TWI387751B - Kits for rhd blood type determination and method for rhd blood type determination by using the same - Google Patents
Kits for rhd blood type determination and method for rhd blood type determination by using the same Download PDFInfo
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本發明關於一種血型檢驗之套組和方法，尤指一種Rh血型檢驗之套組和方法。The present invention relates to a kit and method for blood group testing, and more particularly to a kit and method for Rh blood group testing.
為了避免受血者對於輸入之血液產生激烈的免疫反應，輸血前的常規檢測是非常重要的。Rh血型在臨床輸血醫學是複雜且具多型性的血型系統。Rh血型系統的主要抗原有RhD抗原和RhCcEe抗原，其中RhD抗原的相容性係造成輸血之免疫反應的主要原因，而RhCcEe抗原存在的重要性，則是影響RhD的蛋白質結構，造成RhD抗原的弱化，並不會顯著地引發輸血不相容的免疫反應。In order to prevent the recipient from having a fierce immune response to the input blood, routine testing prior to transfusion is very important. Rh blood type is a complex and polymorphic blood group system in clinical transfusion medicine. The main antigens of the Rh blood group system are RhD antigen and RhCcEe antigen. The compatibility of RhD antigen is the main cause of the immune response of transfusion, and the importance of the presence of RhCcEe antigen is the protein structure of RhD, which causes RhD antigen. Weakening does not significantly trigger an incompatible immune response to transfusion.
因此，目前Rh血型的檢驗主要是針對RhD抗原來判定Rh陽性或陰性。然而，目前的研究指出，RhD血型依據其抗原的弱化程度(即，紅血球表面抗原的多寡)可分為多種次型態(subtype)，如：RhD陽性、Partial D型、Weak D(或稱Du )型、Rh(Del)血型以及RhD陰性。Therefore, the current test of Rh blood type is mainly to determine whether Rh is positive or negative for RhD antigen. However, current research indicates that the RhD blood type can be divided into multiple subtypes depending on the degree of weakening of its antigen (ie, the amount of red blood cell surface antigen), such as: RhD positive, Partial D, Weak D (or D u ), Rh (Del) blood type and RhD negative.
Partial D型的形成主要是因為紅血球表面缺乏某些D抗原的決定位置(epitopes)，導致其在Rh血型的判讀上容易造成偽陰性的誤判。近年來由於單株抗體(monoclone antibody)技術的發展，已可明確地將Partial D型檢測出來。The formation of Partial D type is mainly due to the lack of certain epitopes (epitopes) on the surface of red blood cells, which makes it easy to cause false negative judgment on the interpretation of Rh blood type. In recent years, due to the development of monoclonal antibody technology, Partial D type has been clearly detected.
Weak D型的定義為，該血型的紅血球無法與免疫球蛋白M(IgM)之抗D抗體產生凝集反應，但卻可以藉由免疫球蛋白G(IgG)之抗D抗體於強化血清白蛋白測試(albumin enhanced test)、低離子濃度溶液試驗(Low-ionic strength saline test)或非直接抗免疫球蛋白測試(Indirect antiglobulin test)中檢測出凝集反應。進一步的研究指出，每個正常RhD陽性的紅血球細胞大概有10,000到30,000個RhD抗原，而每個Weak D型的紅血球細胞則大概有300到9,000個RhD抗原。Weak D is defined as the red blood cell of this blood type cannot produce agglutination reaction with anti-D antibody of immunoglobulin M (IgM), but can be used to strengthen serum albumin by anti-D antibody of immunoglobulin G (IgG). The agglutination reaction was detected in an (albumin enhanced test), a low-ionic strength saline test or an indirect antiglobulin test. Further studies indicate that each normal RhD-positive red blood cell has approximately 10,000 to 30,000 RhD antigens, and each Weak D-type red blood cell has approximately 300 to 9,000 RhD antigens.
Rh(Del)血型係在1984年被研究發現，該研究指出，有10%經檢驗為RhD陰性的個體，進一步經吸附沖出法(adsorption and elution method)試驗，可沖出抗D抗體，這意味著這些個體的紅血球存在著少量的RhD抗原，而這些個體因此被稱為具有Rh(D elution)血型(簡稱(Rh(Del))。這種Rh(Del)血型的個體在亞洲區域如日本、香港和台灣出現的頻率相當的高。The Rh(Del) blood group was discovered in 1984. The study indicated that 10% of individuals who were tested to be negative for RhD were further tested for adsorption and elution methods to rush out anti-D antibodies. It means that there are a small amount of RhD antigen in the red blood cells of these individuals, and these individuals are therefore called to have the Rh (D elution) blood type (Rh (Del)). The individuals of this Rh (Del) blood type are in Asian regions such as Japan. The frequency of occurrence in Hong Kong and Taiwan is quite high.
目前於血庫常規檢驗中的新技術有凝膠技術(gel-column technology)、固態技術(solid phase technology)和親合柱技術(affinity-column technology)，這些技術較傳統手工試管測試優異之處是具有標準化、穩定測試結果、減少所需之試驗樣本、提高敏感度和特異性等優點。然而，除了吸附沖出法仍缺乏有效的方法可以檢驗出Rh(Del)血型，而吸附沖出法卻過於耗費時間和人力，並不適用於血庫常規檢驗。而Rh(Del)血型在亞洲區域發明的高頻率，使得Rh(Del)血型成為輸血程序上的一大隱憂。有鑑於此，開發出一種得以迅速檢驗出Rh血型之次型態(尤其是Rh(Del)血型)的檢驗方法和套組，確有其急迫性。The new technologies currently in routine blood bank testing are gel-column technology, solid phase technology, and affinity-column technology. These techniques are superior to traditional manual test tubes. It has the advantages of standardization, stable test results, reduced test samples required, improved sensitivity and specificity. However, in addition to the adsorption and flushing method, there is still no effective method to detect the Rh(Del) blood type, and the adsorption and rushing method is too time-consuming and labor-intensive, and is not suitable for routine blood bank testing. The high frequency of the Rh (Del) blood type invented in the Asian region makes the Rh (Del) blood type a major concern in blood transfusion procedures. In view of this, it has been urgency to develop a test method and a kit that can quickly detect the subtype of Rh blood type (especially Rh (Del) blood type).
