAF; diluted 1:4000) was added and allowed to react at 37 ° C for 60 minutes. Subsequently, a goat anti-mouse IgG antibody grafted with wasabi peroxidase was added and allowed to react at 37 ° C for 60 minutes. Finally, the addition of horseradish peroxidase substrate (3,3'5,5'-tetramethylbenzidine) coloring, and the spectrophotometer reading the absorbance at OD _450. Anti-NS1 antibody detection of WNV or DENV-infected serum samples was performed in the same manner, in which NS1 of WNV or DENV prepared in vitro was used, and the dilution factor of the serum samples was changed to 1:1000.

The absorbance of the obtained OD ₄₅₀ was calculated by the following formula, and the P/N ratio (P/N ratio) of each experimental group was converted; wherein the NHS is an abbreviation for a healthy human serum sample (Normal Human Serum). This calculation formula is based on the scientific literature in the field of research (Martin et al.). In order to exclude the non-specific binding effects in the experiment, the P/N≧3 is the same as the convention definition in the field. The result of the read immunological reaction (antibody antigen reaction) is true.

The experimental results are shown in the third figure; (A) is the experimental result of the JEV-infected serum sample, (B) is the experimental result of the WNV-infected serum sample, and (C) is the DENV-infected serum sample. Experimental results (The sample used was infected with a serotype type 2 dengue virus, and a pre-adsorption step was carried out using the envelope protein of the serotype type 2 dengue virus as an antigen).

As can be seen from the third graph (A), since the pre-adsorption step of the present invention was carried out, the P/N ratio obtained by detecting anti-NS1 IgM in the serum sample infected with JEV was raised from 2.8 to 17.2, and the anti-NS1 IgG was detected. The P/N ratio increased from 0.82 to 7.71. As is apparent from the third diagram (B), since the pre-adsorption step of the present invention is carried out, the P/N ratio obtained by detecting anti-NS1 IgM can be raised from 1.24 to 6.76. Although the P/N ratio obtained by detecting anti-NS1 IgG in serum samples infected with WNV did not change significantly, it was judged that the cause of WNV infection was that IgG usually appeared in a large amount in the late infection or more serious infection. Further, in the test of DENV, it is known from the third diagram (C) that the present invention is carried out The pre-adsorption step increased the P/N ratio from anti-NS1 IgM from 1.8 to 4.2, while the P/N ratio from anti-NS1 IgG was increased from 1.62 to 4.71. Therefore, the results of this experiment show that the concept of the present invention incorporating the pre-adsorption step does enhance the detection of anti-NS1 antibodies.

Example 2: The pre-adsorption step of the present invention was carried out by cross-administering antigens of different flaviviruses.

Flaviviruses include Japanese encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus, and dengue virus, which cause various diseases of human diseases. The infections of these viruses all have similar symptoms and are not easily resolved at the beginning of the infection. Therefore, this example is intended to test whether the antigens of various flaviviruses can be cross-administered in the pre-adsorption step of the present invention.

In this example, 16 JEV-infected or WNV-infected serum samples were obtained, and the pre-adsorption steps of the present invention were carried out using JEV and WNV (viral) virus particles prepared in vitro, respectively, and then observed for subsequent anti- The effect of detection of NS1 antibodies. The manner in which the pre-adsorption step is carried out and the subsequent detection steps of the anti-NS1 antibody are all described in the first embodiment.

The fourth panel presents experimental results of JEV-infected serum samples pre-adsorbed with JEV and WNV (class) virus particles, respectively; (A) is a pre-adsorption step using JEV (class) virus particles, and then anti-antigen detection -P1 ratio of NS1 IgM; (B) pre-adsorption step using JEV (class) virus particles, and then P/N ratio of anti-NS1 IgG; (C) pre-preparation using WNV (class) virus particles Adsorption step, then detect the P/N ratio of anti-NS1 IgM; (D) use WNV (class) The virus particles were subjected to a pre-adsorption step, and then the P/N ratio of anti-NS1 IgG was measured.

From the data in the fourth graph (A) and (B), the P/N ratio measured for each of the 16 samples is ≧3, that is, the infection is accurately measured. The data in the fourth (C) and (D) shows that there are 93.8% (15 P/N ratios 163 out of 16 samples) and 81.3% (13 P/N ratios among 16 samples). 3) The accuracy of the infection can be measured. In other words, even in patients infected with JEV, the pre-adsorption step of the present invention can be carried out using the WNV antigen to accurately measure the infection.

