JP2010019739A - Inspection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inspection device for rapidly inspecting a strong toxic type influenza A virus. <P>SOLUTION: A first inspection part and a second inspection part are arranged on a strip centering around a dripping part, and an antibody low in reactivity with respect to strong toxic type influenza A nucleoprotein is used. When both of the first inspection part and the second inspection part develop a color, it is shown that a non-strong toxic type influenza A virus is judged to be positive, and the strong toxic type influenza A virus is judged to be negative. When one of the first inspection part and the second inspection part develops a color and the coloration of the other one of them is weaker than that of one of them, it is shown that the strong toxic type influenza A virus is judged to be positive. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an inspection apparatus capable of rapidly inspecting a highly toxic type A influenza virus.

  Influenza virus is a pathogen that infects and causes influenza in many animals, including humans. When an influenza virus infects humans, after several days of incubation, symptoms such as fever, headache, pain in various parts of the body including joints, weakness, cough, and sore throat are caused. In addition, bronchitis, pneumonia, otitis media and the like are often accompanied, and encephalopathy, myositis, myocarditis, etc. may be caused, resulting in a serious condition. In particular, the elderly, infants, etc. who are poor in physical fitness may be fatal.

  Two types of influenza viruses are known, type A and type B. Of these, type A influenza virus is derived from hemagglutinin (hemagglutinin: HA; H1-H15) and neuramitase NA (N1-N9). There are many subtypes of and many mutant strains can be generated, causing a global pandemic.

  Influenza A virus infects not only humans but also animals such as birds, pigs, horses, minks, and whales. Usually, humans are rarely infected with influenza viruses that infect other animals, and generally humans are infected with A Hong Kong type (H3N2), A Soviet type (H1N1), and the like.

  However, due to mutation of the virus, a virus that has not previously infected humans may be transmitted from animals to humans, or even from humans to humans.

  In particular, in Southeast Asia, a case has been reported in which a highly toxic influenza A virus (H5N1) has been transmitted to humans and many deaths have occurred. Drugs and treatments have been improved for non-virulent type A influenza viruses, but when humans are infected with highly virulent type A influenza viruses, it is difficult to cope with and the mortality rate becomes extremely high.

  A gene amplification method (PCR method) has been conventionally used as a method for practically examining infection with a highly toxic influenza A virus having a high mortality rate (for example, Patent Document 1: Special Table 2008-502362). However, according to this, complicated work is required, and it takes 6 hours to 2 days for inspection alone.

  If the inspection takes a long time in this way, it is not practical. If a patient suspected of having an infection is infected with a highly toxic influenza A virus, symptoms are likely to progress during the examination period and there is a high risk of becoming serious. It is possible that Furthermore, when a highly toxic influenza A virus has infectivity from person to person, the infection spreads from this patient to others, and as a result, the spread of infection is also a concern.

  Now, an inspection apparatus including a reagent by an immunochromatography method is widely used for, for example, a pregnancy test or an infectious disease (hepatitis virus, adenovirus, etc.) test. Furthermore, an inspection apparatus including an immunochromatographic reagent capable of inspecting influenza A virus and influenza B virus has already been put into practical use.

However, there is no known method that can quickly identify a highly toxic influenza A virus in a short time.
Japanese translation of PCT publication No. 2008-502362 JP 2008-14751 A JP 2006-118936 A JP 2000-2533876 A JP-T-2005-519619 JP 2008-83024 A

  Then, an object of this invention is to provide the test | inspection apparatus which can test | inspect a highly toxic type A influenza virus rapidly.

  The inspection apparatus according to the present invention includes a strip, a dripping portion disposed at a central portion of the strip, a first water-absorbing filter paper disposed at a first end portion of the strip, and a first end extending from the dripping portion to the first end portion. A first application section disposed on the downstream side in the flow direction; a first inspection region having a first inspection section disposed between the first application section and the first water-absorbing filter paper; and a second end portion of the strip. The second water absorbent filter paper, the second application part disposed downstream in the second flow direction from the dropping part to the second end, and the second water absorbent filter paper disposed between the second application part and the second water absorbent filter paper. An inspection apparatus comprising a second inspection region having an inspection unit, and when neither the first inspection unit nor the second inspection unit is colored, the non-toxic type A influenza virus and the strong type A It is shown that it is negative for any of the influenza viruses. When both colors are colored, it is shown that it is positive for non-toxic influenza A virus and negative for highly virulent influenza A virus, and one of the first and second test units is When it is colored and the other color is weaker than one, it indicates that it is positive for the highly virulent influenza A virus.

  Preferably, the test apparatus of the present invention is positive for the highly toxic influenza A virus when one of the first test section and the second test section is colored and the other is not colored. .

