TW202212349A - Anit-sars-cov-2 antibodies and application thereof - Google Patents
Anit-sars-cov-2 antibodies and application thereof Download PDFInfo
- Publication number
- TW202212349A TW202212349A TW109131736A TW109131736A TW202212349A TW 202212349 A TW202212349 A TW 202212349A TW 109131736 A TW109131736 A TW 109131736A TW 109131736 A TW109131736 A TW 109131736A TW 202212349 A TW202212349 A TW 202212349A
- Authority
- TW
- Taiwan
- Prior art keywords
- seq
- complementarity determining
- antibody
- determining region
- heavy chain
- Prior art date
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
本發明涉及抗新型冠狀病毒抗體及/或其抗原結合片段，其係用於檢測一生物樣本中的新型冠狀病毒。本發明並提供用於檢測新型冠狀病毒之方法及套組，以及使用如本文所述之抗新型冠狀病毒抗體診斷及治療新冠肺炎之方法及組合物。The present invention relates to anti-novel coronavirus antibodies and/or antigen-binding fragments thereof, which are used to detect the novel coronavirus in a biological sample. The present invention also provides methods and kits for detecting novel coronavirus, as well as methods and compositions for diagnosing and treating novel coronavirus pneumonia using the anti-novel coronavirus antibodies as described herein.
世界衛生組織(World Health Organization, WHO)於2019年12月31日接獲通報，在中國湖北省武漢市出現了幾例肺炎病例。引起該肺炎的病原體病毒與任何其他已知的病毒皆不吻合。該病毒後來被正式命名為“嚴重急性呼吸道症候群冠狀病毒2 (Severe Acute Respiratory Syndrome coronavirus 2, SARS-CoV-2)”，又稱 “新型冠狀病毒”；由新型冠狀病毒(SARS-CoV-2)引起的疾病的正式名稱則為“嚴重特殊傳染性肺炎(COVID-19)”，簡稱新冠肺炎。新冠肺炎(COVID-19)的常見症狀包括發燒、乾咳、疲勞、倦怠等。儘管大多數情況下感染者的症狀輕微，甚至無症狀，但由於細胞激素風暴(cytokine storm)、多器官功能衰竭、敗血性休克，以及血栓等會導致有些患者的病況惡化為急性呼吸窘迫症候群(acute respiratory distress syndrome, ARDS)。The World Health Organization (WHO) was notified on 31 December 2019 of several cases of pneumonia in Wuhan, Hubei Province, China. The causative virus causing this pneumonia does not match any other known virus. The virus was later officially named "Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2)", also known as "new coronavirus"; from the new coronavirus (SARS-CoV-2) The official name of the disease it causes is "Severe Special Infectious Pneumonia (COVID-19)", or COVID-19 for short. Common symptoms of new coronary pneumonia (COVID-19) include fever, dry cough, fatigue, burnout, and more. Although in most cases the symptoms of infected persons are mild or even asymptomatic, due to cytokine storm (cytokine storm, multiple organ failure, septic shock, and blood clots, etc.), the condition of some patients may deteriorate into acute respiratory distress syndrome ( acute respiratory distress syndrome, ARDS).
雖然人類感染新型冠狀病毒(SARS-CoV-2)的首例出現在中國，然而不到半年的時間，全球已有216個國家及地區出現確診案例。截至2020年8月底，全球向WHO報告的確診病例已超過2千萬例，包括超過80萬例患者死亡。確診及死亡人數仍在快速增加中。Although the first case of human infection with the new coronavirus (SARS-CoV-2) appeared in China, in less than half a year, there have been confirmed cases in 216 countries and regions around the world. As of the end of August 2020, more than 20 million confirmed cases have been reported to WHO worldwide, including more than 800,000 patient deaths. The number of confirmed cases and deaths is still increasing rapidly.
迄今為止，尚無有效的抗病毒藥物可用於新冠肺炎(COVID-19)，且疫苗仍處於開發階段。因此，早期診斷新型冠狀病毒(SARS-CoV-2)對於疾病的管理非常重要。新型冠狀病毒(SARS CoV-2)感染的早期診斷可分流感染者，並加以隔離減少病毒傳播的機會。感染新型冠狀病毒(SARS CoV-2)的人類表現出廣泛的臨床表現，從無症狀感染到嚴重的肺炎，使得準確的診斷變得困難。目前新型冠狀病毒(SARS-CoV-2)的檢測方式以實驗室為主，包括病毒分離培養、病毒核酸檢測、病毒抗原及抗體檢測。病毒分離培養需在生物安全第三級(biosafety level 3, BSL-3)實驗室操作，操作不易且耗時。而病毒抗原及抗體檢測方法仍在發展中，因此目前實驗室診斷仍以病毒核酸檢測為主。然而，核酸檢測需要專業的操作人員、特殊的儀器及設備，且需花費4小時以上，不適用於大量篩檢。因此，亟需開發能夠快速檢測新型冠狀病毒(SARS CoV-2)的診斷方法。To date, there are no effective antiviral drugs available for COVID-19, and a vaccine is still in development. Therefore, early diagnosis of novel coronavirus (SARS-CoV-2) is very important for disease management. Early diagnosis of novel coronavirus (SARS CoV-2) infection enables triage of infected individuals and isolation to reduce the chance of virus transmission. Humans infected with the novel coronavirus (SARS CoV-2) exhibit a wide range of clinical manifestations, ranging from asymptomatic infection to severe pneumonia, making accurate diagnosis difficult. At present, the detection methods of the new coronavirus (SARS-CoV-2) are mainly laboratory, including virus isolation and culture, virus nucleic acid detection, virus antigen and antibody detection. Virus isolation and culture need to be operated in a biosafety level 3 (BSL-3) laboratory, which is not easy and time-consuming. The virus antigen and antibody detection methods are still in development, so the current laboratory diagnosis is still mainly based on virus nucleic acid detection. However, nucleic acid detection requires professional operators, special instruments and equipment, and takes more than 4 hours, which is not suitable for mass screening. Therefore, there is an urgent need to develop diagnostic methods that can rapidly detect the novel coronavirus (SARS CoV-2).
本發明基於許多分離的抗體的鑑定，這些抗體出乎意料地表現出對嚴重急性呼吸道症候群冠狀病毒(Severe Acute Respiratory Syndrome coronavirus, SARS-CoV)與新型冠狀病毒(SARS-CoV-2)的優異專一性，且基本上不與其他類型的呼吸道相關病毒交叉反應。本發明提供了這樣的抗體及其抗原結合片段，以及包含它們的組合物及套組，以及使用它們的方法。本發明可用於檢測樣本中的SARS-CoV及/或新型冠狀病毒(SARS-CoV-2)，具體而言是來自懷疑被病毒感染的患者的樣本。The present invention is based on the identification of a number of isolated antibodies that unexpectedly exhibit excellent specificity against Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and novel coronavirus (SARS-CoV-2). Sexuality, and basically does not cross-react with other types of respiratory-associated viruses. The present invention provides such antibodies and antigen-binding fragments thereof, as well as compositions and kits comprising them, and methods of using them. The present invention can be used to detect SARS-CoV and/or novel coronavirus (SARS-CoV-2) in samples, specifically samples from patients suspected of being infected by the virus.
於一方面，本發明提供抗新型冠狀病毒(SARS-CoV-2)的分離的抗體或其抗原結合片段，其中該分離的抗體係選自由下列所組成之群組： (i) 一第一抗體，包含 (a) 一重鏈可變區(heavy chain variable region, V
H )，包含如SEQ ID NO: 1所示之重鏈互補決定區1 (heavy chain complementarity determining region 1, HC CDR1)、如SEQ ID NO: 2所示之重鏈互補決定區2 (HC CDR2)，以及如SEQ ID NO: 3所示之重鏈互補決定區3 (HC CDR3)；以及 (b) 一輕鏈可變區(light chain variable region, V
L )，包含如SEQ ID NO: 5所示之輕鏈互補決定區1 (light chain complementarity determining region 1, LC CDR1)、如SEQ ID NO: 6所示之輕鏈互補決定區2 (LC CDR2)，以及如SEQ ID NO: 7所示之輕鏈互補決定區3 (LC CDR3)；以及(ii) 一第二抗體，包含(a) 一重鏈可變區(V
H )，包含如SEQ ID NO: 9所示之重鏈互補決定區1 (HC CDR1)、如SEQ ID NO: 10所示之重鏈互補決定區2 (HC CDR2)，以及如SEQ ID NO: 11所示之重鏈互補決定區3 (HC CDR3)；以及 (b) 一輕鏈可變區(V
L )，包含如SEQ ID NO: 13所示之輕鏈互補決定區1 (LC CDR1)、如SEQ ID NO: 14所示之輕鏈互補決定區2 (LC CDR2)，以及如SEQ ID NO: 15所示之輕鏈互補決定區3 (LC CDR3)；以及(iii) (i)與(ii)之組合。
In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof against a novel coronavirus (SARS-CoV-2), wherein the isolated antibody system is selected from the group consisting of: (i) a primary antibody , comprising (a) a heavy chain variable region (heavy chain variable region, VH ), comprising a heavy chain complementarity determining region 1 (heavy chain
於某些具體實施例中，該第一抗體包含如SEQ ID NO: 4所示之重鏈可變區(V H )以及包含如SEQ ID NO: 8所示之輕鏈可變區(V L )。 In certain embodiments, the first antibody comprises a heavy chain variable region ( VH ) as set forth in SEQ ID NO:4 and a light chain variable region ( VL ) as set forth in SEQ ID NO:8. ).
於某些具體實施例中，該第二抗體包含如SEQ ID NO: 12所示之重鏈可變區(V H )以及包含如SEQ ID NO: 16所示之輕鏈可變區(V L )。 In certain embodiments, the second antibody comprises a heavy chain variable region ( VH ) as set forth in SEQ ID NO:12 and a light chain variable region ( VL ) as set forth in SEQ ID NO:16. ).
本發明之抗新型冠狀病毒(SARS-CoV-2)的抗體可為全長抗體。本發明抗體之抗原結合片段可為scFv、(scFv) 2、Fab、Fab’、F(ab’) 2。 The anti-novel coronavirus (SARS-CoV-2) antibody of the present invention can be a full-length antibody. The antigen-binding fragment of the antibody of the present invention can be scFv, (scFv) 2 , Fab, Fab', F(ab') 2 .
於另一方面，本發明提供一種核酸，其包含編碼抗體重鏈可變區(V H )或抗體輕鏈可變區(V L )或兩者的核苷酸序列，其中該V H 及V L 如本文所述。 In another aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody heavy chain variable region ( VH ) or an antibody light chain variable region ( VL ) or both, wherein the VH and V L is as described herein.
本發明還提供包含本文所述之任何核酸的載體(例如，表現載體)及包含這種載體的宿主細胞。The invention also provides vectors (eg, expression vectors) comprising any of the nucleic acids described herein and host cells comprising such vectors.
本發明進一步提供一種製備抗新型冠狀病毒(SARS-CoV-2)的抗體之方法，包含(i)在允許表現抗體的條件下培養如本文所述之宿主細胞，及可選擇地(ii)從該細胞陪養中收穫抗體。The present invention further provides a method of preparing an antibody against a novel coronavirus (SARS-CoV-2), comprising (i) culturing a host cell as described herein under conditions that allow expression of the antibody, and optionally (ii) from Antibodies are harvested from the cell culture.
於另一方面，本發明提供一種組合物，包含(a)本文所述之抗新型冠狀病毒(SARS-CoV-2)的任何抗體，如本文所述之任何核酸，或如本文所述之任何載體(vector)；及(b)載體(carrier)，例如，醫藥上可接受之載體。In another aspect, the present invention provides a composition comprising (a) any antibody to the novel coronavirus (SARS-CoV-2) as described herein, any nucleic acid as described herein, or any of the and (b) a carrier, eg, a pharmaceutically acceptable carrier.
於某些具體實施例中，本發明之組合物為用於治療新冠肺炎(COVID-19)之醫藥組合物。In certain embodiments, the compositions of the present invention are pharmaceutical compositions for the treatment of novel coronavirus pneumonia (COVID-19).
於某些具體實施例中，本發明之組合物為用於診斷新冠肺炎(COVID-19)之診斷組合物。In certain embodiments, the compositions of the present invention are diagnostic compositions for diagnosing novel coronavirus pneumonia (COVID-19).
於另一方面，本發明提供一種在懷疑含有新型冠狀病毒(SARS-CoV-2)的樣本中檢測新型冠狀病毒(SARS-CoV-2)之方法，包含使該樣本與如本文所述之抗新型冠狀病毒(SARS-CoV-2)的分離的抗體或其抗原結合片段接觸，並測定該抗體與該樣本的結合。該結合的結果用於確定樣本中新型冠狀病毒(SARS-CoV-2)的存在。於某些具體實施例中，本發明之抗新型冠狀病毒(SARS-CoV-2)的分離的抗體相互配對以進行夾心式測定。In another aspect, the present invention provides a method for detecting a novel coronavirus (SARS-CoV-2) in a sample suspected of containing the novel coronavirus (SARS-CoV-2), comprising subjecting the sample to an anti-virus as described herein An isolated antibody to the novel coronavirus (SARS-CoV-2) or antigen-binding fragment thereof is contacted and the binding of the antibody to the sample is determined. The combined results were used to determine the presence of the novel coronavirus (SARS-CoV-2) in the sample. In certain embodiments, the isolated antibodies against novel coronavirus (SARS-CoV-2) of the present invention are paired with each other for sandwich assays.
於另一方面，本發明提供一種用於在樣本中檢測新型冠狀病毒(SARS-CoV-2)的存在之套組，包含一種以上之本文所述之抗新型冠狀病毒(SARS-CoV-2)的抗體。In another aspect, the present invention provides a kit for detecting the presence of a novel coronavirus (SARS-CoV-2) in a sample, comprising more than one anti-novel coronavirus (SARS-CoV-2) as described herein of antibodies.
於某些具體實施例中，該套組中的至少一種抗體包含可偵測到的標記。In certain embodiments, at least one antibody in the panel comprises a detectable label.
該可偵測到的標記之實例包括，但不限於，酶標記、螢光標記、金屬標記，以及放射性標記。Examples of such detectable labels include, but are not limited to, enzymatic labels, fluorescent labels, metal labels, and radioactive labels.
於某些具體實施例中，該套組為免疫測定套組。In certain embodiments, the kit is an immunoassay kit.
該免疫測定之實例包括，但不限於，酶聯免疫吸附測定(enzyme-linked immunosorbent assay，ELISA)、放射免疫測定(radioimmunoassay，RIA)、螢光免疫測定(fluorescence immunoassay，FIA)、發光免疫測定(luminescence immunoassay，LIA)，或免疫螢光測定(immunoluminometric assay, ILMA)。Examples of such immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay ( luminescence immunoassay, LIA), or immunoluminescence assay (immunoluminometric assay, ILMA).
於某些具體實施例中，該免疫測定係以側流測定形式進行。In certain embodiments, the immunoassay is performed in a lateral flow assay format.
具體而言，該免疫測定為夾心式測定。Specifically, the immunoassay is a sandwich assay.
於下面的描述中闡述了本發明之一個或多個具體實施例之細節。從以下幾個具體實施例的詳細描述以及所附申請專利範圍中，本發明之其他特徵或優點將變得顯而易見。The details of one or more specific embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will become apparent from the following detailed description of several specific embodiments and the scope of the appended claims.
本發明涉及抗新型冠狀病毒(SARS-CoV-2)的抗體。本發明提供這樣的抗體及其抗原結合片段，其可用於在生物樣本中檢測新型冠狀病毒(SARS-CoV-2)。本發明進一步提供製備該抗體或其抗原結合片段的方法及載體，以及包含它們的套組及組合物，以及使用它們來檢測新型冠狀病毒(SARS-CoV-2)及診斷及治療新冠肺炎(COVID-19)之方法。The present invention relates to antibodies against novel coronavirus (SARS-CoV-2). The present invention provides such antibodies and antigen-binding fragments thereof, which can be used to detect novel coronavirus (SARS-CoV-2) in biological samples. The present invention further provides methods and vectors for preparing the antibodies or antigen-binding fragments thereof, as well as kits and compositions comprising them, and using them to detect novel coronavirus (SARS-CoV-2) and diagnose and treat novel coronavirus pneumonia (COVID-19) -19) method.
以下描述僅旨在說明本發明之各種具體實施例。因此，本文討論的具體具體實施例或修改不應解釋為對本發明範圍的限制。對於本領域技術人員顯而易見的是，在不脫離本發明之範圍的情況下，可以進行各種改變或等同物。The following description is intended only to illustrate various specific embodiments of the present invention. Therefore, specific embodiments or modifications discussed herein should not be construed as limitations on the scope of the invention. It will be apparent to those skilled in the art that various changes or equivalents can be made without departing from the scope of the present invention.
I.i. 定義definition
為了提供對本發明清楚及快速的理解，首先定義某些術語。在整個詳細描述中闡述了另外的定義。除非另外定義，否則本文所用之所有技術及科學術語具有與本發明所屬領域的技術人員通常理解的含義之相同的含義。In order to provide a clear and quick understanding of the present invention, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
如本文所用，單數形式「一」、「一個」及「該」包括複數指示物，除非上下文另有明確說明。因此，例如，提及「一個組件」包括多個這樣的組件及其本領域技術人員已知的等同物。As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an element" includes a plurality of such elements and equivalents thereof known to those skilled in the art.
「包含(動詞)」或「包含(動名詞)」等詞通常以包括(動詞)/包括(動名詞)的含義使用，其表示允許存在一種或多種特徵、成分或組分。術語「包含(動詞)」或「包含(動名詞)」包括術語「組成」或「由......組成」。Words such as "comprises (verb)" or "comprises (gerund)" are often used in the sense of including (verb)/includes (gerund), which means that one or more features, ingredients or components are permitted to be present. The term "comprising (verb)" or "comprising (gerund)" includes the term "consisting of" or "consisting of."
