TW200424524A - Reduction of the hook effect in membrane-based assay devices - Google Patents
Reduction of the hook effect in membrane-based assay devices Download PDFInfo
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- TW200424524A TW200424524A TW092133144A TW92133144A TW200424524A TW 200424524 A TW200424524 A TW 200424524A TW 092133144 A TW092133144 A TW 092133144A TW 92133144 A TW92133144 A TW 92133144A TW 200424524 A TW200424524 A TW 200424524A
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Description
200424524 玖、發明說明: 【發明所屬之技術領域】 各種不同分析程序及裝置通常運用於可流入式檢驗,以測定試驗 樣本中分析物的存在與/或者濃度。例如,免疫分析·免疫系統的機構, 其中抗體對病原或外在有機的的抗原產生感應。這些抗體及抗原(即免疫反 應)可與另一個連結,藉以引起高度明確的反應機構,此可使用於測定特殊 抗原在生物樣本中的存在或濃度。 有數個著名使用標有可探知成分之免疫反應的免疫分析方法,因 此了刀析且出分析物。舉例來說,“爽層類型”(s⑽檢驗一般 牽涉將試驗樣本與可偵測的探針混合,比如染色乳膠或放射性同位素,此 與特足連結構件結合以分析之。結合探針形成具有分析物的合成物。然後 這些合成物抵達一固定抗體地區,此處在抗體及分析物之間發生連結,以 形成三元“夾層合成物”(Sandwichc〇mplexes)。夾層合成物位於偵測分析 物的地區。此技術可使用於獲得定量或半定量結果。此夾層類型檢驗的一 些範例由描述於美國專利編號第4,168,146號,以及Tom等人 的美國專利編號第4,366,241號。 無論如何,許多傳統的“夾層類型,,檢驗方式在受到較高分析濃 度時會遭遇相當錯誤。換句話說,當分析物存於高濃度時,試驗樣本中分 析物的很多部分無法形成具有結合探針的合成物。因此,根據抵達偵測地 區’未合成分析物與連結處的合成分析物競爭。因為未合成分析物不標有 探偵’則無法偵測。因此,假使相當數目的連結處變得被未合成分析物佔 領’檢驗可顯示“假陰性”(false negative)。此問題通常稱為“鉤狀效應” (hook effect) 〇 【先前技術】 已提出減少免疫分析中“鉤狀效應,,的各種不同技術。例如,
Mavis-D:\ 專和J\PkOO 1.08〜\088〇\PK-001 -0880.doc2004/5/20 ^ 200424524 Φ_·#人專利編號第6,184,G42號描述減少婦檢驗中釣狀效 應的技術。技術轉將樣本在_中以能連結至分析物的至少二個感受體 產生ϋ受體為選自抗體的連結分子之齊聚體⑽职時第二感受㈣ 連結或可連結至-固態。所謂的可溶寡聚抗體可減少“鉤狀效應- 捭論如何,仍需存有在簡單、有效率及較便宜方式中減少‘ 效應”的改善技術。 【發明内容】 本發明的一實施例,揭發試驗樣本中為了侧分析物存在或 數量的可流入式檢驗裝置。可流入式檢驗裝置包含多孔薄膜,此與能產生 偵測信·結合_探針職。多孔_定義為多雜孔洞_粒内固定 的分層賴。齡_財梅之_乡_,糊具有大於微孔 洞平均尺寸的平均尺寸。在一些實施例中,微孔洞的平均尺寸至少約為 25〇/。此比工間的平均尺寸小。微孔洞顆粒可選自由聚苯乙婦、聚酿 胺、聚丙 1珠及其組合敝成,且其表鱗分浦可被學純。 多孔薄膜進步定義為位於分層地區下方的偵測地區。捕捉劑固 疋於侧地區内,«錢結至結合侧探針。#在_地區⑽,結合 可產生_信號’其中試驗樣本内的分析物數量可由該侧信號 置卿4=Γ的另—實施例,揭發試驗樣本中以流人式夾層檢驗裝 二° _裝置包含與可產生偵測信號之結合偵測 析物二使:rS ’結合偵測探針構成可與試驗樣本中的分 4,使的成分聽/_合纽未合如 多數微孔赚層地區。構成微編維,使得== 以比分析物/探針合成物缓慢的速率流經分層地區。微孔洞薄膜進-步包含