爰是，本發明之主要目的為提供一種得以迅速檢驗出所有Rh血型之次型態的檢驗方法和套組，以避免輸血程序造成免疫不相容的風險。Accordingly, it is a primary object of the present invention to provide a test method and kit for rapidly detecting the subtypes of all Rh blood types to avoid the risk of immunological incompatibility caused by blood transfusion procedures.
為達到上述目的，本發明提供一種用於檢驗RhD血型的套組，其係包含第一試劑及第二試劑；前述第一試劑包含：抗RhD抗原之免疫球蛋白G抗體；抗RhD抗原之免疫球蛋白M抗體；抗免疫球蛋白G之抗體；生物素；及溶劑；前述第二試劑包含：抗生物素之分子，其係包含卵白素(avidin)、抗生物素卵白(streptavidin)或其組合；及溶劑。In order to achieve the above object, the present invention provides a kit for testing RhD blood type, which comprises a first reagent and a second reagent; the first reagent comprises: an anti-RhD antigen immunoglobulin G antibody; and an anti-RhD antigen immunization a globulin M antibody; an antibody against immunoglobulin G; biotin; and a solvent; the second reagent comprises: an avidin molecule comprising avidin, astreptavidin or a combination thereof ; and solvent.
較佳地，前述第一試劑包含：0.000001~20.0wt%的抗RhD抗原之免疫球蛋白G單株抗體；0.000001~20.0wt%的抗RhD抗原之免疫球蛋白M單株抗體；0.000001~20.0wt%的抗免疫球蛋白G之抗體；0.000001~40wt%的生物素；及40~99.999996wt%的溶劑。Preferably, the first reagent comprises: 0.000001 to 20.0% by weight of an anti-RhD antigen immunoglobulin G monoclonal antibody; 0.000001 to 20.0% by weight of an anti-RhD antigen immunoglobulin M monoclonal antibody; 0.000001~20.0wt % anti-immunoglobulin G antibody; 0.000001 to 40 wt% biotin; and 40 to 99.999996 wt% solvent.
較佳地，前述第二試劑包含：0.000001~20.0wt%的抗生物素之分子；及80~99.999999wt%的溶劑。Preferably, the second reagent comprises: 0.000001 to 20.0% by weight of a molecule of avidin; and 80 to 99.999999% by weight of a solvent.
較佳地，前述第一試劑及/或前述第二試劑進一步包含：酵素、血清白蛋白、凝聚胺(polybrene)、魚精蛋白(protamine)、補體C3與抗C3抗體、補體C4與抗C4抗體、運鐵蛋白(transferring)與抗運鐵蛋白抗體或其組合。Preferably, the first reagent and/or the second reagent further comprise: an enzyme, a serum albumin, a polybrene, a protamine, a complement C3 and an anti-C3 antibody, a complement C4 and an anti-C4 antibody. , transferrin and anti-transferrin antibodies or combinations thereof.
較佳地，前述第一試劑中的前述抗RhD抗原之免疫球蛋白G抗體接枝(conjugated)有前述生物素，及/或前述血清白蛋白接枝有前述生物素。Preferably, the anti-RhD antigen immunoglobulin G antibody in the first reagent is conjugated with the biotin, and/or the serum albumin is grafted with the biotin.
較佳地，前述第一試劑中的前述抗RhD抗原之免疫球蛋白M抗體接枝(conjugated)有前述生物素，及/或前述血清白蛋白接枝有前述生物素。Preferably, the anti-RhD antigen immunoglobulin M antibody in the first reagent is conjugated with the biotin, and/or the serum albumin is grafted with the biotin.
較佳地，前述第一試劑中的前述抗免疫球蛋白G之抗體接枝(conjugated)有前述生物素，及/或前述血清白蛋白接枝有前述生物素。Preferably, the anti-immunoglobulin G antibody in the first reagent is conjugated with the biotin, and/or the serum albumin is grafted with the biotin.
較佳地，前述酵素為木瓜酵素。Preferably, the aforementioned enzyme is papaya enzyme.
較佳地，前述溶劑為阿氏液(Alsever’s solution)、磷酸緩衝溶液(PBS)或其組合。Preferably, the aforementioned solvent is Alsever's solution, phosphate buffer solution (PBS) or a combination thereof.
本發明再提供一種RhD血型的檢驗方法，其係包含下述步驟：(a)提供一血液樣本；(b)將前述血液樣本製成0.5~5.0%(v/v)的紅血球溶液；(c)將前述紅血球溶液與前述之第一試劑置入空白的第一凝膠管柱中；(d)將前述紅血球溶液與前述之第一試劑及第二試劑置入空白的第二凝膠管柱中；(e)離心前述第一凝膠管柱和前述第二凝膠管柱後；及(f)判讀血型。The invention further provides a method for testing a RhD blood type, comprising the steps of: (a) providing a blood sample; (b) preparing the blood sample to a 0.5 to 5.0% (v/v) red blood cell solution; Putting the red blood cell solution and the first reagent described above into a blank first gel column; (d) placing the red blood cell solution and the first reagent and the second reagent into a blank second gel column (e) after centrifuging the first gel column and the second gel column; and (f) interpreting the blood type.
較佳地，前述步驟c中，前述紅血球溶液與前述第一試劑的體積比值為0.5~2.0。Preferably, in the step c, the volume ratio of the red blood cell solution to the first reagent is 0.5 to 2.0.
較佳地，前述步驟d中，前述紅血球溶液與前述第一試劑的體積比值為0.5~2.0；且前述紅血球溶液與前述第二試劑的體積比值為0.01~100。Preferably, in the step d, the volume ratio of the red blood cell solution to the first reagent is 0.5 to 2.0; and the volume ratio of the red blood cell solution to the second reagent is 0.01 to 100.
