TWI491880B - A method for improving antibody detection and applications in detecting flavivirus infection - Google Patents
A method for improving antibody detection and applications in detecting flavivirus infection Download PDFInfo
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Description
本發明關於一種增強抗體檢測的方法,尤指一種增強抗黃病毒之非結構蛋白1的抗體的檢測方法。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.
黃病毒(Flaviviruses),如日本腦炎病毒(Japanese encephalitis virus)、西尼羅病毒(West Nile virus)、黃熱病毒(Yellow fever virus)、聖路易斯腦炎病毒(St.Louis encephalitis virus;SLEV)、及登革病毒(Dengue virus)等,會引發多種人類的重大疾病。這些病毒引發的疾病在多數國家中皆有紀錄,且不斷的擴散蔓延。因此,在臨床照護上需要能夠準確地檢測出病毒感染的方法。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.
目前領域中採用的病毒分離檢測法或病毒RNA檢測法皆提供可信賴的檢測結果,但病毒或病毒的RNA通常只存在患者體內短暫的時間,而限制了該些檢測法的實益。因此,抗體檢測法在實務上是更具實用性的替代方式。抗體檢測法通常檢測一病毒專一(virus-specific)的免疫球蛋白M/G(IgM及IgG)以診斷或辨識一個體的感染狀況。研究顯示,黃病毒專一的免疫球蛋白M會在感染症狀出現後五天內產生並持續數月,之後則逐漸減 少到檢測不到的程度。世界衛生組織(World Health Organization;WHO)建議採用凝血抑制試驗(hemagglutination inhibition(HI)test)來進行檢測。血凝抑制試驗也常用於黃病毒感染的血清學分類作業。然而,血凝抑制試驗過於耗費時間,且多種黃病毒之間的交叉反應(cross-reactions among Flavivirus)也是另一個影響該試驗的實用性的重點。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.
另一方面,病毒斑抑制試驗法(The plaque reduction neutralization test;PRNT)被認為是檢驗中的黃金標準法(gold-standard method),但這種試驗法對於多數實驗室而言是難以進行的,並且其往往需要數天才能取得試驗結果,而限制了其臨床上的應用。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.
綜上所述,為了防疫及加速臨床上的檢測,領域中需要一種用於檢測黃病毒感染之精確、省時且符合成本考量的檢測法。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.
本發明之一目的為提供一種檢測黃病毒感染的方法,其藉由提高抗體檢測靈敏度的方式,使檢測作業更加精確。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.
本發明之另一目的為提供一種檢測黃病毒感染的方法,其藉由提高抗體檢測的靈敏度,而足以確定抗體來源是自然感染或是由疫苗所產生。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.
本發明之又一目的為提供一種檢測黃病毒感染的套組,其具有更優異的抗體檢測靈敏度,因而具有省時及符合成本考量的優點。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.
為達到上述目的,本發明提供一種增強抗體檢測的方法,其中前述抗體為抗黃病毒之非結構蛋白1的抗體;前述方法包含去除一樣本中抗黃病毒之套膜蛋白的抗體。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.
較佳地,前述去除一樣本中抗黃病毒之套膜蛋白的抗體的步驟包含使用一黃病毒之套膜蛋白作為抗原來捕捉前述抗黃病毒之套膜蛋白的抗體。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.
較佳地,前述方法係藉由免疫分析法、親和層析法、抗原磁珠法、或其組合來完成。Preferably, the foregoing method is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof.
較佳地,前述免疫分析法為抗原捕捉型酵素免疫分析法。Preferably, the aforementioned immunoassay is an antigen capture type enzyme immunoassay.
本發明又提供一種檢測黃病毒感染的方法,其包含以下步驟:(i)去除一樣本中抗黃病毒之套膜蛋白的抗體;及(ii)檢測前述樣本中抗黃病毒之非結構蛋白1的抗體。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.
較佳地,前述步驟(i)包含使用一黃病毒之套膜蛋白作為抗原來捕捉前述樣本中抗黃病毒之套膜蛋白的抗體。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.
較佳地,前述步驟(i)係藉由免疫分析法、親和層析法、抗原磁珠法、或其組合來完成。Preferably, the aforementioned step (i) is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof.
較佳地,前述免疫分析法為抗原捕捉型酵素免疫分析法。Preferably, the aforementioned immunoassay is an antigen capture type enzyme immunoassay.
較佳地,前述步驟(ii)係藉由免疫分析法、親和層析法、抗原磁珠法、或其組合來完成。Preferably, the aforementioned step (ii) is carried out by immunoassay, affinity chromatography, antigen magnetic beads, or a combination thereof.
較佳地,前述免疫分析法為抗體捕捉型酵素免疫分 析法。Preferably, the aforementioned immunoassay is an antibody-collecting enzyme immunological fraction. Analysis method.
較佳地,前述抗體捕捉型酵素免疫分析法為:對黃病毒之非結構蛋白1專一的IgM抗體捕捉型酵素免疫分析法、對黃病毒之非結構蛋白1專一的IgG抗體捕捉型酵素免疫分析法、或其組合。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.
