直接競爭ELISA檢測擬柱孢藻毒素的方法建立和評價

楊 丹,劉金平,賴淑燕,肖利娟,雷臘梅

直接競爭ELISA檢測擬柱孢藻毒素的方法建立和評價

楊 丹,劉金平,賴淑燕,肖利娟,雷臘梅*

(暨南大學生態(tài)學系,廣東 廣州 510632)

擬柱孢藻毒素(CYN)對人和動物具有高毒性而在世界上廣受關(guān)注,快速檢測方法的建立可為藍藻毒素的風險評估提供有效的技術(shù)手段.本研究基于前期制備的CYN的單克隆抗體N8,旨在建立一種CYN的快速免疫檢測方法-直接競爭ELISA.在對HRP-CYN酶標記物和N8單抗的稀釋度進行優(yōu)化后,最適反應(yīng)條件下建立的直接競爭ELISA在0～200ng/mL CYN濃度下呈現(xiàn)典型的S型反應(yīng)曲線,該方法的靈敏度為0.069ng/mL,經(jīng)logit-log擬合的線性范圍為0.1～10ng/mL,檢出限遠低于WHO建議的0.7ng/mL CYN的安全限值.本研究建立的ELISA方法對水庫水樣和小球藻稀釋液中加標回收率分別為80.44%～125.4%和52.97%～155.19%,在測定CYN時與商業(yè)Beacon試劑盒的檢測結(jié)果高度一致.對大沙河水庫中CYN的監(jiān)測表明該毒素在水體中常年存在,但未超過WHO建議的安全限值.本研究發(fā)展的ELISA可實現(xiàn)水樣和藻樣中CYN的快速檢測,為供水安全保障提供一種新的技術(shù)手段.

直接競爭ELISA；擬柱孢藻毒素；單克隆抗體；HRP-CYN連接物；加標回收率

水體富營養(yǎng)化所導致的藍藻水華現(xiàn)象在全球范圍內(nèi)普遍存在[1-2],許多水華藍藻會產(chǎn)生一類次生代謝物-藍藻毒素,如微囊藻毒素(MCs),擬柱孢藻毒素(Cylindrospermopsins,CYN),節(jié)球藻毒素, anatoxin-a,和antoxin,不僅對水體造成污染,還對公眾健康構(gòu)成嚴重威脅[3-4].近20年來,隨著熱帶特征性藍藻-擬柱孢藻在全球范圍內(nèi)的快速擴張,其所產(chǎn)生的CYN成為繼微囊藻毒素后廣受關(guān)注的另一類藍藻毒素[5-6].擬柱孢藻是世界上第一個被發(fā)現(xiàn)能產(chǎn)CYN的藍藻[7],隨后陸續(xù)發(fā)現(xiàn)束絲藻、長孢藻、顫藻、尖頭藻等藍藻也能產(chǎn)生CYN.據(jù)報導,除了南極洲,CYN目前已呈全球分布態(tài)勢[6].

CYN是一種分子量為415Da的生物堿,由三環(huán)胍部分與羥甲基尿嘧啶結(jié)合形成[8].1992年首次確定了它的化學結(jié)構(gòu)[9],科科辛斯基(Kokociński)等發(fā)現(xiàn)了CYN存在5種類似物,分別是CYN、7-deoxydesulfo-CYN,7-deoxydesulfo-12-acetyl-CYN,7-epi-CYN,7-deoxy-CYN[10].研究發(fā)現(xiàn)CYN具有肝毒性、細胞毒性、神經(jīng)毒性、繁殖毒性等多種毒性效應(yīng)[11-12],其進入細胞后能夠抑制蛋白質(zhì)和谷胱甘肽的合成,通過共價修飾的方式改變DNA和RNA,從而造成基因損傷,具有潛在的致癌作用[8],CYN結(jié)構(gòu)已被證明非常穩(wěn)定,在可見光和紫外光下、極端pH(pH4-12)以及反復煮沸的高溫下都難以降解[7,14],由此有研究者認為CYN比微囊藻毒素對人類和動物的危害更大.鑒于CYN的危害性,澳大利亞首先提出1μg/L作為飲用水中CYN的安全限值[15-16], 2016年美國環(huán)保署也發(fā)布的CYN安全指導值為:對于6歲以下兒童為0.7ng/mL,對于6歲以上人群則為3ng/mL[16-17];2022年,世界衛(wèi)生組織(WHO)也對飲用水中CYN限值進行了建議,其長期暴露濃度為不超過0.7ng/mL.