Mavis-m 專称細〇8 〜_〇vp議獅加2〇〇4/5/2〇 200424524 位於分層地區下方的偵測地區。捕捉劑固定於偵測地區内,此構成連奸至 分析物/探偵合成物,因此合成物在偵測地區内時產生偵測信號,其中試驗 樣本内的分析物數量由偵測信號測定。 揭發試驗樣本中分析物的存在或數量之偵測方法。方法包含: i) 提供包含多孔薄膜的可流入式檢驗裝置,多孔薄膜與 可產生偵測信號的結合偵測探針聯繫,多孔薄膜定義 為在微孔洞顆粒内固定的分層地區,且偵測地區位於 分層地區下方,其中捕捉劑固定於偵測地區内。 ii) 將含有分析物的試驗樣本與結合偵測探針接觸,因此 形成分析物/探針合成及未合成分析物;以及 iii) 允许分析物/探針合成及未合成分析物抵達分層地區 及之後偵測地區,其中分析物/探彳貞合成物在未合成分 析物之前抵達偵測地區。 本發明的其他特性及觀點將更詳述探討於下。 【實施方式】 定義 此處所使用“分析物”(analyte)一詞意謂一般表示偵測的物質。 例如,分析物可包括抗原物質、半抗原、抗體及其組合。分析物包括(但不 受限)毒素、有機化合物、蛋白質、縮氨酸、微生物、氨基酸、核酸、賀爾 蒙、類固醇、維他命、藥劑(包括那些執行治療目的以及執行不法目的)、 藥劑媒介物或副產品、細菌、病毒粒子及代謝物或任何上面物質的抗體。 一些分析物的特定範例包括血清蛋白鐵、肌氨酸酐肌腾MIB(CK-MB)、毛 地只(digoxin)、抗癲癇藥(phenyt〇in)、phen〇barbitol、卡巴氮平 (carbamazepine)、萬古黴素、慶大黴素、茶鹼、帝拔癲(valpr〇ic add)、異 金雞納驗、黃體化荷爾蒙(leutinizinghormone) (LH)、促卵泡激素(FSH)、
Mavis-D:\ 專利\PkOO 1 ·08~\0880\ΡΚ-001 -0880. doc2004/5/20 7 200424524
強力求偶素、黃體脂酮、C-反應蛋白、lipocalins、IgE抗體、維他命B2微 球蛋白、糖化血色素(glycated hemoglobin)(Gly· Hb)、腎上腺皮質素 (cortisol)、洋地黃毒素、N-acetylprocainamide(NAPA)、procainamide、德國 麻瘆的抗體(比如德國麻療-IgG及德國痲療IgM)、住血原病蟲的抗體(比如 住血原病蟲IgG(毒素-IgG)及住血原病蟲IgM(毒素-IgM))、睪丸激素、水揚 酸鹽、乙醯基氨基苯(acetaminophen)、B型肝炎病毒表面抗原(HbsAg)、B型 肝炎核心抗體(hepatitis B core antigen)(比如B型肝炎核心抗體IgG及 IgM)(Anti-HBC)、人類免疫缺陷病毒1及2(HIV 1及2)、人類T細胞血癌 病毒 1 及 2(HTLV)、乙型肝炎 e 抗原(hepatitis B e antigen)(HBeAg)、乙型 肝炎e抗原的抗體(Anti-HBe)、促甲狀腺激素(TSH)、甲狀腺素(T4)、總三 碘甲狀腺素(Total Τ3)、活性態三碘甲狀腺素(Free Τ3)、carcinoembryoic抗 原(CEA)以及α胎兒蛋白(AFP)。濫用及控制物質的藥劑包括(但不受限)安 非他命、甲基安非他命、巴必妥酸鹽(比如戊巴必妥、西可巴必妥、戊巴必 妥、豕巴必妥以及巴必妥)、非巴比妥類(比如1化rium及valium)、大麻驗(比 如印度大麻葉及大麻煙)、古柯鹼、吩坦尼、LSD、甲酮、鎮靜劑(比如海 洛英、嗎钟、可待因、一氫嗎啡嗣、氫可酮、美沙酮、可待因酮、幾二氫 嗎啡酮及鴉片)、環烴六胺及pr〇p〇xyhene。其他可能分析物可描述於 的美國專利編號第號以及Tom等人的專利編 號第 4,366,241 號。 此處所使用試驗樣本”(test sample)—詞一般意謂疑有分析物 的材料。試驗#本可直接獲自來源或下面預先處理方式,以變更樣本的特 質。試驗樣本可衍生於任何生物來源,比如生理流體,此包括血液、組織 液垂液眼睛水θ日體成體、腦脊體液(cerebral Spinal也如)、汗水、尿液、 乳汁、腹水、娜OUS、關節液、腹膜液、陰道流體、羊水等等。試驗樣本 可在使用前事先處理,比如自血液準備血漿、稀_液等等。處理方法可
Mavis-D:\ 專和J\PkOO 丨.08〜\〇880\PJC-001 -0880, d〇c2004/5/20 200424524 牽涉過濾、沉澱、稀釋、蒸餾、濃縮、減除阻礙成分的活性及加入試劑。 除了生理流體外,可使用其他液體樣本,比如水、食物產品及環境或食物 製造裝置作業的相似物。另外,疑有分析物的固體材料可使用作為試驗樣 本。在一些實例中,有益於變更固體試驗樣本,以形成液體媒介物或分離 分析物。 詳述 目前將參照詳述發明的各種不同實施例,下面發表一或更多範 例。經由發明說明提供每個範例,對發明並無限制。事實上,顯然本發明 播須達反發明範圍或精神可而由精通技藝做各種不同變更及變動。例如, 了在另實施例使用說明或描述部分實施例的特性,以產生更進一步實施 例。因此,本發明意圖在附加申請專利範圍及同等物内涵蓋此變更及變動。 -般而言,本發明為針對侧試驗樣本巾分析物存在或數量的薄 膜裝置。汉備利用多數微孔洞顆粒上的分層地區。分層地區可在簡 單、有效及概宜对忖效減少“姗賴”。尤其,乡雜孔洞顆粒 允許較大尺寸的分析物/探針合成物在未合成分析物之前抵達侧地區。因 為未合成分析物大致不能與侧顧巾魏處的合成減爭可限制“假 陰性”發生,即使在較高的分析物濃度。 51用弟一圖,例如 ,可根據本發明形成的可流入式檢驗裝置(20) 目前將做更詳細的描述。如所示,裝綱含有可任意由硬性材刪支撐 勺多孔薄膜(23)般而δ ’多孔薄膜(23)可由任何各種不同材料經由能通 過的試驗樣本製造。_錢,仙卿❹孔雜(23)的冊可包括# =限)天然、合成或自然發生合成變更的材料,比如_(例如纖維素 ’比如紙及纖維物生物,比如纖維素醋酸鹽及硝化纖維素)、聚 薄ΓΓΓ無機材科(比如無_礬土、讀土、或 /、夕孔水口基貝中的—律無機纖細分割材料,以及比如氯化埽、氯化