較佳地,前述步驟(ii)借助過氧化酶呈色反應或鹼性磷酸酶呈色反應來判讀前述檢測之結果。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.
較佳地,前述樣本是一待測定個體的血液。Preferably, the aforementioned sample is the blood of an individual to be determined.
較佳地,前述黃病毒為日本腦炎病毒、西尼羅河病毒、黃熱病毒、聖路易斯腦炎病毒、或登革病毒。Preferably, the aforementioned flavivirus is Japanese encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus, or dengue virus.
較佳地,前述黃病毒為登革病毒,且前述作為抗原之黃病毒之套膜蛋白為:血清型第2型之登革病毒的套膜蛋白、血清型第3型之登革病毒的套膜蛋白、或其組合、或其與其他血清型之登革病毒的套膜蛋白之組合。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.
本發明再提供一種檢測黃病毒感染的套組,其包含:一抗體去除單元,其係用於去除一樣本中的抗黃病毒之套膜蛋白的抗體;及一抗黃病毒之非結構蛋白1抗體的檢測單元。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.
較佳地,前述抗體去除單元包含使用一黃病毒之套膜蛋白作為抗原。Preferably, the aforementioned antibody removal unit comprises a membrane protein using a flavivirus as an antigen.
較佳地,前述抗體去除單元係為一第一免疫分析裝置、一第一親和層析管柱、一第一抗原磁珠裝置、或其組合。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.
較佳地,前述第一免疫分析裝置為一抗原捕捉型酵素免疫分析裝置。Preferably, the first immunoassay device is an antigen capture type enzyme immunoassay device.
較佳地,前述抗黃病毒之非結構蛋白1抗體的檢測單元係為一第二免疫分析裝置、一第二親和層析管柱、一第二抗原磁珠裝置、或其組合。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.
較佳地,前述第二免疫分析裝置係為一對黃病毒之非結構蛋白1專一的IgM抗體捕捉型酵素免疫分析裝置、一對黃病毒之非結構蛋白1專一的IgG抗體捕捉型酵素免疫分析裝置、或其組合。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.
較佳地,前述抗黃病毒之非結構蛋白1抗體的檢測單元係借助過氧化酶呈色反應或鹼性磷酸酶呈色反應來判讀結果。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.
較佳地,前述黃病毒為日本腦炎病毒、西尼羅病毒、黃熱病毒、聖路易斯腦炎病毒、或登革病毒。Preferably, the aforementioned flavivirus is Japanese encephalitis virus, West Nile virus, yellow fever virus, St. Louis encephalitis virus, or dengue virus.
較佳地,前述黃病毒為登革病毒,且前述作為抗原之黃病毒之套膜蛋白為:血清型第2型之登革病毒的套膜蛋白、血清型第3型之登革病毒的套膜蛋白、或其組合、或其與其他血清型之登革病毒的套膜蛋白之組合。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.
綜上所述,本發明提供一種增強抗體檢測的方法及採用該方法之精神所進行之檢測黃病毒感染的方法及套組。藉由增強抗體檢測的靈敏度,本發明方法及套組的檢測結果精確,且具有省時及成本低廉的優點。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‧‧‧ELISA試驗盤的底部10‧‧‧ bottom of ELISA test tray
11‧‧‧兔抗JEV(類)病毒顆粒的多株抗體11‧‧‧Multiple antibodies against rabbit anti-JEV (class) virus particles
12‧‧‧(類)病毒顆粒12‧‧‧(class) virus particles
13‧‧‧anti-E抗體13‧‧‧anti-E antibody
E‧‧‧黃病毒之套膜蛋白(envelop)E‧‧‧Flavor envelope protein (envelop)
NS1‧‧‧黃病毒之非結構蛋白1(nonstructural protein)Nonstructural protein of NS1‧‧‧ flavivirus
Anti-E抗體‧‧‧抗套膜蛋白抗體(anti-envelop antibody)Anti-E antibody ‧‧‧anti-envelop antibody
Anti-NS1抗體‧‧‧抗非結構蛋白1抗體Anti-NS1 antibody ‧‧‧Anti-structural protein 1 antibody
VLP‧‧‧類病毒蛋白(virus-like protein)VLP‧‧‧ virus-like protein
IgM‧‧‧免疫球蛋白M(Immunoglobulin M)IgM‧‧‧ immunoglobulin M (Immunoglobulin M)
IgG‧‧‧免疫球蛋白G(Immunoglobulin M)IgG‧‧‧ immunoglobulin G (Immunoglobulin M)
MHIAF‧‧‧鼠高免疫腹水(murine hyper-immune ascitic fluid)MHIAF‧‧‧murine hyper-immune ascitic fluid
JEV‧‧‧日本腦炎病毒(Japanese encephalitis virus)JEV‧‧‧Japanese encephalitis virus
WNV‧‧‧西尼羅河病毒(West Nile virus)WNV‧‧·West Nile virus
YFV‧‧‧黃熱病毒(Yellow fever virus)YFV‧‧‧Yellow fever virus
SLEV‧‧‧聖路易斯腦炎病毒(St.Louis encephalitis virus)SLEV‧‧‧St.Louis encephalitis virus
DENV‧‧‧登革病毒(Dengue virus)DENV‧‧ Dengue virus
HRP‧‧‧山葵過氧化酶(horseradish peroxidase)HRP‧‧‧Mountain Peroxidase (horseradish peroxidase)
NHS‧‧‧健康個體的血液樣本(Normal Human Serum)NHS‧‧‧ Blood samples from healthy individuals (Normal Human Serum)
MAC-ELISA‧‧‧IgM抗體捕捉型酵素免疫分析法(IgM antibody capture ELISA)MAC-ELISA ‧ ‧ IgM antibody capture enzyme immunoassay (IgM antibody capture ELISA)
GAC-ELISA‧‧‧IgG抗體捕捉型酵素免疫分析法(IgG antibody capture ELISA)GAC-ELISA ‧ ‧ IgG antibody capture ELISA
第一圖係示意本發明的預吸附步驟。The first figure illustrates the pre-adsorption step of the present invention.
