201013186 九、發明說明： •【發明所屬之技術領域】 •本發明是有關於一種檢驗試片，特別是指一種能快速 且有效地檢測出人體血清中是否含有蛇毒的蛇毒快速檢驗 試片。 【先前技術】 毒蛇咬傷是熱帶及亞熱帶地區常見的醫療問題之一， 台灣地處亞熱帶，氣候溫暖潮濕，多高山丘陵且森林茂密， 〇 地形與氣候上的特徵使台灣這麼小的島嶼上就有四十多種的 陸生蛇類，其中，更有六種含有劇毒的毒蛇對住民有危及生 命的威脅，分別是眼鏡蛇（Α/α/α 、雨傘節（Swwgijrws multicinctus)、氧蔽 H (Protobothrops mucrosquamatus) ' 赤 尾，舍合{Trimeresurus stejnegeri)、百步蛇{Deinagkistrodon acutus) ' ^ ^^{Daboia rwMe/z·/ 。雖然這六種毒 蛇分屬於不同科及屬，但被咬傷後的臨床表徵卻多所重疊。 假如病人是在突然之間或天色昏暗的情況下被蛇咬傷，往往 © 會因為沒有清楚看到、或無法抓到咬人的蛇而無法指認正確 的毒蛇種類，若只根據臨床表徵，例如：咬痕、局部傷口外 ，觀及患者的主觀描述來判別蛇種，易造成錯誤判斷，而錯失 治療的黃金時機。為了作出正確的診斷，通常要依賴實驗室 的檢驗結果。 雖然利用目前已開發的三明治式ELISA方法可以較準 確地檢測並確認病人檢體中的眼鏡蛇、龜殼花、赤尾鲐或鎖 鏈蛇蛇毒，但實際上此種檢測方法仍存有下列缺失： 201013186 應用三明治式ELISA方法，需要花費5個小時左右的 檢驗時間，才能鑑別出咬傷的毒蛇種類’並評估被毒蛇咬傷 病人的臨床嚴重度、預後及血清治療效果。此外，進行 ELISA時的實驗步驟繁瑣，配合檢驗用的儀器不容易普及 於各實驗室，因此，還要訓練可操作儀器的專業技術人員， 使現有的蛇毒檢驗方法具有檢驗程序繁瑣而時間耗時、需要 使用特定的儀器且須由受過專業訓練的人員操作的缺點，加 上無法快速得知結果，使臨床醫療人員無法即時作出正確的 診斷而有延誤治療時機及誤診的風險。 【發明内容】 因此，本發明的目的，是在提供一種利用免疫分析檢 驗技術及層析原理，而具有高專一性並能節省檢驗時間， 以快速且有效地確認血清檢體中的蛇毒的蛇毒快速檢驗試 片。 於是，本發明蛇毒快速檢驗試片是用以檢測一生物檢 體中的特定蛇毒，包含一試卡本體，及一設置於該試卡本 體的植入單元。 該試卡本體包括相對應接合的一基底層及一載體層， 該載體層具有相間隔設置的一檢體墊片、一吸收墊片、一 與該檢體墊片相鄰接的金標結合墊片，及一連接在該金標 結合墊片與該吸收墊片之間的多孔薄膜，在該多孔薄膜上 並形成相間隔設置的一測試線區及一控制線區。 該植入單元包括多數個分布在該試卡本鱧的金標結合 墊片内的抗蛇毒金標抗體、多數個固定在該試卡本體的多 201013186 ?匕薄骐的控制線區，用以與該等金標抗體相結合呈色的第 抗體，及多數個固定在該多孔薄膜的測試線區，用以與 結合有蛇毒的金標抗體相結合呈色的第二抗體。 本發明的有益效果在於：當將該生物檢體滴加至該試 卡本體的載體層#檢體墊片上時，該檢體會藉由毛細作用 移動至該墊體金墊片，並與該墊體金墊片内的抗蛇毒金標 抗體相互作用，再繼續沿該多孔薄膜移動至該測試線區與 該控制線區，以分別與該等第二、第一抗體相作用，就能 ® 分別藉由判讀該控制線區是否呈色判讀該檢驗試片是否有 效，及藉由判讀該測試線區是否呈色判讀該檢體是否含有 蛇毒藉此，使本發明成快速且有效地檢驗出生物檢體中 是否含有特定的蛇毒，以即時進行治療。 【實施方式】 本發明蛇毒快速檢驗試片的前述以及其他技術内容、 特點與功效，在以下配合參考圖式的一較佳實施例的詳細 說明中，將可清楚地明白。 ® 參閱圖1、圖3與圖5,本發明蛇毒快速檢驗試片2的 較佳實施例是用以檢測一生物檢體10中的特定蛇毒1〇〇， ，在該實施例中是以該檢驗試片2偵測該生物檢體1〇中的眼 鏡蛇（iVq/α aira)蛇毒，該檢驗試片2包含一試卡本體3、— 設置於該試卡本體3的植入單元4,及一圍繞該試卡本體3 設置的卡匡5。 參閱圖1與圖2,該試卡本體3包括相對應接合的一基 底層（Backing)31及一載體層32’該載體層32具有相間隔 7 201013186 設置的一檢體塾片（Sample pad)321、一吸收墊片（Absorbent pad)322、一與該檢體墊片321相鄰接的金標結合墊片 (Conjugated release pad)323，及一連接在該金標結合塾片 323與該吸收墊片322之間的多孔薄膜324，在該多孔薄膜 324上並形成相間隔設置的一測試線區325及一控制線區 326。 其中，該檢體墊片321、吸收墊片322及金標結合墊片 323皆是由玻璃纖維材質所製成。且該多孔薄膜324是屬於 石肖化纖維膜（cellulose nitrate membrane，簡稱為NC膜）。 參閱圖4與圖5,該植入單元4包括多數個分布在該試 卡本體3的金標結合墊片323内的抗蛇毒金標抗體41、多 數個固定在該試卡本體3的多孔薄膜324的控制線區326， 用以與該等金標抗體41相結合呈色的第一抗體42,及多數 個固定在該多孔薄膜324的測試線區325，用以與結合有蛇 毒100的金標抗體41相結合呈色的第二抗體43。 其中，該等抗蛇毒金標抗體41實質上為兔抗眼鏡蛇蛇 毒（Rabbit anti-cobra IgG)的金標抗體，且是使用多數個兔抗 眼鏡蛇毒的抗體411分別與一膠體金溶液中的多數個金顆粒 412相結合而製成。該等第一抗體42實質上為羊抗兔抗體 (Goat anti-rabbit IgG)，及該等第二抗體43實質上為鴨抗眼 鏡蛇蛇毒抗體（Duck anti-cobra IgY)。 參閱圖3，該卡匣5具有一包覆在該試卡本體3外的殼 體51、一對應該載體層32的檢體墊片321貫穿形成於該殼 體51的加樣孔52，及一對應該多孔薄膜324的控制線區 201013186 326與測試線區325貫穿形成於該殼體51的判讀視窗53。 在該實施例中，還在該判讀視窗53旁的殼體51外表面分 別設置對齊該測試線區325、控制線區326的一第_標呓 511及一第二標記512，以分別透過該第一、第二標記5ιι 、512快速地判讀呈色反應是發生在該測試線區325或該控 制線區326。在此是以T表示該第一標記5 u，並以c表示 該第二標記512。 參閱圖3、圖4與圖5 ’檢驗時，是將所取得的生物檢 體10透過該加樣孔52滴加至該檢體墊片321，該檢體1〇 會流動通過該金標結合墊片323，並沿著該多孔薄膜324流 動，流動過程中，檢體10内若含有眼鏡蛇蛇毒1〇〇(見圖5) ，則該金標結合墊片323中的金標抗體41將追蹤並結合檢 體10中屬於眼鏡蛇蛇毒蛋白的特異性分子，並形成金二抗 體-眼鏡蛇蛇毒蛋白的結合體41〇，再藉由固定於該測試線 區325的第二抗體43與該結合體41〇進行結合後呈色的機 制，判別檢體10中是否含有眼鏡蛇蛇毒，進而能達到檢驗 鑑定的功效。其中，當該生物檢體1〇〇中的蛇毒濃度大於 等於5ng/ml時，就能利用發生在該測試線區3乃且容易為 肉眼所觀察到的呈色反應判別該生物檢體1〇〇内含有眼鏡 蛇蛇毒。 <檢驗試片的製備> (1)抗體 該等金標抗體41中的兔抗眼鏡蛇蛇毒的抗體是取自抗 台灣眼鏡蛇蛇毒之多株兔血清。血清中的多株抗體再使用 201013186201013186 IX. Description of the invention: • [Technical field to which the invention pertains] The present invention relates to a test strip, and more particularly to a test strip for rapid detection of snake venom which can quickly and effectively detect the presence or absence of snake venom in human serum. [Prior Art] Snake bite is one of the common medical problems in tropical and subtropical regions. Taiwan is located in the subtropical zone, with warm and humid climate, many alpine hills and dense forests. The