如本文所用，「多胜肽」係指由透過胜肽鍵連接的胺基酸殘基組成的聚合物。「蛋白質」通常係指相對較大的多胜肽。「胜肽」通常係指相對短的多胜肽(例如，含有至多100、90、70、50、30，或20個胺基酸殘基)。As used herein, "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds. "Protein" generally refers to relatively large polypeptides. "Peptide" generally refers to a relatively short polypeptide (eg, containing up to 100, 90, 70, 50, 30, or 20 amino acid residues).
如本文所用，「約」或「近似」等詞係指本領域普通技術人員將理解的可接受偏差程度，其可在一定程度上變化，這取決於其所用之上下文。通常，「約」或「近似」可表示在引用值附近具有±10%範圍的數值。As used herein, words such as "about" or "approximately" refer to an acceptable degree of deviation as would be understood by one of ordinary skill in the art, which may vary to some extent depending upon the context in which it is used. In general, "about" or "approximately" can mean a numerical value having a range of ±10% around the recited value.
如本文所用，「基本上相同」係指兩個序列具有80%或更多，較佳為85%或更多，更佳為90%或更多，甚至更佳為95%或更多的同源性。As used herein, "substantially identical" means that two sequences are 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more identical origin.
如本文所用，「抗體」(可互換地以複數形式使用)係指具有專一性結合特定目標抗原之能力的免疫球蛋白分子。如本文所用，「抗體」不僅包括完整的(亦即，全長的)抗體分子，還包括其保留抗原結合能力的抗原結合片段，例如，Fab、Fab'、F(ab') 2及Fv。此類片段也是本領域熟知的，且通常在體外及體內使用。「抗體」還包括人源化抗體、嵌合抗體、雙抗體、線性抗體、單鏈抗體、多專一性抗體(例如，雙專一性抗體)，以及免疫球蛋白分子的任何其他修飾構型，其包含具有所需專一性的抗原識別位點，包括抗體的糖基化變體、抗體的胺基酸序列變體，以及共價修飾的抗體。 As used herein, "antibody" (used interchangeably in the plural) refers to an immunoglobulin molecule that has the ability to specifically bind to a specific target antigen. As used herein, "antibody" includes not only intact (ie, full-length) antibody molecules, but also antigen-binding fragments thereof that retain antigen-binding capacity, eg, Fab, Fab', F(ab') 2 , and Fv. Such fragments are also well known in the art and are commonly used in vitro and in vivo. "Antibody" also includes humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (eg, bispecific antibodies), and any other modified configuration of an immunoglobulin molecule, which Antigen recognition sites with the desired specificity are included, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
一個完整或完全的抗體包含兩條重鏈及兩條輕鏈。每條重鏈含有可變區(V H )及第一、第二及第三恆定區(C H 1、C H 2及C H 3)；每條輕鏈含有一可變區(V L )及一恆定區(C L )。抗體具有「Y」形狀，Y的莖部由透過雙硫鍵結合在一起的兩條重鏈的第二及第三恆定區所組成。Y的每個臂部包括與一單個輕鏈的可變區及恆定區結合的一單個重鏈的可變區及第一恆定區。該輕鏈的可變區及重鏈的可變區負責抗原結合。兩條鏈中的可變區通常包含三個高度可變區，稱為互補決定區(complementarity determining regions，CDRs)；亦即，輕(L)鏈CDRs包括LC CDR1、LC CDR2，以及LC CDR3，以及重(H)鏈CDRs包括HC CDR1、HC CDR2，以及HC CDR3。三個CDRs由框架區(framework region, FR1、FR2、FR3，以及FR4)標記，其比該CDRs更高度保守並形成支持高變異區的支架。該重鏈及輕鏈的恆定區不負責抗原結合，但涉及各種效應子的功能。取決於其重鏈恆定結構域的抗體胺基酸序列，可將免疫球蛋白分配到不同的類別。免疫球蛋白有五大類：IgA、IgD、IgE、IgG，以及IgM，其中一些可進一步分為亞類(同種型)，例如：IgG1、IgG2、IgG3、IgG4、IgA1，以及IgA2。對應於不同類免疫球蛋白的重鏈恆定結構域分別稱為α、δ、ε、γ，以及μ。 An intact or complete antibody contains two heavy chains and two light chains. Each heavy chain contains a variable region ( VH ) and first, second and third constant regions ( CH1, CH2 and CH3 ) ; each light chain contains a variable region ( VL ) and a constant region ( CL ). Antibodies have a "Y" shape with the stem of the Y consisting of the second and third constant regions of two heavy chains held together by disulfide bonds. Each arm of Y includes the variable and first constant regions of a single heavy chain combined with the variable and constant regions of a single light chain. The variable region of the light chain and the variable region of the heavy chain are responsible for antigen binding. The variable regions in both chains typically contain three hypervariable regions, called complementarity determining regions (CDRs); that is, light (L) chain CDRs include LC CDR1, LC CDR2, and LC CDR3, And heavy (H) chain CDRs include HC CDR1, HC CDR2, and HC CDR3. The three CDRs are marked by framework regions (FR1, FR2, FR3, and FR4), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable regions. The constant regions of the heavy and light chains are not responsible for antigen binding, but are involved in various effector functions. Depending on the antibody amino acid sequence of their heavy chain constant domains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
如本文所用，「抗原結合結構域」或「抗原結合片段」等詞係指完整抗體分子中負責抗原結合的部分或區域。抗原結合片段能夠結合親本抗體結合的相同抗原。抗原結合片段的實例包括，但不限於：(i) Fab片段，其可為由V H -C H 1鏈以及V L - C L 鏈組成的單價片段；(ii) F(ab') 2片段，其可為由在鉸鏈區透過雙硫鍵連接的兩個Fab片段組成的二價片段；(iii) Fv片段，由透過非共價相互作用結合在一起的抗體分子的V H 及V L 結構域組成；(iv) 單鏈Fv(scFv)，其可為透過胜肽連接子由V H 結構域及V L 結構域組成的單一多胜肽鏈；(v) (scFv) 2，其可包含透過一胜肽連接子連接的兩個V H 結構域，以及兩個V L 結構域，其透過雙硫鍵與兩個V H 結構域連結。 As used herein, the terms "antigen-binding domain" or "antigen-binding fragment" refer to the portion or region of an intact antibody molecule that is responsible for antigen binding. Antigen-binding fragments are capable of binding the same antigen to which the parent antibody binds. Examples of antigen-binding fragments include, but are not limited to: (i) Fab fragments, which may be monovalent fragments consisting of VH - CH1 chains and VL - CL chains; (ii) F(ab') 2 fragments , which can be a bivalent fragment consisting of two Fab fragments linked by disulfide bonds at the hinge region; (iii) an Fv fragment, which consists of the VH and VL structures of antibody molecules bound together by non-covalent interactions domain composition; (iv) a single-chain Fv (scFv), which can be a single polypeptide chain consisting of a VH domain and a VL domain through a peptide linker; (v) (scFv) 2 , which can be Contains two VH domains linked by a peptide linker, and two VL domains linked to the two VH domains by disulfide bonds.
如本文所用，「嵌合抗體」係指含有來自不同來源(例如，不同物種)的多胜肽之抗體。於某些具體實施例中，在這些嵌合抗體中，輕鏈及重鏈的可變區可模擬源自一種哺乳動物(例如，非人類哺乳動物，例如小鼠、兔及大鼠)的抗體的可變區，而恆定部分可以與衍生自另一種哺乳動物例如人類的抗體中的序列同源。As used herein, a "chimeric antibody" refers to an antibody that contains polypeptides from different sources (eg, different species). In certain embodiments, in these chimeric antibodies, the variable regions of the light and heavy chains can mimic antibodies derived from a mammal (eg, non-human mammals such as mice, rabbits, and rats) The variable region, while the constant portion may be homologous to sequences in an antibody derived from another mammal, such as a human.
如本文所用，「人源化抗體」係指包含來自一人類抗體的一框架區以及來自一非人類(通常是小鼠或大鼠)免疫球蛋白的一個或多個互補決定區(CDRs)的抗體。As used herein, a "humanized antibody" refers to an antibody comprising a framework region from a human antibody and one or more complementarity determining regions (CDRs) from a non-human (usually mouse or rat) immunoglobulin Antibody.
如本文所用，「人類抗體」係指其中基本上輕鏈及重鏈序列的完整序列(包括互補決定區(CDRs))來自人類基因的抗體。該人類抗體可包括一個或多個不由人類種系免疫球蛋白序列編碼的胺基酸殘基，例如，透過一個或多個互補決定區(CDRs)中的突變，或在一個或多個框架區(FRs)中的突變，以便例如降低可能的免疫原性、增加親和力、消除可能導致不期望的折疊的半胱胺酸等。As used herein, "human antibody" refers to an antibody in which substantially the entire light and heavy chain sequences, including complementarity determining regions (CDRs), are derived from human genes. The human antibody may include one or more amino acid residues not encoded by human germline immunoglobulin sequences, eg, by mutation in one or more complementarity determining regions (CDRs), or in one or more framework regions Mutations in (FRs) to eg reduce possible immunogenicity, increase affinity, eliminate cysteines that may lead to undesired folding, etc.
如本文所用，「分離的」物質意指透過人類的手其已被從天然狀態改變。於某些具體實施例中，若本發明之多胜肽(例如，抗體)或核酸基本上不含細胞材料或化學前驅物或可能涉及胜肽/核酸製備過程的其他化學物/組成分時，則可說其為「分離的」或「純化的」。應當理解的是，「分離的」或「純化的」等詞不一定反映該胜肽被「絕對」分離或純化的程度，例如，透過去除所有其他物質(例如，雜質或細胞成分)。於某些情況下，例如，分離的或純化的多胜肽包括含有具有少於50%、40%、30%、20%或10% (按重量計)的其他蛋白質(例如，細胞蛋白質)的多胜肽的製劑，具有少於50%、40%、30%、20%或10% (按體積計)的培養基，或具有少於50%、40%、30%、20%或10% (按重量計)的化學前驅物或其他合成程序中涉及的化學物/組成分。As used herein, "isolated" material means that it has been altered from its natural state by the human hand. In certain embodiments, if the polypeptide (eg, antibody) or nucleic acid of the invention is substantially free of cellular material or chemical precursors or other chemicals/components that may be involved in the peptide/nucleic acid production process, It can then be said to be "isolated" or "purified". It should be understood that words such as "isolated" or "purified" do not necessarily reflect the degree to which the peptide has been "absolutely" isolated or purified, eg, by removing all other materials (eg, impurities or cellular components). In some cases, for example, isolated or purified polypeptides include those containing less than 50%, 40%, 30%, 20%, or 10% (by weight) of other proteins (e.g., cellular proteins). A formulation of a polypeptide having less than 50%, 40%, 30%, 20% or 10% (by volume) of medium, or having less than 50%, 40%, 30%, 20% or 10% ( by weight) of chemical precursors or other chemicals/components involved in the synthesis procedure.
如本文所用，「專一性的結合」或「專一性地結合」等詞係指兩個分子之間的非隨機結合反應，例如抗體與其標靶抗原的抗原決定位之結合。「專一性地結合」標靶抗原或抗原決定位的抗體為本領域熟知的術語，確定這種專一性地結合的方法也是本領域熟知的。如果分子與其他標靶/抗原決定位相比更頻繁、更快速地、更長時間地及/或與特定標靶抗原或抗原決定位的親和力更強地反應或結合，則稱其表現出「專一性地結合」。如果抗體以比與其他物質結合的更大的親和力、親留力，更容易，及/或持續更長的時間結合，則抗體「專一性地結合」標靶抗原。換句話說，透過閱讀該定義還可以理解，例如，專一性結合第一標靶抗原的抗體可以或可以不專一性或優先的結合第二標靶抗原。因此，「專一性的結合」或「優先的結合」不一定需要(儘管它可以包括)排他性結合。通常但非必要地，對結合的提及意指專一性的/優先的結合。結合的親和力根據解離常數(dissociation constant, K D )來定義。通常，當針對抗體使用時專一性地結合可指專一性地結合(識別)其標靶抗體，其K D 值小於約10 ~7M，例如約10 ~8M或更低，例如約10 ~9M以下，約10 ~10M以下，約10 ~11M以下，約10 ~12M以下，或甚至更低，以與K D 對應的親和性結合特定標靶，比其與非專一性抗原(例如BSA或酪蛋白)結合的親和力低至少10倍，例如低至少100倍，例如低至少1,000倍，例如低至少10,000倍。 As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope of its target antigen. An antibody that "specifically binds" a target antigen or epitope is a term well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specificity" if it reacts or binds more frequently, more rapidly, for longer, and/or with greater affinity to a particular target antigen or epitope than other targets/epitopes Sexually combined". An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, more readily, and/or for a longer period of time than it binds to other substances. In other words, it is also understood by reading this definition that, for example, an antibody that specifically binds to a first target antigen may or may not bind specifically or preferentially to a second target antigen. Thus, "specific binding" or "preferred binding" does not necessarily require (although it may include) exclusive binding. Usually, but not necessarily, reference to binding means specific/preferential binding. The affinity of binding is defined in terms of the dissociation constant (K D ). Generally, specifically binding when used against an antibody may refer to specifically binding (recognizing) its target antibody with a KD value of less than about 10 ~ 7M, such as about 10 ~ 8M or less, such as about 10 ~8M 9 M or less, about 10-10 M or less, about 10-11 M or less, about 10-12 M or less, or even lower, to bind to a specific target with an affinity corresponding to KD than it does with nonspecific antigens (eg BSA or casein) binds with at least 10-fold lower affinity, eg, at least 100-fold lower, eg, at least 1,000-fold lower, eg, at least 10,000-fold lower.
如本文所用，「交叉性反應」一詞係指抗體與不同蛋白質上的類似抗原位點反應的能力。As used herein, the term "cross-reactivity" refers to the ability of an antibody to react with similar antigenic sites on different proteins.
如本文所用，「冠狀病毒(Coronavirus)」一詞係指冠狀病毒科( Coronavirinae)的病毒。這類病毒為球形的包膜RNA病毒，在電子顯微鏡下可看到表面冠狀尖刺，因此被稱為冠狀病毒。冠狀病毒可進一步分為四個亞群：α、β、γ以及δ冠狀病毒。人類共有7種冠狀病毒株，其中包括兩種α冠狀病毒(HCov-229E以及HCoV-NL63)、兩種β冠狀病毒(HCov-HKU1以及HCov-OC43)、中東呼吸症候群冠狀病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)，嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)，以及新發現的新型冠狀病毒(SARS CoV-2)。 As used herein, the term "Coronavirus" refers to a virus of the Coronavirinae family. This type of virus is a spherical enveloped RNA virus with crowned spikes on the surface under an electron microscope, so it is called a coronavirus. Coronaviruses can be further divided into four subgroups: alpha, beta, gamma and delta coronaviruses. There are 7 strains of coronavirus in humans, including two alphacoronaviruses (HCov-229E and HCoV-NL63), two betacoronaviruses (HCov-HKU1 and HCov-OC43), Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome) coronavirus, MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and the newly discovered novel coronavirus (SARS CoV-2).
如本文所用，「新型冠狀病毒(SARS CoV-2) 」一詞係指造成嚴重特殊傳染性肺炎(COVID-19)的病原菌。新型冠狀病毒(SARS CoV-2)為一種正股單鏈RNA病毒，其為冠狀病毒科(Coronavirinae)，beta冠狀病毒屬(Betacoronavirus)的成員。每個新型冠狀病毒(SARS CoV-2)的RNA序列的長度約為30,000個鹼基，其病毒顆粒直徑為50-200奈米。如同其他冠狀病毒一樣，新型冠狀病毒(SARS CoV-2)具有四種結構蛋白，分別稱為棘(Spike, S)、套膜(envelope, E)、膜(membrane, M)，以及核鞘(nucleocapsid, N)蛋白。核鞘(N)蛋白保留RNA基因組，而棘(S)、套膜(E)，以及膜(M)蛋白共同形成病毒包膜。As used herein, the term "novel coronavirus (SARS CoV-2)" refers to the causative bacterium that causes severe special infectious pneumonia (COVID-19). The novel coronavirus (SARS CoV-2) is a positive-stranded single-stranded RNA virus, which is a member of the family Coronavirinae, the genus Betacoronavirus. The RNA sequence of each novel coronavirus (SARS CoV-2) is about 30,000 bases in length, and its viral particles are 50-200 nanometers in diameter. Like other coronaviruses, the new coronavirus (SARS CoV-2) has four structural proteins, called spine (Spike, S), envelope (envelope, E), membrane (membrane, M), and nucleocapsule ( nucleocapsid, N) protein. The nucleosheath (N) proteins retain the RNA genome, while the spine (S), envelope (E), and membrane (M) proteins together form the viral envelope.