第二圖係顯示確認本發明之預吸附步驟的效果的實驗結果;其中(+)表示已感染。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.
第三圖係顯示本發明之預吸附步驟於檢測anti-NS1 抗體之影響;(A)JEV感染之樣本;(B)WNV感染之樣本;(C)DENV感染之樣本;其中(+)表示有進行預吸附步驟,(-)表示未進行預吸附步驟。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.
第四圖呈現分別以JEV及WNV的(類)病毒顆粒進行預吸附步驟的經JEV感染之血清樣本的實驗結果;其中(A)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-JEV NS1 IgM的P/N比值;(B)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-JEV NS1 IgG的P/N比值;(C)為使用WNV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-JEV NS1 IgM的P/N比值;(D)為使用WNV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-JEV NS1 IgG的P/N比值。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.
第五圖呈現分別以JEV及WNV的(類)病毒顆粒進行預吸附步驟的經WNV感染之血清樣本的實驗結果;其中(A)為使用WNV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-WNV NS1 IgM的P/N比值;(B)為使用WNV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-WNV NS1 IgG的P/N比值;(C)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-WNV NS1 IgM的P/N比值;(D)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-WNV NS1 IgG的P/N比值。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.
第六圖係測試使用不同血清型之登革病毒的套膜蛋白於本發明方法的影響。(A1)(A2)為樣本ID#100,其係為經第1型感染之樣本;(B1)(B2)為樣本ID#56,其係為經第2型感染之樣本;(C1)(C2)為樣本ID#76,其係為經第2型感染之樣本。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.
第七圖係顯示在登革感染之急性期樣本中,本發明之預吸附步驟於檢測anti-NS1抗體之影響。(A)未進行 預吸附步驟;(B)經進行預吸附步驟。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.
第八圖係顯示在登革感染之恢復期樣本中,本發明之預吸附步驟於檢測anti-NS1抗體之影響。(A)未進行預吸附步驟;(B)經進行預吸附步驟。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.
黃病毒,包括日本腦炎病毒(Japanese encephalitis virus)、西尼羅河病毒(West Nile virus)、黃熱病毒(Yellow fever virus)、聖路易斯腦炎病毒(St.Louis encephalitis virus;SLEV)、或登革病毒(Dengue virus),皆屬正鏈單股RNA病毒,其正鏈單股RNA在宿主細胞內被轉譯為單一複合蛋白。該複合單白接著被截切為三個結構蛋白,包括殼體蛋白(capsid(C))、前膜蛋白(premembrane (prM))、及套膜蛋白(envelope(E)),及七個非結構蛋白,包括非結構蛋白1(NS1)、非結構蛋白2A(NS2A)、非結構蛋白2B(NS2B)、非結構蛋白3(NS3)、非結構蛋白4A(NS4A)、非結構蛋白4B(NS4B)、及非結構蛋白5(NS5)。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).
宿主經感染之後產生的抗體絕大多數是針對黃病毒的套膜蛋白產生專一的免疫反應,因此,領域中已有多種針對抗套膜蛋白之抗體(以下簡稱anti-E抗體,包括IgM及IgG)檢測的酵素連結免疫吸附測定法套組。這一類針對抗套膜蛋白之抗體的IgM抗體捕捉型酵素免疫分析裝置(E-MAC-ELISA)或IgG捕捉型酵素免疫分析裝置(E-GAC-ELISA)被認為與HI檢測法具有同等的可信度。另一方面,抗非結構蛋白1之抗體(以下簡稱anti-NS1抗體)亦是另一個研究中的檢測標的。然而, anti-NS1抗體在患者血液中的含量較低,且容易受到患者血液中其他抗體的干擾。因此,即便領域中發展多樣針對anti-NS1抗體的免疫分析技術,在抗非結構蛋白1之抗體數量少,及檢測靈敏度不理想的狀況下,這些免疫分析技術仍未能完善地滿足臨床上的運用。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.
本發明經實驗證實檢測anti-NS1抗體的靈敏度不佳的原因主要來自於患者血液中存在太多anti-E抗體。因此,本發明提供一種增強anti-NS1抗體檢測的方法,其包含去除一樣本中的anti-E抗體。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.
