邴 欣， 李 鵬， 王 軍， 汝少國??

(1.山東省產品質量檢驗研究院，山東 濟南 250102；2.山東省環(huán)境保護科學研究設計院，山東 濟南 250013；

斑馬魚卵黃原蛋白的純化鑒定及其2種ELISA的開發(fā)?

邴 欣1， 李 鵬2， 王 軍3， 汝少國3??

(1.山東省產品質量檢驗研究院，山東 濟南 250102；2.山東省環(huán)境保護科學研究設計院，山東 濟南 250013；

3.中國海洋大學海洋生命學院，山東 青島 266003)

斑馬魚卵黃原蛋白(Vitellogenin, VTG)是檢測環(huán)境雌激素活性的重要生物標志物。為了確定最佳的斑馬魚VTG檢測方法，本研究利用純化的斑馬魚VTG及其多克隆抗體同時建立了競爭ELISA與夾心ELISA，并比較了2種方法對VTG的敏感度與精確度。采用凝膠過濾與離子交換層析相結合的方法從17β-雌二醇暴露后的斑馬魚整體勻漿液中純化獲得了2種高分子量的糖磷脂蛋白，SDS變性電泳顯示分子量為180與143 kDa的兩條主帶，證實純化的蛋白為斑馬魚VTG。Western blot結果表明制備的抗血清對斑馬魚VTG具有很高的特異性，利用純化的VTG及其多克隆抗體建立了定量斑馬魚VTG的競爭ELISA和夾心ELISA。與競爭ELISA相比，夾心ELISA不需要抗原抗體共孵育的過程，操作更加省時、簡便，其工作范圍為3.9～250 ng/mL，檢出限約為2.2 ng/mL，組內與組間變異系數則分別為2.1～5.7和3.0～7.3，表現出更高的敏感度與精確度，推薦該方法用于環(huán)境雌激素活性的檢測。

斑馬魚；卵黃原蛋白；多克隆抗體；競爭ELISA；夾心ELISA

水環(huán)境中的野生動物表現出了性腺發(fā)育異常、性別比例失調與種群數量下降等跡象[1-2]。這些異?，F象的發(fā)生被證實與水體中的環(huán)境雌激素相關，該類物質能夠通過模擬內源雌激素，干擾野生動物的內分泌系統(tǒng)[3-4]。鑒于環(huán)境雌激素對水生生物的危害，經濟合作與發(fā)展組織(OECD)建立了以魚類為受試生物，以卵黃原蛋白(Vitellogenin, VTG)為主要指標的篩選測試導則[5]。斑馬魚(Daniorerio)是導則中推薦的3種模式魚種之一，已廣泛地應用于環(huán)境雌激素活性的檢測[6-7]。

斑馬魚VTG的檢測通常采用導則推薦的酶聯免疫吸附法(ELISA)，在這些ELISA方法，待測樣品均需要孵育過夜，操作步驟繁瑣，通常需要1～4 d才能完成樣品的測定[8-10]。魚類VTG是一種易降解的蛋白，孵育時間過長會加快樣品中VTG的降解，而降解后的VTG通常表現出更多的免疫源性，因此會造成樣品VTG的測定值偏高[11-12]。為了準確評價化學物質的雌激素活性，應該開發(fā)敏感、精確、簡便的斑馬魚VTG ELISA檢測方法。魚類VTG的ELISA方法主要有競爭ELISA與夾心ELISA兩種，其中競爭ELISA方法被更多的研究者所采用[13-14]。為了確定最佳的VTG檢測方法，本研究利用同一條件下純化的抗原，制備了高特異性的多克隆抗體，建立了斑馬魚VTG的競爭ELISA與夾心ELISA，比較了兩種方法的精確度與敏感度，以期為斑馬魚VTG在環(huán)境雌激素活性研究中的應用提供靈敏、簡便的檢測方法。