如本文所用，「嚴重特殊傳染性肺炎」、「新冠肺炎」或「COVID-19」在本文中可互換使用，係指由新型冠狀病毒(SARS CoV-2)感染引起的疾病，主要透過緊密接觸並透過咳嗽或打噴嚏產生的呼吸道飛沫在人與人之間傳播。患有新冠肺炎(COVID-19)的人類可能沒有任何徵兆，或有從輕微到嚴重的症狀。感染新型冠狀病毒(SARS CoV-2)後1至14天開始出現症狀。新冠肺炎(COVID-19)的最常見症狀包括發燒、乾咳，以及疲勞，稍不常見的症狀包括肌肉或身體疼痛、喉嚨痛、腹瀉、結膜炎、頭痛、味覺或嗅覺喪失，皮膚出疹，以及呼吸急促，嚴重症狀則包括呼吸困難或呼吸急促、胸痛或壓迫，以及言語或行動不便，更嚴重的情況下可能導致多器官功能衰竭、敗血性休克、急性呼吸窘迫症候群(ARDS)，甚至死亡。As used herein, "Severe Idiopathic Pneumonia", "COVID-19" or "COVID-19" are used interchangeably herein to refer to disease caused by infection with the novel coronavirus (SARS CoV-2), primarily through close contact It is spread from person to person through respiratory droplets produced by coughing or sneezing. Humans with COVID-19 may have no symptoms or have symptoms ranging from mild to severe. Symptoms start 1 to 14 days after infection with the novel coronavirus (SARS CoV-2). The most common symptoms of COVID-19 include fever, dry cough, and fatigue, with less common symptoms including muscle or body aches, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash, and breathing Shortness of breath, severe symptoms include difficulty breathing or shortness of breath, chest pain or pressure, and difficulty speaking or moving, and in more severe cases can lead to multiple organ failure, septic shock, acute respiratory distress syndrome (ARDS), and even death.
「棘(spike)蛋白」、「S多胜肽」及「S蛋白」等詞在本文中可互換使用，係指由新型冠狀病毒(SARS CoV-2)編碼的一種結構性糖蛋白，負責使病毒附著在宿主細胞膜上並與其融合的蛋白。三聚體S蛋白的每個單體約為180 kDa，並包含S1與S2亞基，分別調節附著與膜融合。S蛋白主要透過與人類細胞上的受體血管緊張素轉換酶2 (angiotensin converting enzyme 2, ACE2)結合而進入人體細胞。新型冠狀病毒(SARS CoV-2) S蛋白的胺基酸序列為本領域已知且可獲得的，例如GenBank登錄號QJH92179.1、QHD43416.1、QJH92167.1、QKU37045.1等。The terms "spike protein", "S polypeptide" and "S protein" are used interchangeably herein to refer to a structural glycoprotein encoded by the novel coronavirus (SARS CoV-2) responsible for the A protein that a virus attaches to and fuses with the host cell membrane. Each monomer of the trimeric S protein is approximately 180 kDa and contains S1 and S2 subunits that regulate attachment and membrane fusion, respectively. The S protein enters human cells mainly by binding to the receptor angiotensin converting enzyme 2 (ACE2) on human cells. The amino acid sequence of the novel coronavirus (SARS CoV-2) S protein is known and available in the art, such as GenBank accession numbers QJH92179.1, QHD43416.1, QJH92167.1, QKU37045.1, etc.
「核酸」或「多核苷酸」等詞可指由核苷酸單元組成的聚合物。多核苷酸包括天然存在的核酸，例如去氧核糖核酸(Deoxyribonucleic acid, DNA)及核糖核酸(ribonucleic acid, RNA)以及核酸類似物，包括具有非天然存在的核苷酸的核酸類似物。多核苷酸可例如使用自動DNA合成儀合成。應當理解的是，當核苷酸序列由DNA序列(即A、T、G、C)表示時，這也包括其中「U」取代的「T」 (即A、U、G、C)的RNA序列。「cDNA」係指以單鏈或雙鏈形式與mRNA互補或相同的DNA。The terms "nucleic acid" or "polynucleotide" can refer to polymers composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and nucleic acid analogs, including nucleic acid analogs with non-naturally occurring nucleotides. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. It should be understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), this also includes RNAs in which "U" is substituted for "T" (ie, A, U, G, C) sequence. "cDNA" refers to DNA that is complementary or identical to mRNA in single- or double-stranded form.
「互補」係指兩個多核苷酸的相互作用表面的拓撲相容性或匹配在一起。因此，該兩個分子可描述為互補的，此外接觸表面特徵彼此互補。當第一多核苷酸的核苷酸序列與第二多核苷酸的多核苷酸結合配偶體的核苷酸序列相同時，第一多核苷酸與第二多核苷酸互補。因此，序列5'-TATAG-3'的多核苷酸與序列為5'-CTATA-3'的多核苷酸互補。"Complementary" refers to the topological compatibility or matching together of the interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, furthermore the contact surface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide when the nucleotide sequence of the first polynucleotide is the same as the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, a polynucleotide of sequence 5'-TATAG-3' is complementary to a polynucleotide of sequence 5'-CTATA-3'.
「編碼」係指多核苷酸中特定核苷酸序列(例如，基因，cDNA，或mRNA)的固有特性，以作為在生物過程中合成其他聚合物及巨分子的模板，這些生物過程具有給定的核苷酸序列(即，rRNA、tRNA及mRNA)或給定的胺基酸序列以及由此產生的生物學特性。因此，如果由基因產生的mRNA的轉錄及轉譯在細胞或其他生物系統中產生蛋白質，則該基因編碼該蛋白質。本領域技術人員應當理解，由於遺傳密碼的簡併性，許多不同的多核苷酸及核酸可以編碼相同的多胜肽。還應理解的是，技術人員可使用常規技術進行不影響由那裡描述的多核苷酸編碼的多胜肽序列的核苷酸取代，以反映其中表現多胜肽的任何特定宿主生物體使用的密碼子。因此，除非另有說明，「編碼胺基酸序列的核苷酸序列」包括彼此簡併形式且編碼相同胺基酸序列的所有核苷酸序列。"Encoding" refers to the inherent property of a particular nucleotide sequence (eg, gene, cDNA, or mRNA) in a polynucleotide to serve as a template for the synthesis of other polymers and macromolecules in biological processes with a given nucleotide sequence (ie, rRNA, tRNA and mRNA) or a given amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein if the transcription and translation of the mRNA produced by the gene produces the protein in a cell or other biological system. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. It will also be appreciated that the skilled artisan can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described therein to reflect the code used by any particular host organism in which the polypeptide is expressed. son. Thus, unless otherwise stated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate to each other and that encode the same amino acid sequence.
「重組核酸」係指具有非天然連接在一起的序列的多核苷酸或核酸。重組核酸可以載體的形式存在。「載體」可含有給定的目標核苷酸序列及調節序列。載體可用於表現給定的核苷酸序列(表現載體)或維持給定的核苷酸序列以使其複製、操作或在不同位置之間(例如，在不同生物體之間)轉移。為了上述目的，可將載體引入合適的宿主細胞中。「重組細胞」係指已經向其中引入重組核酸的宿主細胞。「轉形細胞」係指透過重組DNA技術將編碼的目標蛋白質的DNA分子引入其中的細胞。"Recombinant nucleic acid" refers to a polynucleotide or nucleic acid having sequences that are not naturally linked together. The recombinant nucleic acid can exist in the form of a vector. A "vector" may contain a given nucleotide sequence of interest and regulatory sequences. A vector can be used to express or maintain a given nucleotide sequence (expression vector) so that it can be replicated, manipulated, or transferred between different locations (eg, between different organisms). The vector can be introduced into a suitable host cell for the above purpose. A "recombinant cell" refers to a host cell into which a recombinant nucleic acid has been introduced. A "transformed cell" refers to a cell into which a DNA molecule encoding a protein of interest has been introduced by recombinant DNA technology.
載體可為各種類型，包括質體、黏質體、F型黏接質體(fosmids)、附加體、人工染色體、噬菌體、病毒載體等。通常，在載體中，給定的核苷酸序列與調節序列可操作地連接，使得當載體導入宿主細胞時，給定的核苷酸序列可在調節序列的控制下在宿主細胞中表現。調節序列可包含，例如，但不限於，啟動子序列(例如，巨細胞病毒(cytomegalovirus，CMV)啟動子、猿猴病毒40 (simian virus 40，SV40)早期啟動子、T7啟動子，以及醇氧化酶基因(AOX1)啟動子)、起始密碼子、複製起點、增強子、分泌訊號序列(例如，一α-交配因子訊號)、終止密碼子，以及其他控制序列(例如，Shine-Dalgarno序列及終止序列)。較佳地，載體可進一步含有標記序列(例如，一抗生素抗性標記序列)，用於隨後的篩選/選擇程序。為了蛋白質產生的目的，在載體中，給定的目標核苷酸序列可與除上述調節序列之外的另一核苷酸序列連接，進而產生融合多胜肽並有益於隨後的純化過程。該融合多胜肽包括用於純化的標籤，例如，His-tag。Vectors can be of various types, including plastids, cosmids, fosmids, episomes, artificial chromosomes, bacteriophages, viral vectors, and the like. Typically, in a vector, a given nucleotide sequence is operably linked to regulatory sequences such that when the vector is introduced into a host cell, the given nucleotide sequence can be expressed in the host cell under the control of the regulatory sequence. Regulatory sequences can include, for example, but are not limited to, promoter sequences (e.g., cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, T7 promoter, and alcohol oxidase gene (AOX1) promoter), initiation codon, origin of replication, enhancer, secretion signal sequence (eg, an alpha-mating factor signal), stop codon, and other control sequences (eg, Shine-Dalgarno sequence and termination sequence). Preferably, the vector may further contain marker sequences (eg, an antibiotic resistance marker sequence) for subsequent screening/selection procedures. For the purpose of protein production, in the vector, a given nucleotide sequence of interest can be linked to another nucleotide sequence in addition to the regulatory sequences described above, thereby producing fusion polypeptides and facilitating subsequent purification processes. The fusion polypeptide includes a tag for purification, eg, His-tag.
本文所用之「個體」或「受試者」等詞包括人類及非人類動物，例如伴侶動物(例如狗、貓及其類似物)，農場動物(例如牛、羊、豬、馬及其類似物)，或實驗室動物(如大鼠、小鼠、豚鼠及其類似物)。The terms "individual" or "subject" as used herein include human and non-human animals, such as companion animals (eg, dogs, cats, and the like), farm animals (eg, cows, sheep, pigs, horses, and the like) ), or laboratory animals (such as rats, mice, guinea pigs, and the like).
本文所用之「治療」係指將包括一種或多種活性劑的組合物施用或投予患有疾病、該疾病的症狀或病症或疾病進展的個體，目的為治療、治癒、減緩、緩解、改變、補救、改善，增進，或影響疾病、疾病的症狀或病症、由疾病引起的殘疾，或疾病的進展或其症狀或狀況。"Treatment" as used herein refers to the administration or administration of a composition comprising one or more active agents to an individual suffering from a disease, a symptom or disorder of the disease, or the progression of the disease for the purpose of treating, curing, slowing, alleviating, altering, Remedy, ameliorate, enhance, or affect a disease, a symptom or condition of a disease, a disability caused by a disease, or the progression of a disease or its symptoms or conditions.
本文所用之「有效量」係指在治療的個體或細胞中賦予所需生物效應的活性成分的量。有效量可根據各種原因而改變，例如給藥途徑及頻率，接受該藥物的個體的體重及種類，以及給藥目的。本領域技術人員可基於本文的公開內容、已建立的方法及他們自身的經驗確定每種情況下的劑量。As used herein, an "effective amount" refers to the amount of active ingredient that confers the desired biological effect in the subject or cell being treated. The effective amount may vary depending on various reasons, such as the route and frequency of administration, the weight and species of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dosage in each case based on the disclosure herein, established methods, and their own experience.
II.II. 抗新型冠狀病毒Anti-coronavirus (SARS CoV-2)(SARS CoV-2) 的抗體the antibody
本發明基於鑑定抗新型冠狀病毒(SARS-CoV-2)的許多分離的抗體，包括單株抗體8a-1以及單株抗體27b-2。發現這些抗新型冠狀病毒(SARS CoV-2)的抗體能夠識別SARS-CoV以及新型冠狀病毒(SARS CoV-2)而不與其他類型的呼吸道相關病毒交叉反應。更特定而言，這些抗新型冠狀病毒(SARS CoV-2)的抗體能識別新型冠狀病毒(SARS CoV-2)的S蛋白。發現這些抗新型冠狀病毒(SARS CoV-2)的抗體在各種形式的免疫測定中是有效且可行的，用以檢測樣本中的新型冠狀病毒(SARS CoV-2)，具有優異的靈敏度及專一性。The present invention is based on the identification of a number of isolated antibodies against the novel coronavirus (SARS-CoV-2), including
因此，本文描述抗新型冠狀病毒(SARS-CoV-2)的抗體，包括單株抗體8a-1以及單株抗體27b-2，以及其功能變體。使用ELISA及西方墨點分析法測定這些單株抗體與新型冠狀病毒(SARS-CoV-2)的反應性。這些單株抗體的特徵如
表 1所示。單株抗體8a-1以及單株抗體27b-2各自的重鏈可變區(V
H )及輕鏈可變區(V
L )的胺基酸序列分別如SEQ ID NOs: 4、8、12、16所示。
Accordingly, antibodies against the novel coronavirus (SARS-CoV-2), including
單株抗體8a-1(也稱為「第一抗體」，如本文所述)的功能變體可包含：
(a) 重鏈可變區(V
H )，其包含SEQ ID NO: 1的重鏈互補決定區(HC CDR1)，SEQ ID NO: 2的重鏈互補決定區2 (HC CDR2)，以及SEQ ID NO: 3的重鏈互補決定區3 (HC CDR3)；以及
(b) 輕鏈可變區(V
L )，其包含SEQ ID NO: 5的輕鏈互補決定區1 (LC CDR1)，SEQ ID NO: 6的輕鏈互補決定區2 (LC CDR2)，以及SEQ ID NO: 7的輕鏈互補決定區3 (LC CDR3)，
或其抗原結合片段。
A functional variant of
於某些具體實施例中，該第一抗體包含含有SEQ ID NO: 4或與其基本上相同的胺基酸序列的V H 以及含有SEQ ID NO: 8或與其基本上相同的胺基酸序列的V L 。特定而言，該第一抗體包括含有與SEQ ID NO: 4具有至少80% (例如82%、84%、85%、86%、88%、90%、92%、94%、95%、96%、98%或99%)同一性的胺基酸序列的V H ，以及含有與SEQ ID NO: 8具有至少80% (例如82%、84%、85%、86%、88%、90%、92%、94%、95%、96%、98%或99%)同一性的胺基酸序列的V L 。該第一抗體還包括由編碼本文所述之相關V H 或V L 胺基酸序列的多核苷酸序列編碼的任何重組(工程化)衍生的抗體。 In certain embodiments, the first antibody comprises a VH comprising SEQ ID NO: 4 or substantially the same amino acid sequence and a VH comprising SEQ ID NO: 8 or substantially the same amino acid sequence VL . In particular, the first antibody comprises an antibody that contains at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%) with SEQ ID NO: 4 %, 98%, or 99%) identical to the VH of the amino acid sequence, and containing at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%) with SEQ ID NO: 8 , 92%, 94%, 95%, 96%, 98%, or 99%) VL of amino acid sequences of identity. The primary antibody also includes any recombinant (engineered) derived antibody encoded by a polynucleotide sequence encoding the relevant VH or VL amino acid sequence described herein.
單株抗體27b-2的功能變體(也稱為「第二抗體」，如本文所述)可包含： (a) 重鏈可變區(V
H )，其包含SEQ ID NO: 9的重鏈互補決定區1(HC CDR1)，SEQ ID NO: 10的重鏈互補決定區2 (HC CDR2)，以及SEQ ID NO: 11的重鏈互補決定區3 (HC CDR3)；以及 (b) 輕鏈可變區(V
L )，其包含SEQ ID NO: 13的輕鏈互補決定區1 (LC CDR1)，SEQ ID NO: 14的輕鏈互補決定區2 (LC CDR2)，以及SEQ ID NO: 15的輕鏈互補決定區3 (LC CDR3)。
A functional variant of
於某些具體實施例中，該第二抗體包含含有SEQ ID NO: 12或與其基本上相同的胺基酸序列的V H 以及含有SEQ ID NO: 16或與其基本上相同的胺基酸序列的V L 。特定而言，該第二抗體包括含有與SEQ ID NO: 12具有至少80% (例如82%、84%、85%、86%、88%、90%、92%、94%、95%、96%、98%或99%)同一性的胺基酸序列的V H ，以及含有與SEQ ID NO: 16具有至少80% (例如82%、84%、85%、86%、88%、90%、92%、94%、95%、96%、98%或99%)同一性的胺基酸序列的V L 。該第二抗體還包括由編碼本文所述之相關V H 或V L 胺基酸序列的多核苷酸序列編碼的任何重組(工程化)衍生的抗體。 In certain embodiments, the second antibody comprises a VH comprising SEQ ID NO: 12 or substantially the same amino acid sequence and a VH comprising SEQ ID NO: 16 or substantially the same amino acid sequence VL . In particular, the second antibody comprises an antibody that contains at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%) with SEQ ID NO: 12 %, 98%, or 99%) the VH of the amino acid sequence that is identical, and contains at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%) with SEQ ID NO: 16 , 92%, 94%, 95%, 96%, 98%, or 99%) VL of amino acid sequences of identity. The second antibody also includes any recombinant (engineered) derived antibody encoded by a polynucleotide sequence encoding the relevant VH or VL amino acid sequence described herein.