本發明所述「樣本」,例如一潛在患者的血液;更明確地說,係指一潛在患者的血清。本發明所述「抗體」,例如免疫球蛋白M(IgM)、免疫球蛋白G(IgG)、或其組合。本發明所謂「去除一樣本中抗黃病毒之套膜蛋白的抗體」及「去除一樣本中的anti-E抗體」係指將一樣本中的anti-E抗體去除到相當程度,以使檢測anti-NS1抗體的靈敏度提高到有助於臨床運用的程度。原則上前述「相當程度」無需特別定義,概本發明的精神在於證實並教示當去除一樣本中的anti-E抗體後,可以提升檢測anti-NS1抗體的靈敏度,所屬領域中具有通常知識當可理解在最佳狀況下,將樣本中的anti-E抗體越完整的去除越有助於anti-NS1抗體的檢測。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.
前述「去除一樣本中抗黃病毒之套膜蛋白的抗體」及前述「去除一樣本中的anti-E抗體」可藉由領域中習知的技術手段來完成。舉例來說,可藉由免疫分析法(Immunoassay)、親和層析法(Affinity chromatography)、抗原磁珠法、或其組合來完成。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.
在本發明的一個實施態樣中,採取免疫分析法來去除樣本中的anti-E抗體。明確地說,將一黃病毒之套膜蛋白作為抗原固定在一基材上(如一ELISA分析盤),借助免疫分析法的原理來去除樣本中的anti-E抗體。舉例來說,可採用抗原捕捉型酵素免疫分析法(Antigen-capture ELISA),藉由黃病毒之套膜蛋白以免疫性地結合樣本中的anti-E抗體,達到去除樣本中的anti-E抗體的目的。前述「免疫性地結合」係指藉由抗原抗體的專一性關係將兩種物質相結合。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.
在本發明的另一個實施態樣中,使用親和層析法來去除樣本中的anti-E抗體。明確地說,將一黃病毒之套膜蛋白固定於層析管柱的固定相,使樣本流過該層析管柱時,專一性地將anti-E抗體吸附於該層析管柱中而與樣本分離,藉此達到去除樣本中的anti-E抗體的目的。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.
在本發明的又一個實施態樣中,利用抗原磁珠法來去除樣本中的anti-E抗體。明確地說,將表面固著有一黃病毒之套膜蛋白的磁珠與樣本反應,使樣本中的anti-E抗體與表面固著有黃病毒之套膜蛋白的磁珠形成複合物,達到去除樣本中的anti-E抗體的目的。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.
在本發明之增強檢測anti-NS1抗體之靈敏度的精神下,本發明提供一種檢測黃病毒感染的方法。本發明提供之檢測方法的關鍵之一在於,先去除待測樣本中的anti-E抗體,藉此提升檢測anti-NS1抗體的靈敏度。明確來說,本發明提供之檢測方法包含以下步驟:(i)去除一樣本的anti-E抗體;及(ii)檢測前述樣本中的anti-NS1抗體;其中步驟(i)便是依據本發明精神的預吸附(pre-absorption)步驟,即預先處理樣本,以去除待測 樣本中的anti-E抗體。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.
前述步驟(i)可藉由免疫分析法、親和層析法、抗原磁珠法、或其組合來完成;其中前述免疫分析法、前述親和層析法、及前述抗原磁珠法的操作細節係如前述段落中所述。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.
前述步驟(ii)可藉由領域中習知的技術手段來完成。舉例來說,可採用免疫分析法、親和層析法、抗原磁珠法、或其組合來完成。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.
在本發明的一個實施態樣中,採取免疫分析法來檢測前述樣本中的anti-NS1抗體。簡單來說,先將抗人IgM或IgG抗體固定在一基材上(如一ELISA分析盤),使受測者的血清中的IgM或IgG因前述抗體而結合於前述基材上,爾後加入NS1抗原,以辨認anti-NS1的IgM或IgG。最後加入可辨認anti-NS1之抗體(以本實驗中為例,使用的是老鼠的免疫血清),並藉由山葵過氧化酶(horseradish peroxidase;HRP)呈色反應或鹼性磷酸酶(alkaline phosphatase;AP)呈色反應來判讀結果。舉例來說,可採取對NS1專一的IgM抗體捕捉型酵素免疫分析法(NS1-specific IgM-capture ELISA)、對NS1專一的IgG抗體捕捉型酵素免疫分析法(NS1-specific IgG-capture ELISA)、或其組合來檢測前述樣本中的anti-NS1抗體。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.
在本發明的另一個實施態樣中,使用親和層析法來檢測前述樣本中的anti-NS1抗體。明確地說,將一黃病毒之非結構蛋白1固定於層析管柱的固定相,使樣本流過該層析管柱時,專一性地將anti-NS1抗體吸附於該層析管柱中。爾後,藉由合適之緩衝液使吸附之anti-NS1 抗體流出,並使流出液與接枝有過氧化酶或鹼性磷酸酶的NS1接觸,以借助過氧化酶呈色反應或鹼性磷酸酶呈色反應來判讀結果。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.