藍藻毒素的潛在危害使得其檢測技術(shù)長期以來一直是國內(nèi)外研究熱點[3,6,17],利用藍藻毒素的產(chǎn)生基礎(chǔ)和生理化學特性如分子量、生色團和反應(yīng)性,已發(fā)展了化學分析、分子分析、免疫分析和生物分析等一系列方法用于產(chǎn)毒藍藻的甄別和藍藻毒素的定量檢測,其中以微囊藻毒素的檢測分析體系發(fā)展得最為成熟[19-21].在CYN的檢測中,HPLC[21](高效液相色譜法)和LC-MS/MS[23-24](液相色譜串聯(lián)質(zhì)譜技術(shù))為主要的化學檢測方法,它們具有檢測結(jié)果準確、能區(qū)分不同異構(gòu)體等優(yōu)點[23],但這類檢測技術(shù)操作復雜,儀器昂貴,對操作人員要求高,不適合用于檢測次數(shù)頻繁的常規(guī)和應(yīng)急監(jiān)測[26-29].免疫學分析具有高靈敏、快速度、高通量的優(yōu)點,近二十多年來在藍藻毒素尤其是MCs檢測領(lǐng)域應(yīng)用十分廣泛,基于MCs的多克隆和單克隆抗體發(fā)展的方法有酶聯(lián)免疫吸附法(ELISA)、時間分辨免疫分析法(TRFIA)、化學發(fā)光免疫分析法、現(xiàn)代的電信號免疫分析法、光信號免疫分析法免疫熒光試紙條等[30-33]等,這些免疫檢測技術(shù)已應(yīng)用于水體、藻樣或食品中微囊藻毒素的定量分析.

與微囊藻毒素相比,擬柱胞藻毒素抗體制備的研究極為稀少,目前僅有兩例,2013年 Elliott等[34]在國際上最先報道成功制備了CYN單克隆抗體,并建立了CYN的間接競爭ELISA方法;隨后Lei等[35]也成功篩選到多株CYN的特異性單抗,為國內(nèi)首次,并基于其中的N8單抗建立了TRFIA方法.由于ELISA在藍藻毒素的檢測中應(yīng)用更為廣泛,本研究以N8單抗為基礎(chǔ),發(fā)展CYN的直接競爭ELISA方法,以實現(xiàn)對水體中擬柱孢藻毒素濃度的快速測定,為保障供水安全提供一種新的技術(shù)手段.

1 材料與方法

1.1 材料和試劑

羊抗鼠二抗購自美國Biodesign公司,BSA(牛血清白蛋白)為MBCHEM公司產(chǎn)品,HRP(辣根過氧化物酶)為Sigma-Aldrich公司產(chǎn)品,CYN標準品購自ALEXIS公司,10kd超濾離心管購自廣州普智生物有限公司,96微孔板為Corning公司產(chǎn)品,其他試劑均為國產(chǎn)分析純.

1.2 方法

1.2.1 酶標記抗原HRP-CYN的制備 HRP-CYN的制備參考文獻[34],將辣根過氧化物酶(HRP)溶解在磷酸鹽緩沖液(PBS)中,向其中加入CYN和甲醛并將混合物避光室溫下攪拌,最后通過0.01mol/L磷酸鹽緩沖液對偶聯(lián)物進行透析,于-20℃冷凍保存?zhèn)溆?