「基本上相同」可意指變體的相關胺基酸序列(例如，在FRs、CDRs、V H 或V L 中)相較於參考抗體無基本上不同，使得該變體具有基本相似於該參考抗體的結合活性(例如，親和力、專一性或兩者)及生物活性。這種變體可包括微小的胺基酸變化。可以理解的是，多胜肽可以具有有限數量的變化或修飾，這些變化或修飾可以在與其活性或功能無關的多胜肽的某一部分內進行，且仍然產生具有可接受程度的等同或類似生物活性或功能的變體。於某些實例中，胺基酸殘基的變化為保守胺基酸取代，係指與另一個胺基酸殘基相似的化學結構的胺基酸殘基及多胜肽的功能、活性或其他生物學效應對性質的影響較小或基本沒有影響。通常，相較於CDR區域，可在FR區域中進行相對更多的取代，只要它們不會對抗體的結合功能及生物活性產生不利影響(例如，相較於原始抗體，降低結合親和力超過50%)。於某些具體實施例中，參考抗體及變體之間的序列同一性可為約80%、82%、84%、85%、86%、88%、90%、92%、94%、95%、96%、98%或99%，或更高。可以根據本領域普通技術人員已知的改變多胜肽序列的方法製備變體，例如在依循這些方法的參考文獻中發現的那些，例如， Molecular Cloning: A Laboratory Manual, J. Sambrook等人編輯，第二版，冷泉港實驗室出版社，冷泉港，紐約，1989年。例如，胺基酸的保守取代包括在下列群組中的胺基酸之間進行：(i) A、G；(ii) S、T；(iii) Q、N；(iv) E、D；(v) M、I、L、V；(vi) F、Y、W；以及(vii) K、R、H。 "Substantially the same" can mean that the variant does not have substantially different related amino acid sequences (eg, in FRs, CDRs, VH , or VL ) compared to the reference antibody, such that the variant has substantially similar properties to the Binding activity (eg, affinity, specificity, or both) and biological activity of a reference antibody. Such variants may include minor amino acid changes. It will be appreciated that a polypeptide can have a limited number of changes or modifications that can be made within a portion of the polypeptide unrelated to its activity or function and still result in an acceptable degree of equivalent or similar organisms. Active or functional variants. In some instances, changes in amino acid residues are conservative amino acid substitutions, which refer to amino acid residues with a similar chemical structure to another amino acid residue and the function, activity, or other function of a polypeptide. Biological effects have little or no effect on properties. Generally, relatively more substitutions can be made in the FR regions than in the CDR regions, as long as they do not adversely affect the binding function and biological activity of the antibody (eg, reduce binding affinity by more than 50% compared to the original antibody) ). In certain embodiments, the sequence identity between the reference antibody and the variant can be about 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95% %, 96%, 98%, or 99%, or higher. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references that follow these methods, e.g., Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, conservative substitutions of amino acids include between amino acids in the following groups: (i) A, G; (ii) S, T; (iii) Q, N; (iv) E, D; (v) M, I, L, V; (vi) F, Y, W; and (vii) K, R, H.
本文描述的抗體可為動物抗體(例如，小鼠衍生的抗體)、嵌合抗體(例如，小鼠-人類嵌合抗體)、人源化抗體，或人類抗體。本文描述的抗體可為單株抗體或多株抗體。「單株抗體」係指同源抗體群，「多株抗體」係指異源抗體群。本文描述的抗體還可包括其抗原結合片段，例如，Fab片段、F(ab') 2片段、Fv片段，單鏈Fv (scFv)及(scFv) 2。抗體或其抗原結合片段可透過本領域已知之方法製備。 The antibodies described herein can be animal antibodies (eg, mouse-derived antibodies), chimeric antibodies (eg, mouse-human chimeric antibodies), humanized antibodies, or human antibodies. The antibodies described herein can be monoclonal or polyclonal. "Monoclonal antibody" refers to a population of homologous antibodies, and "polyclonal antibody" refers to a population of heterologous antibodies. Antibodies described herein may also include antigen-binding fragments thereof, eg, Fab fragments, F(ab') 2 fragments, Fv fragments, single chain Fv (scFv) and (scFv) 2 . Antibodies or antigen-binding fragments thereof can be prepared by methods known in the art.
III.III. 抗體之製備Preparation of Antibodies
本領域常規的許多方法可用於獲得抗體或其抗原結合片段。Numerous methods routine in the art can be used to obtain antibodies or antigen-binding fragments thereof.
於某些具體實施例中，本文提供的抗體可透過常規融合瘤技術製備。通常，目標抗原，例如，新型冠狀病毒(SARS CoV-2) S蛋白，可選擇地與一載體蛋白耦合，例如，鑰孔血藍蛋白(keyhole limpet hemocyanin，KLH)及/或與佐劑混合，例如完全弗氏佐劑，可用於免疫宿主動物以產生與該抗原結合的抗體。收穫分泌單株抗體的淋巴細胞並與骨髓瘤細胞融合以產生融合瘤。然後篩選以這種方式形成的融合瘤細胞株以鑑定及選擇分泌所需單株抗體的那些細胞株。In certain embodiments, the antibodies provided herein can be prepared by conventional fusion tumor techniques. Typically, the target antigen, eg, the novel coronavirus (SARS CoV-2) S protein, is optionally coupled to a carrier protein, eg, keyhole limpet hemocyanin (KLH) and/or mixed with an adjuvant, Complete Freund's adjuvant, for example, can be used to immunize host animals to produce antibodies that bind to the antigen. Monoclonal antibody secreting lymphocytes are harvested and fused with myeloma cells to generate fusion tumors. Fusionoma cell lines formed in this manner are then screened to identify and select those that secrete the desired monoclonal antibody.
於某些具體實施例中，本文提供之抗體可透過重組技術製備。於相關方面，還提供編碼所公開的胺基酸序列的分離的核酸，以及包含此類核酸的載體及以該核酸轉型或轉染之宿主細胞。In certain embodiments, the antibodies provided herein can be prepared by recombinant techniques. In related aspects, isolated nucleic acids encoding the disclosed amino acid sequences are also provided, as well as vectors comprising such nucleic acids and host cells transformed or transfected with the nucleic acids.
例如，可將包含編碼這種抗體的重鏈及輕鏈可變區的核苷酸序列的核酸選殖到表現載體中(例如，細菌載體，例如大腸桿菌載體、酵母載體、病毒載體或哺乳動物載體)，透過常規技術，可將任何載體引入合適的細胞(例如，細菌細胞、酵母細胞、植物細胞，或哺乳動物細胞)中以表現該抗體。編碼如本文所述之單株抗體8a-1與單株抗體27b-2的重鏈及輕鏈可變區的核苷酸序列的實例分別如SEQ ID NOs: 17、18、19、20所示。哺乳動物宿主細胞株的實例為人類胚腎細胞株(293細胞)、幼倉鼠腎細胞(BHK細胞)、中國倉鼠卵巢細胞(CHO細胞)、非洲綠猴腎細胞(VERO細胞)，以及人類肝細胞(Hep G2細胞)。用於表現本文所述抗體的重組載體通常含有編碼該抗體胺基酸序列的核酸，該核酸與一啟動子，無論是組成型還是誘導型，可操作地連接。典型的載體含有轉錄及轉譯終止子、起始序列，以及可用於調節編碼抗體的核酸表現的啟動子。載體可選擇地含有原核及真核系統的選擇標記。於某些實例中，重鏈及輕鏈編碼序列都包括在相同的表現載體中。在其他實例中，將抗體的每個重鏈及輕鏈選殖到單個載體中並單獨產生，然後可以在合適的條件下培養抗體組裝。For example, nucleic acids comprising nucleotide sequences encoding the heavy and light chain variable regions of such antibodies can be cloned into expression vectors (eg, bacterial vectors such as E. coli vectors, yeast vectors, viral vectors, or mammalian vectors) vector), any vector can be introduced into suitable cells (eg, bacterial cells, yeast cells, plant cells, or mammalian cells) to express the antibody by conventional techniques. Examples of nucleotide sequences encoding the heavy and light chain variable regions of
用於表現本文所述抗體的重組載體通常含有編碼該抗體胺基酸序列的核酸，該核酸與一啟動子，無論是組成型還是誘導型，可操作地連接。重組抗體可以在原核或真核表現系統中產生，例如細菌、酵母、昆蟲及哺乳動物細胞。典型的載體含有轉錄及轉譯終止子、起始序列及可用於調節編碼抗體的核酸表現的啟動子。載體可選擇地含有原核及真核系統的選擇標記。可進一步分離或純化產生的抗體蛋白，以獲得基本上同質的製劑，用於進一步的測定及應用。合適的純化方法，例如，可包括在免疫親和或離子交換管柱上的分餾、乙醇沉澱、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)、高效液相色層分析(high-performance liquid chromatography，HPLC)，硫酸銨沉澱，以及凝膠過濾。Recombinant vectors used to express the antibodies described herein typically contain nucleic acid encoding the amino acid sequence of the antibody operably linked to a promoter, whether constitutive or inducible. Recombinant antibodies can be produced in prokaryotic or eukaryotic expression systems, such as bacterial, yeast, insect and mammalian cells. A typical vector contains transcriptional and translational terminators, initiation sequences, and promoters that can be used to modulate the expression of the nucleic acid encoding the antibody. Vectors optionally contain selectable markers for prokaryotic and eukaryotic systems. The antibody protein produced can be further isolated or purified to obtain a substantially homogeneous preparation for further assays and applications. Suitable purification methods, for example, may include fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), High-performance liquid chromatography (HPLC), ammonium sulfate precipitation, and gel filtration.
當需要全長抗體時，本文所述之任何V H 及V L 鏈的編碼序列可與免疫球蛋白的Fc區的編碼序列連接，且所得到的編碼全長抗體重鏈及輕鏈的基因可在合適的宿主細胞中表現及組裝，例如植物細胞、哺乳動物細胞、酵母細胞，或昆蟲細胞。 When full-length antibodies are desired, the coding sequences for any of the VH and VL chains described herein can be linked to the coding sequences for the Fc region of an immunoglobulin, and the resulting genes encoding the full-length antibody heavy and light chains can be found in appropriate Expressed and assembled in host cells, such as plant cells, mammalian cells, yeast cells, or insect cells.
可透過常規方法製備抗原結合片段。例如，可透過胃蛋白酶消化全長抗體分子產生F(ab’) 2片段，並可透過還原F(ab’) 2片段的雙硫鍵來製備Fab片段。或者，此類片段的製備亦可透過重組技術透過在合適的宿主細胞中表現重鏈及輕鏈片段並使它們在體內或體外組裝以形成所需的抗原結合片段。透過連接編碼重鏈可變區的核苷酸序列及編碼輕鏈可變區的核苷酸序列，可透過重組技術製備單鏈抗體。較佳地，於兩個可變區之間引入柔性連接子。 Antigen-binding fragments can be prepared by conventional methods. For example, F(ab') 2 fragments can be produced by pepsin digestion of full-length antibody molecules, and Fab fragments can be prepared by reducing the disulfide bonds of F(ab') 2 fragments. Alternatively, such fragments can also be prepared by recombinant techniques by expressing the heavy and light chain fragments in suitable host cells and assembling them in vivo or in vitro to form the desired antigen-binding fragments. Single-chain antibodies can be prepared by recombinant techniques by linking the nucleotide sequence encoding the variable region of the heavy chain and the nucleotide sequence encoding the variable region of the light chain. Preferably, a flexible linker is introduced between the two variable regions.
IV.IV. 抗體之應用Application of Antibodies
本發明之抗新型冠狀病毒(SARS-CoV-2)的抗體可辨識SARS-CoV以及新型冠狀病毒(SARS CoV-2)，且不與其他類型的呼吸道相關病毒交叉反應，如人類冠狀病毒(HCoV)、A型流感病毒(Flu A)、B型流感病毒(Flu B)、腺病毒、副流感病毒、鼻病毒、呼吸道融合病毒(RSV)。因此，本發明提供一種使用任何揭露的抗體或其任何組合之方法，其可有效地用於檢測樣本中的新型冠狀病毒(SARS CoV-2)。The antibody against novel coronavirus (SARS-CoV-2) of the present invention can recognize SARS-CoV and novel coronavirus (SARS CoV-2), and does not cross-react with other types of respiratory related viruses, such as human coronavirus (HCoV) ), influenza A virus (Flu A), influenza B virus (Flu B), adenovirus, parainfluenza virus, rhinovirus, respiratory syncytial virus (RSV). Accordingly, the present invention provides a method using any of the disclosed antibodies or any combination thereof, which can be effectively used to detect the novel coronavirus (SARS CoV-2) in a sample.
通常，本發明之方法包含使樣本與任何揭露的抗體或其任何組合接觸，並測定抗體與該樣本的結合。具體而言，抗體與該樣本的結合包括 (i) 第一抗體與新型冠狀病毒(SARS CoV-2) S蛋白的結合並形成第一抗體-新型冠狀病毒(SARS CoV-2) S蛋白複合物，以及(ii) 第二抗體與新型冠狀病毒(SARS CoV-2) S蛋白的結合並形成第二抗體-新型冠狀病毒(SARS CoV-2) S蛋白複合物。本發明之方法還包含基於抗體與樣本的結合確定是否存在新型冠狀病毒(SARS CoV-2)。Generally, the methods of the invention comprise contacting a sample with any disclosed antibody, or any combination thereof, and determining the binding of the antibody to the sample. Specifically, the binding of the antibody to the sample includes (i) the binding of the primary antibody to the novel coronavirus (SARS CoV-2) S protein and the formation of a primary antibody-novel coronavirus (SARS CoV-2) S protein complex , and (ii) the binding of the secondary antibody to the novel coronavirus (SARS CoV-2) S protein and the formation of a secondary antibody-novel coronavirus (SARS CoV-2) S protein complex. The method of the present invention also includes determining the presence or absence of a novel coronavirus (SARS CoV-2) based on the binding of the antibody to the sample.
本領域普通技術人員已知使用抗體以檢測樣本中的抗原或病原體的各種測定形式。使用對目標抗原/病原體專一的抗體的這些測定通常稱為免疫測定。免疫測定的實例包括，但不限於，酶聯結免疫吸附測定(ELISA)、放射免疫測定(RIA)、螢光免疫測定(FIA)、發光免疫測定(LIA)，或免疫螢光測定(ILMA)。這些測定法可用於檢測生物樣本中新型冠狀病毒(SARS CoV-2)的存在，包括血液、血清、血漿、唾液、腦脊髓液、尿液及其他組織樣本。Various assay formats are known to those of ordinary skill in the art that use antibodies to detect antigens or pathogens in a sample. These assays using antibodies specific for the antigen/pathogen of interest are commonly referred to as immunoassays. Examples of immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), luminescence immunoassay (LIA), or immunofluorescence assay (ILMA). These assays can be used to detect the presence of the novel coronavirus (SARS CoV-2) in biological samples, including blood, serum, plasma, saliva, cerebrospinal fluid, urine, and other tissue samples.
在某些具體實施例中，該測定為夾心式測定。In certain embodiments, the assay is a sandwich assay.
於某些具體實施例中，透過首先將捕獲抗體固定在固體支持物上來進行測定。然後將固定的抗體與生物樣本一起培養，且該新型冠狀病毒(SARS CoV-2)或其目標抗原例如，使S蛋白(如果存在於樣本中)與該抗體結合，以形成抗體-病毒/抗原複合物或綴合物。然後可以透過以合適的緩衝液，例如含有0.05% Tween的磷酸鹽緩衝溶液(phosphate-buffered saline, PBS)，洗滌該固體支持物來除去未結合的樣本，且添加偵測抗體至該固體支持物中，該偵測抗體可結合該固定的抗體-病毒/抗原複合物並包含可偵測到的標記。特定而言，該捕獲抗體及該偵測抗體需要在不同抗原決定位結合該病毒/抗原，使得該病毒/抗原可「夾在」這兩種抗體之間。較佳的可偵測到的標記包括酶標記(例如辣根過氧化物酶)、螢光標記、金屬標記以及放射性標記。可偵測到的標記的一些特定實例包括金奈米顆粒、有色乳膠珠、磁性顆粒、碳奈米顆粒，以及硒奈米顆粒。將偵測抗體與固定的抗體-病毒/抗原複合物一起培養至足以檢測結合的病毒/抗原的一段時間。然後除去未結合的偵測抗體，並基於可偵測到的標記檢測結合的偵測抗體。例如，通常可以透過添加基質，然後對反應產物進行光譜或其他分析來檢測酶標記。In certain embodiments, the assay is performed by first immobilizing the capture antibody on a solid support. The immobilized antibody is then incubated with the biological sample, and the novel coronavirus (SARS CoV-2) or its target antigen, e.g., the S protein (if present in the sample), binds to the antibody to form an antibody-virus/antigen complex or conjugate. Unbound sample can then be removed by washing the solid support with a suitable buffer, such as phosphate-buffered saline (PBS) containing 0.05% Tween, and adding detection antibodies to the solid support , the detection antibody binds to the immobilized antibody-virus/antigen complex and contains a detectable label. In particular, the capture antibody and the detection antibody need to bind the virus/antigen at different epitopes so that the virus/antigen can be "sandwiched" between the two antibodies. Preferred detectable labels include enzymatic labels (eg, horseradish peroxidase), fluorescent labels, metal labels, and radioactive labels. Some specific examples of detectable labels include gold nanoparticles, colored latex beads, magnetic particles, carbon nanoparticles, and selenium nanoparticles. The detection antibody is incubated with the immobilized antibody-virus/antigen complex for a period of time sufficient to detect bound virus/antigen. Unbound detection antibody is then removed and bound detection antibody is detected based on the detectable label. For example, enzymatic labels can often be detected by adding a matrix followed by spectroscopic or other analysis of the reaction product.
為了確定樣本中目標新型冠狀病毒(SARS CoV-2)的存在或不存在，通常將從與固體支持物結合的可偵測到的標記檢測到的訊號與對應於截斷值的訊號進行比較。該截斷值通常是當固定化抗體與來自未感染個體的樣本一起培養時獲得的平均值訊號。通常，產生高於截斷值訊號的樣本被認為是對樣本中存在新型冠狀病毒(SARS CoV-2)的陽性反應。To determine the presence or absence of the novel coronavirus of interest (SARS CoV-2) in a sample, the signal detected from the detectable label bound to the solid support is typically compared to the signal corresponding to a cutoff value. The cutoff value is typically the mean signal obtained when the immobilized antibody is incubated with a sample from an uninfected individual. Typically, a sample that produces a signal above the cutoff value is considered positive for the presence of the novel coronavirus (SARS CoV-2) in the sample.