在本發明的又一個實施態樣中,利用抗原磁珠法來檢測前述樣本中的anti-NS1抗體。可利用如同Khan等人於2006年發表之學術文獻(Imran H.Khan et al.,CLINICAL AND VA CCINE IMMUNOLOGY ,Vol.13,No.1,2006)中所載之抗原磁珠法的基本原理,以前述親和層析法類似的設計邏輯,藉由NS1抗原捕捉anti-NS1抗體來進行偵測。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.
在本發明之增強檢測anti-NS1抗體之靈敏度的精神下,本發明提供一種檢測黃病毒感染的套組。本發明之套組包含:一抗體去除單元,其係用於去除一樣本中的抗黃病毒之套膜蛋白的抗體;及一抗黃病毒之非結構蛋白1抗體的檢測單元。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.
前述抗體去除單元及前述抗黃病毒之非結構蛋白1抗體的檢測單元可分別為一免疫分析裝置、一親和層析管柱、一抗原磁珠裝置、或其組合,其係分別依循免疫分析法、親和層析法、或抗原磁珠法的原理。前述免疫分析法、親和層析法、或抗原磁珠法的原理係如前述段落中所述。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.
舉例來說,前述抗體去除單元可為一抗原捕捉型酵素免疫分析裝置,其包含使用一黃病毒之套膜蛋白;前述抗體去除單元可為一親和層析管柱,其固定相包含一黃病毒之套膜蛋白;前述抗體去除單元可為一抗原磁珠裝置,其所用磁珠的表面固著有一黃病毒之套膜蛋白。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.
舉例來說,前述抗黃病毒之非結構蛋白1抗體的檢測單元可為一對黃病毒之非結構蛋白1專一的IgM抗體捕捉型酵素免疫分析裝置、一對黃病毒之非結構蛋白1專一的IgG抗體捕捉型酵素免疫分析裝置、或其組合;前述抗黃病毒之非結構蛋白1抗體的檢測單元可為一親和層析管柱的固定相,其包含一黃病毒之非結構蛋白1;前述抗黃病毒之非結構蛋白1抗體的檢測單元可為一抗原磁珠裝置,其所用磁珠的表面固著有一黃病毒之非結構蛋白1。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.
以下實施例將記載本發明的研究結果及特定實施態樣,以使發明所屬技術領域中具有通常知識者更容易理解本發明的精神。需注意的是,以下內容僅屬示範性質,而不應對本發明的申請專利範圍產生限制。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.
本實驗所用血清樣本是採集自美國疾管局病媒傳染病研究組節肢動物媒介病毒疾病部的診斷及代檢實驗室(Diagnostic and Reference Laboratory,Arboviral Diseases Branch,Division of Vector-Borne Infectious Diseases,Centers for Disease Control and Prevention)。血清樣本係收集西元1999至2003年間經90%之溶菌斑中和試驗(plaque reduction neutralization test)檢測具有對西尼羅河病毒(WNV;n=21)、聖路易斯腦炎病毒(SLEV; n=6)、或α節肢動物媒介病毒(alphaviruses;n=12)之中和抗體力價(NT antibody titers)者。經證實受DENV(n=24)或JEV(n=16)感染之血清樣本則採集自台灣疾管局(the Center for Disease Control in Taiwan)提供的國人血清樣本。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.
本實驗使用COS-1細胞(ATCC CRL 1650;Manassas,VA)以活體外(in vitro )製得後續實驗所需的(類)病毒顆粒(VLP;virus-like particles)及水溶性NS1蛋白(sNS1)。簡單來說,將帶有可以轉譯為黃病毒之套膜蛋白及前膜蛋白、或非結構蛋白的基因序列的質體轉殖至COS-1細胞(ATCC CRL 1650;Manassas,VA)中,再使COS-1細胞表現並分泌所需之(類)病毒顆粒及sNS1於培養液中。收集培養液並純化其中的(類)病毒顆粒及sNS1備用。由於套膜蛋白為黃病毒結構上最外層的部分,因此由COS-1細胞表現之套膜蛋白及前膜蛋白所組合成的(類)病毒顆粒其免疫學上意義即等同於黃病毒顆粒上的套膜蛋白。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.
於本實施例中,借助抗原捕捉型酵素免疫分析法來去除血清樣本中的anti-E抗體。以感染JEV之血清樣本為例,請參第一圖,先將兔抗JEV(類)病毒顆粒的多株抗體(polyclonal rabbit anti-JEV VLP antibody;11)固著於ELISA試驗盤的底部(10),接著,將活體外製得之JEV 的(類)病毒顆粒(12)與經稀釋的血清樣本(稀釋倍數為1:4000)混合後,再置入ELISA試驗盤,並放置於37℃下60分鐘,使血清樣本中的anti-E抗體(13)與類病毒顆粒(12)的抗原所形成的免疫複合體充分反應,而被多株抗體抓住。最後,移除試驗盤中的多餘的血清樣本以供後續試驗。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.