  Preferably, the inspection apparatus according to the present invention includes a strip, a drip portion disposed at a central portion of the strip, a first absorbent filter disposed at a first end portion of the strip, and a first end portion rather than the dripping portion. A first coating unit that is disposed downstream of the first flow direction and is labeled with a first antibody that is reactive with a nucleoprotein antigen of influenza A virus; A first test region that is disposed between the water-absorbing filter paper and has a first test unit on which a second antibody reactive to a nucleoprotein antigen of influenza A virus is immobilized; and a second end of the strip A second water-absorbing filter paper, and a third antibody that is arranged downstream of the dropping portion and reaches the second end in the second flow direction and is reactive to the nucleoprotein antigen of influenza A virus. To keep it flowable A second test unit disposed between the coating unit, the second coating unit, and the second water-absorbing filter paper, on which a fourth antibody reactive to a nucleoprotein antigen of influenza A virus is solid-phased; An inspection device comprising an inspection region, wherein one of the first, second, third, and fourth antibodies is more reactive with a highly toxic influenza A virus than other antibodies. Low.

  Preferably, the inspection apparatus of the present invention is an inspection apparatus including a first strip and a second strip, and the first strip includes a first dripping portion and a first dripping portion. 1st water absorption filter paper arrange | positioned in the position away in 1 flow direction, and 1st antibody which is arranged in the 1st flow direction downstream from the 1st dripping part, and is reactive with the nucleoprotein antigen of influenza A virus A first application part that retains the one labeled to be flowable, and a second antibody that is disposed between the first application part and the first water-absorbing filter paper and is reactive to a nucleoprotein antigen of influenza A virus. A first inspection region having a first inspection portion to be solid-phased, and the second strip is disposed at a position away from the second dropping portion and the second dropping portion in the second flow direction. 2 water-absorbing filter paper and the second flow direction downstream side of the second dripping part A second coating part that holds the labeled third antibody that is reactive to the nucleoprotein antigen of influenza A virus and is disposed between the second coating part and the second water-absorbing filter paper. And a second examination region having a second examination part on which a fourth antibody reactive to the nucleoprotein antigen of influenza A virus is solid-phased, and the first, second, third and fourth antibodies Of these, one antibody is less reactive to the highly toxic influenza A virus than the other antibodies.

  Preferably, in the inspection apparatus of the present invention, the strip is held by the case, and the case includes a funnel portion that opens corresponding to the dripping portion, and an observation window corresponding to the inspection region.

  According to the test apparatus of the present invention, the positive for non-virulent type A influenza virus due to the intensity of coloration and the toxic type of influenza A influenza in the same test time as the test apparatus containing the reagent by immunochromatography. It can be clearly determined by distinguishing from positive by virus.

  In a short period of time, it is possible to identify positives due to highly toxic influenza A virus, so that the patient can be isolated and the spread of toxic influenza A virus can be prevented, and patients can be treated intensively at an early stage. Can be improved.

(Background of development)
First, the background until the present inventors have completed the present invention will be described.

  The present inventors considered that when examining the presence or absence of influenza A virus infection, an antigenic protein with few mutations should be targeted so that various types of viruses can be detected. Specifically, since hemagglutinin HA and neuraminitase NA itself have many subtypes, they are not suitable as targets. On the other hand, since nucleoprotein (NP) has few mutations, it is considered suitable for the target.

Therefore, the present inventors selected two types of influenza A viruses, H1N1 and H3N2, that are supposed to infect humans, as antigens among influenza A viruses. In addition, as an anti-influenza nucleoprotein monoclonal antibody,
Ab1 (manufactured by Fitzgerald, USA, catalog number: No. 10-I50, clone number: M322221) and Ab2 (manufactured by Fitzgerald, USA, catalog number: No. 10-I50, clone number: M2110169)
A first prototype of an inspection apparatus including a reagent by immunochromatography was prepared.

We have several influenza A virus subtypes H1N1 (A / New Caledonia / 20/1999),
H3N2 (A / Panama / 2007/1999),
H3N2 (A / New York / 55/2004) and H3N2 (A / Wyoming / 03/2003)
Were examined using the first prototype, and all obtained equally good reaction results.

Furthermore, the present inventors used the first prototype and confirmed the reactivity of the bird-derived strain, but the same reactivity could be confirmed for most strains of H1 to H15.
H5N1 (A / Hong Kong / 483/97) and H5N1 (A / Viet Nam / 1203/2004)
The reactivity with respect to was found to be extremely low compared to other cases.

  These H5N1 types are highly toxic type A influenza viruses. This result suggests that the combination of Ab1 and Ab2 is weakly reactive to highly toxic H5N1.

As the anti-influenza nucleoprotein monoclonal antibody, the present inventors
Ab3 (manufactured by Bios Pacific, USA, clone number: A60010044P)
Was added, Ab1 and Ab3 were replaced, and a second prototype was produced by a combination of Ab2 and Ab3. The first prototype and the second prototype are the same except that the combination of antibodies is different.

  The present inventors confirmed the reactivity of the bird-derived strain using the second prototype, and H5N1 (A / Hong Kong / 483/97) and H5N1 (A / Viet Nam / 1203/2004). The same reactivity could be confirmed for all strains H1 to H15.