較佳地，前述離心包含：第一離心步驟，其係於300~4000 r.p.m.下離心1~5分鐘；及第二離心步驟，其係於300~4000 r.p.m.下離心1~5分鐘。較佳地，前述第一離心步驟的轉速係低於前述第二離心步驟的轉速。Preferably, the centrifugation comprises: a first centrifugation step of centrifugation for 1 to 5 minutes at 300 to 4000 r.p.m.; and a second centrifugation step of centrifugation for 1 to 5 minutes at 300 to 4000 r.p.m. Preferably, the rotational speed of the first centrifugation step is lower than the rotational speed of the second centrifugation step.
較佳地，前述第一凝膠管柱之凝集現象判讀為4+時，屬於RhD陽性血型；前述第一凝膠管柱之凝集現象判讀為3+、2+或1+時，屬於Partial D血型；前述第一凝膠管柱之凝集現象判讀為0+，且前述第二凝膠管柱之凝集現象判讀為為3+或2+時，屬於Rh(Del)血型；前述第一凝膠管柱之凝集現象判讀為0+，且前述第二凝膠管柱之凝集現象判讀為為1+或0+時，屬於RhD陰性血型；前述第一凝膠管柱之凝集現象判讀為0+，且前述第二凝膠管柱之凝集現象判讀為為4+時，須重新檢驗。Preferably, when the agglutination phenomenon of the first gel column is 4+, it belongs to the RhD-positive blood type; when the agglutination phenomenon of the first gel column is 3+, 2+ or 1+, it belongs to Partial D. Blood type; the agglutination phenomenon of the first gel column is 0+, and the agglutination phenomenon of the second gel column is 3+ or 2+, belonging to the Rh (Del) blood type; the first gel The agglutination phenomenon of the column is judged as 0+, and when the agglutination phenomenon of the second gel column is judged as 1+ or 0+, it belongs to the RhD-negative blood type; the agglutination phenomenon of the first gel column is 0+ And when the agglutination phenomenon of the second gel column is judged to be 4+, it is necessary to re-inspect.
綜上所述，本發明之用於檢驗RhD血型的套組及使用該試驗組以檢驗RhD血型的方法，係使用本發明配方之雙試劑，搭配使用習知的凝膠技術，藉判讀抗體抗原交互作用產生的凝集現象，達到迅速並精確地檢測RhD血型之所有次型態的目的。In summary, the kit for testing the RhD blood type of the present invention and the method for testing the RhD blood type using the test group are based on the use of the conventional gel technique and the reading of the antibody antigen. The agglutination phenomenon produced by the interaction achieves the purpose of quickly and accurately detecting all subtypes of the RhD blood type.
本發明之檢驗RhD血型的方法係搭配運用本發明套組之雙試劑配方。於第一試劑中，藉著一抗-二抗之原理，放大抗RhD抗體與RhD抗原的交互作用所產生的凝集現象，而得以分辨出RhD陽性與Partial D血型。而於合併使用第二試劑時，藉卵白素及/或抗生物素卵白與生物素的交互作用，更進一步地放大抗RhD抗體與RhD抗原的交互作用所產生的凝集現象[Teramura et al.,Biomaterials,2009]。據此，習所難以於血庫常規檢測中辨識的Rh(Del)血型，也得以藉本發明快速地辨識出來，減少輸血的風險。The method of the present invention for testing RhD blood type is in combination with a two-reagent formulation using the kit of the present invention. In the first reagent, the agglutination phenomenon caused by the interaction between the anti-RhD antibody and the RhD antigen was amplified by the principle of the primary antibody and the secondary antibody, and the RhD-positive and Partial D blood groups were distinguished. When the second reagent is used in combination, the interaction between the avidin and/or avidin and biotin further amplifies the agglutination caused by the interaction between the anti-RhD antibody and the RhD antigen [Teramura et al., Biomaterials, 2009]. Accordingly, the Rh (Del) blood type that is difficult for the laboratory to recognize in the blood bank routine detection can also be quickly identified by the present invention to reduce the risk of blood transfusion.
本發明所述之「抗RhD抗原之免疫球蛋白G抗體」係指一種可以與人類紅血球表面的RhD抗原形成專一性鍵結(binding)的蛋白質(抗體)；其可從人、大鼠、鼠、兔、牛、山羊、羊、雞或其它哺乳類物種經免疫反應產生；其可為單株抗體、多株抗體、嵌合抗體或擬人化抗體；較佳地，其係為抗RhD抗原之小鼠免疫球蛋白G單株抗體(monoclone mouse anti-D IgG antibody)。The "anti-RhD antigen immunoglobulin G antibody" as used in the present invention refers to a protein (antibody) which can form a specific binding with the RhD antigen on the surface of human red blood cells; it can be obtained from human, rat and mouse. , rabbit, cow, goat, sheep, chicken or other mammalian species are produced by an immune reaction; it may be a single antibody, a plurality of antibodies, a chimeric antibody or a humanized antibody; preferably, it is a small anti-RhD antigen Monoclonal mouse anti-D IgG antibody.
本發明所述之「抗RhD抗原之免疫球蛋白M抗體」係指一種可以與人類紅血球表面的RhD抗原形成專一性鍵結(binding)的蛋白質(抗體)；其可從人、大鼠、鼠、兔、牛、山羊、羊、雞或其它哺乳類物種經免疫反應產生；其可為單株抗體、多株抗體、嵌合抗體或擬人化抗體；較佳地，其係為抗RhD抗原之小鼠免疫球蛋白M單株抗體(monoclone mouse anti-D IgM antibody)。The "anti-RhD antigen immunoglobulin M antibody" as used in the present invention refers to a protein (antibody) which can form a specific binding with the RhD antigen on the surface of human red blood cells; it can be obtained from human, rat and mouse. , rabbit, cow, goat, sheep, chicken or other mammalian species are produced by an immune reaction; it may be a single antibody, a plurality of antibodies, a chimeric antibody or a humanized antibody; preferably, it is a small anti-RhD antigen Monoclonal mouse anti-D IgM antibody.
