丄 M3479 100年3月29日修正替換頁 玖、發明說明： 一^ 【發明所屬之技術領域】 ‘ 本發明係有關於一種偵測體液中結核菌抗原之方法尤指一種可以1 . 接檢測體液中結核菌所分泌特異性抗原之偵測方法。 【先前技術】 結核病+的感染途徑主要是經由空氣中飛沫傳染，而造成結核病的 主要致病菌為結核菌（細咖ciery⑽驗則/〇咖，根據世界衛生 組織的報告，目前每年約有八百多萬的結核病新增病例，且每年約有3 百萬人因結核病死亡，而全世界約有3测的人口為潛伏性結核病患，因 此如何快速正確的偵測診斷出結核病患是非常重要的。 馨 目前常見之結核病診斷方法不外乎下列幾種： 1、f膚測試（Skin test):這是檢測結核病最t見的初步診斷 法，在前臂以皮下注射約〇. lml的結核菌的純化抗原（Tub⑽丄M3479 March 29, 100 revised replacement page 发明, invention description: a ^ [Technical field of the invention] ' The present invention relates to a method for detecting tuberculosis antigen in body fluids, especially one can be used to detect body fluids A method for detecting a specific antigen secreted by tuberculosis. [Prior Art] The infection route of tuberculosis+ is mainly transmitted through droplets in the air, and the main pathogen causing tuberculosis is tuberculosis. (10) The test is based on the report of the World Health Organization. More than one million new cases of tuberculosis, and about 3 million people die from tuberculosis each year, and about 3 people in the world are latent tuberculosis patients. So how to quickly and correctly detect and diagnose tuberculosis patients is Very important. Xin's current common diagnosis methods for tuberculosis are nothing more than the following: 1. Skin test: This is the most common diagnostic method for detecting tuberculosis. In the forearm, subcutaneous injection is about 〇. Purified antigen of tuberculosis (Tub(10)
Punfled protein derivate，ppD) ’在注射48〜72小時後該處皮膚會 f見f服的情狀’此方法_為陽性的病患代表此人曾受過結核菌感 v ^ ’若要確^是否為活動性結核病(active TB)則需再配合χ光檢查及 ^ 八他診斷方法。結核素皮膚測試的靈敏度大約65%。 — -^ i胸部Χ *X—ray):胸部Χ光檢查對於肺部結核病的 1疋十分重要的’此方法除可以判斷是否感染肺部結核病外，也可 以助皮膚測試陽性的病患做更進一步的確認，但對於非肺部結核病 則無法沴斷。胸部X光檢測的靈敏度大約50%。 人3、抗酸性染色（Acid_fast stain，:由於結核菌的細胞壁 a有較厚的脂質層’故當以酸性的染劑進行染色後，染劑較不易脫 可在顯微鏡下觀察到粉紅色的桿菌存在，且此方法 液，最好是從肺部咳出之深層錢，辦患無法自行㊁ 以霧狀兩渗透壓的食鹽水幫助引流，或可以胃部抽取液、 液為檢测物。抗酸性染色的靈敏度約50%。 彳1 方、、細菌培養（Culture):為了正確的診斷結核病目前最準確的 /為細菌培養’且細菌培養後可做抗藥性測試，提供醫師給藥的 5 1343479 100年3月29日修正兰j备百 I_^ /4 uu U4 依據，但難_生長十分緩慢，實驗室診斷㈣時4〜6週 ί抗f測試約需2~3週，[種十分⑽·斷方法。細菌培養的 特異度大約95%。 @ 5 '聚合酵素連鎖反應⑽ymemse Chain Reaction，PCR):聚 合酵素連鎖反應是目前積極絲的診_具之―，它對於診_期的 結核病感錄其财法錄，因此錢狀麟也開細此方法做為 結核病#斷方法，但此方法所需花f較高，操作人員需經訓練，故此 =仍錄適合應驗研私作上。聚合酵素義反應㈣敏度大約 9〇% 〇 6 '血清顺（Serology test):近十年來很多的研發图隊都在 的血清檢驗，但發現此方法無法分辨活動性結核病患 ί^ ΐ、··，。核病患；且對於接種過卡介苗的人們容易產生偽陽性 勺情形。血峋測试的靈敏度大約6〇〜85%。 全世界約有三分之-的人σ受到結核菌的感染，因此如何早 及，胃疾病是非常重要的，特別是如何隔離結核病患者 w k祕結核菌的病患是控制結核病傳雜—大難題，但由 於目前在臨床上频結核觸方法仍不夠完備，故造成辑正確率偏 低。 緣疋於此，本發日月人為了改善目前結核病診斷方法正確率偏低的 2點選擇了結分泌的蛋⑽特異抗原，並製作其多株和單株抗 體。由於結核細分泌的蛋白可經過體液交換後而存在於人體的各種 體液中’因此特異性的抗原抗體反應，本發明係為-種可以直 接檢測體液中結核菌所分泌特異抗原之方法。 【發明内容】 本發明之主要目的在提供-種可以直接檢測體液中結核菌所分泌 特異抗原並提高檢赃確率之結_侧方法。 基於上述目地’本發明所制的各種體液包括全血、血製、血清、 痰液、尿液、腦脊騎、肋酿及_料各敝祕·，此方法 的内涵如下： 6 1343479 100年3月29日修正替換頁 1、 結核菌分泌之特異抗原的選擇是轉譯於結核菌基因中的 RD卜RD2或RD3等基因群。 2、 抗源的製備可由基因工程的方式由大腸桿菌生產重組蛋白而 來。 〇〇 3、重組蛋白抗源可能是由RD1、RD2或RD3等基因群轉殖出的 早一蛋白或融合蛋白。 4、 主要選定的抗原為RDs基因上所轉譯的培養濾液蛋白1〇 (CFP-10(culturefiltratedprotein-10))、早期分泌性抗原 6((ESA丁_6)) 及 CFPl〇/ESAT6(early secreting antigen-6)融合蛋白。 5、 單株抗體及多株抗體可經由重組蛋白或其片段做為 疫動物而製造出來。 6、 本發明製備了 anti-CFP-l〇、antUESAT-6 及 ami- CFP_SAT6 的單株及多株抗體。 7、 本發明之單株及多株抗體可義各式免疫檢财法，包括酵 素免疫分析法、勞統疫分析法、冷歧疫分析法、免錢膠法、快 速免疫色層分析及抗體晶片等。 ’ ' 8、 此研發最終理想的免疫分析法為酵素免疫分析法或快速免疫 色層分析，將一級之反應抗體吸附在固體上，並以顏色物質或酵 合於另一抗體做為呈色偵測之用。 