  By summing up the results of the first and second prototypes, it is estimated that Ab1 shows good reactivity with normal influenza A virus but is extremely weak with the highly toxic H5N1 type. The

  In order to further clarify the properties of Ab1, the present inventors performed the following confirmation work.

  A cell culture was prepared by selecting H3N2 (A / New York / 55/2004) as an example of a general influenza A virus, and H5N1 (A / Viet Nam / 1203) as an example of a highly toxic H5N1 type. / 2004) gene recombinant protein was prepared.

Here, the amino acid sequence of H5N1 (A / Viet Nam / 1203/2004) is
MASQGTKRSYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEIRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGYDFEREGYSLVGIDPFRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRVVPRGQLSTRGVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKATNPIVPSFDMNNEGSYFFGDNAEEYDN
And
The amino acid sequence of H3N2 (A / New York / 55/2004) is
MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDHEGRLIQNSLTIEKMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELVLYDKEEIRRIWRQANNGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMVKRGINDRNFWRGENGRKTRSAYERMCNILKGKFQTAAQRAMVDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACAYGPAVSSGYNFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIASNENMDNMGSSTLELRSGYWAIRTRSGGNTNQQRASAGQTSVQPTFSVQRNLPFEKSIIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDN
It is.

  Each of these fabricated products was treated by the following procedure.

  (Treatment 1) 50 μl of Ab1, Ab2, and Ab3 diluted to 1 μg / ml in 50 mM sodium carbonate buffer (pH 9.0) were added to a 96-well plate for ELISA, and allowed to stand at room temperature for 1 hour.

  (Treatment 2) Each hole was washed once with 400 μl PBS (containing 0.02% twin 20. PBS-T) and blocked with 200 μl blocking agent.

  (Treatment 3) Each well was washed once with 400 μl of PBS-T, and then 50 μl of H3N2 antigen and H5N1 antigen solution diluted 20-fold with PBS-T was added and allowed to stand at room temperature for 1 hour.

  (Treatment 4) After washing each well 3 times with 400 μl PBS-T, 50 μl of rabbit antinuclear protein polyclonal antibody diluted to 1 μl / ml with PBS-T was added and allowed to stand at room temperature for 1 hour.

  (Treatment 5) Each well was washed 3 times with 400 μl of PBS-T, 50 μl of horseradish peroxidase-labeled anti-rabbit IgG antibody diluted 5000-fold with PBS-T was added, and allowed to stand at room temperature for 30 minutes.

  (Treatment 6) After washing each well 3 times with 400 μl PBS-T, color was developed by adding peroxidase substrate. After 10 minutes, 1M sulfuric acid was added to stop the reaction, and the ratio was measured at a wavelength of 450 nm using a spectrophotometer. The color was determined.

  As a result of the above treatment, the reactivity as shown in the following table was obtained for each of Ab1, Ab2, and Ab3. In the following table, the reactivity of Ab2 is 100%.

  (Table 1) revealed that Ab1 has extremely low reactivity (1.4%) against H5N1 compared to other antibodies Ab2 and Ab3. As a result of the experiment, it was found that the reaction site of Ab1 recognizes any of the 1st to 188th sites from the N-terminal side of the nucleoprotein amino acid sequence.

  Here, the “low-reactivity antibody” against the highly toxic H5N1 nucleoprotein referred to in the present specification is not limited to Ab1, and is very reactive to H5N1 compared to other antibodies used in combination. If it is low, there is no problem. The “low-reactivity antibody” preferably has a reactivity to H5N1 of 1/10 or less with respect to other antibodies used in combination.

(principle)
Hereinafter, the principle of the inspection apparatus of the present invention will be described with reference to FIGS. To conclude, in short, this principle is based on the fact that a low-reactivity antibody is much less reactive to H5N1 than other antibodies, and that the highly virulent influenza A virus is released in a short time. The point is to identify quickly.

<Pattern 1>
A system for inspecting influenza A virus is prepared as shown in FIG. 1 (a), and a system for inspecting highly toxic influenza A is prepared as shown in FIG. 1 (b). The flow direction of the strips 1 and 2 mainly composed of a long nitrocellulose membrane is set from left to right (arrows N1 and N2) in FIGS. 1 (a) and 1 (b).

  As shown in FIG. 1A, an application part is set on the upstream side in the flow direction N1, and the antibody 6 (Ab3) labeled with, for example, gold colloid 3 is downstream in the flow direction N1 in the application part. It is held so that it can flow with the liquid sample toward the side. The inspection region 10 is set downstream of the application portion in the flow direction N1, and the antibody 5 (Ab2) is solid-phased in the inspection region 10 to form the inspection portion.

  As shown in FIG. 1 (b), an application part is set on the upstream side in the flow direction N2, and a low-reacting antibody 4 (Ab1) having low reactivity with a highly toxic influenza virus is applied to the application part, for example, colloidal gold. The one labeled with 3 or the like is held so that it can flow with the liquid sample toward the downstream side in the flow direction N2. An inspection region 10 is set downstream of the application portion in the flow direction N2, and the antibody 5 (Ab2) is solid-phased in the inspection region 10 to form an inspection portion.