改使用兔抗WNV(類)病毒顆粒的多株抗體及活體外製得之WNV的(類)病毒顆粒進行上述步驟以預吸附經WNV感染之血清樣本(稀釋倍數為1:1000)中的anti-E抗體,以供後續試驗。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.
本實驗是為了確認本發明之前述預吸附步驟是否確實將經感染之血清樣本中的anti-E抗體移除。以經JEV感染之血清樣本實驗為例,取得經前述預吸附步驟後的ELISA試驗盤,分別加入接枝有山葵過氧化酶(HRP)的經JEV感染之鼠高免疫腹水(MHIAF)、anti-human IgM/IgG抗體,並使其於37℃下反應60分鐘。接著,加入山葵過氧化酶之基質(3,3’5,5’-tetramethylbenzidine)呈色,並以分光光度計讀取OD450 的吸光值。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.
實驗結果如第二圖中所示。處理過健康個體的血清樣本(Normal Human Serum;NHS)的ELISA試驗盤,再以經JEV感染之鼠高免疫腹水、及anti-human IgM/IgG抗體分別反應後,所得OD450 的吸光值分別為1.08、0.4、及0.1。而處理過經感染JEV之血清樣本的ELISA試驗盤所得OD450 的吸光值分別為2.64、1.22、0.32,皆有顯 著提升,意味著本發明之預吸附步驟確實有效地將血清樣本中的anti-E抗體移除。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.
本實驗將分別使用未經前述預吸附步驟及經過前述預吸附步驟處理的血液樣本來進行anti-NS1抗體的檢測,並比較其差異。本實驗將採用對NS1專一的IgM抗體捕捉型酵素免疫分析法(NS1-specific IgM-capture ELISA)、及對非結構蛋白1專一的IgG抗體捕捉型酵素免疫分析法(NS1-specific IgG-capture ELISA),分別檢測經JEV、WNV、及DENV感染的血清樣本。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.
以檢測經JEV感染的血清樣本為例,首先,於ELISA試驗盤的底部固著有羊抗人IgM之抗體。接著,置入經稀釋的血清樣本(稀釋倍數為1:4000;包括經JEV感染的血清樣本作為對照組及健康個體的血清樣本做為控制組),並使其於37℃下反應120分鐘。然後,投入活體外製得之JEV的NS1,並使其於37℃下反應60分鐘。爾後,加入經JEV感染之鼠高免疫腹水(MHIAF;經1:4000稀釋),並使其於37℃下反應60分鐘。接著加入接枝有山葵過氧化酶之羊抗鼠IgG抗體,並使其於37℃下反應60分鐘。最後加入山葵過氧化酶之基質(3,3’5,5’-tetramethylbenzidine)呈色,並以分光光度計讀取OD450 的吸光值。以同樣的方式進行經WNV或DENV感染的血清樣本的anti-NS1抗體檢測,其中改用活體外製得之WNV或DENV的NS1,並將血清樣本的稀釋倍數改為1:1000。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.
以下列公式計算所得之OD450 的吸光值,換算出各實驗組的P/N比值(positive/negative ratio;P/N ratio);其中NHS為健康個體之血清樣本(Normal Human Serum)的簡稱。此計算公式係參酌領域中的科學文獻(Martin et al.),為了排除實驗中的非專一性結合(nonspecific binding)影響實驗結果,領域中慣例定義若一樣本的P/N≧3,則表示所讀得的免疫反應結果(抗體抗原反應)為真。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.
實驗結果如第三圖中所示;其中(A)為經JEV感染的血清樣本的實驗結果,(B)為經WNV感染的血清樣本的實驗結果,(C)為經DENV感染的血清樣本的實驗結果(所用樣本係經血清型第2型的登革病毒感染,並使用血清型第2型之登革病毒的套膜蛋白作為抗原來進行預吸附步驟)。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).
從第三圖(A)中可知,因為進行本發明之預吸附步驟,檢測感染JEV之血清樣本中anti-NS1 IgM所得的P/N比值由2.8提升到17.2,而檢測anti-NS1 IgG所得的P/N比值由0.82提升到7.71。從第三圖(B)中可知,因為進行本發明之預吸附步驟,可使檢測anti-NS1 IgM所得的P/N比值由1.24提升到6.76。雖然檢測感染WNV之血清樣本中anti-NS1 IgG所得的P/N比值並沒有顯著變化,但判斷其原因是WNV的感染中,IgG通常在感染後期或較嚴重的感染情況中才會大量出現。此外,在DENV的試驗中,從第三圖(C)可知,因為進行本發明之 預吸附步驟,可使檢測anti-NS1 IgM所得的P/N比值由1.8提升到4.2,而檢測anti-NS1 IgG所得的P/N比值由1.62提升到4.71。因此,此實驗結果顯示本發明納入預吸附步驟的概念確實地提升了對於anti-NS1抗體的檢測。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.
黃病毒包括日本腦炎病毒、西尼羅病毒、黃熱病毒、聖路易斯腦炎病毒、及登革病毒等多種會造成人類疾病的病毒。這些病毒的感染皆具有類似的病徵而不容易在感染初期分辨。因此,本實施例欲試驗各種黃病毒之抗原是否可以交叉使用於本發明之預吸附步驟。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.