9 '本發明可檢測結核g分泌於體液中之特異抗原，體液括錢、 血毁、血清、痰液、尿液、腦脊髓液、肋獏液及組織液等各種生物性 體液。 接下來舉一較佳實施例做進一步之說明，以使貴審查委員對 發明有更詳盡的瞭解，惟以下所述者僅為用來解釋本發明:較佳實^ 例，並非企圖據以對本發明做任何形式上之限制，是以凡有以本= 明之創作精神為基礎，而為本發明任何形式的修飾或變更 於本發明意_護之範4。 【實施方式】 請配合參關-至圖七所示，為提高結核病檢測之準確性近年 7 1343479 100年3月29日修正替換頁 來不=有研究再尋找結核菌特異性抗原，研究報告指出結核菌在感染 ESAT-6 (early-secreted antigenic target 6-kDa ΡΓ〇_蛋自’此蛋白屬分泌性蛋自，且可引起T細胞發线疫反應， 而esat- 土因p、存在於M iubercu!〇sis和會致病的μ咖在M㈣ BCG疫苗和其他許多非典型肺結核菌種則不具此基因，因此推論 ESAT-6的存在可以區分真正的結核病與BCG疫苗接種的不同。為了更 清楚地知道ESAT-6的·表職轉，便有學者时子生物的方法分析 會致病的M W與M “ BCG，發現二者在则（regi〇n 〇f ddeti〇n 1)、RD2與RD3S區的基因有所差異，這些咖只存在於致病性分支 桿菌，在所有的BCG中皆沒有RDs的存在，RDs〇per〇n⑽多基因， 而鍾-6基因即位於RD1，另外發現跡6基因的啟動子除調控碟6 # 基因外還調控另-％基因’此基因可轉擇出另—蛋白培養濾液蛋白 10 (CFP-I〇(cultureflltrateprotein))’此蛋白亦為分泌性蛋白，可引起 T細胞引發之免疫反應。本發明中以基因選殖的方法表現出ESAT_6及 CFP-10 s種蛋白，並製造對抗此兩種蛋白的單株及雙株抗體，進而研 發出各式免疫檢測方法，包括酵素免疫分析法、免疫凝膠法、快速免 疫色層分析及抗體晶片等’這些體外檢測方法可以在人體各種體液中 偵測到結核騎分泌之特紐抗原，進而提高辑的速度及正確性。 本發明所使用的結核菌抗原包含了重組蛋白CFp丨〇、ESAT 6及 CFP10/ESAT_6融合蛋白。湘聚合酵素連鎖反應(pCR⑽特異性的？丨 子放大結核菌上/如及以扣-6基因。將PCR產物接入pGEM_T載體並 送入£.cO/i(DH5a)，接著在具有抗生素的培養基挑取代有/;^及__6 基因單一菌落，並以基因定序方法確認序列無誤，以限制酵素切下基 因並進行接合反應’使兩基因接合在__起，最後分麟咖、咖_6集 結核後的基因與表現載體pET29b結合送入瓦co//(BL21DE3)。 ' /很多研究顯示了結核菌'BCG菌株及環境中的結核桿菌類菌種具 有很多相同的抗原，而這些抗原或許能夠應用於疫苗的設計上，但可 月b因為父又反應所以無法作為診斷用途上的抗原，因此，越來越多的 研究在尋找結核菌的特異抗原，而目前所使用的結核菌純化抗原(ppD) 8 1343479 正替換頁 ~~~- ' ^ ;=Γΐ發生交，故如要預測抗原是否是否會與疫苗或環 二X生父又反應疋較困難的’因此’所要尋找作為診斷結核病之 几原必須具有極高的特異性，需為疫苗或環賴株所沒有的。 用Za=as以_卿的生物技較麟核_級抗原，並利 土雜父反應比對出致病性的結核桿菌屬和1925年所發展出的BCV 菌株在某録目是有料刺，紐在BCG _摘轉 別被命名為㈣、腿及聰，因此，有很多研究便想利用 所轉譯出的蛋白作為疫苗或檢驗診斷上的候選抗原1 RD2尚可轉出 -種名為MPT64的分泌蛋白，此蛋白大小約為32kD，很多研究發現 此蛋白可刺激T細胞而發生遲發性過敏反應（犯吵以加^ hypersensitivity, DTH)’由於BCG菌株不具有寧料基因，故利用Μτρ64 作為皮膚測試的抗原’可分辨受結核祕染的病患及接受卡介苗注射 之健康者。坤似基因可存在於下列菌株：从如心卿/〇此H37Rv、似 祕謂/_ H37Ra ϋ吣 BCG substmns Tokyo, Moreau，Russian 等；. 而下列菌株則不具有《砂料基因：MM bcg subStrainsPunfled protein derivate, ppD) 'After 48 to 72 hours of injection, the skin will see the condition of f.' This method is positive for the patient who has been affected by tuberculosis v ^ 'If you want to confirm whether it is Active tuberculosis (active TB) is required to cooperate with the sputum examination and the diagnostic method. The sensitivity of the tuberculosis skin test is approximately 65%. — -^ i chest Χ *X-ray): Chest stenosis is very important for tuberculosis in the lungs. 'This method can also help patients with positive skin tests to be able to determine whether they are infected with tuberculosis. Further confirmation, but for non-pulmonary tuberculosis can not be cut off. The sensitivity of chest X-ray detection is approximately 50%. Human 3, Acid_fast stain: Since the cell wall a of tuberculosis has a thick lipid layer, when dyed with an acidic dye, the dye is less likely to be removed, and pink bacillus can be observed under the microscope. Exist, and the method liquid, preferably the deep money coughed out from the lungs, can not be used to help the drainage by the two osmotic saline, or the stomach can be used as the test substance. The sensitivity of acid staining is about 50%. 