1.2.2 固相包被二抗的制備 將濃度為5μg/mL的羊抗鼠IgG按200μL/孔分裝到96孔微孔板中,室溫震蕩10min,37℃恒溫箱放置過夜,傾倒包被液并洗板,加入封閉液進行封閉,4℃過夜,傾去封閉液并洗滌后真空抽干,－20℃保存?zhèn)溆?

1.2.3 抗原抗體效價優(yōu)化測定 通過棋盤格滴定法對單克隆抗體N8和酶標抗原HRP-CYN的效價進行摸索確定,在包被二抗的微孔板中依次加入50μL不同稀釋度的HRP-CYN(1:2000,1:4000, 1:8000,1:16000)和50μL不同稀釋度的N8單抗(1:2500, 1:5000, 1:7500, 1:10000, 1:15000, 1:20000), 37℃下反應(yīng)1h,洗滌6次后加入顯色液,37℃反應(yīng)30min后加入終止液,通過多功能酶標儀測定OD450nm的吸光值.

1.2.5 評價指標 (1)靈敏度

計算8組標準曲線中零濃度點的OD450nm的平均值()和標準差(s),根據(jù)-2s從標準曲線計算出相對應(yīng)的濃度.

(2)穩(wěn)定性

比較該體系不同時間段的50,根據(jù)多次檢測判斷劑量反應(yīng)曲線的位置漂移從而考核該方法的穩(wěn)定性.

(3)加標回收率的測定

收集小球藻裂解上清100倍稀釋液和沒有發(fā)生藍藻水華的流溪河水庫水為空白水樣,過濾后加入CYN標準品,終濃度分別為0.1,0.5,5,8ng/mL,隨后用直接競爭ELISA進行濃度測定,每個濃度做3個平行測定.

(4)與商業(yè)ELISA試劑盒檢測結(jié)果的比較

用美國Beacon公司的ELISA試劑盒和我們建立的ELISA方法對22份樣品(水庫水樣和產(chǎn)毒擬柱孢藻藻樣)同時進行檢測,比較兩種方法對CYN濃度測定的一致性.

1.2.6 大沙河水庫中CYN濃度的測定 大沙河水庫位于廣東省開平市,前期研究表明該水庫擬柱孢藻已形成季節(jié)性優(yōu)勢[35],為了解該水庫中CYN的發(fā)生情況,于2021年9月～2022年8月期間(2021年12月樣品缺失)每月定期采集取水口和浮臺表層0.5m的水樣,經(jīng)反復凍融處理后采用上述ELISA法測定上清液中的總CYN濃度.

2 結(jié)果

2.1 酶標抗原HRP-CYN的稀釋度和單克隆抗體效價確定

由圖1可知,在相同的HRP-CYN稀釋度下, OD450nm的吸光值均隨抗體稀釋度的升高而降低, HRP-CYN在1:8000的稀釋后的吸光值較1:4000顯著下降,為保證較高的吸光值,在標準曲線的制作中,酶標抗原選用1:4000的稀釋度.競爭ELISA反應(yīng)中酶標抗原HRP-CYN需與游離CYN競爭性結(jié)合單克隆抗體,因此抗體濃度必須適量,因此在進一步研究中選用1:10000的稀釋度.

圖1 不同稀釋度下酶標抗原HRP-CYN和CYN單抗反應(yīng)曲線

2.2 直接競爭ELISA標準曲線

以CYN為標準品制作的直接競爭ELISA的標準曲線見圖2,在0～200ng/mL的范圍下,標準曲線呈典型的S型(圖2a);采用logit-log法進行線性擬合發(fā)現(xiàn),本研究建立的直接競爭ELISA在0.1～10ng/mL的范圍內(nèi)呈良好的線性(圖2b).進一步從圖2b可知該標準曲線的的標準差極小,板內(nèi)3個標樣重復測定的變異系數(shù)在0.54%～7.8%,說明本研究建立的ELISA標準曲線具有較好的重現(xiàn)性.

a, CYN濃度在0～200ng/mL范圍內(nèi)OD450nm吸光值變化的反應(yīng)曲線; b,相對應(yīng)的logit-log 線性標準曲線和批內(nèi)精確度

2.3 方法的靈敏度與穩(wěn)定性

CYN濃度為0ng/mL時在OD450nm處的吸光值均值減去2倍標準差的值,在線性標準曲線中所對應(yīng)的濃度,即為該直接競爭ELISA方法的靈敏度,經(jīng)計算為0.069ng/mL.IC50在8條不同時間段的均值為(0.48±0.053)ng/mL,SD值相對較小說明該方法的穩(wěn)定性好.