1 材料與方法

1.1 實驗材料

性成熟斑馬魚(平均體重(0.31±0.08)g，平均體長(2.9±0.2)cm)，雌、雄斑馬魚分開養(yǎng)殖于50 L水族箱中，實驗用水為連續(xù)暴氣24 h的自來水，水溫為(27±1)℃，溶解氧為(7.0±0.1) mg/L，光周期為16 h∶8 h。每天換水1/2，早晚投喂適量新鮮孵化的豐年蝦。

1.2 儀器和試劑

DYCZ-40B型轉印電泳槽為(北京六一儀器廠)，Multiskan MK3酶標儀(美國Thermo公司)，凝膠成像系統(tǒng)(上海培清科技有限公司)，HD21-2型紫外檢測儀(上海青浦滬西儀器廠)，AE-8130型電泳儀與AE-6450型電泳槽(日本Atto公司)，UV-2000型分光光度計(上海尤尼柯儀器有限公司)，TGL-16M臺式高速冷凍離心機(上海盧湘儀離心機儀器有限公司)。

17β-雌二醇(E2,純度≥90%)、抑肽酶、辣根過氧化物酶(HRP)、弗氏完全佐劑、弗氏不完全佐劑、牛血清白蛋白均為美國Sigma公司產品；Sephacryl S-300 high resolution型填料與DEAE-Sepharose Fast Flow型填料購自瑞典Amersham Biosciences公司；HitrapTMProtein G親和層析柱購自美國GE Healthcare公司；HRP標記山羊抗兔二抗IgG、DAB顯色劑與TMB單組分顯色劑購自Solarbio公司；SDS-PAGE分子量標準品購自美國Therom公司；其他試劑為進口分裝或國產分析純。

1.3 實驗方法

1.3.1 VTG的誘導與整體勻漿液的制備 采用水體暴露E2的方法誘導斑馬魚VTG的生成[9]。將10尾雄性斑馬魚與雌性斑馬魚分別置于5 L燒杯中，E2的暴露濃度為200 μg/L。每天將水全部換掉，并加入相應體積的E2。一周后，用MS-222麻醉斑馬魚，將雄魚和去除卵巢的雌魚稱重后裝入1.5 mL EP管，加入3倍體積含有2 mmol/L PMSF的勻漿緩沖液(25 mmol/L Tris-HCl,0.07 mol/L NaCl,pH=7.5)。在冰浴條件下用塑料研磨棒勻漿，8 000×g離心20 min，上清液經0.45 μm過濾后，用于VTG的分離純化。

1.3.2 VTG的分離純化 采用此前報道的方法[15]略加修改。簡言之，將1 mL E2誘導組斑馬魚與對照組斑馬魚勻漿液分別進行凝膠過濾層析，將可能含有VTG的洗脫組分收集，進一步進行陰離子交換層析。結合的蛋白用分別含有0.1、0.2和1 mol/L NaCl的25 mmol/L Tris-HCl緩沖液(pH=7.5)洗脫，收集0.2 mol/L NaCl下的洗脫峰，裝入離心超濾管。3 000×g離心20 min后，將超濾樣品用0.01 mol/L PBS緩沖液透析24 h。純化的VTG利用Brandford法，以牛血清白蛋白為標準品測定蛋白濃度后，保存于-80 ℃。

1.3.3 電泳 Native-PAGE(4%～7.5%)按照Davis[16]的方法，上樣量10 μL，樣品與緩沖液(0.20 mol/L Tris-HCl, pH=6.8,25%甘油,0.1%溴酚蘭)等體積混合，4 ℃電泳，電壓為150 V。電泳結束后，對凝膠分別進行考馬斯亮藍染色(CBB)、Schiff試劑，甲基綠和蘇丹黑B染色。

SDS-PAGE(4%～9%)按照Laemmli[17]的方法，樣品與樣品緩沖液(0.16 mol/L Tris-HCl,pH=6.8,25%甘油,0.1%溴酚蘭,4% SDS,5%巰基乙醇)等體積混合，上樣前煮沸5 min，室溫下電泳，電壓200 V。電泳結束后，進行考馬斯亮藍染色，采用未預染蛋白Marker測定蛋白的分子量。