  Here, in the system for inspecting influenza A virus, a combination of antibodies that are not low-reacting antibodies as shown in FIG. 1 (a) is used. In the system for inspecting highly toxic influenza A, FIG. The point is that at least one of the antibodies is a low-reactivity antibody as in b).

"Positive test for non-virulent type A influenza virus"
Now, when the liquid sample contains a non-toxic A-type influenza virus nucleoprotein 8, when the sample liquid reaches the coating part, the nucleoprotein 8 binds to both the antibody 6 and the low-reacting antibody 4. To do. Further, when reaching the examination region 10, the nucleoprotein 8 binds to either the antibody 5 in FIG. 1 (a) or the antibody 5 in FIG. 1 (b).

  Therefore, as shown in FIG. 2 (a), in the system for testing influenza A virus, a sandwich-like conjugate of antibody 5-nucleoprotein 8-antibody 6-label 3 is immobilized, and label 3 is labeled in the test region 10. The coloration due to appears. In addition, as shown in FIG. 2 (b), a sandwich-like conjugate of antibody 5-nucleoprotein 8-low-reactivity antibody 4-label 3 is immobilized even in a system for examining virulent type A influenza virus. The coloration by the marker 3 appears in the inspection area 10.

"Positive test for highly virulent influenza A virus"
Next, in the case where the liquid sample contains the nucleoprotein 7 of highly toxic influenza A virus, when the sample liquid reaches the application part, the nucleoprotein 7 binds to the antibody 6 but has a low reaction. It does not bind to antibody 4. Further, when reaching the examination region 10, the nucleoprotein 7 binds to either the antibody 5 in FIG. 1 (a) or the antibody 5 in FIG. 1 (b).

  Therefore, as shown in FIG. 3 (a), in the system for testing influenza A virus, a sandwich-like conjugate of antibody 5-nucleoprotein 7-antibody 6-label 3 is immobilized and labeled 3 in the test region 10 The coloration due to appears. However, as shown in FIG. 3 (b), in the system for examining highly virulent influenza A virus, the low-reactive antibody 4 and the nucleoprotein 7 do not bind, so the low-reactive antibody 4 is labeled with the label 3. Things will flow further downstream than the inspection region 10. As a result, in the system for inspecting highly virulent influenza A virus, the coloration by the marker 3 does not appear at all, or the coloration becomes very weak.

"Test results by coloration"
From the above description, the following points will be understood.

  (Test result 1) In both the system for testing influenza A viruses and the system for testing highly toxic influenza A viruses, if coloration is observed, the patient is infected with a non-toxic influenza A virus Can be positively determined.

  (Test result 2) Coloration is recognized only in the system for examining influenza A virus, and coloration is weak in the system for examining highly toxic influenza A virus (including the case where no coloration is observed at all). ), A positive determination can be made that the patient is infected with a highly toxic type A influenza virus.

  (Test result 3) If no coloration is observed in both the system for testing influenza A virus and the system for testing highly toxic influenza A virus, the virus is not infected with any influenza A virus. Negative judgment can be made. It should be noted that viruses that are not subject to inspection, such as type B influenza virus, are not subject to determination, but this does not preclude inspection.

  In order to obtain the above test results, it takes almost the same time as a test apparatus including a reagent by a general immunochromatography method. That is, the test result can be obtained in about 15 minutes. The advantage will be clear compared to the previous method, which took longer than 6 hours.

  For example, it is possible to make a quick diagnosis at the bedside of a hospital or the like, and inspecting a traveler at a quarantine station such as an airport so that the spread of infection can be prevented by the water.

  When the above test result 2 is applicable, it can be expected that the patient is immediately isolated to prevent the spread of highly toxic influenza virus. It can also be expected that patients will be treated intensively to improve the patient's prognosis.

  In fact, regarding the virus cultures isolated in Vietnam from 2004 to 2007, the non-virulent type A influenza virus and the highly virulent type A influenza virus were tested with the prototype according to Pattern 1, When the results were confirmed by the PCR method, the results in the following table were obtained.

  It will be understood from Table 2 that it is possible to discriminate between a non-toxic A-type influenza virus and a highly-toxic A-type influenza virus with 100% accuracy.

  Furthermore, when the present inventors tested the prototype according to Pattern 1 while changing the dilution ratio, the results shown in the following table were obtained.

  It will be understood from Table 3 that good test results are obtained when diluted 10 times or diluted 100 times.

  In the above description, the case where the system for inspecting the influenza A virus and the system for inspecting the highly toxic A virus are used in combination. This is advantageous because the inspection results 1 to 3 can be clearly identified.

  However, it is not unforeseen that an emergency such as the spread of a highly toxic influenza A virus may occur. In such a case, it may be the first priority to make testing easier and to isolate patients suspected of infection as soon as possible. In such a case, use only a system that tests for highly toxic influenza A virus, and if no color appears, it is possible to immediately isolate the patient, thus preventing the spread of infection. And are encompassed by the present invention.