terrain and climate characteristics make it such a small island in Taiwan. More than forty species of terrestrial snakes, including six highly virulent snakes, pose a life-threatening threat to the inhabitants: cobra (Α/α/α, umbrella festival (Swwgijrws multicinctus), oxygen mask H (Protobothrops mucrosquamatus) ) 'Red tail, rounded {Trimeresurus stejnegeri), hundred steps snake {Deinagkistrodon acutus) ' ^ ^^{Daboia rwMe/z·/ . Although these six species of snakes belong to different families and genera, the clinical signs after being bitten overlap. If the patient is bitten by a snake suddenly or in the darkness of the day, often © will not be able to identify the correct snake species because they do not clearly see or can not catch the biting snake, if only based on clinical characterization, such as: In addition to bite marks, local wounds, and subjective descriptions of patients to identify snake species, it is easy to cause misjudgment, and miss the golden opportunity of treatment. In order to make a correct diagnosis, it is usually dependent on the laboratory test results. Although the currently developed sandwich ELISA method can more accurately detect and confirm the cobra, turtle flower, red-tailed hawksbill or chain snake venom in the patient's specimen, in fact, the following methods still exist in this test method: 201013186 Application The sandwich ELISA method takes about 5 hours to verify the bite of the snake species' and evaluates the clinical severity, prognosis and serum treatment of the snake bite. In addition, the experimental procedures for performing ELISA are cumbersome, and the instruments for testing are not easily popularized in various laboratories. Therefore, it is necessary to train professional technicians who can operate the instruments, so that the existing snake venom test method has complicated inspection procedures and time consuming. The disadvantages of using a specific instrument and being operated by trained personnel, coupled with the inability to quickly learn the results, make it impossible for clinical staff to make a correct diagnosis immediately and to delay the timing of treatment and the risk of misdiagnosis. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a snake venom which is highly specific and saves inspection time by using immunoassay inspection technology and chromatographic principle to quickly and efficiently confirm snake venom in serum samples. Quickly test the test piece. Thus, the snake venom rapid test strip of the present invention is for detecting a specific snake venom in a biological specimen, comprising a test card body, and an implant unit disposed on the test card body. The test card body includes a base layer and a carrier layer which are correspondingly joined, and the carrier layer has a sample spacer spaced apart from each other, an absorbing pad, and a gold mark adjacent to the sample pad. a spacer, and a porous film connected between the gold bond pad and the absorbing pad, and forming a test line area and a control line area spaced apart from each other on the porous film. The implant unit comprises a plurality of anti-venom gold-labeled antibodies distributed in the gold-labeled gasket of the test card, and a plurality of control lines of the multi-201013186 thin crucible fixed on the test card body. A first antibody that binds to the gold-labeled antibody, and a plurality of second antibodies that are immobilized in the test line region of the porous membrane for binding to a gold-labeled antibody that binds to snake venom. The beneficial effects of the present invention are: when the