本發明之抗新型冠狀病毒(SARS-CoV-2)的抗體可作為捕獲或偵測抗體，且可與本發明之另一抗新型冠狀病毒(SARS-CoV-2)的抗體配對，以形成抗體對而可進行夾心式測定。如本文所述之另一抗體能夠結合目標病毒/抗原，但是與用於進行夾心式測定之配對的本發明之任何抗新型冠狀病毒(SARS-CoV-2)的抗體不同的抗原決定位結合。The antibody against novel coronavirus (SARS-CoV-2) of the present invention can be used as a capture or detection antibody, and can be paired with another antibody against novel coronavirus (SARS-CoV-2) of the present invention to form an antibody Instead, sandwich assays can be performed. Another antibody as described herein is capable of binding the target virus/antigen, but a different epitope than any of the anti-novel coronavirus (SARS-CoV-2) antibodies of the invention paired to perform the sandwich assay.
於某些具體實施例中，用於檢測樣本中的新型冠狀病毒(SARS CoV-2)的本發明之方法包含 (i) 使該樣本與本發明之抗新型冠狀病毒(SARS CoV-2)的第一抗體接觸，作為捕獲抗體，形成一第一抗體-S蛋白複合物； (ii) 使該第一抗體-S蛋白複合物與如本文所述之抗新型冠狀病毒(SARS-CoV-2)的第二抗體接觸，作為偵測抗體，以形成一第一抗體-S蛋白-第二抗體複合物；以及 (iii) 檢測該第一抗體-S蛋白-第二抗體複合物的存在，進而檢測樣本中新型冠狀病毒(SARS CoV-2)的存在。 In certain embodiments, the methods of the invention for detecting a novel coronavirus (SARS CoV-2) in a sample comprise (i) contacting the sample with the primary antibody against novel coronavirus (SARS CoV-2) of the present invention as a capture antibody to form a primary antibody-S protein complex; (ii) contacting the primary antibody-S protein complex with a secondary antibody against a novel coronavirus (SARS-CoV-2) as described herein as a detection antibody to form a primary antibody-S protein - a secondary antibody complex; and (iii) Detecting the presence of the primary antibody-S protein-second antibody complex, and then detecting the presence of the novel coronavirus (SARS CoV-2) in the sample.
於某些具體實施例中，用於檢測樣本中的新型冠狀病毒(SARS CoV-2)的本發明之方法包含 (i) 使該樣本與本發明之抗新型冠狀病毒(SARS CoV-2)的第二抗體接觸，作為捕獲抗體，形成一第二抗體-S蛋白複合物； (ii) 使該第二抗體-S蛋白複合物與如本文所述之抗新型冠狀病毒(SARS-CoV-2)的第一抗體接觸，作為偵測抗體，以形成一第二抗體-S蛋白-第一抗體複合物；以及 (iii) 檢測該第二抗體-S蛋白-第一抗體複合物的存在，進而檢測樣本中新型冠狀病毒(SARS CoV-2)的存在。 In certain embodiments, the methods of the invention for detecting a novel coronavirus (SARS CoV-2) in a sample comprise (i) contacting the sample with the anti-novel coronavirus (SARS CoV-2) secondary antibody of the present invention as a capture antibody to form a secondary antibody-S protein complex; (ii) contacting the secondary antibody-S protein complex with a primary antibody against a novel coronavirus (SARS-CoV-2) as described herein as a detection antibody to form a secondary antibody-S protein - the primary antibody complex; and (iii) Detecting the presence of the secondary antibody-S protein-primary antibody complex, and then detecting the presence of the novel coronavirus (SARS CoV-2) in the sample.
於一實例中，本發明之方法在ELISA夾心式測定中進行。在該測定中，將捕獲抗體包覆在ELISA盤上。阻隔後，將該ELISA盤與生物樣本一起培養、洗滌，然後與偵測抗體一起培養。例如，捕獲抗體為本發明之抗新型冠狀病毒(SARS CoV-2)的第一抗體，且偵測抗體為本發明之抗新型冠狀病毒(SARS CoV-2)的第二抗體，且使用一ELISA比色的3,3',5,5'-四甲基聯苯胺(3,3′,5,5′-Tetramethylbenzidine, TMB)試劑進行該ELISA盤的顯色。In one example, the methods of the invention are performed in an ELISA sandwich assay. In this assay, capture antibodies are coated on ELISA plates. After blocking, the ELISA plate was incubated with the biological sample, washed, and then incubated with detection antibodies. For example, the capture antibody is the primary antibody against the novel coronavirus (SARS CoV-2) of the present invention, and the detection antibody is the secondary antibody against the novel coronavirus (SARS CoV-2) of the present invention, and an ELISA is used The colorimetric 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) reagent was used to develop the color of the ELISA plate.
於另一實例中，本發明之方法以流通或側流形式進行。在該測定中，將具有可偵測到的標記，如比色標記(例如，膠體金)之偵測抗體固定在膜上，例如硝酸纖維素膜(作為條帶)。將懷疑含有新型冠狀病毒(SARS CoV-2)的生物樣本施加到存在偵測抗體的膜上。該生物樣本沿著該膜遷移，透過含有該偵測抗體的區域，其中如果該新型冠狀病毒(SARS CoV-2)存在於生物樣本中，該偵測抗體會與該新型冠狀病毒(SARS CoV-2)的S蛋白結合。然後，偵測抗體以及其所結合的抗原的複合物遷移到固定有捕獲抗體的測試區域，該捕獲抗體並結合新型冠狀病毒(SARS CoV-2)的S蛋白，進而形成偵測抗體、抗原及捕獲抗體的夾心。測試(捕獲)區域處的偵測抗體的濃度/聚集表示該樣本中存在新型冠狀病毒(SARS CoV-2)的S蛋白。這種測試通常可以使用非常少量的生物樣本進行。In another example, the methods of the present invention are carried out in a flow-through or side-flow format. In this assay, a detection antibody with a detectable label, such as a colorimetric label (eg, colloidal gold), is immobilized on a membrane, such as a nitrocellulose membrane (as a strip). A biological sample suspected of containing the novel coronavirus (SARS CoV-2) was applied to a membrane in the presence of detection antibodies. The biological sample migrates along the membrane through the region containing the detection antibody, which, if the novel coronavirus (SARS CoV-2) is present in the biological sample, will interact with the novel coronavirus (SARS CoV-2) 2) S protein binding. Then, the complex of the detection antibody and its bound antigen migrates to the test area where the capture antibody is immobilized, and the capture antibody binds to the S protein of the new coronavirus (SARS CoV-2) to form the detection antibody, antigen and Sandwich of capture antibody. The concentration/aggregation of detection antibodies at the test (capture) area indicates the presence of the S protein of the novel coronavirus (SARS CoV-2) in this sample. This test can usually be performed using a very small amount of biological sample.
在一相關方面，本發明還提供用於實施本發明方法之套組，其包含如本文所述之任何抗體或其組合。該套組可進一步包含使用該套組檢測樣本中新型冠狀病毒(SARS CoV-2)的說明書。In a related aspect, the invention also provides kits for practicing the methods of the invention comprising any of the antibodies or combinations thereof as described herein. The kit may further comprise instructions for using the kit to detect the novel coronavirus (SARS CoV-2) in the sample.
於某些具體實施例中，該免疫測定為夾心形式。In certain embodiments, the immunoassay is in a sandwich format.
具體而言，該套組包含一對抗新型冠狀病毒(SARS-CoV-2)的抗體，其為該第一抗體與該第二抗體，兩者配對以進行夾心式測定。Specifically, the kit includes an antibody against a novel coronavirus (SARS-CoV-2), which is the first antibody and the second antibody, which are paired for sandwich assay.
於某些具體實施例中，本發明之抗新型冠狀病毒(SARS-CoV-2)的第一抗體作為捕獲抗體，與作為偵測抗體的第二抗體配對。In certain embodiments, the anti-novel coronavirus (SARS-CoV-2) primary antibody of the present invention is used as a capture antibody and paired with a secondary antibody as a detection antibody.
於其他具體實施例中，本發明之抗新型冠狀病毒(SARS-CoV-2)的第一抗體作為偵測抗體，與作為捕獲抗體的第二抗體配對。In other specific embodiments, the anti-novel coronavirus (SARS-CoV-2) primary antibody of the present invention is used as a detection antibody, and is paired with a secondary antibody as a capture antibody.
作為一偵測抗體，該抗體可包含可偵測到的標記，例如酶標記、螢光標記、金屬標記及放射性標記。As a detection antibody, the antibody may comprise detectable labels such as enzymatic labels, fluorescent labels, metal labels and radioactive labels.
於某些實例中，在一側向流動形式中，該套組包含測定條帶(例如，硝酸纖維素膜)；偵測抗體可以與該條帶的反應區結合，且捕獲抗體可以結合於該條帶的測試區中。該條帶還包含樣本墊，其中放置體液樣本，然後該樣本透過毛細管作用向條帶的相對端遷移，藉此該樣本首先在該反應區中與該偵測抗體結合，其中在該樣本中的抗原與該偵測抗體結合形成抗原-偵測抗體複合物，然後該抗原-偵測抗體複合物在該測試(捕獲)區中與該捕獲抗體結合。使用膠體金作為偵測抗體的可偵測到的標記，例如，在該測試(捕獲)區域的檢測標記的濃度/聚集顯示紅色，表示該樣本中存在專一性抗原。或者，該測試區可具有發色基質，且當該抗原-偵測抗體複合物與該捕獲抗體結合時，該發色基質轉化為可見的有色產物。In certain examples, in a lateral flow format, the kit includes an assay strip (eg, a nitrocellulose membrane); a detection antibody can bind to the reactive region of the strip, and a capture antibody can bind to the strip. in the test area of the strip. The strip also includes a sample pad in which a sample of bodily fluid is placed, which then migrates by capillary action toward the opposite end of the strip, whereby the sample first binds to the detection antibody in the reaction zone, wherein the sample in the sample first binds to the detection antibody Antigen binds to the detection antibody to form an antigen-detection antibody complex, which then binds to the capture antibody in the test (capture) zone. Using colloidal gold as the detectable label for the detection antibody, eg, the concentration/aggregation of the detection label in the test (capture) area appears red, indicating the presence of a specific antigen in the sample. Alternatively, the test zone can have a chromonic matrix, and when the antigen-detection antibody complex binds to the capture antibody, the chromogenic matrix is converted to a visible colored product.
於某些實例中，在ELISA形式中，該套組包含微量滴定盤，其具有固定有捕獲抗體的孔；含有偵測抗體的溶液；以及發色顯影劑。In certain examples, in an ELISA format, the kit comprises a microtiter plate with wells immobilized with capture antibodies; a solution containing detection antibodies; and a chromogenic developer.
具體而言，該套組可進一步包含額外的試劑或緩衝液、用於從個體收集生物樣本的醫療裝置，及/或用於保持及/或儲存樣本的容器。In particular, the kit may further comprise additional reagents or buffers, medical devices for collecting biological samples from individuals, and/or containers for holding and/or storing samples.
於進一步的具體實施例中，本發明提供包含一種或多種如本文所述抗體及醫藥上可接受的載體之組合物。In further embodiments, the present invention provides compositions comprising one or more antibodies as described herein and a pharmaceutically acceptable carrier.
於某些具體實施例中，本發明之組合物係用於治療新冠肺炎(COVID-19)之醫藥組合物。In certain embodiments, the composition of the present invention is a pharmaceutical composition for the treatment of novel coronavirus pneumonia (COVID-19).
於某些具體實施例中，本發明之組合物係用於診斷新冠肺炎(COVID-19)之診斷組合物。In certain embodiments, the compositions of the present invention are diagnostic compositions for diagnosing novel coronavirus pneumonia (COVID-19).
如本文所用，「醫藥上可接受的」係指載體與組合物中的活性成分相容，且較佳地可以穩定該活性成分並對接受治療的個體是安全的。該載體可為活性成分的稀釋劑、載體、賦形劑或基質。合適的賦形劑的一些實例包括乳糖、右旋糖、蔗糖、山梨糖、甘露糖、澱粉、***膠、磷酸鈣、藻酸鹽、黃蓍膠、明膠、矽酸鈣、微晶纖維素、聚乙烯吡咯烷酮、纖維素、無菌水、糖漿及甲基纖維素。該組合物可另外包含潤滑劑，例如滑石、硬脂酸鎂及礦物油；潤濕劑；乳化劑及懸浮劑；防腐劑，如甲基及羥基苯甲酸丙酯；甜味劑；及調味劑。本發明之組合物可以在給予患者後提供活性成分的快速、持續或延遲釋放的效果。As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, and preferably stabilizes the active ingredient and is safe for the subject being treated. The carrier can be a diluent, carrier, excipient or base for the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, sterile water, syrup and methylcellulose. The composition may additionally contain lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, such as methyl and propyl hydroxybenzoate; sweeteners; and flavoring agents . The compositions of the present invention may provide rapid, sustained or delayed release of the active ingredient after administration to a patient.
根據本發明，該組合物的形式可以為片劑、丸劑、粉末、錠劑、小包、片劑、酏劑、懸浮液、洗劑、溶液、糖漿、軟及硬明膠膠囊、栓劑、無菌注射液及包裝粉末。According to the present invention, the composition may be in the form of tablets, pills, powders, lozenges, sachets, tablets, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions and packaging powder.
本發明之組合物可透過任何生理學上可接受的途徑遞送，例如口服、腸胃外(例如肌肉內、靜脈內、皮下及腹膜內)、透皮、栓劑及鼻內方法。關於腸胃外給藥，較佳以無菌水溶液的形式使用，其可包含足以使溶液與血液等滲的其他物質，例如鹽或葡萄糖。根據需要，可以適當地緩衝水溶液(較佳pH值為3至9)。在無菌條件下製備合適的腸胃外組合物可以使用本領域技術人員熟知的標準藥理學技術完成，且不需要額外的創造性勞動。The compositions of the present invention can be delivered by any physiologically acceptable route, such as oral, parenteral (eg, intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. For parenteral administration, it is preferably used in the form of a sterile aqueous solution, which may contain other substances sufficient to make the solution isotonic with blood, such as salt or dextrose. The aqueous solution may be appropriately buffered (preferably at pH 3 to 9) as needed. The preparation of suitable parenteral compositions under sterile conditions can be accomplished using standard pharmacological techniques well known to those skilled in the art and does not require additional inventive step.
透過以下實施例進一步說明本發明，提供這些實施例是為了說明而非限制。根據本發明公開的內容，本領域技術人員應當理解，在不脫離本發明之精神及範圍的情況下，可以對所公開的特定具體實施例進行許多改變並仍然獲得相同或相似的結果。The present invention is further illustrated by the following examples, which are provided by way of illustration and not limitation. Based on this disclosure, those of skill in the art should appreciate that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
實施例Example
實施例一Example 1 抗anti- SARS-CoV-2SARS-CoV-2 抗體之製備Preparation of Antibodies
1.1. 重組reorganization SS 蛋白之製備Preparation of protein
將編碼新型冠狀病毒(SARS CoV)的S蛋白的第268個至第1255個胺基酸(如SEQ ID NO: 21所示)的DNA序列選殖至表現載體pET，以獲得質體pET-S 268-1255，並將質體pET-S 268-1255轉型至大腸桿菌( Escherichia coli) BL21(DE3)中。將該帶有質體pET-S 268-1255的 E. coli以異丙基-β-D-硫代吡喃半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG)誘導帶有組胺酸標籤的重組S蛋白表現後，收穫 E. coli細胞並進行超音波處理以裂解細胞。將裂解的細胞離心後以緩衝液B (8 M尿素、0.1 M磷酸鈉[pH 8.0]，以及10 mM Tris)重新懸浮沈澱物，並於室溫下攪拌1小時後，離心回收上清液。使用Ni 2+-氮基三醋酸(nitrilotriacetic acid, NTA)複合物(Qiagen公司，希爾登，德國)將上清液以金屬螯合親和色層分析法純化。簡言之，Ni 2+-NTA瓊脂管柱先以緩衝液B、緩衝液C (8 M尿素、0.1 M磷酸鈉，以及10 mM Tris [pH 6.3])，以及緩衝液F (6 M鹽酸胍、0.2 M醋酸)預洗，並以緩衝液B預先平衡。然後依序以10倍體積的緩衝液B、緩衝液C清洗，再將蛋白以緩衝液D (8 M尿素、0.1 M磷酸鈉，以及10 mM Tris [pH 5.9])以及緩衝液E (8 M尿素、0.1 M磷酸鈉，以及10 mM Tris [pH 4.5])以0.5 mL的流份流洗。含有蛋白的流份以十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)以及考馬斯藍(Coomassie blue)染色確認。於4 oC下對含有逐步降低濃度的尿素(4、2、1、0.5、0.25、0.125，以及0.05 M)的緩衝液B及對緩衝液D (20 mM HEPES [pH 8.0]、20%甘油、0.1 M KCl、0.2 mM EDTA、0.5 mM 苯基甲基磺醯氟、0.5 mM二硫蘇糖醇[dithiothreitol, DTT]，以及0.2% Nonidet P-40)進行逐步透析，以使蛋白復性。透析後於4 oC下以14,000 x g離心5分鐘，並將上清液以液態氮快速冷凍並存於-80 oC。 The DNA sequence encoding the 268th to 1255th amino acids of the S protein of the novel coronavirus (SARS CoV) (as shown in SEQ ID NO: 21) was cloned into the expression vector pET to obtain plastid pET-S 268-1255 and transformed the plastid pET-S 268-1255 into Escherichia coli BL21(DE3). The E. coli with pET-S 268-1255 was induced with histidine tag by isopropyl-β-D-thiogalactopyranoside (IPTG). After expression of the recombinant S protein, E. coli cells were harvested and sonicated to lyse the cells. Lysed cells were centrifuged and the pellet was resuspended in buffer B (8 M urea, 0.1 M sodium phosphate [pH 8.0], and 10 mM Tris), and after stirring at room temperature for 1 hour, the supernatant was recovered by centrifugation. The supernatant was purified by metal chelation affinity chromatography using Ni 2+ -nitrilotriacetic acid (NTA) complex (Qiagen, Hilden, Germany). Briefly, a Ni 2+ -NTA agar column was pretreated with buffer B, buffer C (8 M urea, 0.1 M sodium phosphate, and 10 mM Tris [pH 6.3]), and buffer F (6 M guanidine hydrochloride , 0.2 M acetic acid) and pre-equilibrated with buffer B. Then wash with 10 volumes of buffer B, buffer C, and then wash the protein with buffer D (8 M urea, 0.1 M sodium phosphate, and 10 mM Tris [pH 5.9]) and buffer E (8 M urea, 0.1 M sodium phosphate, and 10 mM Tris [pH 4.5]) in 0.5 mL fractions. Fractions containing proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. Buffer B with decreasing concentrations of urea (4, 2, 1, 0.5, 0.25, 0.125, and 0.05 M) and buffer D (20 mM HEPES [pH 8.0], 20% glycerol) at 4 oC , 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol [DTT], and 0.2% Nonidet P-40) were progressively dialyzed to renature the protein. After dialysis, centrifugation was performed at 14,000 x g for 5 min at 4 ° C, and the supernatant was snap-frozen in liquid nitrogen and stored at -80 ° C.