2.4 加標回收率

將CYN標準品分別加至流溪河水庫濾水和小球藻裂解稀釋液中,采用本研究建立的ELISA法對加標樣品中的毒素濃度進行測定,發(fā)現(xiàn)水庫濾水的回收率在80.4%～125.4%,CV值小于9.05%(表1),這表明環(huán)境水樣中的基質(zhì)對ELISA測定的影響較小.CYN在小球藻稀釋液中的回收率在52.9%～ 155.2%間,CV值小于8.16%(表1),表明藻樣中的基質(zhì)對ELISA測定的存在一定干擾.

表1 CYN加標回收率和變異系數(shù)

2.5 與商業(yè)ELISA試劑盒的比較

采用自行研制的ELISA方法和商業(yè)ELISA試劑盒(Beacon公司)同時分析22個樣品中的CYN濃度,由圖3可知,兩種方法所獲得CYN濃度間具極顯著相關(guān)(2=0.9638,<0.05).整體來看商業(yè)試劑盒的檢測濃度要低于自制直接競爭ELISA的檢測濃度,進一步對兩組數(shù)據(jù)進行了one-way ANOVA分析發(fā)現(xiàn)它們不存在顯著差異(>0.05),表明自行研制的ELISA方法和商業(yè)ELISA試劑盒所獲得的CYN濃度高度一致.

圖3 自制ELISA方法與商業(yè)ELISA試劑盒的線性關(guān)系

2.6 廣東省大沙河水庫中CYN濃度的季節(jié)變化

圖4 大沙河水庫取水口與浮臺表層擬柱胞藻毒素濃度

由圖4可知,2021年9月～2022年8月間大沙河水庫取水口和浮臺表層的CYN濃度分別0.1～0.426ng/mL和0.147～0.459ng/mL變化,僅取水口2021年9月的CYN濃度低于ELISA的線性檢測限0.1ng/mL.與2021年相比,兩個采樣點的CYN濃度在2022年1月起即顯著上升,隨后基本維持在0.4ng/mL的水平;除2021年9月和2022年1月外,大沙河水庫兩個采樣點的CYN濃度基本一致.

3 討論

作為近年來備受關(guān)注的藍藻毒素,CYN的抗體制備研究極少,因此基于抗體的快速免疫監(jiān)測方法也相對較少[20,33-34].本研究建立了基于CYN的特異性單克隆抗體N8的直接競爭ELISA,該方法靈敏度為0.069ng/mL,線性檢測范圍在0.1～10ng/mL,靈敏度與Elliott et al(2013)發(fā)展的間接競爭ELISA以及兩種商業(yè)試劑盒(美國Abraxis和Beacon公司)相當[20].上述兩種商業(yè)試劑盒的線性范圍分別為0.05～ 2ng/mL和0.1～2ng/mL,因此本研究建立的ELISA的線性范圍(0.1～10ng/mL)優(yōu)于兩種商業(yè)試劑盒,但比同樣基于N8單抗建立的時間分辨熒光免疫技術(shù)(TRFIA法)的范圍窄(0.1～50ng/mL)[33].而以稀土離子作為標記物的時間分辨熒光免疫分析法被認為是更先進的免疫標記方法,是優(yōu)于酶聯(lián)免疫吸附法的一種超微量定量免疫分析檢測技術(shù),其靈敏度、重復性、檢測范圍等均要優(yōu)ELISA[36].