1.3.4 多克隆抗體的制備 將800 μg純化的VTG與弗氏完全佐劑(初次注射)或不完全佐劑(接下來的4次注射)等體積混勻后，皮下多點注射新西蘭大白兔。當效價達到要求后取血，分離獲得抗血清。向3 mL抗血清中加入1 mL對照雄魚勻漿液，以去除非特異性成分，混合物在冰浴條件下震蕩4 h后，離心(5 000×g,5 min,4 ℃)，收集上清。隨后利用Protein G親和層析柱從抗血清中純化獲得IgG組分。

1.3.5 Western blot 將雄魚勻漿液、雌魚勻漿液與純化的VTG各10 μL進行SDS-PAGE后，電轉至PVDF膜，利用Towbin等[18]的方法進行Western blot。一抗為1∶1 000倍稀釋的兔抗VTG多克隆抗血清，二抗為1∶2 000倍稀釋的HRP標記羊抗兔IgG。檢測采用新鮮配制的DAB底物顯色液，待條帶清晰時，用蒸餾水洗膜終止反應。

1.3.6 兩種ELISA的建立與驗證

1.3.6.1 競爭ELISA的建立 采用Roy等[19]的方法略加修改。將純化的VTG經碳酸鈉包被緩沖液(0.05 mol/L,pH=9.6)稀釋至200 ng/L后，按100 μL/孔加入96孔板中，4 ℃孵育過夜；次日，以200 μL PBST(0.01 mol/L PBS，0.05% Tween-20)洗板3次后，每孔加入200 μL封閉緩沖液(PBST-2% BSA)，37 ℃封閉2 h；再次洗板3次后，每孔加入50 μL VTG標準品溶液(1.95～2 000 ng/mL)或稀釋后的斑馬魚整體勻漿液，再加入50 μL 1∶5 000倍稀釋的斑馬魚多克隆抗體，搖晃3 min后，將板置于37 ℃孵育2 h；PBST洗板3次，向每孔加入100 μL 1∶2 000倍稀釋的HRP標記羊抗兔IgG，37 ℃孵育1 h；PBST洗板5次后，每孔加入100 μL TMB單組分顯色液，37 ℃顯色10 min后，加入100 μL 2N H2SO4，終止顯色，測定450 nm的吸光值。最佳的抗原包被濃度與一抗稀釋倍數在預實驗中確定。

ELISA吸光值采用Lomax等[13]的方法轉化為百分結合率(B%)，將B(%)VsVTG濃度的Log值線性化獲得標準曲線，以20%～80%結合率之間的VTG濃度作為ELISA的工作范圍。

1.3.6.2 夾心ELISA的建立 HRP的標記與夾心ELISA的建立采用此前報道的方法[15]，HRP標記抗體用作夾心ELISA的二抗。首先，向96孔板中加入100 μL純化的VTG抗體(5 μg/mL)，4 ℃包被過夜；用0.1% Tween-20的PBS(PBST)洗板3次后，每孔加入200 μL封閉緩沖液(2% BSA-PBST)，37 ℃封閉1 h；向板孔中加入100 μL純化的VTG(1.95～500 ng/mL)或稀釋后的斑馬魚整體勻漿液，于37 ℃孵育1 h。

用TPBS洗板5次后，加入1∶5 000稀釋HRP-標記抗體，于37 ℃孵育1 h。洗板5次后，每孔加入100 μL TMB單組分顯色液，37 ℃顯色10 min后，加入100 μL 2 mol/L H2SO4終止反應，測定450 nm波長下的吸光值。標準品與樣品各設2個平行，HRP標記抗體的最佳稀釋倍數經預實驗確定。

1.3.7 ELISA的性能驗證 精確度通過組內差異與組間差異來評價。組內差異：將不同濃度的VTG標準品在同一酶標板上進行8個重復，測定其吸光值；組間差異：將VTG標準品加入到6塊不同的酶標板上，進行ELISA，測定其吸光值。利用公式CV%=(S.D./mean)×100計算組內與組間的變異系數(CV)。