前述抗RhD抗原之免疫球蛋白G抗體與前述抗RhD抗原之免疫球蛋白M抗體的不同在於，免疫球蛋白M抗體的結構係由五個免疫球蛋白G抗體藉由雙硫鍵連結在一起所成為的五角環狀結構。The anti-RhD antigen immunoglobulin G antibody differs from the aforementioned anti-RhD antigen immunoglobulin M antibody in that the immunoglobulin M antibody is composed of five immunoglobulin G antibodies linked by a disulfide bond. Become a pentagonal ring structure.
研究指出，紅血球之間的凝集現象(agglutination)與抗體和紅血球表面抗原之間的下錨距離(anchoring distance)習習相關。由於紅血球細胞膜帶負電的特性，使得紅血球在血漿或生理食鹽水中會被帶正電的陽離子雲包圍，造成單一紅血球之間因為帶正電陽離子雲的排斥而至少保持約25奈米的距離。而免疫球蛋白G抗體接在兩個紅血球抗原之間的距離約為14奈米；免疫球蛋白M抗體接在兩個紅血球抗原之間的距離則約為30奈米，因此單獨使用免疫球蛋白G抗體使紅血球產生凝集現象的效果並不顯著，而必須同時使用免疫球蛋白M抗體及/或藉由其他方式的輔助，如經酵素處理、添加二級抗體(secondary antibody)、血清白蛋白(albumin)、凝聚胺(polybrene)、魚精蛋白(protamine)、補體C3與抗C3抗體、補體C4與抗C4抗體、運鐵蛋白(transferring)與抗運鐵蛋白抗體或其組合；該酵素可為木瓜酵素(papain)。Studies have shown that agglutination between red blood cells is associated with an anchoring distance between antibodies and red blood cell surface antigens. Due to the negatively charged nature of the red blood cell membrane, the red blood cells are surrounded by positively charged cation clouds in plasma or physiological saline, resulting in a distance of at least about 25 nm between the single red blood cells due to the rejection of the positively charged cation cloud. The distance between the two erythrocyte antigens of the immunoglobulin G antibody is about 14 nm; the distance between the two erythrocyte antigens of the immunoglobulin M antibody is about 30 nm, so the immunoglobulin is used alone. The effect of G antibody on the agglutination of red blood cells is not significant, and it is necessary to use both immunoglobulin M antibodies and/or other means of assistance, such as enzyme treatment, addition of secondary antibodies, serum albumin ( Albumin), polybrene, protamine, complement C3 and anti-C3 antibodies, complement C4 and anti-C4 antibodies, transferrin and anti-transferrin antibodies, or combinations thereof; Papaya enzyme (papain).
本發明所述之「抗免疫球蛋白G之抗體」係指對前述抗RhD抗原之免疫球蛋白G抗體具有專一性的抗體，更明確地，前述抗免疫球蛋白G之抗體會與前述抗RhD抗原之免疫球蛋白G抗體之結晶端(Fc site)專一性鍵結，因此又稱為二級抗體(secondary antibody)。前述抗免疫球蛋白G之抗體與前述抗RhD抗原之免疫球蛋白G抗體較佳地係來自於不同的來源；較佳地，前述抗免疫球蛋白G之抗體係為兔抗小鼠免疫球蛋白G之抗體(rabbit anti-mouse IgG antibody)。前述二級抗體可為單株抗體、多株抗體、嵌合抗體或擬人化抗體；較佳地，係為單株抗體。The "anti-immunoglobulin G antibody" according to the present invention means an antibody specific for the anti-RhD antigen immunoglobulin G antibody, and more specifically, the anti-immunoglobulin G antibody may be associated with the aforementioned anti-RhD. The Fc site of the antigen immunoglobulin G antibody is specifically bonded, and is therefore also referred to as a secondary antibody. The anti-immunoglobulin G antibody and the anti-RhD antigen immunoglobulin G antibody are preferably from different sources; preferably, the anti-immunoglobulin G anti-system is rabbit anti-mouse immunoglobulin Rabbit anti-mouse IgG antibody. The aforementioned secondary antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a humanized antibody; preferably, it is a monoclonal antibody.
本發明配方之血清白蛋白的來源包括，但不限於：人、大鼠、鼠、兔、牛、山羊、羊、雞。本發明配方之血清白蛋白係用以強化紅血球細胞表面抗原與抗體的鍵結能力。Sources of serum albumin of the present invention include, but are not limited to, human, rat, rat, rabbit, cow, goat, sheep, chicken. The serum albumin of the present invention is used to enhance the binding ability of red blood cell cell surface antigen to an antibody.
生物素，即維生素B7(又稱維生素H)，在醣類、蛋白質、脂肪等營養素代謝過程中扮演重要輔酶(coenzyme)的角色。藉著運用生物素與抗生物素之分子之間的專一性結合，生物素廣泛運用於細胞檢測的技術中。較佳地，前述抗免疫球蛋白G之抗體接枝(conjugated)有前述生物素，及/或前述血清白蛋白接枝有前述生物素。Biotin, vitamin B7 (also known as vitamin H), plays an important role as a coenzyme in the metabolism of nutrients such as sugars, proteins, and fats. Biotin is widely used in cell detection technology through the use of specific binding between biotin and avidin molecules. Preferably, the anti-immunoglobulin G antibody is conjugated with the aforementioned biotin, and/or the serum albumin is grafted with the biotin.