The fifth panel presents experimental results of WNV-infected serum samples pre-adsorbed with JEV and WNV (class) virus particles, respectively; (A) is a pre-adsorption step using WNV (class) virus particles, and then anti-antigen detection - P1 ratio of NS1 IgM; (B) pre-adsorption step using WNV (class) virus particles, and then P/N ratio of anti-NS1 IgG; (C) pre-preparation using JEV (class) virus particles The adsorption step is followed by detection of the P/N ratio of anti-NS1 IgM; (D) is the pre-adsorption step using JEV (class) virus particles, and then the P/N ratio of anti-NS1 IgG is detected.

From the data in the fifth (A) and (B), the P/N ratio of 13 and 11 samples in each of the 16 samples is ≧3, and the accuracy is 86.7% and 73.3%, respectively. The data in the fifth (C) and (D) data shows that when using JEV (class) virus particles for the pre-adsorption step, there are 40% (6 of 16 samples, P/N ratio ≧3) and 0%. The accuracy of the 16-sample P/N ratio <3 can be measured for WNV virus infection.

From the above experimental results, it is known that in the case of infection with JEV, no pre-adsorption step is performed using JEV or WNV (viral) virus particles. Affects the detection of subsequent anti-NS1 antibodies. In the case of WNV infection, although the pre-adsorption step of JEV (type) virus particles was used, the anti-NS1 IgG for detecting WNV infection was not practical, but the anti-NS1 IgM for detecting WNV infection still had 40%. Accuracy. That is to say, if the condition of a patient shows the possibility of infection with flavivirus, in principle, the pre-adsorption step with other antigens of flavivirus can effectively improve the antibody detection of the subsequent anti-NS1 IgM; and among them, WNV The antigen is most suitable for the detection of infections of various flaviviruses.

Example 3: Application of the method of the invention to dengue viruses of different serotypes.

It is currently known that there are four serotypes of dengue virus, and their clinical symptoms are very similar. Therefore, it must be confirmed that the pre-adsorption step of the method of the present invention is different in the case of an infected serotype of an unknown infected person. Whether the antigen still has an effect. Accordingly, the present embodiment employs three samples: ID#100 (DENV type-1), ID#56 (DENV type-2), ID#76 (DENV type-2), and is as contained in the first embodiment. The method of separately testing the antigens using different serotypes or a combination thereof for the pre-adsorption step, whether the P/N ratio of the subsequent anti-NS1 antibody detection can still be improved.

Please refer to Figure 6 A1 and A2. In sample ID#100, no matter which serotype antigen is used for the pre-adsorption step, the detection efficiency of anti-NS1 antibody can be effectively improved; in addition, the second or third is used. The pre-adsorption step of the serotype antigen has the most significant effect, and obvious effects can be observed whether used alone or in combination with antigens of other serotypes. Similar experimental results are also available from sample ID#56M (p. Six figures B1 and B2) and sample ID #76 are shown in the sixth figure C1 and C2). In general, in the detection of dengue infection, it is not necessary to confirm the serotype of the infected patient in advance, and the detection efficiency of the anti-NS1 antibody can be effectively improved by the method of the present invention.

Example 4: Comparison of the detection method of the present invention and the conventional detection method for anti-E antibody (JEV & WNV).

The E-MAC-ELISA for anti-E antibodies is currently a method of detection of arthropod-mediated viral infections recommended by the International Health Organization. This example is intended to compare the effects of the method of the present invention with the conventional E-MAC-ELISA assay (without the preadsorption step of the present invention). In this example, a total of 185 serum samples of individuals confirmed to be infected with flavivirus arthropod-mediated virus were obtained by the method of the present invention (using JEV-NS1 antigen or WNV-NS1 antigen) and the conventional E-MAC-ELISA method ( The test was performed using JEV-VLP antigen or WNV-VLP antigen, and the test results were recorded as shown in Table 1 below (P/N≧3 was defined as positive for detection, ie, infection was detected).

There were 16 serum samples infected with JEV in this experiment. The results of the NS1-MAC-ELISA using the method of the present invention were all 16 samples were detected (16/16, 16 samples, the lowest P/N ratio was 4.07, the highest was 23.53, and the average was 16.5). The results of the NS1-GAC-ELISA using the method of the present invention were all 16 samples (16/16, 16 samples, the lowest P/N ratio was 4.15, the highest was 13.23, and the average was 9.42). The number of detected E-MAC-ELISA and E-GAC-ELISA was 16/16 and 15/16, respectively.