檢出限：競爭ELISA的檢出限定義為90%結合率所對應的標準品濃度[20]；夾心ELISA的檢出限定義為12個0標準品吸光值的平均值加上兩倍標準差所對應的標準品濃度[21]。

特異性：將連續(xù)稀釋的對照組雄魚與E2誘導雄魚勻漿液進行ELISA，比較它們的稀釋曲線與VTG標準

曲線的平行性。

2 結果

2.1 斑馬魚VTG的純化與鑒定

凝膠過濾與離子交換層析結果如圖1所示，E2誘導組雄魚與去除卵巢雌魚的勻漿液在80 mL洗脫體積處出現了洗脫峰，而對照組雄魚和去除卵巢的雌魚勻漿液在該洗脫體積處未檢測到洗脫峰，收集該峰洗脫液進行離子交換層析，在0.2 mol/L NaCl洗脫下出現主洗脫峰。

Native-PAGE結果如圖2所示，與對照組雄魚相比，E2誘導組雄性斑馬魚勻漿液增加了兩條清晰的條帶，并且兩步層析法成功純化出了E2誘導后的蛋白。經計算，斑馬魚VTG的天然分子量分別為483與458 kDa，并且還檢測到一條分子量約為236 kDa的條帶。

特異性染色顯示，純化的2種蛋白能夠同時被蘇丹黑B、Schiff試劑和甲基綠著色(見圖3)，表明它們均含有糖磷脂基團。

( A,B. 凝膠過濾圖譜；C. 離子交換圖譜。A and B. Sephacryl S-300 column chromatograph; C. DEAE-sepharose chromatograph.)

圖1 斑馬魚勻漿液的洗脫圖譜

Fig.1 Elution profiles of zebrafish whole-body homogenates

(M.蛋白Marker；1.對照雄魚勻漿液；2.E2誘導雄魚勻漿液；3,4.凝膠過濾層析第二峰；5.離子交換層析的主洗脫峰(0.2 mol/L NaCl)。M.Protein Marker；Lane； 1.WBH from control male; lane 2.WBH of E2-treated male; lanes 3 and 4.VTG fractions from Sephacryl S-300 chromatography; lane 5.VTG fractions from DEAE-Sepharose chromatography.)

圖2 斑馬魚勻漿液與層析洗脫液的電泳結果

Fig.2 (A) Native-PAGE (4%～7.5%) of whole body

homogenate (WBH) and fractions of zebrafish WBH

(1.脂蛋白染色；2.糖蛋白染色；3.磷蛋白染色。1.Lipoprotein stained by Sudan black B; 2.Glycoportein stained by Schiff reagent; 3.Phosphoprotein stained by Methyl green.)

圖3 斑馬魚VTG的特異性染色結果

Fig. 3 Determination of carbohydrate, phosphorus,

and lipid components in Zebrafish VTG

2.2 抗體的特異性

在SDS-PAGE中，VTG顯示分子量為143與180 kDa的2條主帶，以及3條分子量分別為114、103和96 kDa的模糊條帶(見圖4A)。Western blot結果顯示，制備的抗血清能夠與E2誘導組雄性斑馬魚勻漿液和純化的VTG反應，檢測到相似的條帶，而與雄魚勻漿液無任何反應(見圖4B)。

(A. 蛋白Marker(條帶1)與純化VTG(條帶2)的SDS-PAGE電泳結果; B. 對照組雄魚(條帶1)，E2誘導組雄魚(條帶2)與純化VTG(條帶3)的Western blot結果。A. SDS-PAGE pattern of protein marker (Lane 1) and purified VTG (Lane 2); B. Western blot result of homogenates from E2-treated male zebrafish (Lane 1), control male zebrafish (Lane 2), and purified VTG (Lane 3).)