前述抗生物素之分子係指能與前述生物素形成專一性鍵結之分子。本發明所述之抗生物素之分子包括，但不限於：卵蛋白(avidin)、抗生物素卵白(streptavidin)或其組合，其可與生物素產生強大的專一性鍵結能力，每一分子的抗生物素卵白或卵白素均可吸附一個生物素，並且四個卵白素或抗生物素卵白彼此會形成緊密且穩定的結合，即便在酸性溶液、鹼性溶液或有機溶劑下，其結合能力也不受影響。卵白素與抗生物素卵白的最大差異在於卵白素的對生物素的專一性遠比抗生物素卵白來的高，也就是說，抗生物素卵白可能會在檢測中產生非專一性鍵結(non-specific binding)。The aforementioned avidin molecule refers to a molecule capable of forming a specific bond with the aforementioned biotin. The avidin molecule of the present invention includes, but is not limited to, avidin, streptavidin or a combination thereof, which can generate strong specific binding ability with biotin, each molecule Avidin or avidin can adsorb a biotin, and four avidin or avidin egg white will form a tight and stable combination with each other, even in acidic solution, alkaline solution or organic solvent, its binding ability Also unaffected. The biggest difference between avidin and avidin is that the specificity of avidin to biotin is much higher than that of avidin, that is, avidin may cause non-specific binding in the test ( Non-specific binding).
本發明所述之溶劑可在不影響本發明配方之功效的前提下選用所屬領域習知之溶劑，該溶劑包括，但不限於：阿氏液(Alsever’s solution)、磷酸緩衝溶液(PBS)或其組合。前述阿氏液(Alsever’s solution)每10毫升包含葡萄糖410毫克、檸檬酸鈉160毫克、檸檬酸11毫克、氯化鈉84毫克、疊氮化鈉10毫克；前述磷酸緩衝溶液(PBS)包含氯化鈉(8 mg/mL)、氯化鉀(0.2 mg/mL)、磷酸鈉(1.15 mg/mL)、磷酸二氫鉀(0.2 mg/mL)。前述阿氏液(Alsever’s solution)和磷酸緩衝溶液(PBS)的成分比例僅列舉以供參考，並非用以限制本發明所述之溶劑的成分比例關係，熟習該項技術者可依實際實驗需求，調整為其他適當比例。The solvent of the present invention may be selected from the prior art without affecting the efficacy of the formulation of the present invention, including but not limited to: Alsever's solution, phosphate buffer solution (PBS) or a combination thereof. . The aforementioned Alsever's solution contains 410 mg of glucose, 160 mg of sodium citrate, 11 mg of citric acid, 84 mg of sodium chloride, and 10 mg of sodium azide per 10 ml; the aforementioned phosphate buffer solution (PBS) contains chlorination. Sodium (8 mg/mL), potassium chloride (0.2 mg/mL), sodium phosphate (1.15 mg/mL), potassium dihydrogen phosphate (0.2 mg/mL). The composition ratios of the aforementioned Alsever's solution and phosphate buffer solution (PBS) are only for reference, and are not intended to limit the composition ratio of the solvent of the present invention. Those skilled in the art can meet the actual experimental needs. Adjust to other appropriate ratios.
本發明之試驗組可由傳統上商業可取得之檢驗試劑為基礎，再依據本發明之配方加以調整其成分及成分比例來配製，以符合本案發明之精神。The test group of the present invention can be formulated on the basis of a test reagent which has been conventionally commercially available, and is further adjusted according to the composition of the present invention to adjust the composition and the proportion of the ingredients to conform to the spirit of the invention.
舉例來說，本發明套組之第一試劑可由下列所述之A、B和C溶液於適當比例下混合而得：For example, the first reagent of the kit of the present invention can be obtained by mixing the A, B and C solutions described below at an appropriate ratio:
A溶液係一般捐血中心和血庫習用之Rh血型檢驗用常規試劑，其係包含溶於阿氏液(Alsever’s solution)的下列成分：0.01~5.0mg/dL的抗RhD抗原之免疫球蛋白G單株抗體；0.01~5.0mg/dL的抗RhD抗原之免疫球蛋白M單株抗體；及0.01~10.0wt%的血清白蛋白。The A solution is a conventional blood donation center and a blood reagent for the routine blood test for Rh blood type, which comprises the following components dissolved in Alsever's solution: 0.01 to 5.0 mg/dL of an anti-RhD antigen immunoglobulin G single plant. Antibody; 0.01 to 5.0 mg/dL of anti-RhD antigen immunoglobulin M monoclonal antibody; and 0.01 to 10.0% by weight of serum albumin.
B溶液係商業可取得之二級抗體接枝生物素試劑(biotin conjugated secondary antibody)，其係包含溶於磷酸緩衝溶液的下列成分：0.01~11.2mg/mL的接枝有生物素的抗免疫球蛋白G之抗體；其中，每一個前述接枝有生物素的抗免疫球蛋白G之抗體接枝有1~32個生物素。The B solution is a commercially available biotin conjugated secondary antibody comprising the following components dissolved in a phosphate buffer solution: 0.01 to 11.2 mg/mL of biotin-conjugated anti-immune spheres An antibody of protein G; wherein each of the anti-immunoglobulin G antibodies grafted with biotin is grafted with 1 to 32 biotin.
C溶液係商業可取得之血清白蛋白接枝生物素試劑，其係包含溶於氯化鈉溶液的下列成分：0.01~9.6mg/mL的接枝有生物素的血清白蛋白；其中每一個前述接枝有生物素的血清白蛋白接枝有6~8個生物素。C試劑並進一步以氯化鈉稀釋5×10-8 倍以供後續配製本發明第一試劑之用。The C solution is a commercially available serum albumin-grafted biotin reagent comprising the following components dissolved in a sodium chloride solution: 0.01 to 9.6 mg/mL of biotin-loaded serum albumin; each of the foregoing The serum albumin grafted with biotin is grafted with 6-8 biotin. The C reagent is further diluted 5 x 10 -8 times with sodium chloride for subsequent preparation of the first reagent of the present invention.