彳1,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, On March 29, 100, the revised Lan j prepared 100 I_^ /4 uu U4 basis, but difficult _ growth is very slow, laboratory diagnosis (four) 4 to 6 weeks ί anti-f test takes about 2 to 3 weeks, [species very (10) · Breaking method. The specificity of bacterial culture is about 95%. @ 5 'Polymerase chain reaction (10) ymemse Chain Reaction, PCR): The polymerase chain reaction is the current diagnosis of positive silk - it is a diagnosis of tuberculosis Record its financial records, so Qian Zilin also This method is fine as tuberculosis #-off method, but this method required a high flower f, operators need to be trained, therefore = still recorded for the fulfillment of research for the private. Polymerase-like reaction (4) Sensitivity is about 9〇% 〇6 'Serology test: A lot of research and development teams in the past decade have been in the serum test, but found that this method can not distinguish active TB patients ί, ··,. Nuclear patients; and people who have been vaccinated with BCG are prone to false positive scoops. The sensitivity of the blood test is about 6 〇 85%. About three-thirds of the world's people are infected with tubercle bacilli, so how to get sick early, stomach disease is very important, especially how to isolate patients with tuberculosis patients with tuberculosis is a big problem in controlling tuberculosis. However, due to the fact that the current method of frequency-frequency tuberculosis is still not complete enough, the correct rate is low. Because of this, the Japanese and Japanese people chose the secreted egg (10) specific antigen to improve the current low rate of tuberculosis diagnosis, and produced multiple strains and individual antibodies. Since the finely secreted proteins of tuberculosis can be present in various body fluids of the human body after body fluid exchange, the specific antigen-antibody reaction, the present invention is a method for directly detecting specific antigens secreted by tuberculosis in body fluids. SUMMARY OF THE INVENTION The main object of the present invention is to provide a method for directly detecting a specific antigen secreted by tuberculosis in a body fluid and improving the detection rate. Based on the above objectives, the various body fluids prepared by the present invention include whole blood, blood, serum, sputum, urine, cranial riding, ribbing, and sputum. The connotation of this method is as follows: 6 1343479 100 years Correction of the replacement page on March 29, the selection of the specific antigen secreted by Mycobacterium tuberculosis is to translate into the RD RD2 or RD3 gene group in the tuberculosis gene. 2. Preparation of anti-sources Recombinant proteins can be produced from E. coli by genetic engineering. 〇〇 3. Recombinant protein resistance may be an early protein or fusion protein that is transferred from a gene group such as RD1, RD2 or RD3. 4. The main selected antigen is the culture filtrate protein 1 (CFP-10 (culturefiltrated protein-10)), the early secretory antigen 6 ((ESA D_6)) and CFPl〇/ESAT6 (early secreting) translated on the RDs gene. Antigen-6) fusion protein. 5. Individual antibodies and multiple antibodies can be produced as recombinant animals via recombinant proteins or fragments thereof. 6. The present invention prepares single and multiple antibodies of anti-CFP-l, antUESAT-6 and ami-CFP_SAT6. 