2.2. 抗anti- SARS-CoV-2 SSARS-CoV-2S 蛋白單株抗體之產生及特徵描述Production and Characterization of Protein Monoclonal Antibodies
所有實驗均使用購自國家實驗動物中心(臺灣)並在預防醫學研究所的動物飼養設施(臺灣)中飼養的BALB/c小鼠。將4週齡的BALB/c小鼠進行腹腔免疫，以存在於完全弗氏佐劑中的10 µg重組S蛋白進行第一次接種，然後以存在於不完全弗氏佐劑中的10 µg重組S蛋白用於隨後的加強免疫。最後一次加強免疫以腹腔注射10 µg抗原，並於5天後進行融合。簡言之，除去免疫小鼠的脾臟。將脾細胞與NSI/1-Ab4-1骨髓瘤細胞融合以產生融合瘤細胞，其根據標準程序選擇(Kohler及Milstein，1975年)。融合細胞以RPMI培養基洗滌兩次，然後在15 mL錐形管中混合，並於1分鐘內在溫和攪拌下加入1 mL 50% (w/v) PEG 1500 (Roche公司，Penzberg，德國)。透過緩慢(1分鐘)加入1 mL RPMI培養基稀釋混合物兩次，然後緩慢加入(2分鐘) 8 mL無血清RPMI培養基。然後將混合物在400 xg下離心5分鐘。將融合的細胞沉澱物重新懸浮於補充有20% FBS的次黃嘌呤-胺基蝶呤-胸苷 (hypoxanthine-aminopterin-thymidine, HAT) 培養基 (Life technologies公司，Burlington，安大略省，加拿大)以及高果糖玉米糖漿(High-fructose corn syrup (HFCS)溶液(Roche公司，Mannhein，德國)的RPMI培養基中。接下來，以每孔200 µL的量將重新懸浮混合物分配在96微孔盤中。透過間接ELISA (使用純化的重組S蛋白作為包覆抗原)鑑定分泌針對重組S蛋白的專一性抗體的融合瘤細胞株。透過有限稀釋產生單株細胞。進行來自分別以SARS-CoV、SARS-CoV-2、人類冠狀病毒(human coronavirus, HCoV) 229E、HCoV OC43、HCoV NL63、A型流感病毒(influenza A, Flu A) A/CA/07/2009、B型流感病毒(influenza B, Flu B) B/T/81863/2014、副流感病毒(Parainfluenza)-3、腺病毒(Adenovirus)-7a、鼻病毒(Rhinovirus)、呼吸道融合病毒(respiratory syncytical virus, RSV)感染的Vero E6細胞的裂解物的西方墨點分析以確定 (a) 抗S蛋白單株抗體的專一性，以及 (b) 抗體識別的抗原決定位是否為構象或線性。使用市售的小鼠單株抗體同種型套組(IsoStrip TM，Roche公司，Mannheim，德國)對單株抗體進行同種型分型。將融合瘤細胞腹腔注射到姥鮫烷引發的BALB/c小鼠中以產生腹水。然後根據製造商的說明書使用蛋白G-sepharose管柱(HiTrap protein G，GE Healthcare公司，Uppsala，瑞典)從腹水中純化單株抗體。 All experiments used BALB/c mice purchased from the National Laboratory Animal Center (Taiwan) and housed in the Animal Breeding Facility of the Institute of Preventive Medicine (Taiwan). 4-week-old BALB/c mice were immunized intraperitoneally with 10 µg recombinant S protein in complete Freund's adjuvant first, followed by 10 µg recombinant in incomplete Freund's adjuvant The S protein was used for subsequent booster immunizations. The last booster was 10 µg of antigen by intraperitoneal injection and fusion was performed 5 days later. Briefly, the spleens of immunized mice were removed. Splenocytes were fused with NSI/1-Ab4-1 myeloma cells to generate fusion tumor cells, which were selected according to standard procedures (Kohler and Milstein, 1975). Confluent cells were washed twice with RPMI medium, then mixed in a 15 mL conical tube, and 1 mL of 50% (w/v) PEG 1500 (Roche, Penzberg, Germany) was added within 1 minute with gentle agitation. The mixture was diluted twice by slow (1 min) addition of 1 mL of RPMI medium followed by slow (2 min) addition of 8 mL of serum-free RPMI medium. The mixture was then centrifuged at 400 xg for 5 minutes. The confluent cell pellet was resuspended in hypoxanthine-aminopterin-thymidine (HAT) medium (Life technologies, Burlington, Ontario, Canada) supplemented with 20% FBS and high High-fructose corn syrup (HFCS) solution (Roche, Mannhein, Germany) in RPMI medium. Next, the resuspension mixture was dispensed at 200 µL per well in 96 microwell dishes. ELISA (using purified recombinant S protein as coating antigen) identified fusion tumor cell lines secreting specific antibodies against recombinant S protein. Single cells were generated by limiting dilution. Performed from SARS-CoV, SARS-CoV-2 , human coronavirus (human coronavirus, HCoV) 229E, HCoV OC43, HCoV NL63, influenza A virus (influenza A, Flu A) A/CA/07/2009, influenza B virus (influenza B, Flu B) B/ Western blotting of lysates of Vero E6 cells infected with T/81863/2014, Parainfluenza-3, Adenovirus-7a, Rhinovirus, Respiratory syncytical virus (RSV) Dot analysis to determine (a) the specificity of the anti-S protein monoclonal antibody, and (b) whether the epitope recognized by the antibody is conformational or linear. A commercially available mouse monoclonal antibody isotype kit (IsoStrip ™ , Monoclonal antibodies were isotyped by Roche Corporation, Mannheim, Germany. Fusion tumor cells were injected intraperitoneally into pristane-primed BALB/c mice to generate ascites. Protein G-sepharose was then used according to the manufacturer's instructions Monoclonal antibodies were purified from ascites fluid by column (HiTrap protein G, GE Healthcare, Uppsala, Sweden).
3.3. 間接免疫螢光染色分析Indirect immunofluorescence staining analysis (Indirect immunofluorescence assay, IFA)(Indirect immunofluorescence assay, IFA)
以PBS清洗感染SARS-CoV-2的Vero E6單層細胞三次，並於室溫下以丙酮-甲醇混合物(1:1)固定細胞3分鐘。隨後以阻隔緩衝液(PBS，0.05% Tween，5%脫脂奶粉)於37 oC下阻隔細胞1小時，並以洗滌緩衝液(PBS，0.05% Tween)清洗。以阻隔緩衝液稀釋單株抗體並於37 oC下作用1小時。清洗培養盤後加入山羊抗小鼠-異硫氰酸螢光素(Fluorescein isothiocyanate, FITC)抗體，並於37 oC下作用1小時。最後，以免疫螢光顯微鏡檢視每個染色的單層細胞。 Vero E6 monolayers infected with SARS-CoV-2 were washed three times with PBS and fixed with an acetone-methanol mixture (1:1) for 3 min at room temperature. Cells were then blocked with blocking buffer (PBS, 0.05% Tween, 5% nonfat dry milk) for 1 hour at 37 ° C and washed with wash buffer (PBS, 0.05% Tween). Monoclonal antibodies were diluted in blocking buffer and incubated at 37 ° C for 1 hour. After washing the culture plate, goat anti-mouse-fluorescein isothiocyanate (FITC) antibody was added and incubated at 37 ° C for 1 hour. Finally, each stained monolayer was examined by immunofluorescence microscopy.
4.4. 單株抗體可變區之序列分析Sequence Analysis of Monoclonal Antibody Variable Regions
使用RNeasy mini套組(Qiagen公司，Valencia，加州，美國)根據製造商的方法從融合瘤細胞株8a-1以及27b-2中分離總RNA，然後使用oligo dT引子進行反轉錄以產生cDNA。使用PCR擴增抗體的重鏈及輕鏈可變鏈，並確認該序列為功能可變區。Total RNA was isolated from the
5.5. 結果result
針對SARS-CoV-2的單株抗體之產生及特徵描述Generation and characterization of monoclonal antibodies against SARS-CoV-2
如
表 1所示，選殖株8a-1以及27b-2 (單株抗體8a-1及27b-2)皆對SARS-CoV與SARS-CoV-2具有專一性，且與其他類型的呼吸道相關病毒，例如人類冠狀病毒、A型流感病毒、B型流感病毒、副流感病毒、腺病毒、鼻病毒、呼吸道融合病毒，不具有交叉反應。
As shown in Table 1 ,
表 1與SARS-CoV-2的S蛋白反應的單株抗體之特徵描述
間接免疫螢光染色分析(IFA)結果如
圖 1所示，單株抗體8a-1及27b-2以及患者的血清皆可辨識感染SARS-CoV-2的Vero E6細胞(分別如
圖 1A、
圖 1B、
圖 1C所示)。而作為負對照的抗登革病毒單株抗體anti-D2 NS1則無法辨識感染SARS-CoV-2的Vero E6細胞(如
圖 1D所示)。
The results of indirect immunofluorescence staining analysis (IFA) are shown in Figure 1.
單株抗體8a-1的重鏈可變區(V
H )胺基酸序列如SEQ ID NO: 4所示，其相應的核酸序列如SEQ ID NO: 17所示，包含如SEQ ID NO: 1所示之重鏈互補決定區1 (HC CDR1)、如SEQ ID NO: 2所示之重鏈互補決定區2 (HC CDR2)，以及如SEQ ID NO: 3所示之重鏈互補決定區3 (HC CDR3)；單株抗體8a-1的輕鏈可變區(V
L )胺基酸序列如SEQ ID NO: 8所示，其相應的核酸序列如SEQ ID NO: 18所示，包含如SEQ ID NO: 5所示之輕鏈互補決定區1 (LC CDR1)、如SEQ ID NO: 6所示之輕鏈互補決定區2 (LC CDR2)，以及如SEQ ID NO: 7所示之輕鏈互補決定區3 (LC CDR3)。
The heavy chain variable region ( VH ) amino acid sequence of
單株抗體27b-2的重鏈可變區(V
H )胺基酸序列如SEQ ID NO: 12所示，其相應的核酸序列如SEQ ID NO: 19所示，包含如SEQ ID NO: 9所示之重鏈互補決定區1 (HC CDR1)、如SEQ ID NO: 10所示之重鏈互補決定區2 (HC CDR2)，以及如SEQ ID NO: 11所示之重鏈互補決定區3 (HC CDR3)；單株抗體27b-2的輕鏈可變區(V
L )胺基酸序列如SEQ ID NO: 16所示，其相應的核酸序列如SEQ ID NO: 20所示，包含如SEQ ID NO: 13所示之輕鏈互補決定區1 (LC CDR1)、如SEQ ID NO: 14所示之輕鏈互補決定區2 (LC CDR2)，以及如SEQ ID NO: 15所示之輕鏈互補決定區3 (LC CDR3)。
The heavy chain variable region ( VH ) amino acid sequence of
實施例二
1. HRP1. HRP 結合combine
為了將單株抗體與辣根過氧化物酶(Horseradish peroxidase, HRP) (Innova Biosciences公司，劍橋，英國)綴合，將100 μg HRP及20 μL等分試樣的修飾試劑與200 μL單株抗體27b-2 (1 mg/mL)混合。於室溫(20-25
oC)下將混合物培養3小時後，以20 μL等份的猝滅劑終止反應。於室溫下再作用30分鐘後，加入260 μL甘油，最終溶液於-20
oC保存。單株抗體27b-2-HRP的終濃度為400 μg/mL。
For conjugation of monoclonal antibody to Horseradish peroxidase (HRP) (Innova Biosciences, Cambridge, UK), 100 μg HRP and 20 μL aliquots of modification reagent were combined with 200 μL
2. SARS-CoV-22. SARS-CoV-2 捕獲capture ELISAELISA 檢測試劑之製備Preparation of detection reagents
以單株抗體8a-1作為捕獲抗體，並以單株抗體27b-2-HRP作為偵測抗體。透過棋盤滴定測定最合適的實驗條件，例如捕獲抗體的包覆濃度及偵測抗體的稀釋度。將96孔微孔盤(Nunc Immuno
TMMaxisorp
TM，Thermo Fisher Scientific公司，羅斯基勒，丹麥)以100 μL的10 μg/mL捕獲抗體(單株抗體8a-1)包覆，並於4
oC培養整夜。隨後以阻隔緩衝液(PBS，0.05% Tween，5%脫脂奶粉)於37
oC下阻隔孔井1小時，並以洗滌緩衝液(PBS，0.05% Tween)洗滌後備用。
3. SARS-CoV-23. SARS-CoV-2 捕獲capture ELISAELISA 檢測試劑之專一性分析Specificity Analysis of Detection Reagents
以阻隔緩衝液稀釋SARS-CoV或SARS-CoV-2感染細胞的裂解液、各病毒培養上清液(HCoV 229E、HCoV OC43、A型流感病毒H1N1、A型流感病毒H3N2、B型流感病毒、腺病毒-7a、副流感病毒-3、鼻病毒、呼吸道融合病毒(RSV)-A2)。將稀釋後的病毒裂解液及病毒培養上清液加入上述第2部分之備用的微孔盤中，並於37
oC下培養1小時。洗滌該微孔盤後，加入100 μL的0.8 μg/mL單株抗體27b-2-HRP，將微孔盤於37
oC下培養1小時。然後，再次洗滌微孔盤，加入100 μL 3,3',5,5'-四甲基聯苯胺(TMB)試劑，將微孔盤於室溫下培養10分鐘。然後以1N硫酸終止反應，並使用微孔盤自動讀取儀在450 nm波長處讀取吸光度(OD
450)。
Dilute the lysate of SARS-CoV or SARS-CoV-2 infected cells, each virus culture supernatant (HCoV 229E, HCoV OC43, Influenza A H1N1, Influenza A H3N2, Influenza B, adenovirus-7a, parainfluenza virus-3, rhinovirus, respiratory syncytial virus (RSV)-A2). Add the diluted virus lysate and virus culture supernatant to the prepared microplates in
4. SARS-CoV-24. SARS-CoV-2 捕獲capture ELISAELISA 檢測試劑之靈敏度Sensitivity of detection reagents (( 偵測極限detection limit )) 分析analyze
以阻隔緩衝液連續稀釋SARS-CoV-2 S蛋白。將稀釋後的蛋白加入上述第2部分之備用的微孔盤中，並於37
oC下培養1小時。洗滌微孔盤後，加入100 μL的0.8 μg/mL單株抗體27b-2-HRP，將微孔盤於37
oC下培養1小時。然後，再次洗滌微孔盤，加入100 μL TMB試劑，將微孔盤於室溫下培養10分鐘。然後以1N硫酸終止反應，並使用微孔盤自動讀取儀在450 nm波長處讀取吸光度(OD
450)。
Serial dilution of SARS-CoV-2 S protein in blocking buffer. The diluted protein was added to the prepared microplate in
為了確定本發明之SARS-CoV-2捕獲ELISA檢測試劑的基準線偵測極限，以連續稀釋的牛血清白蛋白(BSA)建立檢測試劑的基準線。樣本的OD 450值至少為BSA基準線的二倍者視為陽性反應。 In order to determine the baseline detection limit of the SARS-CoV-2 capture ELISA detection reagent of the present invention, serially diluted bovine serum albumin (BSA) was used to establish the baseline detection reagent. Samples with an OD 450 value at least twice the BSA baseline were considered positive.
55 .. 統計分析Statistical Analysis
使用GraphPad Prism 6.0版(GraphPad軟體公司，聖地牙哥，加州，美國)進行每種ELISA的專一性、靈敏度，以及相應的95%信賴區間(CI 95)，顯著性標準設定為P值＜0.05。 Specificity, sensitivity, and corresponding 95% confidence intervals (CI 95 ) for each ELISA were performed using GraphPad Prism version 6.0 (GraphPad Software, Inc., San Diego, CA, USA), with significance criteria set at P-value < 0.05.