In this experiment, two batches of serum samples infected with WNV were obtained. There are 70 and 25 samples. In the first 70 samples, the detection of the conventional E-MAC-ELISA was 70/70, and the detection of the E-GAC-ELISA was 28/70. The detection of the NS1-MAC-ELISA of the present invention was 65/70 (the lowest P/N ratio was 1.07, the highest was 18.9, the average was 7.32 in 70 samples), and the NS1-GAC-ELISA was detected as 30/70 (70). In the samples, the P/N ratio is at least 0.59, the highest is 10.9, and the average is 3.32).

In the second 25 samples of WNV, the detection of the conventional E-MAC-ELISA was 20/25, and the detection of the E-GAC-ELISA was 2/25. The NS1-MAC-ELISA of the present invention was detected as 24/25 (25 samples, the lowest P/N ratio was 1.12, the highest was 14.11, the average was 6.16), and the NS1-GAC-ELISA was detected as 10/25 (25). In the sample, the P/N ratio is at least 1.24, the highest is 7.23, and the average is 2.94).

In addition, the method of the present invention does not detect infections other than viruses infected with flaviviruses (such as Hanta virus infection (HTN) and rheumatic fever (RF) patients in Table 1), and it is apparent that the present invention has detection of flavivirus infection. Excellent specificity, with few false positive results.

Next, the obtained experimental data is analyzed by receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity of the conventional method and the method of the present invention. The results obtained are shown in Table 2 below.

In the samples infected with JEV, both the conventional MAC-ELISA and the NS1-MAC-ELISA of the present invention achieved 100% sensitivity and 88.9% specificity. The NS1-GAC-ELISA of the present invention has a sensitivity of 100% and a specificity of 96%, which is superior to the sensitivity of 93.7% of the conventional GAC-ELISA and the specificity of 92.9%. In the sample infected with WNV, the abuse Both the method and the method of the present invention are not far from sensitivity and specificity in both MAC-ELISA and GAC-ELISA.

Table 3 summarizes the samples that are inconsistent with the detection of the method of the present invention.

In sample ID #183, ID #224, ID#WHO PR 1148, the MAC-ELISA of the method of the present invention measured the result of no infection, while the other three tests all detected positive. In addition to these three samples, the conventional methods and the methods of the present invention are very similar in both MAC-ELISA and GAC-ELISA.

The sample ID #J9300198 is a 52-year-old JEV patient whose blood sample was collected on the 14th day after the onset of symptoms. This sample was not detected in the GAC-ELISA assay of the conventional method, and the possible reason is that the E protein is still in production. Similar to this sample, seven other samples were not detected in the GAC-ELISA test of the conventional method, but were positive in the other three assays.

Only one sample (ID #214) was not detected in the GAC-ELISA of the method of the invention, but was positive in the other three assays. Accordingly, the GAC-ELISA of the method of the invention has the best detection accuracy.

Example 5: Comparison of the detection method of the present invention with the conventional detection method for anti-E antibody (DENV).

As in the fourth embodiment, this example is intended to test the difference between the method of the present invention and the conventional method in the detection of dengue infection. A total of 36 serum samples (covering acute phase serum and convalescent serum) were obtained in the present invention and tested in the same manner as described in Example 4.

In the present example, it was first confirmed whether the pre-adsorption step of the present invention can enhance the detection efficiency of the anti-NS1 antibody in the infection state in the acute phase and the recovery phase. Please refer to the first example for the test method. In this test, the pre-adsorption step was carried out using Type 2 and Type 3 DENV envelope viruses as antigens. Experimental Results As shown in the seventh and eighth figures, the pre-adsorption step of the present invention significantly improved the detection efficiency of the anti-NS1 antibody in the acute phase sample (seventh image) and the recovery phase sample (eighth image). .

In addition, in comparison with the conventional detection method, the test results showed that among the 36 samples, the detection method using the anti-E antibody method detected 26 positive infections (detection rate: 72.2%), and the present invention detected 25 were positive for infection (detection rate: 69.4%). By calculation, the correlation between the method of the present invention and the conventional anti-E antibody detection method is 96.2%, which represents that the method of the present invention is comparable to the detection ability of the anti-E antibody detection method recommended by the International Health Organization.

in conclusion

Summarizing the above experiments, the sensitivity and specificity of the method of the present invention is sufficient to match the E-MAC/GAC-ELISA recommended by the International Health Organization. In addition, since NS1 is the only non-structural protein released by the infected virus in the flavivirus, the anti-NS1 antibody is the detection target. The antibody in a body can be distinguished by the infection or by the vaccine, and the value of the clinical application is better than that of the anti-E antibody as the detection method.