加標準樣品回收實驗是評價ELISA、TRFIA等免疫學檢測方法準確度的一個重要指標,一般認為80%～120%的回收率為正常波動范圍[37-39].本研究中水庫濾水的加標回收率80.4%～125.4%,此前我們基于N8單抗建立的TRIFA法評價了無水華的池塘濾水的加標回收率[33],與許多基于微囊藻毒素免疫檢測的研究結(jié)果一致,即自然河流和湖泊的濾水中藍藻毒素的加標回收率高[30-40],但也有研究發(fā)現(xiàn)水樣中微囊藻毒素的回收率只有62%[41],這可能是來源不同的水樣中存在的干擾物質(zhì)差異所致[42].而小球藻稀釋液在0.1和0.5ng/mL兩個低濃度的回收率分別為155.2%和52.9%,比正常波動范圍出現(xiàn)較大偏離,這表明小球藻中存在的基質(zhì)對ELISA檢測有更大的干擾,尤其在低濃度時,YU et al.(2001)在藻樣中微囊藻毒素的檢測中也發(fā)現(xiàn)類似的現(xiàn)象.因此采用ELISA分析藻類樣品中的CYN時,低濃度的測值可能需要固相微萃取等前處理,以適當?shù)娜コ寮毎械母蓴_物質(zhì)[43].

目前市場上主要有兩種商業(yè)ELISA試劑盒(分別來自美國Abraxis和Beacon公司)在國內(nèi)外廣泛應(yīng)用于CYN檢測[20,22,44].兩者的檢測模式均為直接競爭模式,與本研究建立的ELISA方法相似.然而Abraxis和Beacon兩種商業(yè)試劑盒中的抗體都為兔多克隆抗體,而作者建立的ELISA則是基于特異性的單克隆抗體N8,一般認為多克隆抗體特異性相對要低于單克隆抗體[45],這可能是Beacon試劑盒所測得的CYN濃度要稍低于我們建立的ELISA方法的原因,在此前TRFIA與Beacon試劑盒的對比中也發(fā)現(xiàn)類似的現(xiàn)象[33].一般認為LC-MS/MS為復雜樣品中小分子化合物分析的金標準方法,但由于該方法所需儀器極為昂貴,且需專業(yè)人員操作,這極大地限制了質(zhì)譜技術(shù)在藍藻毒素檢測中的推廣應(yīng)用[25,27].基于商業(yè)試劑盒在我國的廣泛使用[33,44-47],本研究選擇與Beacon公司試劑盒所獲得的測值進行對比,結(jié)果發(fā)現(xiàn)盡管兩種方法獲得的CYN濃度存在差異,但統(tǒng)計分析不顯著,這表明本實驗研究的ELISA方法是可信賴的,為進一步開發(fā)試劑盒奠定了堅實基礎(chǔ).

大沙河水庫地處廣東省開平市,屬典型的亞熱帶氣候.大沙河水庫基本常年可檢到CYN,但均沒有超過WHO建議的0.7ng/mL的安全限值,對飲用水安全不存在威脅.此前我國巢湖夏季水華期的CYN檢出率也非常高,在6～7月達100%,最高濃度為0.328ng/mL,均未超出安全限值[46].結(jié)合我們前期對廣東省水庫的研究[47-48],推測CYN在我國的湖泊和水庫可能呈廣泛分布,但目前除廣東省外還缺乏監(jiān)測數(shù)據(jù).我國水體中較低的CYN濃度可能是擬柱孢藻的產(chǎn)毒基因型優(yōu)勢度低所致,Jiang等[49]發(fā)現(xiàn)產(chǎn)CYN的擬柱孢藻在我國呈零星分布,本課題組此前的研究也發(fā)現(xiàn)廣東省水庫是非產(chǎn)毒基因型占據(jù)優(yōu)勢,CYN濃度與產(chǎn)毒擬柱孢藻豐度和比例的相關(guān)性要高于總擬柱孢藻豐度[48].盡管目前大沙河水庫中CYN濃度未超過0.7ng/mL,但該水庫自2016年以來擬柱孢藻的相對優(yōu)勢度和持續(xù)時間不斷增加[35],因此需對該水庫加強監(jiān)測,以防范產(chǎn)毒擬柱孢藻優(yōu)勢增加而導致CYN超標問題.