66 .. 結果result
SARS-CoV-2 捕獲ELISA檢測試劑之專一性分析的結果如 圖 2所示。該結果證明本發明之SARS-CoV-2 捕獲ELISA檢測試劑對SARS-CoV與SARS-CoV-2具有專一性，且與其他類型的呼吸道相關病毒，例如人類冠狀病毒、A型流感病毒、B型流感病毒、副流感病毒、腺病毒、鼻病毒、呼吸道融合病毒，不具有交叉反應。 The results of the specificity analysis of the SARS-CoV-2 capture ELISA detection reagents are shown in Figure 2 . This result proves that the SARS-CoV-2 capture ELISA detection reagent of the present invention has specificity for SARS-CoV and SARS-CoV-2, and is compatible with other types of respiratory related viruses, such as human coronavirus, influenza A virus, type B virus Influenza virus, parainfluenza virus, adenovirus, rhinovirus, respiratory syncytial virus, no cross-reactivity.
SARS-CoV-2 捕獲ELISA檢測試劑之靈敏度(偵測極限)分析的結果如 圖 3所示。該結果顯示，本發明之SARS-CoV-2 捕獲ELISA檢測試劑的最小檢測量介於3.9至7.8 ng/mL。 The results of the sensitivity (detection limit) analysis of the SARS-CoV-2 capture ELISA detection reagent are shown in Figure 3 . The results show that the minimum detection amount of the SARS-CoV-2 capture ELISA detection reagent of the present invention is between 3.9 and 7.8 ng/mL.
實施例三Embodiment 3 SARS-CoV-2SARS-CoV-2 快速檢測試劑Rapid detection reagent (( 檢測條帶detection band )) 之製備preparation
本發明之抗SARS-CoV-2單株抗體8a-1、27b-2也用於開發SARS-CoV-2的檢測條帶。The anti-SARS-CoV-2
1.1. 膠體金探針之製備Preparation of colloidal gold probes
使用35 +/- 5 nm的膠體金(TANBead NanoGold-40，Taiwan Advanced Nanotech公司，臺灣)進行IgG的綴合。以0.2mM K
2CO
3調節膠體金溶液(1% w/v)的pH值，並將抗SARS-CoV-2單株抗體8a-1 (在PBS中，pH7.4)加入到pH調節的膠體金溶液中。用於綴合的優化抗體濃度為1 μg/條帶。將抗體/膠體金混合物輕輕混合90分鐘，以1% BSA溶液阻隔30分鐘，並以7000 rpm離心15分鐘。離心後以1% BSA (含有1%[w/v] BSA的20 mM Tris/HCl緩衝液[pH7.2])洗滌一次後，將金顆粒懸浮在1% BSA中。將這種抗SARS-CoV-2單株抗體8a-1塗層膠體金探針放入墊中乾燥，然後於4
oC下保存整夜。
Conjugation of IgG was performed using 35 +/- 5 nm colloidal gold (TANBead NanoGold-40, Taiwan Advanced Nanotech, Taiwan). The pH of the colloidal gold solution ( 1 % w/v) was adjusted with 0.2 mM K2CO3, and the anti-SARS-CoV- 2
2.2. 側流試紙之製備Preparation of lateral flow test paper
以0.2 μg/條帶的山羊抗小鼠IgG (Jackson ImmunoResearch公司，賓州，美國)作為對照線抗體。以1 μg/條帶的抗SARS-CoV-2單株抗體27b-2作為測試線(捕獲抗體)(在PBS (pH 7.4)中)，分別施加到醋酸纖維素負載的硝酸纖維素膜條帶(孔徑：12 μm直徑)的頂端附近，並在室溫下乾燥1小時。組成分處理後，組裝SARS-CoV-2檢測條帶裝置。Goat anti-mouse IgG (Jackson ImmunoResearch, Pennsylvania, USA) at 0.2 μg/band was used as a control line antibody. Anti-SARS-CoV-2
圖 4A所示為使用本發明之單株抗體27b-2作為偵測抗體，並以綴合膠體金的單株抗體8a-1作為捕獲抗體的條帶基本設計。
圖 4B所示為使用本發明之單株抗體8a-1作為偵測抗體，並以綴合膠體金的單株抗體27b-2作為捕獲抗體的條帶基本設計。
Figure 4A shows the basic design of the strip using the
3.3. 檢測條帶上detection strip SARS-CoV-2SARS-CoV-2 病毒之檢測及檢測靈敏度Virus detection and detection sensitivity
將含有適當測試SARS-CoV-2病毒S蛋白(250 ng/mL、125 ng/mL、62.5 ng/mL、0 ng/mL)的樣本(100 μL)施加到裝置的樣本墊以進行測定。測試SARS-CoV-2病毒及檢測試劑的組合溶液沿著條帶往另一端移動，於室溫下20分鐘後膠體金沉積在固相抗體的位點。A sample (100 μL) containing the appropriate test SARS-CoV-2 virus S protein (250 ng/mL, 125 ng/mL, 62.5 ng/mL, 0 ng/mL) was applied to the sample pad of the device for the assay. The combined solution of the test SARS-CoV-2 virus and detection reagent moved along the strip to the other end, and colloidal gold was deposited on the site of the solid-phase antibody after 20 minutes at room temperature.
4.4. 其他類型的呼吸道相關病毒條帶的交叉反應性Cross-reactivity of other types of respiratory-associated viral bands (( 檢測專一性Detection specificity ))
以其他類型的呼吸道相關病毒(人類冠狀病毒HCoV-229E、HCoV-OC43、A型流感病毒Flu A/CA/07/2009、B型流感病毒Flu B B/T/81863/2014、副流感病毒-3、腺病毒-7a、鼻病毒、呼吸道融合病毒RSV)作為樣本評估SARS-CoV-2 快速檢測試劑(檢測條帶)的交叉反應性(專一性)，並以來自未感染的Vero E6細胞的上清液作為陰性對照組。Other types of respiratory-associated viruses (human coronavirus HCoV-229E, HCoV-OC43, influenza A virus Flu A/CA/07/2009, influenza B virus Flu B B/T/81863/2014, parainfluenza virus-3 , Adenovirus-7a, Rhinovirus, Respiratory Fusion Virus (RSV) were used as samples to evaluate the cross-reactivity (specificity) of SARS-CoV-2 rapid detection reagents (detection bands), and were used to evaluate the cross-reactivity (specificity) of SARS-CoV-2 rapid detection reagents (detection bands) from uninfected Vero E6 cells. Serum was used as negative control group.
5. 結果5. Results
如 表 2及 圖 5所示，本發明之單株抗體(27b-2)被證明成功地作為捕獲抗體，與本發明之另一單株抗體(8a-1)配對作為偵測抗體，在S蛋白條帶中，對SARS-CoV-2表現出優異的專一性，而不與其他類型的呼吸道相關病毒(人類冠狀病毒(HCoV)、A型流感病毒、B型流感病毒、副流感病毒、腺病毒、鼻病毒、呼吸道融合病毒(RSV))交叉反應。 As shown in Table 2 and Figure 5 , the monoclonal antibody of the present invention (27b-2) was proved to be successfully used as a capture antibody, paired with another monoclonal antibody of the present invention (8a-1) as a detection antibody, in S Among the protein bands, it showed excellent specificity for SARS-CoV-2, but not for other types of respiratory-associated viruses (human coronavirus (HCoV), influenza A virus, influenza B virus, parainfluenza virus, adenovirus virus, rhinovirus, respiratory syncytial virus (RSV)) cross-reactivity.
表 2本發明之SARS-CoV-2檢測條帶的專一性測試結果
此外，測定本發明之檢測條帶的偵測極限。結果如 圖 6所示，使用本發明之單株抗體的檢測條帶展現出優異的靈敏度，可偵測到62.5 ng/mL以上的SARS-CoV-2 S蛋白。 In addition, the detection limit of the detection strip of the present invention was determined. The results are shown in FIG. 6 , the detection band using the monoclonal antibody of the present invention exhibited excellent sensitivity, and could detect more than 62.5 ng/mL of SARS-CoV-2 S protein.
上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實施或變更，均應包含於本案之專利範圍中。The above detailed description is a specific description of a feasible embodiment of the present invention, but this embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the technical spirit of the present invention shall be included in the within the scope of the patent in this case.
綜上所述，本案所揭露之技術特徵已充分符合新穎性及進步性之法定發明專利要件，爰依法提出申請，懇請 貴局核准本件發明專利申請案，以勵發明，至感德便。To sum up, the technical features disclosed in this case have fully met the requirements of the statutory invention patent for novelty and progress, and an application is filed in accordance with the law.
當結合圖式閱讀時，將更好地理解前述發明內容以及本發明之以下詳細描述。出於說明本發明之目的，在附圖中示出了目前較佳的具體實施例。然而，應當理解的是，本發明不限於所示的精確佈置及手段。The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings specific embodiments that are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
於圖式中：In the schema:
圖 1所示為間接免疫螢光染色分析(Indirect immunofluorescence assay, IFA)的結果(400x)。使用本發明之抗新型冠狀病毒(SARS-CoV-2)的單株抗體(monoclonal antibody, mAb) 8a-1 ( 圖 1A)及27b-2 ( 圖 1B)、患者的血清( 圖 1C)，以及抗登革病毒單株抗體anti-D2 NS1( 圖 1D，負對照)辨識感染SARS-CoV-2的Vero E6細胞。 Figure 1 shows the results of an Indirect immunofluorescence assay (IFA) (400x). Using the anti-novel coronavirus (SARS-CoV-2) monoclonal antibodies (monoclonal antibody, mAb) 8a-1 ( Fig. 1A ) and 27b-2 ( Fig. 1B ) of the present invention, patient serum ( Fig. 1C ), and The anti-dengue monoclonal antibody anti-D2 NS1 ( Fig. 1D , negative control) recognized Vero E6 cells infected with SARS-CoV-2.
圖 2所示為使用本發明之抗新型冠狀病毒(SARS-CoV-2)的單株抗體8a-1作為捕獲抗體，並與作為偵測抗體的抗新型冠狀病毒(SARS-CoV-2)的單株抗體27b-2配對的夾心式(捕獲) ELISA的專一性測定結果。在該測定中，來自分別以嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)或新型冠狀病毒(SARS-CoV-2)感染的Vero E6細胞的細胞裂解液、人類冠狀病毒(human coronavirus, HCoV) 229E、HCoV OC43、A型流感病毒(influenza A, Flu A) H1N1、A型流感病毒(influenza A, Flu A) H3N2、B型流感病毒(influenza B, Flu B)、腺病毒(Adenovirus)-7a、副流感病毒(Parainfluenza)-3、鼻病毒(Rhinovirus)、呼吸道融合病毒(respiratory syncytial virus, RSV)-A2病毒培養上清液作為抗原之來源，並以來自未感染的Vero E6細胞的上清液作為陰性對照組。
Figure 2 shows the use of the anti-new coronavirus (SARS-CoV-2)
圖 3所示為使用本發明之抗新型冠狀病毒(SARS-CoV-2)的單株抗體8a-1作為捕獲抗體，並與作為偵測抗體的抗新型冠狀病毒(SARS-CoV-2)的單株抗體27b-2配對的夾心式(捕獲) ELISA的靈敏度測定結果。以連續稀釋的SARS-CoV-2 S2亞基蛋白進行捕獲ELISA，以確定本發明之抗新型冠狀病毒(SARS-CoV-2)的單株抗體的偵測極限。以牛血清白蛋白(bovine serum albumin, BSA)建立基準線。每個數據點代表三次重複測試的平均值±標準差(Standard Deviation, SD)。
Figure 3 shows the use of the anti-novel coronavirus (SARS-CoV-2)
圖 4A所示為使用本發明之抗新型冠狀病毒(SARS-CoV-2)的單株抗體8a-1作為捕獲抗體，並與作為偵測抗體的抗新型冠狀病毒(SARS-CoV-2)的單株抗體27b-2配對的夾心式條帶的基本設計。
Figure 4A shows the use of the anti-novel coronavirus (SARS-CoV-2)
圖 4B所示為使用本發明之抗新型冠狀病毒(SARS-CoV-2)的單株抗體27b-2作為捕獲抗體，並與作為偵測抗體的抗新型冠狀病毒(SARS-CoV-2)的單株抗體8a-1配對的夾心式條帶的基本設計。
Figure 4B shows the use of the anti-novel coronavirus (SARS-CoV-2)
圖 5所示為本發明之夾心式檢測條帶之特性。將抗新型冠狀病毒(SARS-CoV-2)的單株抗體8a-1與抗新型冠狀病毒(SARS-CoV-2)的單株抗體27b-2配對，分別作為偵測及捕獲抗體，然後測試結合專一性。在該測定中，以SARS-CoV-2 (CoV19/34005-MK2/20200227病毒株)、SARS-CoV-2 (CoV19/73611-MK2/20200314病毒株)、人類冠狀病毒HCoV-229E、HCoV OC43、A型流感病毒(Flu A) (A/CA/07/2009病毒株)、B型流感病毒(Flu B) (B/T/81863/2014病毒株)、副流感病毒、腺病毒-7a、鼻病毒、呼吸道融合病毒(RSV)的病毒培養上清液作為抗原之來源，並以來自未感染的Vero E6細胞的上清液作為陰性對照組。箭頭指示陽性反應。
FIG. 5 shows the characteristics of the sandwich-type detection strip of the present invention. The anti-novel coronavirus (SARS-CoV-2)
圖 6所示為本發明之夾心式檢測條帶之特性。將抗新型冠狀病毒(SARS-CoV-2)的單株抗體8a-1與抗新型冠狀病毒(SARS-CoV-2)的單株抗體27b-2配對，分別作為偵測及捕獲抗體，然後測試結合靈敏度。在該測定中，分別以不同濃度的S蛋白(0、62.5、125、250 ng/mL)作為抗原。箭頭指示陽性反應。
FIG. 6 shows the characteristics of the sandwich-type detection strip of the present invention. The anti-novel coronavirus (SARS-CoV-2)
序列表
<![CDATA[<110> 國防醫學院]]>
<![CDATA[<120> 抗新型冠狀病毒抗體及其應用]]>
<![CDATA[<130> P20-0130]]>
<![CDATA[<160> 21 ]]>
<![CDATA[<170> PatentIn version 3.5]]>
<![CDATA[<210> 1]]>
<![CDATA[<211> 10]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的重鏈可變區的重鏈互補決定區1]]>
<![CDATA[<400> 1]]>
Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser
1 5 10
<![CDATA[<210> 2]]>
<![CDATA[<211> 17]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的重鏈可變區的重鏈互補決定區2]]>
<![CDATA[<40]]>0> 2]]>
<br/>
<br/><![CDATA[Tyr Ile Ser Asn Gly Gly Gly Ser Ile Ser Tyr Pro Asp Thr Val Lys
1 5 10 15
Gly
<![CDATA[<210> 3]]>
<![CDATA[<211> 16]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的重鏈可變區的重鏈互補決定區3]]>
<![CDATA[<400> 3]]>
Glu Gly Leu Arg Pro Gly Asp Arg Asp Arg Tyr Tyr Ala Met Asp Tyr
1 5 10 15
<![CDATA[<210> 4]]>
<![CDATA[<211> 125]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的重鏈可變區]]>
<![CDATA[<400> 4]]>
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Asn Gly Gly Gly Ser Ile Ser Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu His Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr His Cys
85 90 95
Ala Arg Glu Gly Leu Arg Pro Gly Asp Arg Asp Arg Tyr Tyr Ala Met
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<![CDATA[<210> 5]]>
<![CDATA[<211> 12]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的輕鏈可變區的輕鏈互補決定區1]]>
<![CDATA[<400> 5]]>
Ser Val Ser Ser Ser Val Ser Ser Ser Asn Leu His
1 5 10
<![CDATA[<210> 6]]>
<![CDATA[<211> 7]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的輕鏈可變區的輕鏈互補決定區2]]>
<![CDATA[<400> 6]]>
Gly Thr Ser Thr Leu Ala Ser
1 5
<![CDATA[<210> 7]]>
<![CDATA[<211> 9]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223]]>> 第一抗體的輕鏈可變區的輕鏈互補決定區3]]>
<br/>
<br/><![CDATA[<400> 7]]>
<br/>
<br/><![CDATA[Gln Gln Trp Ser Ser Tyr Pro Leu Thr
1 5
<![CDATA[<210> 8]]>
<![CDATA[<211> 109]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的輕鏈可變區]]>
<![CDATA[<400> 8]]>
Asp Ile Val Ile Thr Gln Ser Pro Ala Leu Met Ala Ala Phe Pro Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ser
20 25 30
Asn Leu His Trp Tyr Arg Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp
35 40 45
Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Val Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
85 90 95
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<![CDATA[<210> 9]]>
<![CDATA[<211> 10]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的重鏈可變區的重鏈互補決定區 1]]>
<![CDATA[<400> 9]]>
Gly Phe Thr Phe Ser Thr Tyr Tyr Met Ser
1 5 10
<![CDATA[<210> 10]]>
<![CDATA[<211> 17]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的重鏈可變區的重鏈互補決定區2]]>
<![CDATA[<400> 10]]>
Ala Ile Asn Ser Asn Gly Gly Gly Thr Tyr Tyr Pro Asp Asn Val Lys
1 5 10 15
Gly
<![CDATA[<210> 11]]>
<![CDATA[<211> 10]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的重鏈可變區的重鏈互補決定區3]]>
<![CDATA[<400> 11]]>
Tyr Phe Gly Ser Thr Tyr Ala Met Asp Tyr
1 5 10
<![CDATA[<210> 12]]>
<![CDATA[<211> 119]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的重鏈可變區]]>
<![CDATA[<400> 12]]>
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val
35 40 45
Ala Ala Ile Asn Ser Asn Gly Gly Gly Thr Tyr Tyr Pro Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Thr Asn Tyr Phe Gly Ser Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<![CDATA[<210> 13]]>
<![CDATA[<211> 16]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的輕鏈可變區的輕鏈互補決定區1]]>
<![CDATA[<400> 13]]>
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<![CDATA[<210> 14]]>
<![CDATA[<211> 7]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的輕鏈可變區的輕鏈互補決定區2]]>
<![CDATA[<400> 14]]>
Lys Val Ser Asn Arg Phe Ser
1 5
<![CDATA[<210> 15]]>
<![CDATA[<211> 9]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工]]>序列
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的輕鏈可變區的輕鏈互補決定區3]]>
<![CDATA[<400> 15]]>
Ser Gln Asn Thr His Val Pro Phe Thr
1 5
<![CDATA[<210> 16]]>
<![CDATA[<211> 113]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的輕鏈可變區]]>
<![CDATA[<400> 16]]>
Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Leu Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ile Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn
85 90 95
Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<![CDATA[<210> 17]]>
<![CDATA[<211> 375]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的重鏈可變區的DNA]]>
<![CDATA[<400> 17]]>
gaggtgcaac tgcaggagtc tgggggaggt ttagtgcagc ctggagggtc cctgaaactc 60
tcctgtgcag cctctggatt cactttcagt agctatacca tgtcttgggt tcgccagact 120
ccagagaaga ggctggagtg ggtcgcatac attagtaatg gtggtggtag tatcagctat 180
ccagacactg taaagggccg attcaccatc tccagagaca atgccaagaa caccctgtat 240
ctgcacatga gcagtctgaa gtctgaggac acggccatgt atcattgtgc aagagagggg 300
ttacgacccg gggaccgcga taggtactat gctatggact actggggtca aggaacctca 360
gtcaccgtct cctca 375
<![CDATA[<210> 18]]>
<![CDATA[<211> 327]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第一抗體的輕鏈可變區的DNA]]>
<![CDATA[<400> 18]]>
gatattgtga taacccagtc tccagcactc atggctgcat ttccagggga cagggtcacc 60
atcacctgca gtgtcagctc aagtgtaagt tccagcaact tgcactggta ccgacagaag 120
tcagaaacct cccccaaacc ctggatttat ggcacatcca ccctggcttc tggagtccct 180
gttcgcttca gtggcagtgg atctgggacc tcttactctc tcacaatcag cagcatggag 240
gctgaagatg ctgccactta ttactgtcaa cagtggagta gttacccact cacgttcggt 300
gctgggacca agctggagct gaaacgg 327
<![CDATA[<210> 19]]>
<![CDATA[<211> 357]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的重鏈可變區的DNA]]>
<![CDATA[<400> 19]]>
gaggtgcagc tgcaggagtc tgggggaggc ttagtgaagc ttggagggtc cctgaaactc 60
tcctgtgcag cctctggatt cactttcagt acttattaca tgtcttgggt tcgccagact 120
ccagagaaga ggctggagtt ggtcgcagcc attaatagta atggtggtgg cacctactat 180
ccagacaatg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240
ctgcaaatga gcagtctgaa gtctgaggac acagccttgt attactgtac aaattacttc 300
ggtagtacct acgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357
<![CDATA[<210> 20]]>
<![CDATA[<211> 339]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> 第二抗體的輕鏈可變區的DNA]]>
<![CDATA[<400> 20]]>
gacattgtgc tgacccaatc tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120
tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggattaggga cagatttcac actcaagatc 240
atcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaaatac acatgttcca 300
ttcacgttcg gctcggggac aaagttggaa ataaagcgg 339
<![CDATA[<210> 21]]>
<![CDATA[<211> 988]]>
<![CDATA[<212> PRT]]>
<![CDATA[<213> 人工序列]]>
<![CDATA[<220>]]>
<![CDATA[<223> SARS CoV棘蛋白的第268至第1255個胺基酸殘基]]>
<![CDATA[<400> 21]]>
Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu
1 5 10 15
Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile
20 25 30
Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg
35 40 45
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
50 55 60
Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn
65 70 75 80
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr
85 90 95
Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe
100 105 110
Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg
115 120 125
Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys
130 135 140
Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn
145 150 155 160
Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu
165 170 175
Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro
180 185 190
Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr
195 200 205
Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr
210 215 220
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro
225 230 235 240
Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln
245 250 255
Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr
260 265 270
Pro Ser Ser Lys Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val
275 280 285
Ser Asp Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu
290 295 300
Asp Ile Ser Pro Cys Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly
305 310 315 320
Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys
325 330 335
Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp
340 345 350
Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys
355 360 365
Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro
370 375 380
Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg
385 390 395 400
Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala
405 410 415
Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn
420 425 430
Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys
435 440 445
Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
450 455 460
Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
465 470 475 480
Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val
485 490 495
Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe
500 505 510