Mavis-DA 專称 Pk001.08〜\0880\PK-001-0880.doc2004/5/20 9 200424524 烯-丙晞共聚物及氯化♦醋酸乙埽共聚物的聚合物)、天然發生(例如棉花) 及合成(例如尼龍或人造絲)的布料、多孔凝膠(比如碎凝膠、洋菜膠、右旋 醣及骨膠)、聚合雜(比如聚合憐_)等等。在一特膽施例中,多孔 薄膜(23)由硝化纖維素與/或者聚醋續胺材料形成。需了解此項‘‘硝化纖維 素”⑽賴她se)麵纖維素的硝,旨,此可單獨為硝化纖維素,或酷類 與硝酸及其他義合,比如具有〗至7個碳原子的肺驗酸。 裝置(20)也可含有-芯吸襯塾(28)。芯吸觀塾(28)一般引入流體, 此流體移往整個多孔薄膜(23)。如著名技藝,芯吸襯塾⑽可幫助促進毛細 管作用及流體流經薄膜(23)。 欲著手偵測試驗樣本内的分析物,使用者可直接經由移動而將試 驗樣本運用於多孔、薄膜(23)。或者,試驗樣本首先可運用於取樣襯塾(無圖 π) ’此與多孔薄膜(23)有流體聯繫。一些可使用於形成取樣襯塾的適當材 料包括(但不受限)硝化纖維素、纖維素、多孔聚乙烯襯塾及玻璃纖維過遽 紙。假使理想的話,抽樣鱗也可含有—或更多檢驗預先處理試劑,此不 是擴散性就是無擴散性附著。 在圖解的實施例中,試驗樣本從取樣襯塾(無圖示)移至結合槪塾 ⑽’此與取樣襯塾-端聯繫。結合襯塾(22)由能通過試驗樣本的材料形 成。舉例«,在-實施财,結合錄可由玻顧_成。雖然僅顯示 一結合襯墊,需了解其他結合襯墊也可使用於本發明中。 為了促進準確_試驗樣本内是否有分析物存在,可在裝置(2〇) 的各種不同位置運用探針。隨更詳述於下,探針可使用於侧分析物及校 準。-般可產生錢的任何物質可依視覺铜,或藉儀器裝政用作為探 針。各種不同適當物質可包括色元、催化個、螢光化合物、化合光化合 物、磷光化合物、放射性化合物、直視標籤(包括膠質金屬(例如金)及非金 屬顆粒、染料顆粒、酵素或基質或有機聚合乳膠顆粒)、微脂粒或其他含有
Mavis-D:\^^|j\Pk〇〇1.08*-\0880\PK-001-0880.d〇c2004/5/20 j 〇 200424524 ㈣產生物質的倾等等。·,適合使用作膽針的_些酵素由仏薩 等人描述於美國專利編號第4,275,M9號,其為了所有目的而完全合併於 此作為參考。酵素/基免系統的範例為酵素驗性活性鱗酸酿臃及基質硝藍四 峻溴 4 亂引心鱗酸(ni仕〇 blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate)或衍生物或其相似物,或基質本methylumbemf㈣磷酸。其他 適當探針可由描述於美國專利編號第5,670,381號以及Tarcha等 Λ描述於美國專利編號第5,252,459號,其為了所有目的而完全合併於此 作為參考。
在一些實施例中,探真可含有製造偵測信號的螢光化合物。螢光 化合物可為螢光分子、聚合物、樹狀分子、粒子等等。例如,適當螢光分 子的些範例包括(但不受限)榮光素、螯化錄、藻膽蛋白、 嗇薇紅染料及衍生物及相似物。可視覺偵測的有色化合物也可使用作為探 針,藉以提供直接色彩讀出樣本中分析物的存在或濃度,此無須進一步信 號產生劑。
碳針(比如上面所述)可單獨使用或與微顆粒(有時稱為“珠,,或 小珠”)結合使用。例如,可使用自然發生的微顆粒,比如細胞核、黴漿 菌、質粒、質體、哺乳動物細胞(例如紅血球複製影像)、單細胞微生物(例 如細菌)、多醣類(例如洋菜)等等。進一步,也可利用合成的微顆粒。舉例 來說,在一實施例中,可利用標有螢光或有色染料的乳膠微顆粒。雖然本 發明可使用乳膠微顆粒,乳膠微顆粒一般由聚苯乙烯、丁二烯苯乙缔、丁 一缔丙締-乙婦二聚物、聚甲基丙稀酸丁脂(polymethylmethacrylate)、聚 曱基丙缔酸乙酯(polyethylmethacrylate)、苯乙晞-順丁晞二肝共聚物、聚乙 締基醋酸鹽、聚乙烯呲啶、聚二乙烯苯、聚丁烯對苯二甲酸酯、丙稀骑、 氯化晞-丙稀酸醋等等或乙酸、致基、氨基、氫氧基或其井化物衍生物。其 他式當微顆粒描述於Jou等人的美國專利編號第5,670,381號及Tarcha筈
Mavis-I>\^ilJ\PkO〇 1,08~\0880\PK-001 -0880.doc200475/20 11 200424524 么的美國專利編號第5,252,459號,其為了所有目的而完全合併於此作為 參考。適當螢光顆粒的一些商業上可用範例包括螢光羧化微粒體,此售自 商標名 FluoSphere”(Red 580/605)及 “TransfluoSphere”(543/620)以及
Texas Red下的分子探針有限公司(Molecular Probes,Inc.),且 6-carboxytetramethylrhodamine 也售自 Molecular Probes,Inc·。適當有色乳 膠微顆粒的商業上可用範例包括羧化乳膠珠,此售自Bang's Laborator, Inc.。