4 結(jié)論與展望

4.1 結(jié)論

本研究建立基于CYN單克隆抗體N8 的直接競爭ELISA方法,該法的檢測限為0.069ng/mL,線性檢測范圍為0.1～10ng/mL,具較好的穩(wěn)定性和回收率,與商業(yè)試劑盒的檢測結(jié)果高度一致,因此我們發(fā)展的ELISA方法有望替代進口試劑盒,為水樣和藻樣中CYN的快速檢測提供可靠的技術(shù)手段,而大沙河水庫中CYN的常年發(fā)生表明需加強對該毒素的常規(guī)監(jiān)測,以保障水庫供水安全.

4.2 展望

本研究表明基于N8單克隆抗體的直接競爭ELISA與Beacon公司試劑盒對CYN的測值高度一致,且我們新建立的ELISA具更寬的線性范圍.由于目前國外的Abraxis和Beacon商業(yè)試劑盒雖然靈敏度高、穩(wěn)定性好,但價格極為昂貴,在進行大量樣品的監(jiān)測時成本較高.我們的目標是進一步優(yōu)化新建立的ELISA方法,提高試劑的穩(wěn)定性,增加試劑的保存期,逐步將本研究開發(fā)的ELISA方法向商業(yè)試劑盒的方向推進,最終實現(xiàn)CYN檢測的ELISA試劑盒的進口替代.

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Establishment and assessment of a competitive direct enzyme-linked immunosorbent assay (cdELISA) for the determination of cylindrospermopsin.

YANG Dan, LIU Jin-ping, LAI Shu-yan, XIAO Li-juan, LEI La-mei*

(Department of Ecology, Jinan University, Guangzhou 510632, China)., 2023,43(10)：5543～5549

Cylindrospermopsins(CYN) has been widely concerned in the world for its high toxicity to humans and animals. The establishment of a rapid detection method can provide an effective tool for the risk assessment of CYN. Based on the monoclonal antibody N8 against CYN prepared, a direct competitive ELISA has been developed. After optimization of the dilution of HRP-CYN conjugate and N8 monoclonal antibody, the newly developed ELISA exhibited a typical sigmoidal response for CYN at concentrations of 0～200ng/mL, with a wide quantitative range between 0.1 and 10ng/mL by using logit-log method for fitting the curve. The detection limit of the method was calculated to be 0.069ng/mL, which is well below guideline value of 0.7ng/mL CYN recommended by WHO. Recoveries ranging from 80.44% to 125.4% and 52.97% to 155.19% were observed upon analyzing CYN-spiked water samples anddiluent, respectively. Moreover, CYN concentrations obtained from our cdELISA method through testing different samples was highly consistent with the Beacon ELISA kit. It was found that CYN can be detected year around in Dashahe reservoir but never beyond the guideline value of 0.7ng/mL.Therefore, the developed ELISA will achieve the rapid detection of CYN in water samples and algae samples and may provide a reliable technical tool for drinking water safety.

direct competitive ELISA；cylindrospermopsin；monoclonal antibody；HRP-CYN；spiked recovery

X171.5;X835

A

1000-6923(2023)10-5543-07

2023-02-20

廣東省自然科學基金項目(2023A1515012361);國基自然科學基金項目(31770507)

* 責任作者, 副教授, tleilam@jnu.edu.cn

楊 丹(1997-),女,湖南邵陽人,暨南大學碩士研究生,主要從事藍藻毒素免疫學檢測相關(guān)研究.914884242@qq.com.

楊 丹,劉金平,賴淑燕,等.直接競爭ELISA檢測擬柱孢藻毒素的方法建立和評價 [J]. 中國環(huán)境科學, 2023,43(10):5543-5549.

Yang D, Liu J P, Lai S Y, et al. Establishment and assessment of a competitive direct enzyme-linked immunosorbent assay (cdELISA) for the determination of cylindrospermopsin [J]. China Environmental Science, 2023,43(10):5543-5549.