<Pattern 2>
As described in Pattern 1, in a system for testing influenza A virus, a combination of antibodies that are not low-reactivity antibodies is used, and in a system for testing highly toxic influenza A, at least one antibody is low. The point is a reactive antibody.

  If this point is provided, various valuations will be established. For example, a system for inspecting influenza A virus is prepared as shown in FIG. 4 (a), and a system for inspecting highly toxic influenza A is prepared as shown in FIG. 4 (b).

  As shown in FIG. 4A, an application part is set on the upstream side in the flow direction N1, and the antibody 5 (Ab2) labeled with, for example, gold colloid 3 is downstream in the flow direction N1 in the application part. It is held so that it can flow with the liquid sample toward the side. An inspection region 10 is set downstream of the application portion in the flow direction N1, and the antibody 6 (Ab3) is solid-phased in the inspection region 10 to form an inspection portion.

  As shown in FIG. 4B, an application part is set on the upstream side in the flow direction N2, and the antibody 5 (Ab2) labeled with, for example, gold colloid 3 is downstream in the flow direction N2 in the application part. It is held so that it can flow with the liquid sample toward the side. A test region 10 is set downstream of the application unit in the flow direction N2, and the low-reacting antibody 4 (Ab1) is solid-phased in the test region 10 to form a test unit.

"Positive test for non-virulent type A influenza virus"
Now, if the liquid sample contains a non-toxic A-type influenza virus nucleoprotein 8, when the sample liquid reaches the application part, the nucleoprotein 8 is converted into the antibody 5 in FIG. It binds to any of the antibodies 5 in (b). Further, when reaching the examination region 10, the nucleoprotein 8 binds to both the antibody 6 in FIG. 4 (a) and the low-reactivity antibody 4 in FIG. 4 (b).

  Therefore, as shown in FIG. 5 (a), in the system for testing influenza A virus, a sandwich-like conjugate of antibody 6-nucleoprotein 8-antibody 5-label 3 is immobilized, and label 3 is labeled in the test region 10. The coloration due to appears. In addition, as shown in FIG. 5 (b), a sandwich-like conjugate of low-reactivity antibody 4-nucleoprotein 8-antibody 5-label 3 is immobilized even in a system for examining virulent type A influenza virus. The coloration by the marker 3 appears in the inspection area 10.

"Positive test for highly virulent influenza A virus"
Next, in the case where the liquid sample contains the nucleoprotein 7 of highly toxic A influenza virus, when the sample liquid reaches the application part, the nucleoprotein 7 is converted into the antibody 5 in FIG. It binds to any of the antibodies 5 of 4 (b). Further, when reaching the examination region 10, the nucleoprotein 7 binds to the antibody 6 in FIG. 4 (a), but does not bind to the low-reactivity antibody 4 in FIG. 4 (b).

  Therefore, as shown in FIG. 6 (a), in the system for testing influenza A virus, a sandwich-like conjugate of antibody 6-nucleoprotein 7-antibody 5-label 3 is immobilized, and label 3 The coloration due to appears. However, as shown in FIG. 6 (b), in the system for examining highly virulent influenza A virus, the low-reactive antibody 4 and the nucleoprotein 7 do not bind, so that the antibody 5 is labeled with the label 3. What the nucleoprotein 7 is bound to is flowed further downstream than the inspection region 10. As a result, no coloration due to the label 3 appears in the system for testing highly toxic influenza A virus.

  In the pattern 1, the nucleoprotein 7 binds to the antibody 5 and is fixed to the examination region 10. However, in the pattern 2, the nucleoprotein 7 flows away together with the antibody 5. However, with regard to the presence or absence of coloration in the inspection region 10, pattern 1 and pattern 2 have the same result.

"Test results by coloration"
From the above description, the following points will be understood in the pattern 2 as in the case of the pattern 1.

  (Test result 1) In both the system for testing influenza A viruses and the system for testing highly toxic influenza A viruses, if coloration is observed, the patient is infected with a non-toxic influenza A virus Can be positively determined.

  (Test result 2) Coloration is observed only in the system for testing influenza A virus, and coloration is weak in the system for testing highly virulent influenza A virus (including cases where no coloration is observed at all). ), A positive determination can be made that the patient is infected with a highly toxic type A influenza virus.

  (Test result 3) If no coloration is observed in both the system for testing influenza A virus and the system for testing highly toxic influenza A virus, the virus is not infected with any influenza A virus. Negative judgment can be made. It should be noted that viruses that are not subject to inspection, such as type B influenza virus, are not subject to determination, but this does not preclude inspection.

  In order to obtain the above test results, it takes almost the same time as a test apparatus including a reagent by a general immunochromatography method. That is, the test result can be obtained in about 15 minutes. The advantage will be clear compared to the previous method, which took longer than 6 hours.

  For example, it is possible to make a quick diagnosis at the bedside of a hospital or the like, and inspecting a traveler at a quarantine station such as an airport so that the spread of infection can be prevented by the water.