biological sample is dropped onto the carrier layer of the test card body, the sample is moved to the pad gold gasket by capillary action, and The anti-venom gold-labeled antibody in the pad gold gasket interacts and continues to move along the porous film to the test line region and the control line region to interact with the second and first antibodies, respectively. Determining whether the test strip is valid by interpreting whether the control line area is colored, and determining whether the sample contains snake venom by interpreting whether the test line area is colored or not, thereby enabling the present invention to quickly and effectively check out Whether the biopsy contains specific snake venom for immediate treatment. [Embodiment] The foregoing and other technical contents, features and effects of the present invention are described in detail in the following detailed description of a preferred embodiment of the accompanying drawings. ® Referring to Figures 1, 3 and 5, a preferred embodiment of the snake venom rapid test strip 2 of the present invention is for detecting a specific snake venom in a biological specimen 10, in this embodiment The test strip 2 detects the cobra (iVq/α aira) snake venom in the biopsy, and the test strip 2 includes a test card body 3, an implant unit 4 disposed on the test card body 3, and A cassette 5 disposed around the test card body 3. Referring to FIG. 1 and FIG. 2, the test card body 3 includes a corresponding backing 31 and a carrier layer 32'. The carrier layer 32 has a sample pad disposed at intervals of 7 201013186. 321 , an absorption pad 322 , a conjugated release pad 323 adjacent to the sample pad 321 , and a connection between the gold standard bond pad 323 and the absorption A porous film 324 between the spacers 322 is formed on the porous film 324 and forms a test line region 325 and a control line region 326 spaced apart from each other. The sample spacer 321, the absorbing pad 322 and the gold bonding pad 323 are all made of fiberglass material. The porous film 324 is a cellulose nitrate membrane (abbreviated as NC membrane). Referring to FIG. 4 and FIG. 5, the implant unit 4 includes a plurality of anti-venom gold standard antibodies 41 distributed in the gold standard bonding pads 323 of the test card body 3, and a plurality of porous films fixed on the test card body 3. a control line region 326 of 324, a first antibody 42 for coloring in combination with the gold standard antibody 41, and a plurality of test line regions 325 fixed to the porous film 324 for gold combined with venom 100 The labeled antibody 41 binds to the colored secondary antibody 43. The anti-venom gold-labeled antibody 41 is substantially a gold-labeled antibody against rabbit anti-cobra IgG, and is a majority of a rabbit anti-cobra venom antibody 411 and a colloidal gold solution. The gold particles 412 are combined to form. The first antibody 42 is substantially a Goat anti-rabbit IgG, and the second antibody 43 is substantially a duck anti-cobra IgY antibody. Referring to FIG. 3, the cassette 5 has a housing 51 covering the outside of the test card body 3, and a pair of sample pads 321 for the carrier layer 32 are inserted through the sample insertion hole 52 formed in the housing 51, and A pair of control line regions 201013186 326 and test line regions 325 that should be porous film 324 are formed through the interpretation window 53 of the housing 51. In this embodiment, an outer surface of the housing 51 adjacent to the reading window 53 is respectively provided with a first _ mark 511 and a second mark 512 aligned with the