7. The single and multiple antibodies of the present invention can be used for various immunoassays, including enzyme immunoassay, labor law analysis, cold plague analysis, money-free glue method, rapid immunochromatography and antibodies. Wafers, etc. ' ' 8, the ultimate ideal immunoassay for enzyme development is enzyme immunoassay or rapid immunochromatography analysis, the first-level reaction antibody is adsorbed on the solid, and the color substance or enzyme is combined with another antibody as color detection. For measurement purposes. 9 'The present invention can detect specific antigens secreted by tuberculosis g in body fluids, body fluids including blood, blood, serum, sputum, urine, cerebrospinal fluid, ribs and tissue fluids and other biological fluids. In the following, a preferred embodiment will be further described to enable the reviewing committee to have a more detailed understanding of the invention, but the following is only for explaining the present invention: a preferred embodiment is not intended to be Any limitation on the form of the invention is based on the spirit of the creation of this invention, and is a modification or modification of any form of the invention. [Embodiment] Please cooperate with the participation - as shown in Figure 7, in order to improve the accuracy of tuberculosis detection in recent years 7 1343479 March 29, 100 revised replacement page does not = research to find tuberculosis specific antigen, research report pointed out Mycobacterium tuberculosis is infected with ESAT-6 (early-secreted antigenic target 6-kDa ΡΓ〇 _ egg from 'this protein is secreted egg, and can cause T cell line-line epidemic response, while esat- soil due to p, exist in M Iubercu! 〇sis and disease-causing μ coffee in the M (four) BCG vaccine and many other atypical tuberculosis species do not have this gene, so the inference of the existence of ESAT-6 can distinguish between true tuberculosis and BCG vaccination. To be clearer Knowing the ESAT-6's turnover, there are scholars who analyze the pathogenic MW and M "BCG" and find that they are both (regi〇n 〇f ddeti〇n 1), RD2 and RD3S The genes in the area are different. These coffees are only present in pathogenic mycobacteria. There is no RDs in all BCG, RDs〇per〇n(10) multiple genes, and the clock-6 gene is located in RD1, and trace 6 is found. The promoter of the gene is in addition to the regulatory dish 6 # gene. Control the other -% gene 'this gene can be transferred to another - culture culture filtrate protein 10 (CFP-I (culture flltrate protein)' This protein is also a secreted protein, can cause immune response triggered by T cells. In the present invention The method of gene selection shows ESAT_6 and CFP-10 s proteins, and produces single and double antibodies against these two proteins, and developed various immunoassays, including enzyme immunoassay and immunogel. , rapid immunochromatographic analysis and antibody wafers, etc. These in vitro detection methods can detect the specific antigens of tuberculosis in various body fluids of human body, thereby improving the speed and correctness of the