  When the above test result 2 is applicable, it can be expected that the patient is immediately isolated to prevent the spread of highly toxic influenza virus. It can also be expected that patients will be treated intensively to improve the patient's prognosis.

  The inventors of the present invention performed a comparative test of the prototypes according to Pattern 1 and Pattern 2 while changing the dilution ratio, and obtained the results shown in the following table.

  It will be understood from Table 4 that good test results are obtained when diluted 10 times or diluted 100 times.

  In the above description, the case where the system for inspecting the influenza A virus and the system for inspecting the highly toxic A virus are used in combination. This is advantageous because the inspection results 1 to 3 can be clearly identified.

  However, it is not unforeseen that an emergency such as the spread of a highly toxic influenza A virus may occur. In such a case, it may be the first priority to make testing easier and to isolate patients suspected of infection as soon as possible. In such a case, use only a system for testing highly virulent influenza A virus, and if no color appears, it is possible to immediately isolate the patient, thus preventing the spread of infection. And are encompassed by the present invention.

<Patterns 3, 4>
As variations of the patterns 1 and 2, FIGS. 7 and 8 show an example using the pattern 3 and an example using the pattern 4, respectively. In the patterns 3 and 4, the same results as those of the patterns 1 and 2 are obtained, and the effects are almost the same.

  Of course, the present invention is not limited to the patterns 1 to 4 described above, and various modifications can be made. In the patterns 1 to 4 or other valuations, a combination that can share antibodies as much as possible is preferable in consideration of manufacturing cost and ease of manufacturing. For example, according to the pattern 1, since the antibody solid-phased in the test region 10 is common to the antibody 5 in FIGS. 1A and 1B, it is preferable in manufacturing. However, the present invention is not limited to the case where antibodies are used in common as described above, but includes various valuations.

(Embodiment 1)
Based on the above principle, a preferred embodiment of the present invention will be described below with reference to the drawings.

  The inspection apparatus according to the first embodiment includes a test piece shown in a sectional view in FIG. 9 and a case (see a plan view of FIG. 10) in which the test piece is housed.

  This test piece has a dropping portion 11 into which a liquid sample is dropped at the center in the longitudinal direction. In FIG. 9, a system for inspecting influenza A virus is arranged from the dropping portion 11 toward the left side. A system for inspecting the highly toxic A-type influenza virus is arranged from the section 11 toward the right side.

  When arranged in this manner, the liquid sample is dropped once on the dropping unit 11 and the inspection by the system for inspecting the influenza A virus and the inspection by the system for inspecting the highly toxic influenza A virus are performed in parallel. And can be handled easily.

  That is, the test piece in the first embodiment has a substantially symmetrical layout, and as shown in FIG. 9, the test piece has a dropping portion 11 that is commonly used for the inspection of the two systems at the center.

  On the left and right sides of the dripping section 11, a long nitro is provided in each of the first flow direction N1 (system for inspecting influenza A virus) and the second flow direction N2 (system for inspecting highly toxic influenza A virus). Strips 1 and 2 mainly composed of a cellulose membrane are arranged. The strips 1, 2 may or may not be lined.

  A first application unit 12 is provided downstream of the dropping unit 11 in the flow direction N1 along the first flow direction N1, and the first application unit 12 is immune to the first flow direction N1. It contains labeled particles (for example, colloidal gold) labeled with a reaction component.

  The 1st water absorption filter paper 14 is provided in the downstream end part of the 1st flow direction N1, absorbs an excess sample, and provides the suction force of the arrow N1 direction.

  In the first inspection region 16 set between the first application unit 12 and the first water-absorbing filter paper 14, a line in which a reaction component related to the first flow direction N1 is fixed is arranged. As a result of an immune reaction, a sandwich reactant Is generated, a colored line derived therefrom is exhibited.

  A test result (for example, positive or negative) regarding the first flow direction N1 is determined based on the state of the coloring line (details will be described later). The coloring line includes a first inspection line 18 and a reference line 20. Since the reference line 20 is always colored when the inspection is properly performed, it is understood that the inspection is inappropriate when the color of the reference line 20 is not recognized.

  The antibodies used for the first application unit 12 and the first test line 18 follow the combinations of the antibodies for the test for influenza A virus described in patterns 1 to 4 or valuation thereof.

  A second application part 13 is provided downstream of the dropping part 11 in the flow direction N2 along the second flow direction N2, and the second application part 13 is immune to the second flow direction N2. It contains labeled particles (for example, colloidal gold) labeled with a reaction component.

  The second water-absorbing filter paper 15 is provided at the downstream end portion in the second flow direction N2, absorbs an excessive sample, and provides a suction force in the arrow N2 direction.

  In the second inspection region 17 set between the second application unit 13 and the second water-absorbing filter paper 15, a line in which reaction components relating to the second flow direction N2 are fixed is arranged, and as a result of the immune reaction, a sandwich reactant. Is generated, a colored line derived therefrom is exhibited.