在一些實例中,理想的是在一些方式中變更探針,使能夠更輕易 連結至分析物。在此實例中,探針可由某特定連結構件變更,此附著形成 結合探針。特定連結構件一般適用於特定連結組的構件,即二個不同分 子,此處其中一分子化學與/或者物理連結至第二分子。例如,免疫反應特 定連結構件可包括抗原、半抗原、適合體、抗體及其合成物,此包括由重 組DNA方法或縮氨酸合成。抗體可為單株或多株抗體、重組蛋白質或混 合物或其片斷以及抗體與其他特定連結構件的混合物。此抗體及適合使用 作為特定連結構件的製備細節為眾所周知的技藝。其他共同特定連結組包 括(但不受限)維生素Η及抗生物性蛋白、碳水化合物及凝集素(lectin)、互 補核甘酸順序(包括探針及使用於DNA雜交檢驗的捕捉核酸順序,以偵測 目標核酸順序)、互補縮氨酸順序(包括由重組方法形成)、作用器及感受 體、賀爾蒙及贺爾蒙連結蛋白、酵素補助因子及酵素、酵素抑制劑及酵素 等等。再者,特定連結組可包括最初特定連結構件相似物的構件。舉例來 說,只要抗原決定素與分析物相同,可使用分析物的衍生物或片斷(即相似 分析物)。 特定連結構件一般可使用任何各種著名技術附著至探針。例如, 特定連結構件至探針(例如微顆粒)的共價附著可使用羧基、氨基、乙醛、 澳化乙酿基、琪化乙醯基、硫醇、環氧基及其他反應或連結官能基以及剩
Mavis-D:\||[ilJ\Pk001 〇8~\0880\PK-001 -0880. doc2004/5/20 12 200424524 餘自由基及自由基陽離子完成,可經由蛋白f連結反應完成。表面官能基 也可隨官能共同單體合併,因為微顆粒的表面可含有極性基的較高表面濃 度。另外,雖然微顆粒探針常常在合成後官能化嶂例來說,引用第二圖, 說明本發明共價結合—探針的施例。如圖示,結合的第—步驟為使用 碳二亞胺(c—ide)在探針表面上活化絲。在第二步驟中活化幾 酸基與抗體的氨基反應,以軸—氨統合物鍵#性鑛/或者抗體連結 可在緩衝财發生,比如猶贿魅(PBS)(㈣pH72)或轉乙基績酸 (順)(例如pH 5.3)。如圖示,例如’然後結果探針可用乙醇胺阻撐,以形 成探針、(^· 了共知键結外’其他附著技術(比如物理吸附)也可利用於 本發明中。 如上所示,在試驗樣本中的一些分析物無法變成在理想方式中合 成探針,尤其當分析物在高濃度下存於試驗樣本中。此未合成分析物可稍 後與捉捕劑之合成分析物在偵測地區⑼(如下描述)競爭,藉以對檢驗裝置 (20)的精確性有反效果。欲抵制此效果,多孔薄膜(23)含有一分層地區 (35),此分布多數微孔洞顆粒。如第三圖至第五圖所示,微孔洞顆粒⑽ 的存在允許分層地區(35)充當“參透,,圓柱,使較大分子以比小分子快速 的速率移經分層地區(35)。換句話說,如第五圖所示,比微孔洞顆粒(5〇) 的微孔(51)有較大尺寸的分子無法流經,因此強制流經顆粒(5〇)之間的空 間,即經過薄膜(23)的孔洞(以方向箭頭L2表示因為顆粒(5〇)的微孔(51) 在微粒狀結構内形成彎曲路徑”(tortuous paths)(即具有合成形狀的路 徑),一般分子行經微孔(51)比經過顆粒(50)之間的空間花費較長時間。因 此’當行經分層地區(35)時,較大尺寸的分子首先退出。中間尺寸的分子 穿過微孔洞顆粒(50),以依照本身尺寸變化範圍。最後,非常小的分子流 經顆粒(50)的微孔(51)(以方向箭頭Li表示),因此最後退出分層地區(35)。 一般而言,分析物/探針合成物比未合成的分析物有較大尺寸。因此,合成
Mavis-DA 專利\Pk001.08〜\0880\PK-001-0880.doc2004/5/20 13 200424524 物可抵達偵測地區(31),並在未合成分析物抵達偵測地區(31)前連結至捕捉 劑。在此方式中,阻止合成及未合成分析物的競爭。 分層地區(35)—般提供單一個別區域(例如直線等等),雖然本發 明必疋考慮多數區域。例如,在圖解的實施例中,利用單線。當利用時, 一般可改變線的寬度。舉例來說,在一些實施例中,分析物流動方向乙中 的線寬約為1〇%至100%,且在一些實施例中約為1〇%至5〇%的總距離, 此距離自分析物位置(例如結合襯墊(22))至偵測地區(31)。進一步,線配置 的方向大致與試驗樣本流經裝置(2〇)垂直。同樣地,在一些實施例中,線 的方向大至於試驗樣本流經裝置(2〇)平行。 使用於給予檢驗中的選擇適當微孔洞顆粒(50)之標準可包括各種 因素’比如感興趣的分析物特質、試驗狀況、利用探針特質等等。一般理 想的是微孔洞顆粒(50)具有較相同的孔洞及顆粒尺寸分布及良好機械與化 學穩疋性。另外,一般同樣理想為微孔洞顆粒(5〇)的表面仍需使化學鈍氣 至檢驗裝置(20)的其他成分。例如,微孔洞顆粒(5〇)的表面一般為關於分析 物的化學鈍氣。可使用於本發明的微孔洞顆粒(5〇)之一些範例包括(但不受 限)合成聚合顆粒,比如聚苯乙烯(例如高度交鍵的聚苯乙烯)、聚丙烯醯 胺、聚丙烯腈、矽石珠等等。描述一些適當微孔洞顆粒(5〇)的特定範例, 例如^i〇y的美國專利編號第4,110,529號、Ley等人的美國專利編號第 4,940,734號以及g2Qke蔓么的美國專利編號第5,314,923號,其為了所有 目的而完全合併於鱗為參考。擁針也為雜讀㈣#施例中,需了 解分層地區(35)的微孔洞顆粒(5〇)可與探針相同。 理想中’一般可變化微孔洞顆粒(50)的平均直徑。舉例來說,再 -些實施例巾’顆粒的平均纽範圍可約為Q1至丨,麵微米在一些實 施例中可約為0.1至1〇〇微米,且在一些實施例中約為〗至1〇微米。一般 顆粒(5〇)大致為球形(即珠狀),雖然其他形狀(包括(但不受限)盤狀、桿狀、