test line area 325 and the control line area 326 to respectively transmit the The first and second indicia 5, 512, 512 quickly interpret the coloring reaction to occur in the test line region 325 or the control line region 326. Here, the first mark 5 u is denoted by T, and the second mark 512 is denoted by c. Referring to FIG. 3, FIG. 4 and FIG. 5', the obtained biological sample 10 is dropped into the sample pad 321 through the sample hole 52, and the sample 1〇 flows through the gold mark. The spacer 323 flows along the porous film 324. If the sample 10 contains cobra venom 1 (see FIG. 5) during the flow, the gold standard antibody 41 in the gold label combined with the spacer 323 will be tracked. And combining the specific molecule belonging to the cobra venom protein in the sample 10, and forming a combination of the gold di-antibody-cobra venom protein 41 〇, and the second antibody 43 and the binding body 41 fixed to the test line region 325 The mechanism of coloration after combining is used to discriminate whether the sample 10 contains cobra venom, thereby achieving the test and identification effect. Wherein, when the concentration of the snake venom in the biological specimen is greater than or equal to 5 ng/ml, the biological specimen can be discriminated by the coloring reaction occurring in the test line region 3 and easily observed by the naked eye. The cockroach contains cobra venom. <Preparation of test strips> (1) Antibody The antibody against rabbit anti-cobra venom in the gold-labeled antibody 41 was obtained from a plurality of rabbit sera resistant to Taiwanese cobra venom. Multiple antibodies in serum are reused 201013186
Protein A Sepharose 4B(Zymed Laboratoriess, Inc·, USA)親合 性管柱純化，透析隔夜之後調整濃度至lmg/ml供製備結合 有抗蛇毒金標抗體41的膠體金墊片323使用。 固定在該測試線區325的第二抗體43是使用鴨抗眼鏡 蛇蛇毒抗體（Duck anti-cobra IgY)，且是鴨多株抗體，固定 在該控制線區326的第一抗艎42則是使用抗兔子抗體之羊 多株抗禮（Jackson ImmunoResearch Laboratories, Inc., USA) (2) 膜與墊片 該多孔薄膜324使用之硝化纖維膜為AE98 (Whatman Θ pic.，England)，該金標結合塾片 323 是 grade 6613(Ahlstrom Corp.，Finland)的玻璃纖維墊片、該檢體墊片321是grade 8964 (Ahlstrom Corp·，Finland)的玻璃纖維塾片，該吸收墊 片 322 則是 grade 205 (Whatman pic.，England)的玻璃纖維 墊片。將該檢體墊片321、金標結合墊片323、多孔薄膜 324及吸收墊片322依圖2所示的配置排列並固定至塑膠材 質的基底層31上，該基底層31為GL-187(G&L Precision Die Cutting Inc.，USA)的塑膠材質所製成。 β (3) 含抗蛇毒金標抗體的膠體金溶液的製備 以氣化金以及檸檬酸鈉製備該膠體金溶液（Frens， ， 1973)，並以光譜波長訂出其直徑為30nm。所配製出的膠體 金溶液再以0.1M碳酸緩衝液（pH9.6)調整pH至7.0。將兔 抗眼鏡蛇蛇毒的兔子抗體（2 mg)加入已調整pH的膠體金溶 液中並溫和攪拌一小時’以和溶液中的金顆粒（lrnl，〇.D.=3) 相結合形成金標抗體41。之後以i%bsA 20 mM的borate 10 201013186 buffer pH 8進行阻斷，並以39000g離心30分鐘。離心後 重新回溶到含2%蔗糖以及1%BSA的PBS緩衝液中，金濃 度在540nm下約為O.D. =40，將該膠體金溶液保存在 〇 (4)結合該試卡本體3與該植入單元4製備一平流層析套組 參閱圖3與圖5，分別將1 mg/ ml的第一抗體（鴨多株 抗體）42、0.5 mg/ml的第二抗體（羊多株抗體）43，以1从i /cm之流速噴到已經固定在該基底層η的多孔薄膜 (AE98)324的測試線區325、控制線區326上。該金標結合 塾片323而先浸泡在含有〇_ι μ Broax buffer (pH8.2)， 0.25% Triton χ-ioo，0.1% pvp_4〇的緩衝溶液中，再乾燥過 夜。之後將已含有該等金標抗體41的膠體金溶液以1 /cm之流速噴灑到經過前處理的金標結合墊片323上面。然 後在溫度37。（：乾燥4小時。該植入單元4中的該等抗體41 、42、43分別對應地植入該試卡主體3的載體層32後，將 該試卡主體3裁切成〇.4mm寬度的細條，並放入塑膝材質 的卡匣5中，就製得該蛇毒快速檢驗試片2成品。該檢驗 試片2成品可儲存在鋁箔袋中並放入乾燥劑密封保存以延 長保存期限。該試片2在室溫中可保存一年。 <使用方法> 2拆封後平放於桌面上，以吸Protein A Sepharose 4B (Zymed Laboratoriess, Inc., USA) was affinity column purified, and the concentration was adjusted to 1 mg/ml after overnight dialysis for preparation of a colloidal gold spacer 323 incorporating anti-venom gold antibody 41. The second antibody 43 immobilized in the test line region 325 is a duck anti-cobra IgY antibody and is a duck polyclonal antibody, and the first antibody 42 immobilized in the control line region 326 is used. Anti-rabbit antibody sheep multi-drug resistance (Jackson ImmunoResearch Laboratories, Inc., USA) (2) Membrane and pad The