  A test result (for example, positive or negative) regarding the second flow direction N2 is determined based on the state of the coloring line (details will be described later). The coloring line includes a second inspection line 19 and a reference line 21. Since the reference line 21 is always colored when the inspection is properly performed, it is understood that the inspection is inappropriate when the color of the reference line 21 is not recognized.

  The antibodies used in the second application part 13 and the second inspection line 19 follow the combinations of the antibodies for the system for inspecting the highly virulent type A influenza virus described in patterns 1 to 4 or the valuation thereof.

  As shown in FIG. 10, the case 22 of the first embodiment has a funnel portion 23 projecting downward in the center portion in accordance with the position of the dropping portion 11 of the test piece, and the lower end surface of the funnel portion 23 is open. And a sample can be dripped at the dripping part 11 downward through there.

  A transparent first observation window 24 and a second observation window 25 are provided in accordance with the positions of the inspection regions 16 and 17 with the funnel portion 23 as the center.

  Next, the determination method based on the pattern of the colored line will be described with reference to FIGS.

  First, before the liquid sample is dropped onto the dropping unit 11 via the funnel 23, no colored line appears in either the first observation window 24 or the second observation window 25 as shown in FIG.

  (Failure) The test was unsuccessful if either of the reference lines 20 and 21 did not color as shown in FIG. become.

  (Negative) When a liquid sample is dropped and a time of about 15 minutes elapses, only the reference lines 20 and 21 are colored as shown in FIG. 12, and either the first inspection line 18 or the second inspection line 19 is displayed. If no color develops, it is determined that the virus is not infected with influenza A virus (negative).

  When a liquid sample (not positively toxic type A influenza positive) is dropped and a time of about 15 minutes elapses, as shown in FIG. 13, the reference lines 20 and 21 are colored, and the first test line 18 and the first test line 18 When both of the two test lines 19 are colored, it is determined that the patient is infected with a non-strongly virulent type A influenza virus (positive for a non-toxic form of influenza A).

  (Poisonous influenza A positive) When a liquid sample is dropped and a time of about 15 minutes has passed, as shown in FIG. 14, the reference lines 20 and 21 are colored and the first test line 18 is presented. If it is colored but the color of the second test line 19 is weak (including the case where it is not colored at all), it is infected with a virulent type A influenza virus (positive for a virulent virulent type A influenza) It is determined.

  As described above, the liquid sample is dropped once on the dropping unit 11 and only about 15 minutes are waited. Thus, the failure, the negative, the non-virulent type A influenza positive, and the virulent type A influenza positive are clearly identified. It is possible to obtain a test result specified in

  If a test result is positive for highly virulent influenza A, the patient should be immediately quarantined and the patient intensively treated.

(Embodiment 2)
In the first embodiment, a preferred example has been described in which the liquid sample only needs to be dropped once onto the dropping unit 11. However, there are cases where this is not always necessary, and such cases are also included in the present invention.

  That is, as shown in FIG. 15, the test piece shown in FIG. 9 may be separately provided in a system for testing influenza A virus and a system for testing highly toxic influenza A virus. (In order to avoid repetition, FIG. 15 shows only a test piece of a system for inspecting influenza A virus).

  When a plurality of test pieces are used in this way, the case 22 may be shared by two test pieces as shown in FIG. 16, or as shown in FIGS. 17 (a) and 17 (b). In addition, the case 22 may be divided for each test piece.

  In any case, when a plurality of test pieces are used, it is necessary to provide a plurality of funnel portions and dropping portions and drop the liquid sample a plurality of times accordingly.

  However, as shown in FIGS. 16, 17 (a), and 17 (b), if the funnels are close to each other, the labor is hardly changed, and the time until obtaining the inspection result is the same as in the first embodiment. It doesn't extend significantly compared to this. Therefore, it can be said that the examples shown in FIGS. 16, 17A, and 17B are practically sufficient.

  Furthermore, when a highly virulent influenza A virus is prevalent, it may be the first priority to isolate a patient suspected of being infected quickly. In such a case, if only the system for inspecting the highly toxic influenza A virus shown in FIG. 17 (b) is used and no coloration appears, there is a certain benefit even if the patient is immediately isolated. It is believed that there is. This is also included in the present invention.