圖4 抗體的特異性分析結果

Fig. 4 Specific analysis of rabbit anti-VTG antibody

2.3 斑馬魚VTG 2種ELISA的建立與比較

以包被抗原的濃度為200 ng/L、一抗稀釋倍數為1∶5 000為最佳反應條件建立了競爭ELISA(見圖5)。該反應的檢出限(90%結合率)約為20 ng/mL，工作范圍為31.25～1 000 ng/mL(y=-41.48x+144.6,R2=0.992)，并且在該工作范圍內E2誘導雄魚勻漿液的連續(xù)稀釋曲線和VTG標準曲線表現出很好的平行性，而雄魚勻漿液與抗體沒有交叉反應。

以anti-VTG IgG包被濃度為5 μg/mL，HRP標記抗體稀釋倍數為1∶5 000建立了最佳的夾心ELISA(見圖6)，其檢出限約為2.2 ng/mL，工作范圍為3.9～250 ng/mL(y=1.399x-0.654,R2=0.989)。同樣，E2誘導雄魚勻漿液的連續(xù)稀釋曲線與VTG標準曲線表現出很好的平行性，而雄魚勻漿液未檢測到VTG。

(A. 斑馬魚VTG競爭ELISA的標準曲線；B. 對照組和E2誘導雄魚勻漿液的稀釋曲線。A. A representative standard curve obtained for zebrafish VTG in the developed competitive ELISA; B. WBH dilution curves of male and E2-induced male zebrafish in the competitive ELISA.)

圖5 斑馬魚VTG的競爭ELISA結果

Fig. 5 A competitive ELISA for zebrafish VTG

(A. A representative standard curve for zebrafish VTG in the developed sandwich ELISA; B. WBH dilution curves of male and E2-induced male zebrafish in the sandwich ELISA.)

圖6 斑馬魚VTG的夾心ELISA結果

Fig. 6 A sandwich ELISA for zebrafish VTG

2種ELISA的精確度測定結果如表1所示，競爭ELISA的組內與組間變異系數分別為3.4～7.8和4.3～9.6；夾心ELISA的組內與組間變異系數則分別為2.1～5.7和3.0～7.3。

3 討論

本研究利用純化的VTG與制備的多克隆抗體同時建立了斑馬魚VTG的競爭ELISA與夾心ELISA，并比較了兩種方法的檢測性能。斑馬魚VTG的純化與特異性抗體的制備是ELISA建立的前提。本研究利用兩步層析法成功純化出了斑馬魚由E2誘導后生成的蛋白，特異性染色結果顯示兩種蛋白均為糖磷脂蛋白，符合魚類VTG的共同特征[15,19]，SDS-PAG測得純化蛋白的2個主要亞基分子量分別為143與180 kDa，這與Fenske等[8]報道的斑馬魚VTG亞基分子量(142與171 kDa)相近，因此可以判定純化的蛋白為斑馬魚VTG。此外，電泳結果還顯示一條分子量較小的條帶，并且該條帶也是E2誘導后生成的糖磷脂蛋白。

表1 競爭ELISA與夾心ELISA的精確度測定

大量的研究已經證實魚類VTG是一種非常不穩(wěn)定的蛋白，在純化過程容易發(fā)生降解，部分二聚體降解為單體，形成低分子量的條帶[22-23]。因此，推測該條帶是斑馬魚VTG的降解條帶。將純化的VTG免疫新西蘭大白兔制備抗血清，Western blot結果顯示制備的抗血清能夠與純化的VTG和E2暴露組雄魚勻漿液反應，而與對照組雄魚勻漿液無交叉反應，表明抗體對VTG具有較高的特異性[14,24]，可以用于斑馬魚VTG ELISA的開發(fā)。