nitrocellulose membrane used in the porous film 324 is AE98 (Whatman Θ pic., England), the gold standard combination The slab 323 is a fiberglass gasket of grade 6613 (Ahlstrom Corp., Finland), the specimen spacer 321 is a glass fiber raft of grade 8964 (Ahlstrom Corp., Finland), and the absorbing gasket 322 is grade 205. (Whatman pic., England) glass fiber gasket. The sample spacer 321 , the gold label bonding pad 323 , the porous film 324 , and the absorbing pad 322 are arranged and fixed to the base layer 31 of the plastic material according to the arrangement shown in FIG. 2 , and the base layer 31 is GL-187. (G&L Precision Die Cutting Inc., USA) made of plastic material. Preparation of colloidal gold solution containing β (3) anti-venom gold-labeled antibody The colloidal gold solution (Frens, 1973) was prepared by gasification of gold and sodium citrate, and its diameter was 30 nm at a spectral wavelength. The prepared colloidal gold solution was adjusted to pH 7.0 with 0.1 M carbonate buffer (pH 9.6). The rabbit anti-cobra venom rabbit antibody (2 mg) was added to the pH-adjusted colloidal gold solution and gently stirred for one hour to form a gold-labeled antibody in combination with gold particles (lrnl, 〇.D.=3) in the solution. 41. This was followed by blocking with i% bsA 20 mM borate 10 201013186 buffer pH 8 and centrifugation at 39,000 g for 30 minutes. After centrifugation, it was re-dissolved into PBS buffer containing 2% sucrose and 1% BSA, and the gold concentration was about OD=40 at 540 nm. The colloidal gold solution was stored in 〇(4) in combination with the test card body 3 and Implantation unit 4 prepares an advection chromatography kit. Referring to Fig. 3 and Fig. 5, 1 mg/ml of the first antibody (duck polyclonal antibody) 42 and 0.5 mg/ml of the second antibody (the sheep polyclonal antibody) 43 is sprayed at a flow rate of i/cm from the test line region 325 and the control line region 326 of the porous film (AE98) 324 which has been fixed to the base layer η. The gold label was combined with the septum 323 and first immersed in a buffer solution containing 〇_ι μ Broax buffer (pH 8.2), 0.25% Triton χ-ioo, 0.1% pvp_4 ,, and dried overnight. Thereafter, the colloidal gold solution containing the gold-labeled antibody 41 was sprayed onto the pretreated gold-labeled spacer 323 at a flow rate of 1 /cm. Then at temperature 37. (: drying for 4 hours. After the antibodies 41, 42, and 43 in the implant unit 4 are respectively implanted into the carrier layer 32 of the test card main body 3, the test card main body 3 is cut into a width of 4 mm. The thin strips are placed in a plastic knee-shaped cassette 5 to prepare the finished product of the snake venom rapid test strip 2. The finished test strip 2 can be stored in an aluminum foil pouch and placed in a desiccant sealed storage to extend the shelf life. The test piece 2 can be stored for one year at room temperature. <Usage method> 2 After unpacking, lay it flat on the table to suck
利用肉眼觀察並判讀與該判讀視窗53 將該蛇毒快速檢驗試片 管吸取檢體1 〇，並透過該4 321 ’靜待20分鐘後，就能 見窗53相對齊的測試線區 11 201013186 325與控制線區326。 <結果判讀> 該檢體ίο内的液體會促使該金標結合墊片323中的金 標抗體41藉毛細作用沿該多孔薄膜324肖前移動，若該檢 體10中未含有眼鏡蛇蛇毒100，則該等金標抗體41會與該 控制線區326中的第-抗體結合42發生特異性結合而被截 留，及聚集在該控制線區326上，進而形成肉眼可觀察到 的色帶。若該檢體10中含有眼鏡蛇蛇毒，則該蛇毒蛋白會 與部分金標抗體41進行特異性結合形成金標抗體_眼鏡蛇蛇〇 毒蛋白的結合體410，該等結合體41〇與部分未結合有蛇毒 蛋白的金標抗體41會同時因毛細作用沿該多孔薄膜324向 前移動，移動過程中，該等未結合蛇毒蛋白的金標抗體41 會與該控制線區326的第一抗體42特異性結合而顯示出色 帶，而該等結合體410則會與該測試線區325的第二抗體 U發生特異性結合而被截留，並呈現出另一條色帶，因此 ’可透過該判讀視窗53看到二條色帶。 (1) 陽性反應（Positive):如圖6-1所示，當測試結果出 © 現分別對齊該第一、第二標記511、512的二條色帶時，表 示此檢體10(見圖3)中艮鏡蛇蛇毒蛋白。 (2) 陰)生反應（Negative).