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr
515 520 525
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
530 535 540
Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn
545 550 555 560
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
565 570 575
Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu
580 585 590
Val Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala
595 600 605
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
610 615 620
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn
625 630 635 640
Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr
645 650 655
Ser Thr Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
660 665 670
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
675 680 685
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala
690 695 700
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
705 710 715 720
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
725 730 735
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
740 745 750
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
755 760 765
Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
770 775 780
Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly
785 790 795 800
Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser
805 810 815
Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr
820 825 830
Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile
835 840 845
Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
850 855 860
Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp
865 870 875 880
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
885 890 895
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
900 905 910
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro
915 920 925
Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met
930 935 940
Val Thr Ile Leu Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
945 950 955 960
Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser
965 970 975
Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
980 985
Sequence Listing <![CDATA[<110> National Defense Medical College]]> <![CDATA[<120> Anti-COVID-19 Antibody and Its Application]]> <![CDATA[<130> P20-0130]]> < ![CDATA[<160> 21 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 10]]> < ![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> The heavy chain of the primary antibody can be Heavy chain complementarity determining region 1]]> <![CDATA[<400> 1]]> Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser 1 5 10 <![CDATA[<210> 2]]> < ![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![ CDATA[<223> Heavy chain complementarity determining region 2 of the heavy chain variable region of the primary antibody]]> <![CDATA[<40]]>0> 2]]> <br/> <br/>< ![CDATA[Tyr Ile Ser Asn Gly Gly Gly Ser Ile Ser Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly <![CDATA[<210> 3]]> <![CDATA[<211> 16]]> < ![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> The heavy chain of the primary antibody can be Heavy chain CDR3]]> <![CDATA[<400> 3]]> Glu Gly Leu Arg Pro Gly Asp Arg Asp Arg Tyr Tyr Ala Met Asp Tyr 1 5 10 15 <![CDATA[< 210> 4]]> <![CDATA[<211> 125]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220 >]]> <![CDATA[<223> Heavy chain variable region of primary antibody]]> <![CDAT A[<400> 4]]> Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Gly Gly Gly Ser Ile Ser Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu His Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr His Cys 85 90 95 Ala Arg Glu Gly Leu Arg Pro Gly Asp Arg Asp Arg Tyr Tyr Ala Met 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 125 <![CDATA[<210> 5]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA [<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Light chain complementarity determining region 1 of light chain variable region of primary antibody]]> <![ CDATA[<400> 5]]> Ser Val Ser Ser Ser Val Ser Ser Ser Asn Leu His 1 5 10 <![CDATA[<210> 6]]> <![CDATA[<211> 7]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Light of primary antibody Light chain complementarity determining region of chain variable region 2]]> <![CDATA[<400> 6]]> Gly Thr Ser Thr Leu Ala Ser 1 5 <![CDATA[<210> 7]]> <![ CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223]]>> Light chain complementarity determining region 3 of the light chain variable region of the primary antibody]]> <br/> <br/> <![CDATA[<400>7]]> < br/> <br/><![CDATA[Gln Gln Trp Ser Ser Tyr Pro Leu Thr 1 5 <![CDATA[<210> 8]]> <![CDATA[<211> 109]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Light chain variable region of primary antibody ]]> <![CDATA[<400> 8]]> Asp Ile Val Ile Thr Gln Ser Pro Ala Leu Met Ala Ala Phe Pro Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ser 20 25 30 Asn Leu His Trp Tyr Arg Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp 35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Val Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <![CDATA [ <210> 9]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[< 220>]]> <![CDATA[<223> Heavy chain complementarity determining region 1 of the heavy chain variable region of the secondary antibody]]> <![CDATA[<400> 9]]> Gly Phe Thr Phe Ser Thr Tyr Tyr Met Ser 1 5 10 <![CDATA[<210> 10]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213 > Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Heavy chain complementarity determining region 2 of the heavy chain variable region of the secondary antibody]]> <![CDATA[< 400> 10]]> Ala Ile Asn Ser Asn Gly Gly Gly Thr Tyr Tyr Pro Asp Asn Val Lys 1 5 10 15 Gly <![CDATA[<210> 11]]> <![CDATA[<211> 10]] > <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Secondary Antibody Heavy chain complementarity determining region 3]]> <![CDATA[<400> 11]]> Tyr Phe Gly Ser Thr Tyr Ala Met Asp Tyr 1 5 10 <![CDATA[<210> 12]] > <![CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> < ![CDATA[<223> heavy chain variable region of secondary antibody]]> <![CDATA[<400> 12]]> Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asn Gly Gly Gly Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Asn Tyr Phe Gly Ser Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <![CDATA[<210 > 13]]> <![CDATA[<211> 16]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Light chain complementarity determining region 1 of the light chain variable region of the secondary antibody]]> <![CDATA[<400> 13]]> Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <![CDATA[<210> 14]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <! [CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Light chain complementarity determining region 2 of light chain variable region of secondary antibody]]> < ![CDATA[<400> 14]]> Lys Val Ser Asn Arg Phe Ser 1 5 <![CDATA[<210> 15]]> <![CDATA[<211> 9]]> <![CDATA[< 212> PRT]]> <![CDATA[<213> Artificial]]> Sequence<![CDATA[<220>]]> <![CDATA[<223> Light chain of the light chain variable region of the secondary antibody Complementarity Determining Region 3]]> <![CDATA[<400> 15]]> Ser Gln Asn Thr His Val Pro Phe Thr 1 5 <![CDATA[<210> 16]]> <![CDATA[<211> 113]]> <![CDATA[<212> PRT]]> <![CDATA[< 213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Light chain variable region of secondary antibody]]> <![CDATA[<400> 16]]> Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Leu Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ile Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <![CDATA[<210> 17]]> <! [CDATA[<211> 375]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA [<223> DNA of heavy chain variable region of primary antibody]]> <![CDATA[<400> 17]]> gaggtgcaac tgcaggagtc tgggggaggt ttagtgcagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatacca tgtcttgggt tcgccagact 120 ccagagaaga ggctggagtg ggtcgcatac attagtaatg gtggtggtag tatcagctat 180 ccagacactg taaagggccg attcaccatc tccagagaca atgccaagaa caccctgtat 240 ctgcacatga gcagtctgaa gtctgaggac acggccatgt atcattgtgc aagagagggg 300 ttacgacccg gggaccgcga taggtactat gctatggact actggggtca aggaacctca 360 gtcaccgtct cctca 375 <![CDATA[<210> 18]]> <![ CDATA[<211> 327]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> 第一抗體的輕鏈可變區的DNA]]> <![CDATA[<400> 18]]> gatattgtga taacccagtc tccagcactc atggctgcat ttccagggga cagggtcacc 60 atcacctgca gtgtcagctc aagtgtaagt tccagcaact tgcactggta ccgacagaag 120 tcagaaacct cccccaaacc ctggatttat ggcacatcca ccctggcttc tggagtccct 180 gttcgcttca gtggcagtgg atctgggacc tcttactctc tcacaatcag cagcatggag 240 gctgaagatg ctgccactta ttactgtcaa cagtggagta gttacccact cacgttcggt 300 gctgggacca agctggagct gaaacgg 327 <![CDATA[<210> 19]]> <![CDATA[<211> 357]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223 > 第二抗體的重鏈可變區的DNA]]> <![CDATA[<400> 19]]> gaggtgcagc tgcaggagtc tgggggaggc ttagtgaagc ttggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt acttattaca tgtcttgggt tcgccagact 120 ccagagaaga ggctggagtt ggtcgcagcc attaatagta atggtggtgg cacctactat 180 ccagacaatg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccttgt attactgtac aaattacttc 300 ggtagtacct acgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357 <![CDATA[<210> 20]]> <![CDATA[<211> 339]]> <![CDATA[<212 > DNA]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> DNA of the light chain variable region of the secondary antibody]] > <![CDATA[<400> 20]]> gacattgtgc tgacccaatc tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggattaggga cagatttcac actcaagatc 240 atcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaaatac acatgttcca 300 ttcacgttcg gctcggggac aaagttggaa a taaagcgg 339 <![CDATA[<210> 21]]> <![CDATA[<211> 988]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]] > <![CDATA[<220>]]> <![CDATA[<223> Amino acid residues 268 to 1255 of SARS CoV spine protein]]> <![CDATA[<400> 21]] > Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu 1 5 10 15 Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile 20 25 30 Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val Arg 35 40 45 Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala 50 55 60 Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn 65 70 75 80 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr 85 90 95 Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe 100 105 110 Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg 115 120 125 Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys 130 135 140 Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn 145 150 155 160 Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu 165 170 175 Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro 180 185 190 Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr 195 200 205 Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr 210 215 220 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro 225 230 235 240 Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln 245 250 255 Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr 260 265 270 Pro Ser Ser Lys Arg Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val 275 280 285 Ser Asp Phe Thr Asp Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu 290 295 300 Asp Ile Ser Pro Cys Ala Phe Gly Gly Val Ser Val Ile Thr Pro Gly 305 310 315 320 Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys 325 330 335 Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp 340 345 350 Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys 355 360 365 Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro 370 375 380 Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg 385 390 395 400 Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala 405 410 415 Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn 420 425 430 Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met Ala Lys 435 440 445 Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 450 455 460 Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 465 470 475 480 Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val 485 490 495 Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe 500 505 510 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr 515 520 525 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 530 535 540 Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn 545 550 555 560 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 565 570 575 Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu 580 585 590 Val Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala 595 600 605 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 610 615 620 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn 625 630 635 640 Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr 645 650 655 Ser Thr Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 660 665 670 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 675 680 685 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala 690 695 700 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 705 710 715 720 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 725 730 735 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser 740 745 750 Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 755 760 765 Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro 770 775 780 Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly 785 790 795 800 Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser 805 810 815 Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr 820 825 830 Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Ile 835 840 845 Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys 850 855 860 Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp 865 870 875 880 Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys 885 890 895 Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu 900 905 910 Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro 915 920 925 Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met 930 935 940 Val Thr Ile Leu Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys 945 950 955 960 Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser 965 970 975 Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr 980 985
Claims (17)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW109131736A TW202212349A (en) | 2020-09-15 | 2020-09-15 | Anit-sars-cov-2 antibodies and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW109131736A TW202212349A (en) | 2020-09-15 | 2020-09-15 | Anit-sars-cov-2 antibodies and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| TW202212349A true TW202212349A (en) | 2022-04-01 |
Family
ID=82197098
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW109131736A TW202212349A (en) | 2020-09-15 | 2020-09-15 | Anit-sars-cov-2 antibodies and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| TW (1) | TW202212349A (en) |
-
2020
- 2020-09-15 TW TW109131736A patent/TW202212349A/en unknown
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6979875B2 (en) | Antibody-mediated neutralization of chikungunya virus | |
| JP7112790B2 (en) | Monoclonal antibody against S protein of MERS coronavirus and use thereof | |
| US10215754B2 (en) | Anti-T. cruzi antibodies and methods of use | |
| CN112851804B (en) | Neutralizing active monoclonal antibody of human source for resisting novel coronavirus (SARS-CoV-2) | |
| IL295310A (en) | Human monoclonal antibodies to severe acute respiratory syndrome coronavirus 2 (sars-cov- 2) | |
| CN110590943B (en) | Anti-dengue virus antibody and application thereof | |
| US20220089691A1 (en) | Anti-sars-cov-2 antibodies and application thereof | |
| WO2013043125A1 (en) | Enterovirus 71 specific antibodies and uses thereof | |
| EP2917731B1 (en) | Diagnostic, prognostic, therapeutic and screening protocols | |
| KR102008609B1 (en) | Hybridomas that produce specific antibodies to non-structural protein 1 of Zika virus and antibodies produced therefrom, and uses thereof | |
| CN116836270B (en) | Monoclonal antibody of anti-bluetongue virus VP7 protein, preparation method and application | |
| CN113045646B (en) | Antibodies against novel coronavirus SARS-CoV-2 | |
| KR20070103548A (en) | Monoclonal antibody against nucleocapsid protein of sars coronavirus and the use thereof | |
| CN116217716A (en) | Monoclonal antibody for identifying Coxsackie viruses A2, A4 and A5 and application thereof | |
| TWI737918B (en) | Anti-dengue virus antibodies and applications thereof | |
| WO2011093217A1 (en) | ANTI-Pandemic (H1N1) 2009 ANTIBODY AND IMMUNOASSAY METHOD USING SAME | |
| TW202212349A (en) | Anit-sars-cov-2 antibodies and application thereof | |
| KR102623999B1 (en) | AN ANTIBODY SPECIFIC FOR N PROTEIN OF SARS-CoV-2 AND AN APPLICATION THEREOF | |
| KR20220055423A (en) | Detection method of SARS-CoV-2 using novel SARS-CoV-2 specific antibody | |
| KR20070103547A (en) | Monoclonal antibody against nucleocapsid protein of sars coronavirus and the use thereof | |
| TW201915016A (en) | Monoclonal antibody or antigen-binding fragment and use of the same | |
| KR102051052B1 (en) | An anti-MERS-CoV monoclonal antibody and use of the same | |
| WO2023240246A1 (en) | Computationally engineered monocolonal antibodies and antigen binding fragments specific for sars-cov-2 spike proteins and uses thereof | |
| KR20230124282A (en) | Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and Use thereof | |
| CN117007808A (en) | African swine fever virus CD2v protein blocking biochip antibody detection kit |