series. The tuberculosis antigen used in the present invention contains Recombinant proteins CFp丨〇, ESAT 6 and CFP10/ESAT_6 fusion protein. The poly-chain reaction of cytokinase (pCR(10)-specific 丨 放大 amplification of tuberculosis / 如 -6 -6 gene. The PCR product was inserted into the pGEM_T vector and Feed in £.cO/i (DH5a), then replace the single colony with /;^ and __6 genes in the medium with antibiotics, and confirm the sequence by gene sequencing to limit the enzyme excision. Because of the joint reaction, the two genes were ligated in the __, and finally the gene after the tuberculosis and the _6 tuberculosis was combined with the expression vector pET29b and sent to the watt co//(BL21DE3). '/Many studies showed tuberculosis The strain 'BCG strain and the Mycobacterium tuberculosis strain in the environment have many identical antigens, and these antigens may be applied to the design of the vaccine, but the monthly b may not be used as an antigen for diagnosis because the father responds, therefore, More and more research is looking for specific antigens of tubercle bacilli, and the currently used tuberculosis purified antigen (ppD) 8 1343479 is replacing the page ~~~- ' ^ ;=Γΐ occurs, so if you want to predict whether the antigen is It is difficult to react with the vaccine or the circadian father. Therefore, it is necessary to look for the diagnosis of tuberculosis. It must have a very high specificity, which is not required for vaccines or circulatory strains. Using Za=as to _qing's biotechnology is better than the nucleus-level antigen, and the soil-dwelling father reaction is better than the pathogenic Mycobacterium tuberculosis and the BCV strain developed in 1925. New Zealand at BCG _ picking is not named (four), leg and Satoshi, therefore, there are many studies that want to use the translated protein as a candidate for vaccine or test diagnostic antigen 1 RD2 can still be transferred out - the name is MPT64 Secreted protein, the protein size is about 32kD, many studies have found that this protein can stimulate T cells and develop delayed allergic reactions (hypersensitivity, DTH) 'Because BCG strain does not have the gene, so use Μτρ64 as The skin test antigen' distinguishes patients who are secreted by tuberculosis and those who receive BCG injection. The Kun-like gene may be present in the following strains: from the heart of the genus / 〇 this H37Rv, like the secret / _ H37Ra ϋ吣 BCG substmns Tokyo, Moreau, Russia, etc.; and the following strains do not have "sand gene: MM bcg subStrains
Glaxo, Pasteur，Canadian, Tice, Danish 1331。但對於原始的 BCG 菌株 (Pasteur)來說’ MPT64是一種非常有效的皮膚測試抗原。 在結核菌的RD1基因群中，ESAT_6是目前特性被了解最多的蛋 白，而在所有的BCG菌株中皆不具有esat_6基因，ESAT_6係為一分泌 性蛋白’經研究發現ESAT-6可以刺激受結核菌感染後的記憶性免疫細 胞，使其釋放出大量的IFN-Y，且ESAT-ό是結核菌中一種主要引起Thl 免疫反應的抗原’在細胞學實驗中發現，ESAT-6可以區分由Μ 祕6腳/〇成或Μ⑽·画所引起的肺部疾病，故ESAT_6極具有作為診 斷結合併之特異性抗原的潛力。 另外，RD1基因群上共有π個基因，除咖卜6基因位於RD卜同 時並發現以加-6基因的啟動子除調控α扣_6基因外還調控另一 /如基 因’此基因可轉譯出另一蛋白CFP-10，此蛋白大小約為10kD，也是一 種分泌性的蛋白。越來越多的研究發現一群屬於ESAT-6家族(ESAT-6、 CFP-10、TB10.4)的蛋白皆具有引發人類免疫反應，刺激大量1|^釋出 9 1343479 100年3月29日修正替換頁 的功能，呵血清職上這些家族成員皆能增加診斷上的特異性因 此本發明選擇早期分泌性抗原6 (ear〗y從⑽如a此獅—6 (ESAT·6 )以及培養濾液蛋白 10 ( culture fiUrated。 