將前述A試劑、前述B試劑以及前述稀釋過後的C試劑以10：1.22：0.52的比例混合均勻，即可得本案發明套組之第一試劑。The A reagent, the B reagent, and the diluted C reagent are uniformly mixed at a ratio of 10:1.22:0.52 to obtain the first reagent of the kit of the present invention.
此外，本發明套組之第二試劑可由以下方式製備：將商業可取得之溶於磷酸緩衝溶液的抗生物素卵白(avidin)以阿氏液(Alsever’s solution)調整前述抗生物素卵白(avidin)之最後濃度(final concentration)為0.01~20mg/ml，即可得本案發明套組之第二試劑。In addition, the second reagent of the kit of the present invention can be prepared by adjusting the avidin avidin in a commercially available avidin dissolved in a phosphate buffer solution in an Alsever's solution. The final concentration of 0.01-20 mg/ml can be used to obtain the second reagent of the invention kit of the present invention.
前述配製方法係為了簡化本發明之套組的配製過程，並非用以限制本案發明套組的配製方法，而所屬技術領域之具有通常知識者，當可在不違背本發明之精神的前提下，以不同方式調配符合本發明之成分配方的套組。The foregoing preparation method is not intended to limit the preparation method of the kit of the present invention in order to simplify the preparation process of the kit of the present invention, and those having ordinary knowledge in the art can, without departing from the spirit of the present invention, The kits of the ingredients of the invention are formulated in different ways.
以下實施例係用於進一步了解本發明之優點，並非用於限制本發明之申請專利範圍。The following examples are intended to further understand the advantages of the present invention and are not intended to limit the scope of the invention.
於本實施例中，本發明之第一試劑係由下列A、B、C三種溶液，以適當比例混合而成。前述A、B、C溶液分別包含下列成分：In the present embodiment, the first reagent of the present invention is prepared by mixing the following three solutions A, B and C in an appropriate ratio. The aforementioned A, B, and C solutions respectively contain the following components:
[A溶液][A solution]
A溶液係一般捐血中心和血庫習用之Rh血型檢驗用常規試劑，其係包含溶於阿氏液(Alsever’s solution)的下列成分：1.83 mg/dL的抗RhD抗原之小鼠免疫球蛋白G單株抗體(mouse anti-D IgG antibody)；0.89 mg/dL的抗RhD抗原之小鼠免疫球蛋白M單株抗體(mouse anti-D IgM antibody)；及7 wt%的血清白蛋白。The A solution is a conventional blood donation center and a blood reagent for the routine blood test for Rh blood type, which contains the following components dissolved in Alsever's solution: 1.83 mg/dL of anti-RhD antigen mouse immunoglobulin G single plant Mouse anti-D IgG antibody; 0.89 mg/dL anti-RhD antigen mouse anti-D IgM antibody; and 7 wt% serum albumin.
[B溶液][B solution]
B溶液係商業可取得之二級抗體接枝生物素試劑(biotin conjugated secondary antibody，JonsonJonson)，其係包含溶於磷酸緩衝溶液的下列成分：1.5 mg/mL的接枝有生物素的兔抗小鼠免疫球蛋白G之抗體(rabbit anti-mouse IgG antibody,biotin conjugates)；其中，每一個前述接枝有生物素的兔抗小鼠免疫球蛋白G之抗體接枝有10~12個生物素。The B solution is a commercially available biotin conjugated secondary antibody (Jonson Jonson) comprising the following components dissolved in a phosphate buffer solution: 1.5 mg/mL rabbit biotin grafted with biotin Rabbit anti-mouse IgG antibody (biotin conjugates); wherein each of the aforementioned rabbit anti-mouse immunoglobulin G grafted with biotin is grafted with 10 to 12 biotin.
[C溶液][C solution]
C溶液係商業可取得之牛血清白蛋白接枝生物素試劑(bovine albumin,biotin conjugates，Sigma)，其係包含溶於0.9 wt%之氯化鈉溶液的下列成分：5 mg/mL的接枝有生物素的血清白蛋白；其中每一個前述接枝有生物素的血清白蛋白接枝有6~8個生物素。C溶液並進一步以0.9 wt%之氯化鈉稀釋到5×10-8 倍以供後續配製本發明第一試劑之用。The C solution is a commercially available bovine albumin-grafted biotin reagent (bovine albumin, biotin conjugates, Sigma) comprising the following components dissolved in a 0.9 wt% sodium chloride solution: 5 mg/mL grafting Biotin-containing serum albumin; each of the aforementioned biotin-loaded serum albumin is grafted with 6-8 biotin. The solution of C was further diluted with 0.9 wt% of sodium chloride to 5 x 10 -8 times for subsequent preparation of the first reagent of the present invention.
[混合][mixing]
將10毫升的前述A溶液、1.22毫升的前述B溶液以及0.52毫升的前述稀釋過後的C溶液混合均勻，即可得本實施例之第一試劑。10 ml of the aforementioned A solution, 1.22 ml of the aforementioned B solution, and 0.52 ml of the diluted C solution described above were uniformly mixed to obtain the first reagent of the present example.
本實施例之第二試劑係將商業可取得之溶於磷酸緩衝溶液的抗生物素卵白(avidin，具有10~15 unit/per protein，Sigma)，以阿氏液(Alsever’s solution)調整前述抗生物素卵白(avidin)之最後濃度(final concentration)為5 mg/ml，即可得本案發明套組之第二試劑。須注意的是，於此實施例中，本發明之第二試劑未添加卵白素，但在其他實施例中，本發明之第二試劑可進一步添加卵白素(10~15 unit/per protein，Sigma)。The second reagent of the present embodiment is a commercially available anti-biotin egg white (avidin, having 10-15 unit/per protein, Sigma) dissolved in a phosphate buffer solution, and the aforementioned antibiotic is adjusted by Alsever's solution. The final concentration of the avidin is 5 mg/ml, and the second reagent of the inventive kit of the present invention can be obtained. It should be noted that in this embodiment, the second reagent of the present invention does not add avidin, but in other embodiments, the second reagent of the present invention may further add avidin (10-15 unit/per protein, Sigma). ).