如圖6-2所示，當測試好果僅 在對齊該第二標記512的控制線區326出現色帶，而該測 D式線區325未出現色帶時，表示此檢體1〇(見圖3)中不含眼 鏡蛇蛇毒蛋白。 (3) 無反應（Invalid):如圖6-3、圖6-4所示，如果測試 12 201013186 結果未出現任何色帶或是該控制線區326未出現色帶，表 ' 示該檢驗試片2已失效、過期，或操作不當，需另做一次 ^ 測試。 <偵測極限試驗> 分別配製不含眼鏡蛇蛇毒的檢體a，及蛇毒濃度分別為 • 5ng/ml、l〇ng/ml、50ng/ml、l〇〇ng/ml 的檢體 b、c、d、e， 共5組檢體，並準備5個本發明的蛇毒快速檢驗試片2，將 a、b、c、d、e檢體分別取3滴滴入該檢驗試片2的檢體墊 ® 片321 ’靜待20分鐘觀察該判讀視窗53中的呈色結果。結 果如圖7所示，當該檢體未含眼鏡蛇蛇毒時，只有該控制 線區326出現色帶，當蛇毒濃度為5ng/ml時，該控制線區 326與該測試線區325皆出現能夠以肉眼觀察到的色帶，且 隨著蛇毒濃度增加，呈現在該測試線區325的色帶顏色也 有加深的趨勢，顯示該蛇毒快速檢驗試片2可偵測的蛇毒 濃度至少為5ng/ml，及本發明的檢驗試片確實可偵測檢體 中是否含有眼鏡蛇蛇毒。 ® <與台灣其他常見毒蛇的蛇毒蛋白的交互作用試驗> 分別調配下列檢體：⑴未含任何蛇毒的正常血清、（η) 含雨傘節蛇毒的血清、（出）百步蛇蛇毒的灰清、（iv)含龜殼 花蛇毒的血清、（v)含赤尾餘蛇毒的血清及⑽含鎖鏈蛇蛇毒 的血清，其中編號（iiHvi)的血清檢體中的蛇毒濃度皆為 雜咖丨，並㈣6財發㈣蛇毒快速檢賴# 2，將編 號⑴〜㈣的檢體分別取3滴滴入該等檢驗試片2的檢體塾 片32卜靜待20分鐘後，所得到的檢驗結果如圖8所示， 13 201013186 根據圖8的結果得知，當檢體中含有非眼鏡蛇的蛇毒時， 則只有該控制線區326出現肉眼可見的色帶’該測試線區 325則未出現色帶，顯示本發明檢驗試劑不會與台灣其他五 種常見毒蛇的蛇毒蛋白產生交互作用，因此，能夠根據檢 驗結果準確地判別檢體中是否含有眼鏡蛇蛇毒。 歸納上述，本發明蛇毒快速檢驗試片2可獲致下述的 功效及優點’故能達到本發明的目的： 一、由於本發明快速檢驗試片2可在2〇分鐘内得知蛇 咬傷病患是否為眼鏡蛇咬傷，且偵測極限達5ng/m卜因此◎ 月b節省許多等待檢驗結果的時間，而且使用本檢驗試片進 行檢驗時’無需專業人士執行，也不需操作使用特殊儀器 ，使本發明容易操作且能快速取得檢驗結果，而具有實用 性高的優點。 二、以本發明檢驗試片2與台灣其他五種常見毒蛇的 蛇毒檢體進行交互仙，結果顯示該檢驗試片2與這五種Using the naked eye to observe and interpret and read the window 53 to take the sample from the snake venom rapid test strip, and wait for 20 minutes through the 4 321 ', then the window 53 can be seen to be aligned with the test line area 11 201013186 325 and Control line area 326. <Result Interpretation> The liquid in the sample ίο causes the gold label antibody 41 in the gold label binding pad 323 to move forward along the porous film 324 by capillary action, if the sample 10 does not contain the cobra venom 100, the gold-labeled antibody 41 is specifically bound to the first-antibody binding 42 in the control line region 326, and is trapped on the control line region 326 to form a visually observable ribbon. . If the sample 10 contains cobra venom, the snake venom protein will specifically bind to a part of the gold standard antibody 41 to form a combination 410 of the gold standard antibody _ cobra snake venom protein, and the combination 41 〇 is partially unbound. The gold-labeled antibody 41 having a snake venom protein moves forward along the porous membrane 324 by capillary action. During the movement, the gold-labeled antibody 41 which does not bind the snake venom protein is specific to the first antibody 42 of the control line region 326. Sexually bound to show an excellent band, and the combination 410 will be specifically bound to the second antibody U of the test line region 325 to be trapped and present another ribbon, so 'through the interpretation window 53 I saw two ribbons. (1) Positive (Positive): As shown in Figure 6-1, when the test results are respectively aligned with the two ribbons of the first and second markers 511, 512, the specimen 10 is shown (see Figure 3). ) 艮 艮 蛇 snake venom protein. (2) Negative reaction. As shown in Fig. 6-2, when the test results, only the color band appears in the control line region 326 aligned with the second mark 512, and the D line region 325 does not appear. In the case of the ribbon, it indicates that the specimen 1 〇 (see Fig. 3) does not contain the cobra venom protein. (3) Invalid: As shown in Figure 6-3 and Figure 6-4, if the test 12 201013186 results in no ribbon or the control line area 326 does not appear in the ribbon, the table ' shows the test Slice 2 has expired, expired, or is not properly operated. You need to do another ^ test. <Detection limit test> A sample a containing no cobra venom, and a sample having a snake venom concentration of 5 ng/ml, l〇ng/ml, 50 ng/ml, l〇〇ng/ml, respectively, c, d, e, a total of 5 groups