Mavis-D:\$ilJ\Pk00l.08~\0880\PK-001-0880.doc2004/5/20 200424524 棒子、不規卿狀等等)適合使本發明中。隨著由那些熟知技藝了解, 可廣泛變化顆粒(50)的合成物、形狀、尺寸與/或者密度。 一般而吕,顆粒(5〇)的微孔(M)具有比顆粒⑽之間的空間(此由 多孔薄膜(23)的孔洞(52)形成)小的平均尺寸(即直徑)。換句話說,微孔⑼ 的平均尺寸-般至少約比空間之_平均尺寸小则%,在—些實施例中 至少约為150%,且在-些實施例中至少約為25〇%。在一些實施例中,例 如,微孔(51)具有約小於100 nm(十億分之一公尺)的平均尺寸的平均尺 寸,在-些實施例中約為5至100nm,且在一些實施例中約為1〇至⑼啦。 隨比較下,多孔薄膜(23)的孔洞(52)-般平均尺寸約大於2〇〇 nm ,在一些 實施例中約為勘至漏腕,且在一些實施例中約為綱至MOO細。一 檢驗裝置⑽也可含有-侧輕⑼,此峡可義至結合探 針的捕捉劑。舉例來說,在-些實施例中,捕捉劑可為生物捕捉劑。齡 物捕捉劑為眾所周知的技藝,並可包括(但不受限)抗原、半抗原、抗體、 蛋白A或G、抗生物性蛋白、抗生蛋白鏈菌素、第二抗體及其合成物。在 許多情形巾,理想的紐些越敝财職藉針上的特歧結構件(例 如抗體)。另外,也可理想將各種不同非生物材料利用於捕捉劑。例如,在 一些實施例中,捕捉劑可包括多電解質。多電解質可具有靜正或負電荷, 且靜電荷-般為中性。例如,具有靜正電荷的多電解質之―些適當範例包 括(但不受限)聚二氨基己酸(商業上獲自密蘇里州路易斯街的 Sigma-AWrich化學公司)、聚次乙亞胺、&環氧_氯_丙垸_官能聚胺與/或者 聚氨基胺(比如聚二甲胺·共_α_環氧4丙垸)、己二晞乙垸遺化銨、陽離 子纖、、隹素衍生物(比如纖維素共聚物或與季铵水溶性單體接枝的纖維素衍 生物)等等。在一特別實施例中,可利用CelQu_ Sea·或Η_ι〇〇(獲自 _c>nal Starch & Chemieal,Ine·) ’此為含有季銨水雜單體_維素衍生 物再者’具有靜負電荷的多電解質之一些適當範例包括(但不受限)聚丙
Mavis-D:\^Wk001.08~\0880\PK-001-0880.doc2004/5/20 15 200424524 烯酸,比如聚乙埽.共-甲基丙稀酸、麵鹽等等。也須了解也可利用其他多 電解質’比如兩性多電解質(即具有極性及非極性部分)。例如,適當兩性 多電解質的-些範例包括(但不受限)聚(苯乙缔基协甲基_2乙缔基雄化 吡旬及聯㈣基切猶),二麵自岭大_摩咖打 Inc. 〇 捕捉劑可作為分析物/探針合成物的固定連結處。換句話說,分析 物(比如抗體、抗原等等)一般具有二個連結處。根據抵達獅也區⑼,其 中-連結處被結合探珍的特定連結構件佔領。無論如何分析物的未固定 連結處可連結纠定_湖。姆麟纠定概劑合雜針形成新 的二兀央層合成物。 偵測地區(31)-般可提供許多個別偵測區域因此使用者更可測 足試驗樣本_特殊精物濃度。侧_可含有姻敝劑,或可含有 不同捕鋪〃敝多數分析物。舉例來說,侧地區⑼可包括二或更 多個別偵趣域(例如線、畴等)1漸域可配置於大致與試驗樣本流 經檢驗裝置(20)為垂直方向中所形成的線。同樣地在一些實施例中偵 測區域可配置於大致與試驗樣本流經檢驗裝置㈣為平行方向中所形成的 線。 雖然偵測地區(31)可表示分析物的存在,此常常困於使用單獨偵 測地區⑼測定試驗樣本内的分析物相對濃度。因此,檢驗裝置⑽也可包 括;^準地區㈤。在此實施例中,在多孔薄膜㈣上形成校準地區㈤, 並位於鮮地區⑼的下方。解地_)提供可連結至任侧下未捕捉探 、十勺捕u此可通過薄膜㈣的長度。利用於校準地區㈤的捕獲劑可與 偵測地區⑼使_捕捉劑_或不同。再者,與侧地_)相似的是; 枚準地區(32)也可在任何方向中提供許多侧校準區域,因此使用者更可 測定試驗樣本⑽特殊分析物濃度。每健域可含有相同捕捉劑 ,或可含
Mavis-D:\ 專利\PkOO 1 ·〇8~\0880\ΡΚ-001 -0880.doc2004/5/20 16 200424524 有不同捕捉劑,以捕捉不同探針。 校準區域可事先裝於具有不同捕捉劑數量的多孔薄膜(Μ)上,因 此由每個鮮區域根據探針働而產生不同信號強度。每鍊準區域内連 結劑的所有數量可利用不同尺寸的校準區域變化,與/或者改變每個校準區 域中捕捉季的濃度或容量。假伽想的話,過乡的探針可運麟檢驗裝置 (2〇)中,因此每個校準區域達到本身充滿及預先測定信號強度的可能性。 亦即,因為運用於校準區域的捕捉劑數量設定為預先測定及已知高度,則 校準區域的探針數量可事先測定。 一般而言,各種可流入式檢驗裝置可根據本發明構成。在此方 面,本發明的各種不同實施例目前將做更進一步的詳述。無論如何,需了 解下面所探討的實施例僅為示範,且本發明也可考慮其他實施例。