prot⑽-IO(CFP-IO)蛋白作為特異抗原模式以描述所研發之試劑在診 斷結核病之新賴性、進步性以及實用性。 本發明抗體之製備主要係包括多株抗體與單株抗體之製備與分 才斤。 卜抗結核菌抗原之多株抗體製備與分析：以結核菌抗原免疫nzw 母兔’第-次免疫以完全性的佐劑混合定量的抗原進行皮下注射之 後美兩週以非完全性劑混合定量的抗原進行肌肉注射，再最後一 次注射後兩週即可大量抽取兔血，已獲取具抗體之兔子金清，並以硫鲁 酸氨沉殿法及透析法使抗體存於PBS中冷;東於_2〇t>a eusa的方法 測定抗體的效價，並與各歡原制抗體讀雜無和性，最後再 將抗體加以定量。 ,2、抗結㈣抗原之單株抗體製備與分析：以結核級錢疫老& 後再取其脾臟’使脾财的淋巴物胞與倾瘤細胞進行細胞融合， 所產生的融合瘤細胞便會分泌紐於培養射，以極限獅法絲合 ‘ 瘤細胞做獅’筛選辨識單一抗原位置的單株抗體，將單一的細胞株 /主射到Balb/e老鼠腹腔，使其產纽水，純水巾即可分_化獲的 单株抗體。以ELISA的方法測定抗體的效價，並與各種抗原侧抗體 之特異性與親和性，錢再毅體加叹量。 冒 當以上述之製備方法以早期分泌性抗原6 (呀❿secreti〇n antlgene-6 (ESAT-6)以及培養濾液蛋白1〇 (culture⑴廿獅 =〇teni-lO(CFP-lO)蛋白作為特異抗原，製造出抗核菌特異性抗原之 早株或多株抗體後，與生物檢體結合形成抗原_抗體複合物，最後再加 ^有標示的該結核義異性抗原之單株❹株抗體透過免疫方法與該 抗原-抗體複合物反應，本發明所使用之免疫方法係為免疫色層分析 法、免疫凝膠法、酵錢疫分析法以及免疫⑼法，兹述如下： 1、免疫色層分析法：免疫色層分析法是一種定性的分析方法， 10 100年3月29日修拙頁 此方法所需的檢體體積很少、只需單一步驟、不需要特殊儀器、非常 的快速且結果十分容易判讀。當檢體滴到分析膜上，檢體中的結核菌 抗原會與接有抗體的有顏色顆粒球做反應，形成抗原抗體複合物，並 藉由毛細管擴散原理將複合物帶到具有二級抗體吸附的固定相，再次 藉由抗原抗體的接合使有色顆粒球在固定相上形成可以判讀的顏色 線。2、免疫凝膠法：免疫凝膠法是一種定性的分析方法，此方法所 需的檢體體積很少、只需單-步驟、不需要特殊賴、非常的快速且 結果十分容易繼。當檢體巾的抗原與表面附著結合_抗體的凝膠發 生反應，由於抗原與抗體的接合或使得微小的凝膠連結在一起，形成 凝集反應’使檢體濁度增加，以吸光度的改變便可得知是否有抗原存 在。 μ 3 '酵素免疫分析法：卿親和性管柱純化獲得兩種對抗結核菌 抗原之抗體’使-種抗體韻在固定相上，加人檢體，檢體中的抗原 會被-級抗體所辨認，因此也會⑽於固定相，接著加人帶有酵素尸 示的二級抗體，二級抗體亦會變是固定相上的抗原而發生接合反應二 最後利麟素特定的受質呈色，以呈色的職進行判讀，此方法^一 級與二級抗體最好來自不同物種所製造，可增加專一性。 4、免疫晶片法：結核g特異性抗體可以特殊技術接合在— 子傳導器上’例如壓電式晶體、液輯導器、聲波共振傳導器、電極、 熱能或光能侧料’當檢體巾的抗原與@定相上的抗體接合 抗體反應造成電子訊號的變動。 μ 歸納上述的·，藉由本判Μ人討以絲正麵糊 出，核病患’並提升檢_正確率。由於本發财有上述眾多的優點 及貫用價值，S此本發明為-創意極佳之發明創作，且在相同的技術 ，域中未見相同或近㈣產品創作或公開制，故本㈣已符合發明 專利的要件，乃依法提出申請。 【圖式簡單說明】 圖-係本發組蛋白腿-6及CFP—1G的製備示意圖。 圖二係本個以西綠驗_巾GFp_1G及ESAT 6的存在之示 1343479 100年3月29曰修正替換頁 意圖。 ^係本發明叫料結合免疫吸附法制 culture filtrate 並愈 PCR及culture結果物比較之表格。 =四係本㈣輯素結合免疫吸附法侧肋驗結果之示意圖。 片法 酵素結合免疫吸附法偵測血清結果之示意圖。 y絲發㈣酵素結合減吸暇_並與 及culture結果迮杆thM之示音圖。 文往朵色抹 圖七係本發明以酵素結合免疫吸附法彳貞測健 【主要原件符號說明】 ' 之血清結 果示意 圖Glaxo, Pasteur, Canadian, Tice, Danish 1331. But for the original BCG strain (Pasteur), MPT64 is a very effective skin test antigen. Among the RD1 gene group of Mycobacterium tuberculosis, ESAT_6 is the most well-known protein, but in all BCG strains, there is no esat_6 gene, and ESAT_6 is a secreted protein. It has been found that ESAT-6 can stimulate tuberculosis. The memory immune cells after infection of the bacteria cause a large amount of IFN-Y to be released, and ESAT-ό is an antigen of the tuberculosis which mainly causes the Th1 immune response. In the cytology experiment, ESAT-6 can be distinguished by Μ Secret 6 feet / 〇 Μ or Μ (10) · The lung disease caused by painting, so ESAT_6 has the potential to be a specific antigen for diagnostic binding. In addition, there are π genes on the RD1 gene group, except that the gab 6 gene is located in the RD and it is found that the promoter of the -6 gene can regulate the other gene, such as the gene, in addition to the regulation of the α-k6 gene. Another protein, CFP-10, which is about 10kD in size, is also a secreted protein. A growing number of studies have found that a group of proteins belonging to the ESAT-6 family (ESAT-6, CFP-10, TB10.4) have a human immune response that stimulates a large number of 1|^ release 9 1343479 March 29, 100 To correct the function of the replacement page, these family members can increase the diagnostic specificity. Therefore, the