本實施例中分別取得16個經JEV感染或經WNV感染之血清樣本,並分別使用活體外製得之JEV及WNV的(類)病毒顆粒進行本發明的預吸附步驟,再觀察對後續anti-NS1抗體之檢測的影響。關於預吸附步驟的進行方式及後續anti-NS1抗體之檢測步驟皆參照前述實施例一中所述。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.
第四圖呈現分別以JEV及WNV的(類)病毒顆粒進行預吸附步驟的經JEV感染之血清樣本的實驗結果;其中(A)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgM的P/N比值;(B)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgG的P/N比值;(C)為使用WNV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgM的P/N比值;(D)為使用WNV(類) 病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgG的P/N比值。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.
從第四圖(A)及(B)的數據可知,16個樣本中每一個測得的P/N比值皆≧3,即準確測得感染。而第四圖(C)及(D)的數據顯示,分別有93.8%(16個樣本中有15個P/N比值≧3)及81.3%(16個樣本中有13個P/N比值≧3)的準確度可測得感染。換言之,即便是感染JEV的患者,也可使用WNV抗原進行本發明的預吸附步驟,而準確地測得感染。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.
第五圖呈現分別以JEV及WNV的(類)病毒顆粒進行預吸附步驟的經WNV感染之血清樣本的實驗結果;其中(A)為使用WNV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgM的P/N比值;(B)為使用WNV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgG的P/N比值;(C)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgM的P/N比值;(D)為使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測anti-NS1 IgG的P/N比值。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.
從第五圖(A)及(B)的數據可知,16個樣本中分別有13個及11個樣本的P/N比值皆≧3,準確度分別為86.7%及73.3%。第五圖(C)及(D)的數據顯示,當使用JEV(類)病毒顆粒進行預吸附步驟時,分別有40%(16個樣本中有6個P/N比值≧3)及0%(16個樣本的P/N比值皆<3)的準確度可測得WNV病毒的感染。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.
從上面的實驗結果可知,在感染JEV的狀況下,不論使用JEV或WNV(類)病毒顆粒進行預吸附步驟,皆不 影響後續anti-NS1抗體的檢測。而在感染WNV的狀況下,雖然使用JEV(類)病毒顆粒進行預吸附步驟,爾後檢測WNV感染之anti-NS1 IgG的實用性不高,但檢測WNV感染之anti-NS1 IgM仍然具有40%的準確度。也就是說,若一患者的病癥顯示出經黃病毒感染的可能性,原則上以其他黃病毒之抗原進行預吸附步驟,皆可有效提升後續anti-NS1 IgM的抗體檢測;而其中又以WNV的抗原最適用於各種黃病毒的感染檢測。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.
目前已知登革病毒共有4種血清型,且其在臨床上的感染症狀非常相似,因此,必須確認在未知感染者受感染的血清型的情況下,本發明方法的預吸附步驟於使用不同抗原時,是否仍然具有效果。據此,本實施例採用三個樣本:ID#100(DENV type-1)、ID#56(DENV type-2)、ID#76(DENV type-2),並以如同實施例一中所載的方法分別測試使用不同血清型之抗原或其組合進行預吸附步驟,是否仍然可以提升後續anti-NS1抗體的檢測的P/N比值。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.
請參第六圖A1及A2,在樣本ID#100中,不論採用哪一種血清型的抗原進行預吸附步驟,皆可以有效提升anti-NS1抗體的檢測效率;其中又以使用第2或第3血清型的抗原進行的預吸附步驟最具顯著效果,且不論單獨使用,或搭配他種血清型的抗原使用,皆可觀察到明顯的效果。類似的實驗結果也可自樣本ID#56M(第 六圖B1及B2)及樣本ID#76第六圖C1及C2)中觀察到。綜合而言,於登革感染的檢測中,並不需要事先確認患者受感染的血清型,採用本發明方法皆可以有效提升anti-NS1抗體的檢測效率。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.
針對anti-E抗體進行的E-MAC-ELISA為目前國際衛生組織建議之節肢動物媒介病毒感染的檢測方法。本實施例欲比較本發明方法與習用E-MAC-ELISA檢測法(不含本發明的預吸附步驟)效果。本實施例共取得185個確認經黃病毒節肢動物媒介病毒感染之個體的血清樣本,分別以本發明之方法(使用JEV-NS1抗原或WNV-NS1抗原)及習用的E-MAC-ELISA方法(使用JEV-VLP抗原或WNV-VLP抗原)加以檢測,並記錄檢測結果如下表一(P/N≧3則定義為檢測陽性,即檢出感染)。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).
本實驗所用經JEV感染的血清樣本有16個。採用本發明方法之NS1-MAC-ELISA的檢測結果為16個樣本皆檢出(16/16,16個樣本中,P/N比值最低為4.07,最高為23.53,平均16.5)。採用本發明方法之NS1-GAC-ELISA的檢測結果為16個樣本皆檢出(16/16,16個樣本中,P/N比值最低為4.15,最高為13.23,平均9.42)。習用E-MAC-ELISA及E-GAC-ELISA的檢出數量則分別為16/16及15/16。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.