例如, 引用第二圖至第四圖,顯示使用於偵測的探針(41)及使用於校準的探針(43) 之特別實施例。在此實施例中,偵測探針(Μ)及校準探針(43)運用於結合襯 塾(22) ’因此在與試驗樣本聯繫時,可流經裝置(2〇)(以方向箭頭[表示)。 根據與分析物A接觸,偵測探針(41)與分析物A的特定連結構件(9〇)結合, 探針(41)連結形成分析物/探針合成物(49)。 如第三圖所示,探針/分析物合成物(49)、任何未固定分析物a及 校準探針(43)可自結合襯墊(22)流經多孔薄膜(23)直到配置多數微孔洞顆 粒(50)的分層地區(35)。較大合成物(49)及校準探針(43)可迅速流經顆粒(5〇) 之間的空間(52),同時較小未合成分析物A在顆粒(50)的微孔内以較緩慢 的速率流動。然後分析物/探針合成物(49)流經裝置(2〇)直到抵達偵測地區 (31),此處連結至捕捉劑(91),比如抗體,以形成夾層合成物(53)。進一步, 杈準探針(43)流至校準地區(32),並連結至一捕捉劑(無圖示),比如多電解 質。之後,如第四圖所示,未合成分析物A行經分層地區(35),以抵達偵 測地區(31)。因為合成物(49)已經連結至捕捉劑,無論如何,分析物a移
Mavis-D:\i|[^IJ\PkO〇l ,08~\0880\PK-001 -0880.doc2004/5/20 17 200424524 經偵測地區(31)及校準地區(32),直到抵達芯吸襯墊(28)。因此,在偵測地 區(31)中,分析物的數量可由偵測探針(41)的信號強度確定。>胺使理想的 話,此信號強度可由校準探針(43)在校準地區(32)中的信號強度校準。信號 強度可用視覺或經由裝置(比如螢光閱讀機)幫助測量。 雖然上面已描述裝置外形的各種不同實施例,需了解本發明的裝 置一般理想中可具有任何形狀,且不需含有上面所述的全部構件。例如, 各種不同其他裝置外形與/或者檢驗方式描述於Lambotte等人的美國專利 編號第5,395,754號;J0U等人的美國專利編號第5,67〇,381號;以及Malick 等人的美國專利編號第6,194,220號,其為了所有目的而完全合併於此作 為參考。 本發明已發現檢驗裝置的多孔薄膜上存有的分層地區可在簡 單、有效率且較便宜方式中能有效減少“鉤狀效應”。尤其,多數微孔洞 顆粒可配置於分層地區上,以允許較大尺寸的分析物/探針合成物在任何未 合成分析物之前達成偵測。因此,未合成分析物不與合成物為了偵測地區 可利用的連結處競爭。因為未合成的分析物不能佔領偵測地區中相當數目 的連結處,可限制“假陰性,,的發生率,即使在較高分析物濃度中。 儘管發明已用特定實施例描述,將了解根據達成對前文的了解而 使精通技藝可輕易想像對這些實施例有所變更、變動及同等物。因此,本 發明的範圍可由附加申請專利範圍及任何同等物評估。 【圖式簡單說明】 本發明朝向一般精通技藝的全部及授權揭發(包括其最佳模式)尤 其更發表於剩下的說明書中,並參照附圖,如下·· 第一圖為本發明可流入式檢驗裝置的一實施例之立體圖; 第二圖為將一抗體共價結合至羧化奈米粒子的一實施例之圖解 實例;
Mavis-D:\^IJ\Pk001.08~\0880\PK-00l-0880.doc2004/5/20 200424524 第三圖為本發明可流入式檢驗裝置的一實施例之圖解實例,此顯 示未合成分析物行經分層地區; 第四圖為第三圖的圖解實例,此顯示未合成分折物行經分層地區; 第五圖微第-圖分層地區的分解圖,且圖不意圖表示發明的相同 或類似特性或要素。 元件簡單說明】 _ _xv, 20 flow-through assay device p-------------- 町流入式檢驗裝置 -------------- _ 21 rigid material 硬性材料 -------------- 22 conjugate pad 一 結合襯墊 23 -__ porous membrane 多孔薄膜 28 ---- wicking pad 芯吸襯整 31 "—__ detection zone 偵測地區 32 -^^__ calibration zone 校準地區 35 ----- chromatographic zone 分層地區 41 -— detection probe 偵測探針 43 calibration probe 校準探針 49 -~~~~一 analyte/probe complexes 分析物/探針合成物 50 ---- microporous particle 微孔洞顆粒 ^^51 micropore 微孔 52 -— space 空間 53 ~^- sandwich complexes 夾層合成物 91 capture reagent 捕捉劑
Mavis-D: \^[^IJ\PkOO 1.08~\0880\PK-〇〇1 -0880. doc2004/5/20
Claims (1)