present invention selects the early secretory antigen 6 (ear y from (10) such as a lion-6 (ESAT·6) and culture filtrate Protein 10 (culture fiUrated. prot(10)-IO(CFP-IO) protein as a specific antigen pattern to describe the new reliance, advancement and practicability of the developed reagents in diagnosing tuberculosis. The preparation of the antibody of the present invention mainly includes multiple antibodies Preparation and analysis of individual antibodies with monoclonal antibodies. Preparation and analysis of multiple antibodies against tuberculosis antigens: immunization with TBw antigens nzw female rabbits' first-immunization with subcutaneous injection of complete adjuvant mixed quantitative antigen After that, the US injected intramuscularly with a non-complete agent mixed antigen for two weeks, and then rabbit blood was collected in two weeks after the last injection. The rabbit with the antibody was obtained, and the method of ammonia sulphate was used. Dialysis The antibody was stored in PBS and cold; the antibody titer was determined by the method of 〇2〇t>a eusa, and the antibody was mismatched with each of the original antibodies, and finally the antibody was quantified. (IV) Preparation and analysis of single antibody of antigen: the spleen is taken after the tuberculosis grade aging & and the spleen cells are fused with the tumor cells, and the resulting fusion tumor cells will secrete Cultivate the shoot, identify the single antibody at the single antigen position with the lion's silk fibroblast 'small cell lion' screening, and shoot a single cell strain/mainly into the abdominal cavity of Balb/e mice to make it fresh water, pure water towel The individual antibodies can be obtained by ELISA. The titer of the antibody is determined by ELISA, and the specificity and affinity with various antigen-side antibodies are added, and the amount of the drug is increased. Early secretory antigen 6 (Ea❿secreti〇n antlgene-6 (ESAT-6) and culture filtrate protein 1〇 (culture(1) 廿 〇=〇teni-lO (CFP-lO) protein as a specific antigen to produce anti-nuclear specificity After the early strain or multiple antibodies of the antigen, combined with the biological sample to form an antigen-antibody And finally, the labeled monoclonal antibody of the tuberculosis sense antigen is reacted with the antigen-antibody complex by an immunological method, and the immunological method used in the present invention is immunochromatography, immunoglobulin. The gel method, the yeast disease analysis method and the immunization (9) method are as follows: 1. Immunochromatographic analysis method: The immunochromatographic analysis method is a qualitative analysis method, and the method is revised on March 29, 10100. The required sample volume is small, requires only a single step, does not require special instruments, is very fast, and the results are very easy to interpret. When the sample is dropped onto the analysis membrane, the tuberculosis antigen in the sample reacts with the colored particle ball with the antibody to form an antigen-antibody complex, and the complex is brought to the secondary antibody by capillary diffusion principle. The adsorbed stationary phase, again by the bonding of the antigenic antibodies, causes the colored particle spheres to form a color line that can be interpreted on the stationary phase. 