本實驗共取得兩批經WNV感染的血清樣本,分別 有70個及25個樣本。在第一批70個樣本中,習用E-MAC-ELISA的檢出為70/70,E-GAC-ELISA的檢出為28/70。本發明NS1-MAC-ELISA的檢出為65/70(70個樣本中,P/N比值最低為1.07,最高為18.9,平均7.32),NS1-GAC-ELISA的檢出為30/70(70個樣本中,P/N比值最低為0.59,最高為10.9,平均3.32)。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).
在WNV的第二批25個樣本中,習用E-MAC-ELISA的檢出為20/25,E-GAC-ELISA的檢出為2/25。本發明NS1-MAC-ELISA的檢出為24/25(25個樣本中,P/N比值最低為1.12,最高為14.11,平均6.16),NS1-GAC-ELISA的檢出為10/25(25個樣本中,P/N比值最低為1.24,最高為7.23,平均2.94)。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).
此外,本發明方法並不會檢測出感染黃病毒以外之病毒)的感染(如表一中的漢他病毒感染(HTN)和風溼熱(RF)患者),顯見本發明對於黃病毒感染的檢測具有優異的專一度(specificity),鮮少有偽陽性的結果產生。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.
接著,借助接收者操作特徵曲線分析法(receiver operating characteristic(ROC)curve analysis)分析所得實驗數據,以判定習用方法及本發明方法的靈敏度(sensitivity)及專一度(specificity)。所得結果係如下表二中所示。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.
在感染JEV的樣本中,習用MAC-ELISA及本發明之NS1-MAC-ELISA皆達到100%的靈敏度及88.9%的專一度。本發明之NS1-GAC-ELISA具有100%的靈敏度及96%的專一度,較習用GAC-ELISA的93.7%的靈敏度及92.9%的專一度來得優異。在感染WNV的樣本中,習用 方法及本發明方法不論在MAC-ELISA及GAC-ELISA中的靈敏度及專一度皆相去不遠。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.
表三係彙整出習用方法與本發明方法之檢測產生不一致的樣本。Table 3 summarizes the samples that are inconsistent with the detection of the method of the present invention.
在樣本ID#183、ID#224、ID#WHO PR 1148中,本發明方法之MAC-ELISA測出未感染的結果,而其他三個檢測則皆檢測出陽性。而除了這三個樣本之外,習用方法及本發明方法不論在MAC-ELISA或GAC-ELISA所得結果皆非常相近。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.
樣本ID #J9300198係為一52歲的JEV患者,其血液樣本係在症狀出現後第14天才採集。此樣本在習用方法之GAC-ELISA檢測中未檢出感染,其可能的原因是E蛋白尚在產出中。與此樣本相似的,另外有七個樣本在習用方法之GAC-ELISA檢測中未檢出感染,但在其他三個檢測法中皆檢測出陽性。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.
只有一個樣本(ID#214)在本發明方法之GAC-ELISA中為未檢出,但在其他三個檢測法中則為陽性。據此,本發明方法之GAC-ELISA具有最好的檢測準確度。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.
如同實施例四,本實施例欲測試在登革感染的檢測中,本發明方法與習用方法的差異。本發明共取得36個血清樣本(涵蓋急性期血清及恢復期血清),並以如同實施例四所記載的實驗方式進行測試。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.
於本實施例中,首先確認本發明預吸附步驟是否得以提升急性期及恢復期之感染狀態下的anti-NS1抗體的檢測效率。試驗方式請參酌實施例一。此試驗係採用第2及3型的DENV套膜病毒作為抗原來進行預吸附步驟。實驗結果如第七圖及第八圖中所示,本發明預吸附步驟在急性期樣本(第七圖)及恢復期樣本(第八圖)中皆顯著地提升了anti-NS1抗體的檢測效率。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). .
此外,在與習用檢測方法的比較中,試驗結果顯示,在36個樣本中,習用anti-E抗體之檢測方法檢測出26個為感染陽性(檢出率:72.2%),而本發明則檢測出25個為感染陽性(檢出率:69.4%)。經計算,本發明方法與習用anti-E抗體之檢測方法的相關性達96.2%,代表本發明方法與國際衛生組織建議之anti-E抗體檢測法的檢測能力不相上下。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.
總結上述實驗可知,本發明方法的靈敏度和專一度足以與國際衛生組織建議的E-MAC/GAC-ELISA相匹配。此外,由於NS1為黃病毒中唯一會被受感染之細胞釋出的非結構蛋白,以anti-NS1抗體為檢測標的更具有 可分辨一個體體內的抗體是因感染所獲致或是因疫苗所獲致,在臨床運用的價值更勝習用以anti-E抗體為檢測標的的檢測方法。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.
所屬領域之技術人員當可了解,在不違背本發明精神下,依據本案實施態樣所能進行的各種變化。因此,顯見所列之實施態樣並非用以限制本發明,而是企圖在所附申請專利範圍的定義下,涵蓋於本發明的精神與範疇中所做的修改。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.
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