(A) Schematic diagram of test piece (A type) according to pattern 1 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 1 of the present invention (A) Schematic diagram of test piece (A type) according to pattern 1 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 1 of the present invention (A) Schematic diagram of test piece (A type) according to pattern 1 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 1 of the present invention (A) Schematic diagram of test piece (A type) according to pattern 2 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 2 of the present invention (A) Schematic diagram of test piece (A type) according to pattern 2 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 2 of the present invention (A) Schematic diagram of test piece (A type) according to pattern 2 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 2 of the present invention (A) Schematic diagram of test piece (A type) according to pattern 3 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 3 of the present invention (A) Schematic diagram of test piece (A type) according to pattern 4 of the present invention (b) Schematic diagram of test piece (strongly toxic type) according to pattern 4 of the present invention 1 is a longitudinal sectional view of a test piece according to Embodiment 1 of the present invention. The top view of the case in Embodiment 1 of this invention The top view of the inspection apparatus in Embodiment 1 of this invention The top view of the inspection apparatus in Embodiment 1 of this invention The top view of the inspection apparatus in Embodiment 1 of this invention The top view of the inspection apparatus in Embodiment 1 of this invention Longitudinal sectional view of a test piece in Embodiment 2 of the present invention The top view of the inspection apparatus in Embodiment 2 of this invention (A) Plan view of the inspection apparatus according to the second embodiment of the present invention (b) Plan view of the inspection apparatus according to the second embodiment of the present invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 2 Strip 3 Label | marker 4 Low reaction antibody 5, 6 Antibody 7, 8 Nucleoprotein 10 Inspection area | region 11 Drop part 12 1st application part 13 2nd application part 14, 15 Water absorption filter paper 16 1st inspection area 17 2nd inspection area 18 First inspection line 19 Second inspection lines 20 and 21 Reference line 22 Case 23 Funnel portion 24 First observation window 25 Second observation window

Claims (6)

A strip, a dripping portion disposed at a central portion of the strip, a first water-absorbing filter paper disposed at a first end portion of the strip, and a first flow direction downstream from the dripping portion to the first end portion. A first application section disposed on the side, a first inspection area having a first inspection section disposed between the first application section and the first water-absorbing filter paper, and a second end of the strip. Between the second water absorption filter paper, the second application part disposed on the downstream side in the second flow direction from the dropping part to the second end part, and between the second application part and the second water absorption filter paper. An inspection apparatus comprising a second inspection region disposed and having a second inspection unit,
When neither the first test part nor the second test part is colored, it is shown that it is negative for both non-toxic A type influenza virus and highly toxic type A influenza virus,
When both the first test part and the second test part are colored, it is shown that it is positive for a non-virulent type A influenza virus and negative for a virulent type A influenza virus,
When one of the first test part and the second test part is colored and the other color is weaker than the one, it is indicated that it is positive for the highly toxic influenza A virus. Inspection device. 2. When one of the first test part and the second test part is colored and there is no coloration of the other, it is indicated that it is positive for a highly toxic type A influenza virus. The inspection device described. A strip, a dripping portion disposed at a central portion of the strip, a first water-absorbing filter paper disposed at a first end portion of the strip, and a first flow direction downstream from the dripping portion to the first end portion. A first coating unit that is disposed on the side and that is labeled with a first antibody that is reactive to a nucleoprotein antigen of influenza A virus, and is flowable, and includes the first coating unit and the first water-absorbing filter paper. A first test region having a first test portion disposed between and a solid phase on which a second antibody reactive to a nucleoprotein antigen of influenza A virus is solid-phased, and disposed at the second end of the strip Flowing through a second water-absorbing filter paper and a third antibody labeled downstream of the dropping portion and extending to the second end in the second flow direction and having reactivity with a nucleoprotein antigen of influenza A virus Hold as possible A second test part, which is disposed between the second application part, the second application part, and the second water-absorbing filter paper, and on which a fourth antibody reactive to a nucleoprotein antigen of influenza A virus is solid-phased A second inspection region having
One of the first, second, third and fourth antibodies has a low reactivity with a highly toxic influenza A virus compared to the other antibodies. An inspection apparatus comprising a first strip and a second strip,
The first strip has a first dripping part, a first water-absorbing filter paper disposed at a position away from the first dripping part in the first flow direction, and a first flow than the first dripping part. A first coating unit that is arranged downstream of the direction and that is labeled with a first antibody that is reactive with a nucleoprotein antigen of influenza A virus, holds the flowable, the first coating unit, and the first water-absorbing filter paper And a first examination region having a first examination part on which a second antibody reactive to the nucleoprotein antigen of influenza A virus is solid-phased,
The second strip has a second dripping part, a second absorbent filter paper disposed at a position away from the second dripping part in the second flow direction, and a second flow than the second dripping part. A second coating unit that is arranged downstream of the direction and that is labeled with a third antibody that is reactive to the nucleoprotein antigen of influenza A virus, and that allows the fluid to flow, the second coating unit, and the second water-absorbing filter paper And a second examination region having a second examination part on which a fourth antibody reactive to the nucleoprotein antigen of influenza A virus is solid-phased,
One of the first, second, third and fourth antibodies has a low reactivity with a highly toxic influenza A virus compared to the other antibodies. 5. The inspection apparatus according to claim 1, wherein the strip is held by a case, and the case includes a funnel portion opening corresponding to the dripping portion and an observation window corresponding to the inspection region. . The first strip is held in a first case;
The second strip is held in a second case configured separately from the first case,
The first case includes a first funnel portion opening corresponding to the first dripping portion, and a first observation window corresponding to the first inspection region,
The inspection apparatus according to claim 4, wherein the second case includes a second funnel portion that opens corresponding to the second dripping portion, and a second observation window corresponding to the second inspection region.

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