[製備紅血球溶液][Preparation of red blood cell solution]
本實施例係使用經常規處理而儲存於血庫之冷凍紅血球儲存液進行RhD血型的檢驗，共計使用五個來自不同個體之前述冷凍紅血球儲存液進行檢驗，分別標記為樣本A、樣本B、樣本C、樣本D及樣本E；其中，樣本A、B、C係分別來自Rh(Del)血型之不同捐贈者；樣本D和E係分別來自Rh陰性血型之不同捐贈者。In this embodiment, the frozen red blood cell storage solution stored in the blood bank by conventional treatment is used for the RhD blood type test, and a total of five frozen red blood cell storage solutions from different individuals are used for testing, and are respectively labeled as sample A, sample B, and sample C. , sample D and sample E; among them, samples A, B, and C are from different donors of Rh (Del) blood type; samples D and E are from different donors of Rh-negative blood type.
首先將冷凍紅血球儲存液移至試管中，並放置於37℃下解凍至紅血球可自然流動之液體狀態。The frozen red blood cell stock solution is first transferred to a test tube and placed at 37 ° C to thaw to a liquid state in which the red blood cells can naturally flow.
接著，取6滴的紅血球儲存液加入2滴12%的氯化鈉溶液，使其混合均勻後靜置5分鐘。Next, 6 drops of red blood cell stock solution were added to 2 drops of 12% sodium chloride solution, and the mixture was uniformly mixed and allowed to stand for 5 minutes.
接著加入10滴1.6%的氯化鈉溶液，每滴加入時都需使其與紅血球儲存液混合均勻，然後於轉速1,000×g離心30秒。重複此步驟2次以進行緩衝液的置換。視情況再重複此步驟直到上清液無溶血現象為止。Then, 10 drops of a 1.6% sodium chloride solution were added, and each drop was mixed with the red blood cell stock solution, and then centrifuged at 1,000 × g for 30 seconds. This step was repeated twice to perform buffer replacement. Repeat this step as appropriate until the supernatant is free of hemolysis.
然後小心地將試管中的上清液抽除，再加入0.9%的氯化鈉溶液，並於轉速1,000×g離心1分鐘以清洗試管中的紅血球。重複此步驟2次。The supernatant in the test tube was then carefully removed, and then 0.9% sodium chloride solution was added and centrifuged at 1,000 x g for 1 minute to wash the red blood cells in the test tube. Repeat this step 2 times.
接著小心地將試管中的上清液抽除，加入阿氏液以配製3%(v/v)的紅血球溶液。此配製好的紅血球溶液可於4℃保存3到4個禮拜。The supernatant in the tube was then carefully removed and the arsenic solution was added to prepare a 3% (v/v) red blood cell solution. This prepared red blood cell solution can be stored at 4 ° C for 3 to 4 weeks.
前述五個來自不同個體之冷凍紅血球儲存液皆以上述方法配置成3%(v/v)的紅血球溶液。The above five frozen red blood cell stock solutions from different individuals were configured in a 3% (v/v) red blood cell solution as described above.
[進行凝集現象檢驗][Perform agglutination test]
本實施例係使用商業可取得之凝膠管柱(Ortho BioVue System,Ortho-Clinical Diagonostics,Inc.)，並搭配使用前述凝膠管柱之套件離心機(Ortho)。In this example, a commercially available gel column (Ortho BioVue System, Ortho-Clinical Diagonostics, Inc.) was used in combination with a kit centrifuge (Ortho) of the aforementioned gel column.
請參下表一(樣本F為空白對照組，即未添加任何紅血球溶液)，將10μl的前述紅血球溶液(樣本A~E)和20μl的實施例一所述之第一試劑分別加入一空白(blank)之前述凝膠管柱(即為各樣本之第一凝膠管柱)中；將10μl的前述紅血球溶液(樣本A~E)、20μl的實施例一所述之第一試劑以及3.66μl的實施例二所述之第二試劑分別加入另一空白(blank)之前述凝膠管柱(即為各樣本之第二凝膠管柱)中。Please refer to the following Table 1 (sample F is a blank control group, that is, no red blood cell solution is added), and 10 μl of the aforementioned red blood cell solution (samples A to E) and 20 μl of the first reagent described in the first embodiment are respectively added to a blank ( Blank) of the aforementioned gel column (ie, the first gel column of each sample); 10 μl of the aforementioned red blood cell solution (samples A to E), 20 μl of the first reagent described in Example 1, and 3.66 μl The second reagent described in the second embodiment is separately added to the other gel column of the blank (that is, the second gel column of each sample).
接著將前述第一凝膠管柱和前述第二凝膠管柱置入前述離心機(Ortho)中，此離心機係設計以供凝膠技術之血庫檢驗之用，其設有兩階段的離心，第一離心為低速離心，目的是為了讓試劑與樣本能夠在凝膠管柱的上端空腔內均勻混合，其轉速為835 rpm，離心時間為3分鐘；第二離心為高速離心，目的是為了讓紅血球通過前述凝膠管柱之內所設置之膠體，依據凝集之紅血球在前述膠體中的垂直分佈所反應出的凝集程度差異以判別血型，其轉速為1525 rpm，離心時間為2分鐘。The first gel column and the second gel column are then placed in the aforementioned centrifuge (Ortho), which is designed for blood bank testing of gel technology, which is provided with two-stage centrifugation. The first centrifugation is low-speed centrifugation, in order to allow the reagent and the sample to be uniformly mixed in the upper end cavity of the gel column, the rotation speed is 835 rpm, the centrifugation time is 3 minutes; the second centrifugation is high-speed centrifugation, the purpose is In order to allow the red blood cells to pass through the colloid disposed in the gel column, the blood type is discriminated according to the difference in the degree of agglutination reflected by the vertical distribution of the agglomerated red blood cells in the colloid, and the rotation speed is 1525 rpm, and the centrifugation time is 2 minutes.