of samples, and prepared 5 snake venom rapid test strips 2 of the present invention, 3 drops of a, b, c, d, e specimens respectively into the test strip 2 The sample pad 321 'waits for 20 minutes to observe the coloring result in the interpretation window 53. As a result, as shown in FIG. 7, when the specimen does not contain cobra venom, only the control line region 326 has a color band. When the snake venom concentration is 5 ng/ml, both the control line region 326 and the test line region 325 can appear. The color band observed by the naked eye, and as the concentration of the snake venom increases, the color of the ribbon present in the test line area 325 also has a tendency to deepen, indicating that the venom rapid test strip 2 can detect a snake venom concentration of at least 5 ng/ml. And the test strip of the present invention can indeed detect whether the sample contains cobra venom. ® <Interaction test with snake venom proteins of other common snakes in Taiwan> The following samples were prepared separately: (1) normal serum without any snake venom, (η) serum containing venom of snake venom, and venom of venom Ash, (iv) serum containing turtle venom, (v) serum containing red snake venom and (10) serum containing venom of snake venom, wherein the venom concentration in the serum of the number (iiHvi) is miscellaneous And (4) 6 Caifa (4) Snake Venom Rapid Detection Lai #2, take the number (1)~(4) of the specimens and take 3 drops into the specimens of the test strips 2, wait for 20 minutes, and then obtain the test. The results are shown in Fig. 8. 13 201013186 According to the results of Fig. 8, when the specimen contains non-cobra venom, only the control line region 326 has a visible color band. The test line region 325 does not appear. The ribbon shows that the test reagent of the present invention does not interact with the snake venom proteins of five other common snakes in Taiwan, and therefore, it is possible to accurately discriminate whether the sample contains cobra venom according to the test results. In summary, the snake venom rapid test strip 2 of the present invention can achieve the following effects and advantages, so that the object of the present invention can be achieved: 1. Since the rapid test strip 2 of the present invention can be known to be a snake bite patient within 2 minutes Whether it is a cobra bite, and the detection limit is 5ng/m. Therefore, ◎ month b saves a lot of time waiting for the test result, and when using this test piece for inspection, 'no need for professional execution, no need to operate using special instruments, so The invention is easy to operate and can quickly obtain test results, and has the advantage of high practicability. 2. The test piece 2 of the present invention interacts with the snake venom samples of five other common snakes in Taiwan, and the test results show that the test piece 2 and the five kinds are
蛇毒無交互反應，因此不需擔心有誤判的情形發生，使太 發明具有檢驗準確性高的特性。 使本 三、本發明容易保存，在室溫中可保存一年，因此， 有可隨時因應不時之需的拉Μ # — 的特性，並適於作為各醫院急診 至之常備檢驗試劑使本發明有較高的實用性。 惟以上所述者，僅為本發明之—較佳實施例而已， 不能以此限定本發明眘祐 利範圍及發明說明内容所作：簡單了依本發明申請 仍属本發明專利涵蓋之範圍内。 <變化與修飾， 14 201013186 【圖式簡單說明】 圖1是一不完整的正視示意圖，說明現有的一蛇毒快 速檢驗試片一較佳實施例的一試卡本體的一載體層的配置 型式； 圖2是一不完整的側視示意圖，說明該較佳實施例的 試卡本體由一基底層與該載體層相對應接合的情形； 圖3是一立體的使用示意圖，說明將一生物檢體滴加 到該較佳實施例的試卡本體的一檢體墊片上的情形；Snake venom has no interactive reaction, so there is no need to worry about the occurrence of misjudgment, so that the invention has the characteristics of high test accuracy. The present invention is easy to store and can be stored for one year at room temperature. Therefore, it has the characteristics of being able to respond to the need of the cockroach # 不 at any time, and is suitable as a routine test reagent for emergency hospitals. The invention has high practicability. The above is only the preferred embodiment of the present invention, and it should not be construed as limiting the scope of the invention and the description of the invention: the invention is still within the scope of the invention. <Changes and Modifications, 14 201013186 [Simplified Schematic Description] FIG. 1 is an incomplete front view showing a configuration pattern of a carrier layer of a test card body of a preferred embodiment of a snake venom rapid test strip. 2 is a schematic side view showing an incomplete side view showing the case where the test card body of the preferred embodiment is joined by the base layer and the carrier layer; FIG. 3 is a perspective view of the use of a biopsy. a case where a body drop is applied to a sample pad of the test card body of the preferred embodiment;
圖4是一側視的使用示意圖，說明將未含有蛇毒的檢 體滴加到該較佳實施例的試卡本體的檢體墊片上的情形； 圖5 —側視的使用示意圖，說明將含有蛇毒的檢體滴 加到該較佳實施例的試卡本體的檢體墊片上的情形； 圖6-1是一局部的正視示意圖，說明以該較佳實施例的 '•式卡本體測試該檢體後呈陽性反應時的顯示結果； 圖6-2是一局部的正視示意圖，說明以該較佳實施例的 試卡本體測試該檢體後呈陰性反應時的顯示結果； 圖6-3疋一局部的正視示意圖，說明以該較佳實施例的 試卡本體測試該檢體後為無反應時的顯示結果； ^圖6-4疋一局部的正視示意圖，說明以該較佳實施例的 試卡本體測試該檢體後為無反應時的顯示結果； 圖 7 β. 、 一視不意圖’說明以該較佳實施例進行偵測 極限試驗的結果；及 〜 at視不意圖 他 一叫 詋明以該較佳實施例與 見毒蛇的蛇毒蛋白進行交互作用試驗的結果 15 201013186 【主要元件符號說明】 2........ •…檢驗試片 41〇 ···· •…結合體 3........ •…試卡本體 411 ···· •…兔抗眼鏡蛇毒的 31…… —基底層 抗體 32…… …·載體層 412 ···· …·金顆粒 321 ···· •…檢體墊片 5 ....... -----^匣 322 ···· •…吸收墊片 51…… …·殼體 323 ···· •…金標結合墊片 511 ·.·· •…第一標記 324 ·.·. •…多孔薄膜 512···· •…第二標記 325 ···· *…測δ式線£ 52…… •…加樣孔 326 ··· •…控制線£ 53…… …·判讀視窗 4........ …··植入單元 10…… •…生物檢體 41…… .....金^抗體 100 ·.·· •…蛇毒 16Figure 4 is a schematic side view showing the use of a sample containing no snake venom to the sample pad of the test card body of the preferred embodiment; Figure 5 - Schematic view of the side view, illustrating The sample containing the snake venom is dropped onto the sample pad of the test card body of the preferred embodiment; FIG. 6-1 is a partial front elevational view showing the '• card body of the preferred embodiment Figure 6-2 is a partial front elevational view showing the results of a negative reaction after testing the sample with the test card body of the preferred embodiment; Figure 6 is a schematic view showing the result of a positive reaction after testing the sample; - a partial front view showing a display result when the test body of the preferred embodiment is tested to be non-reactive; FIG. 6-4 is a partial front view showing the preferred The test card body of the embodiment tests the display result after the sample is unresponsive; FIG. 7 β. , does not intend to 'describe the result of the detection limit test by the preferred embodiment; and ~ at the point of view He called Yu Ming with the preferred embodiment and sees the snake. Results of the interaction test of snake venom protein 15 201013186 [Explanation of main component symbols] 2........ •...Test test strip 41〇····•...Combined body 3........ •...Test card body 411 ·····... Rabbit anti-cobra venom 31... basal layer antibody 32... ...·Carrier layer 412 ······Gold particles 321 ···· •... Sheet 5 ....... -----^匣322 ···· •...Absorption pad 51...... ...·Shell 323 ···· •... Gold standard combined with spacer 511 ···· •...first mark 324 ···. •...porous film 512···· •...second mark 325 ···· *...measured δ line £ 52... •...sample hole 326 ··· •... Control line £53..................Reading window 4................... implant unit 10... •...biological specimen 41...... ..... gold^antibody 100 ·.·· Snake 16