- 拾、申請專利範圍·· h種偵測試驗樣本中分析物存在或數量的可流入式檢驗裝置,該可流 入式檢驗裝置包含多孔舰,該纽賊與能產生_信號的結合偵 測探針聯繫,該多孔薄膜定義為: 一分層地區,此固定多數微孔洞顆粒内;以及 一偵測地區,其位於該分層地區下方,其中捕捉劑固定於該偵測 地區内,此構成連結至該結合偵測探針,其中該結合偵測探針能在該 偵測地區内產生一偵測信號,其中試驗樣本内的分析物數量可由該偵 測信號測定。 2·如申請專利範圍第丨項的可流入式檢驗裝置,其中該微孔顆粒定義為 多數空間,該空間的平均尺寸比該顆粒的微孔平均尺寸大。 3·如申請專利範圍第1項的可流入式檢驗裝置,其中該微孔的平均尺寸 至少100%小於該空間的平均尺寸。 4·如申請專利範圍第1項的可流入式檢驗裝置,其中該微孔的平均尺寸 至少150%小於該空間的平均尺寸。 5·如申請專利範園第1項的可流入式檢驗裝置,其中該微孔的平均尺寸 至少250%小於該空間的平均尺寸。 6·如申請專利範圍第1項的可流入式檢驗裝置,其中該微孔的平均尺寸 小於100 nm。 7·如申請專利範圍第1項的可流入式檢驗裝置,其中該微孔的平均尺寸 為 10 至 60 nm。 8·如申請專利範圍第1項的可流入式檢驗裝置,其中微孔洞顆粒選自由 聚苯乙烯、聚丙烯醯胺、聚丙晞腈、矽石珠及其組合所組成。 9.如申請專利範圍第1項的可流入式檢驗裝置,其中該微孔洞顆粒的表 面對分析物可為化學鈍氣。 Mavis'DA^Wk001.08~\0880\PK-001-0880.d〇c2004/5/20 20 10. 10. 11. 12. 13. 14. 15. 16. 如申請專利範圍第1項的可流入式檢驗裝置,其中該結合偵測探針包 含選自由色元、催化作用、螢光化合物、化合光化合物、磷光化合物、 放射性化合物、直視標籤、微脂粒及其組合。 如申请專利範圍第1項的可流入式檢驗裝置,其中該多孔薄膜進一步 包含可產生一校準信號的校準地區,其中試驗樣本内的分析物數量可 隨校準信號校準而由偵測信號測定。 如申睛專利範圍第11項的可流入式檢驗裝置,其中該多孔薄膜與校準 探針聯繫,該校準探針在存於校準地區時產生該校準信號。 如申請專利範圍第1項的可流入式檢驗裝置,其中裝置為夾層類型的 檢驗裝置。 一種偵測試驗樣本中分析物存在或數量的可流入式夾層檢驗裝置,該 檢驗裝置包含一多孔薄膜,該多孔薄膜與能產生债測信號的結合偵測 探針聯繫,當接觸時,該結合檢測探針構成與試驗樣本中的分析物結 合,使得形成分析物/探針的合成及未合成分析物,該多孔薄膜定義 為: 一分層地區,此固定多數微孔洞顆粒内,該微孔洞顆粒構成該未 合成分析物以比該分析物/探針合成物緩慢的速率流經分層地區;以及 一偵測地區,其位於該分層地區下方,其中捕捉劑固定於該偵測 地區内,此構成連結至該分析物/探針合成物,因此該合成物能在該偵 測地區内產生一偵測信號,其中試驗樣本内的分析物數量可由該偵測 信號測定。 如申請專利範圍第14項的可流入式夾層檢驗裝置,其中該多孔顆粒定 義為之間的多數空間,該空間的平均尺寸大於該顆粒微孔的平均尺寸。 如申請專利範圍第14項的可流入式夾層檢驗裝置,其中該多孔的平均 尺寸至少100%小於該空間的平均尺寸。 Mavis-D:\^^IJ\Pk001 〇8~\0880\PK-001 -0880.doc2004/5/20 21 200424524 17·如申請專利範圍第14項的可流入式夾層檢驗裝置,其中該多孔的平均 尺寸至少150%小於該空間的平均尺寸。 18·如申請專利範圍第14項的可流入式夾層檢驗裝置,其中該多孔的平均 尺寸至少250%小於該空間的平均尺寸。 19·如申請專利範圍第14項的可流入式夾層檢驗裝置,其中該微孔洞顆粒 選自由聚苯乙烯、聚丙埽醯胺、聚丙稀腈、碎石珠及其組合所組成。 2〇·如申請專利範圍第14項的可流入式夾層檢驗裝置,其中該微孔洞顆粒 的表面對分析物可為化學鈍氣。21·如申請專利範圍第14項的可流入式夾層檢驗裝置,其中該多孔薄膜進 一步包含可產生校準信號的校準地區,其中試驗樣本内的分析物數量 可隨該校準信號校準的偵測信號測定。 22·如申請專利範圍第21項的可流入式夾層檢驗裝置,其中該多孔薄膜與 校準探針聯繫,該校準探針在該校準地區内時產生該校準信號。 23· —種偵測試驗樣本中分析物存在或數量的方法,該方法包含: 0 提供包含多孔薄膜的可流入式檢驗裝置,該多孔薄膜與可產生偵測信號的結合偵測探針聯繫,多孔薄膜定義為在微孔洞顆粒 内固定的分層地區,且偵測地區位於該分層地區下方,其中捕 捉劑固定於偵測地區内; 吣 將含有分析物的試驗樣本與結合偵測探針接觸,因此形成分析 物/探針合成及未合成分析物;以及 出)允許該分析物/探針合成及未合成分析物抵達該分層地區及之後 該偵測地區,其中該分析物/探偵合成物在該未合成分析物之前 抵達該偵測地區。 24·如申請專利範圍第23項的方法,其中該微孔洞顆粒定義為之間多數空 間,該空間的平均尺寸比該顆粒微孔的平均尺寸大。 Mavis-D:\$f IJVPkoo χ ,〇8~\〇880\PK-001-0880.doc2004/5/20 22 25·如申請專利範圍第23項的方法,其中該微孔的平均尺寸至少100%小 於該空間的平均尺寸。 26·如申請專利範圍第23項的方法,其中該微孔的平均尺寸至少150%小 於該空間的平均尺寸。 27·如申請專利範圍第23項的方法,其中該微孔的平均尺寸至少250%小 於該空間的平均尺寸。 28·如申請專利範圍第23項的方法,其中該微孔洞顆粒選自由聚苯乙烯、 聚丙埽醯胺、聚丙缔腈、矽石珠及其組合所組成。 