It will be apparent to those skilled in the art that various changes can be made in accordance with the embodiments of the present invention without departing from the spirit of the invention. Therefore, it is to be understood that the invention is not limited by the scope of the invention, and is intended to cover the modifications of the spirit and scope of the invention.

Claims (24)

A method for enhancing antibody detection, wherein the antibody is an antibody against a non-structural protein 1 of flavivirus; and the method comprises: removing an antibody in the sample before detecting an antibody against the non-structural protein 1 of the anti-flavor virus described above; An antibody to the envelope protein of the flavivirus. The method of claim 1, wherein the step of removing the antibody of the anti-flavivirus envelope protein of the present invention comprises using a flavivirus envelope protein as an antigen to capture the aforementioned anti-flavivirus envelope protein Antibodies. The method of claim 1, wherein the method is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof. The method of claim 3, wherein the immunoassay is an antigen capture enzyme immunoassay. A method for detecting a flavivirus infection comprising the steps of: (i) removing an antibody against a fluffy protein of an anti-flavivirus; and (ii) detecting an antibody against the non-structural protein 1 of the flavivirus in the aforementioned sample. The method of claim 5, wherein the step (i) comprises using an envelope protein of a flavivirus as an antigen to capture an antibody against a flavivirus envelope protein in the aforementioned sample. The method of claim 5, wherein the step (i) is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof. The method of claim 7, wherein the immunoassay is an antigen capture enzyme immunoassay. The method of claim 5, wherein the step (ii) is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof. The method of claim 9, wherein the immunoassay is an antibody-trapping enzyme immunoassay. The method according to claim 10, wherein the antibody-trapping enzyme immunoassay is: an IgM antibody-capturing enzyme immunoassay for a non-structural protein 1 of a flavivirus, and a non-structural protein 1 for a flavivirus A specific IgG antibody capture enzyme immunoassay, or a combination thereof. The method of claim 5, wherein the step (ii) is performed by a peroxidase color reaction or an alkaline phosphatase color reaction to interpret the result of the foregoing detection. The method of claim 5, wherein the sample is blood of an individual to be determined. The method of claim 5, wherein the yellow virus is Japanese encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus, or dengue virus. The method of claim 6, wherein the flavivirus is a dengue virus, and the envelope protein of the aforementioned flavivirus as an antigen is a envelope protein and a serotype of a serotype type 2 dengue virus. The envelope protein of dengue virus of type 3, or a combination thereof, or a combination thereof with a envelope protein of dengue virus of other serotypes. A kit for detecting a flavivirus infection, comprising: an antibody removal unit for removing an antibody against a flavivirus envelope protein; and A detection unit for a non-structural protein 1 antibody against flavivirus. The kit of claim 16, wherein the antibody removal unit comprises a envelope protein using a flavivirus as an antigen. The kit of claim 16, wherein the antibody removal unit is a first immunoassay device, a first affinity chromatography column, a first antigen magnetic bead device, or a combination thereof. The kit of claim 18, wherein the first immunoassay device is an antigen capture type enzyme immunoassay device. The kit of claim 16, wherein the detection unit of the non-structural protein 1 antibody against flavivirus is a second immunoassay device, a second affinity chromatography column, and a second antigen magnetic Bead device, or a combination thereof. The kit of claim 20, wherein the second immunoassay device is a pair of flavivirus non-structural protein 1 specific IgM antibody capture type enzyme immunoassay device, a pair of flavivirus non-structural proteins A specific IgG antibody-trapping enzyme immunoassay device, or a combination thereof. The kit according to claim 16, wherein the detection unit of the anti-tumor non-structural protein 1 antibody of the flavivirus is judged by a color reaction of a peroxidase or a color reaction of an alkaline phosphatase. The kit of claim 16, wherein the yellow virus is Japanese encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus, or dengue virus. The kit of claim 17, wherein the flavivirus is a dengue virus, and the envelope protein of the flavivirus as the antigen is: a membrane protein of serum of a serotype type 2 dengue virus, serum Type 3 A membrane protein of a leather virus, or a combination thereof, or a combination thereof with a envelope protein of a dengue virus of other serotypes.