2. Immunogel method: The immunogel method is a qualitative analysis method. The sample required for this method is small in size, requires only single-step, does not require special treatment, is very fast, and the result is very easy to follow. When the antigen of the sample towel is bound to the surface-adhesive gel, the gel reacts with the antibody or the microgel is joined together to form an agglutination reaction, which increases the turbidity of the sample and changes the absorbance. It can be known whether or not an antigen is present. μ 3 'Enzyme immunoassay: Purification of two affinity antibodies against tuberculosis antigens by affinity chromatography. The antibody is applied to the stationary phase, and the antigen is detected by the antibody. Identification, and therefore (10) in the stationary phase, followed by the addition of a secondary antibody with an enzyme nectar, the secondary antibody will also become the antigen on the stationary phase and the conjugation reaction will occur. In the case of coloring, the method of first- and second-level antibodies is preferably produced by different species, which can increase the specificity. 4. Immune wafer method: tuberculosis g-specific antibodies can be specifically bonded to the sub-conductor 'for example, piezoelectric crystal, liquid sequence guide, acoustic resonance transducer, electrode, thermal energy or light energy side material' The antigen of the towel reacts with the antibody-conjugated antibody on the @phase to cause a change in the electronic signal. μ summarizes the above, and by this judgment, the person is confessed to the front, and the nuclear disease is raised and the rate of correctness is improved. Since the present wealth has the above-mentioned numerous advantages and pervasive value, the present invention is an innovative invention, and the same or near (four) product creation or publicity is not found in the same technology, so this (4) The requirements for the invention patent have been submitted in accordance with the law. [Simplified illustration of the diagram] Figure - Schematic diagram of the preparation of the histone leg-6 and CFP-1G. Figure 2 shows the existence of this western green test _ towel GFp_1G and ESAT 6 1343479 March 29, 100 revised replacement page intention. ^ is a table in which the present invention is combined with immunophoresis to prepare culture filtrate and compare PCR and culture results. = four series of this (four) series of combined with immunosorbent assay side rib test results. Schematic diagram of the detection of serum results by enzymes combined with immunosorbent assay. y silk hair (four) enzyme combined with the reduction of 暇 _ and and the result of the culture of the mast thM. Text to the color of the smear Figure 7 is the invention of the enzyme combined with immunosorbent assay 彳贞 健 [main original symbol description] 'the serum results schematic
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