本實施例僅列舉前述Ortho凝膠管柱之套件離心機作為本發明方法離心步驟之範例，然，可以達到前述離心效果之任何離心設備皆適用於本發明，無需加以限制。This embodiment exemplifies only the aforementioned Orthogel column kit centrifuge as an example of the centrifugation step of the method of the present invention. However, any centrifugal apparatus that can achieve the aforementioned centrifugal effect is applicable to the present invention without limitation.
[判別血型][Differentiated blood type]
依據習知凝膠技術的判斷準則，可觀察凝集之紅血球在前述凝膠管柱中垂直分佈的態樣給予4+、3+、2+、1+、0+的分數，該分數越大，代表凝集的程度越大。而根據本發明之方法，結合第一凝膠管柱和第二凝膠管柱即可判讀Rh血型的次型態，請參下表二：According to the judgment criterion of the conventional gel technique, the vertical distribution of the agglutinated red blood cells in the aforementioned gel column can be observed, and the scores of 4+, 3+, 2+, 1+, and 0+ are given, and the score is larger. The greater the degree of agglutination. According to the method of the present invention, the second type of Rh blood type can be read by combining the first gel column and the second gel column, please refer to the following table 2:
更明確地，若第一凝膠管柱判讀為4+、3+、2+或1+，則無需再判讀第二凝膠管柱；反之，若第一凝膠管柱判讀為0+，則需再判讀第二凝膠管柱以分辨Rh(Del)血型和Rh陰型。此外，若前述第一凝膠管柱判別為0+，而前述第二凝膠管柱判別為4+，代表試劑與紅血球溶液放置於凝膠管柱中的時間過久，則須重新檢驗。More specifically, if the first gel column is interpreted as 4+, 3+, 2+ or 1+, then the second gel column need not be interpreted; otherwise, if the first gel column is interpreted as 0+, Then the second gel column needs to be interpreted to distinguish the Rh (Del) blood type and the Rh negative type. In addition, if the first gel column is judged to be 0+, and the second gel column is judged to be 4+, and the reagent and the red blood cell solution are placed in the gel column for too long, it is necessary to re-inspect.
經判讀，前述樣本A~F的第一凝膠管柱皆為0+，因此進一步判讀第二凝膠管柱的凝集情況(圖中箭頭所指示處)。請參第一圖，樣本A的第二凝膠管柱1、樣本B的第二凝膠管柱2、樣本C的第二凝膠管柱3皆有紅血球凝集現象(箭頭標示處)而判讀為3+，即為Rh(Del)血型；樣本D的第二凝膠管柱4、樣本E的第二凝膠管柱5則沒有紅血球凝集現象，因此判讀為0+，即為Rh陰型。另外，樣本F的第二凝膠管柱6也判讀為0+。本實施例之樣本A~E的判讀結果統整如下表三：After interpretation, the first gel column of the aforementioned samples A~F is 0+, so the agglutination of the second gel column (indicated by the arrow in the figure) is further interpreted. Referring to the first figure, the second gel column of sample A, the second gel column of sample B, and the second gel column 3 of sample C all have red blood cell agglutination (arrow indication) and read It is 3+, which is Rh (Del) blood type; the second gel column 4 of sample D, and the second gel column 5 of sample E have no red blood cell agglutination, so it is 0+, which is Rh type . In addition, the second gel column 6 of the sample F was also interpreted as 0+. The interpretation results of the samples A~E of this embodiment are summarized as follows:
判讀結果顯示皆符合原先預期，即，樣本A~C來自Rh(Del)血型的捐贈者，以及樣本D和E來自Rh陰型血型的捐贈者。據此，本發明套組準確地檢驗出難以檢驗的Rh(Del)血型，而且較習用於檢驗Rh(Del)血型的方法來得快速。The results of the interpretations were consistent with the original expectations, ie, samples A~C were from Rh (Del) blood type donors, and samples D and E were from Rh negative blood type donors. Accordingly, the kit of the present invention accurately detects the Rh (Del) blood type which is difficult to test, and is faster than the method for testing the Rh (Del) blood type.
所屬領域之技術人員當可了解，在不違背本發明精神下，依據本案實施態樣所能進行的各種變化。因此，顯見所列之實施態樣並非用以限制本發明，而是企圖在所附申請專利範圍的定義下，涵蓋於本發明的精神與範疇中所做的修改。It will be apparent to those skilled in the art that various changes can be made in accordance with the embodiments of the present invention without departing from the spirit of the invention. Therefore, it is to be understood that the invention is not limited by the scope of the invention, and is intended to cover the modifications of the spirit and scope of the invention.
1．．．樣本A之第二凝膠管柱1. . . Sample A's second gel column
2．．．樣本B之第二凝膠管柱2. . . Sample B of the second gel column
3．．．樣本C之第二凝膠管柱3. . . Sample C of the second gel column
4．．．樣本D之第二凝膠管柱4. . . Sample 2 of the second gel column
5．．．樣本E之第二凝膠管柱5. . . Sample E of the second gel column
6．．．樣本F之第二凝膠管柱6. . . Sample F of the second gel column
第一圖係顯示樣本A~F的第二凝膠管柱中紅血球的凝集現象。The first figure shows the agglutination of red blood cells in the second gel column of samples A~F.
1．．．樣本A之第二凝膠管柱1. . . Sample A's second gel column
2．．．樣本B之第二凝膠管柱2. . . Sample B of the second gel column
3．．．樣本C之第二凝膠管柱3. . . Sample C of the second gel column
4．．．樣本D之第二凝膠管柱4. . . Sample 2 of the second gel column
5．．．樣本E之第二凝膠管柱5. . . Sample E of the second gel column
6．．．樣本F之第二凝膠管柱6. . . Sample F of the second gel column
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