29·如申請專利範圍第23項的方法,其中該微孔洞顆粒的表面對分析物可 為化學鈍氣。 30·如申請專利範圍第23項的方法,進一步包含測量該偵測地區内產生的 偵測信號強度。 31·如申請專利範圍第23項的方法,其中該多孔薄膜進一步包含能產生校 準信號的校準地區,其中試驗樣本内的分析物數量隨該校準信號校準 的偵測信號測定。 32·如申請專利範圍第31項的方法,其中該多孔薄膜與校準探針聯繫,該 校準探針在該校準地區内時產生該校準信號。 33·如申請專利範圍第32項的方法,進一步包含藉標出偵測信號強度而產 生〜校準曲線,對多數預定分析物濃度而言,此曲線由校準信號強度 校準。 Mavis-D:\專利J\Pk001.08〜\0880\PK-0CH-0880.doc2004/5/20 23
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-
2002
- 2002-12-19 US US10/325,614 patent/US7247500B2/en not_active Expired - Fee Related
-
2003
- 2003-10-29 AT AT03783087T patent/ATE408833T1/de not_active IP Right Cessation
- 2003-10-29 AU AU2003290552A patent/AU2003290552A1/en not_active Abandoned
- 2003-10-29 KR KR1020057009930A patent/KR101072756B1/ko active IP Right Grant
- 2003-10-29 MX MXPA05005951A patent/MXPA05005951A/es active IP Right Grant
- 2003-10-29 CN CNB2003801052766A patent/CN100476437C/zh not_active Expired - Fee Related
- 2003-10-29 WO PCT/US2003/034543 patent/WO2004061454A1/en not_active Application Discontinuation
- 2003-10-29 DE DE60323672T patent/DE60323672D1/de not_active Expired - Lifetime
- 2003-10-29 CA CA2508774A patent/CA2508774C/en not_active Expired - Fee Related
- 2003-10-29 ES ES03783087T patent/ES2312837T3/es not_active Expired - Lifetime
- 2003-10-29 EP EP03783087A patent/EP1573326B1/en not_active Expired - Lifetime
- 2003-11-26 TW TW092133144A patent/TW200424524A/zh unknown
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Also Published As
Publication number | Publication date |
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KR101072756B1 (ko) | 2011-10-11 |
EP1573326A1 (en) | 2005-09-14 |
CN1720455A (zh) | 2006-01-11 |
EP1573326B1 (en) | 2008-09-17 |
DE60323672D1 (de) | 2008-10-30 |
ATE408833T1 (de) | 2008-10-15 |
CA2508774A1 (en) | 2004-07-22 |
US20080014659A1 (en) | 2008-01-17 |
US20040121480A1 (en) | 2004-06-24 |
MXPA05005951A (es) | 2005-08-18 |
US7247500B2 (en) | 2007-07-24 |
WO2004061454A1 (en) | 2004-07-22 |
CN100476437C (zh) | 2009-04-08 |
US7662643B2 (en) | 2010-02-16 |
ES2312837T3 (es) | 2009-03-01 |
KR20050085270A (ko) | 2005-08-29 |
AU2003290552A1 (en) | 2004-07-29 |
CA2508774C (en) | 2011-07-05 |
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