本研究建立的競爭ELISA檢出限約為20 ng/mL，具有較寬的工作范圍(31.25～1 000 ng/mL)，這與普氏鰈[19](Pleuronectesputnami)和泥鰍[25](Misgurnusangaillicaudatus)VTG的競爭ELISA相近；夾心ELISA的工作范圍為3.9～250 ng/mL，檢出限約為2.2 ng/mL，明顯低于競爭ELISA的檢出限。特異性檢測顯示，E2誘導雄魚勻漿液的連續(xù)稀釋曲線與VTG標準曲線表現出很好的平行性，而雄魚勻漿液與抗體沒有交叉反應，證實建立的兩種ELISA都可以用于斑馬魚勻漿液中VTG的定量；兩種ELISA的組內差異與組間差異均小于10%，與其它魚類VTG ELISAs的結果相近[10,14,25]，并且夾心ELISA的組內與組間差異明顯低于競爭ELISA，表明夾心ELISA方法具有更高精確度與敏感度，這可能與其測定程序有關。夾心ELISA不需要樣品中VTG與包被的VTG競爭結合抗體，而是通過包被的抗體直接捕獲樣品中的VTG，隨后HRP標記抗體與捕獲的VTG結合，起到信號放大功能，從而提高了檢測敏感度；另外夾心ELISA不包括抗原與抗體共孵育過程，操作相對簡便，有助于保證該方法的精確度。在此之前的研究為了提高夾心ELISA的敏感度，通常將待測樣品孵育過夜[10]，卻忽視了樣品中VTG在該過程中的降解，從而增加了測定的誤差。本研究建立的夾心ELISA將樣品反應時間縮短至1 h，可減少樣品VTG在檢測過程中的降解。因此，高精確度、高敏感度的夾心ELISA能更加準確地定量VTG。

綜上，本研究利用純化的斑馬魚VTG及其多克隆抗體建立了夾心ELISA與競爭ELISA檢測方法，發(fā)現夾心ELISA具有更高的敏感度與精確度，更加適用于VTG的準確定量，推薦該方法用于環(huán)境雌激素活性的檢測。

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責任編輯 高 蓓

Vitellogenin inDaniorerio: Purification, Characterization,and Development of Competitive ELISA and Sandwich ELISA

BING Xin1, LI Peng2, WANG Jun3, RU Shao-Guo3

(1.Shandong Institute for Product Quality Inspection, Jinan 250102, China; 2.Shandong Academy of Environmental Science, Jinan 250013, China; 3.College of Marine Life, Ocean University of China, Qingdao 266003, China)

Vitellogenin (VTG) in zebrafish (Daniorerio) is a core biomarker for screening estrogenic activity of chemicals in the test guidelines of the Organization for Economic Co-operation and Development. Piscine VTG is normally quantified by the enzyme-linked immunosorbent assay (ELISA) including competitive ELISA and sandwich ELISA. Among them, competitive ELISA was the most commonly used method. To determine the optimal method for accurate quantification of zebrafish VTG, both competitive ELISA and sandwich ELISA were developed and their performances were compared in this study. First, VTG was purified from the whole body homogenate (WBH) of zebrafish treated with 17β-estradiol (E2) by gel filtration followed by anion-exchange chromatography. The purified proteins were subjected to native polyacrylamide gel electrophoresis (Native-PAGE) and stained positively with methyl green, Sudan black B, and Schiff’s reagent, thus they were characterized as phospholipoglycoproteins. In sodium dodecyl sulfate-PAGE (SDS-PAGE), the purified proteins were separated into two major polypeptides corresponding to 180 and 143 kDa, which was similar to other reports on the VTGs. Therefore, the purified proteins were identified as zebrafish VTGs. Then, the purified VTGs were used to immunize rabbits by intraperitoneal injection to prepare anti-VTG polyclonal antibody. Western blot revealed that the prepared antibody reacted with homogenate from E2-treated male zebrafish and purified VTG, but no cross-reaction was observed in the homogenate from control male zebrafish, indicating that the antibody was highly specific to zebrafish VTG. Using the purified VTG and polyclonal antibody, competitive ELISA and sandwich ELISA for quantifying VTG concentrations in zebrafish were developed. The specificity test showed that VTG standard curves of both ELISAs were parallel to curves of WBH from E2-treated males, while there was no cross-reactivity with WBH from control males, confirming that both ELISAs could specifically quantify VTG concentrations of WBH in zebrafish. The competitive ELISA had a detection limit of 20 ng/mL and a working range from 31.25 to 1 000 ng/mL, while the sandwich ELISA had a working range from 3.9 to 250 ng/mL and a detection limit of 2.2 ng/mL, which was lower than the competitive ELISA. Moreover, the sandwich ELISA was simple and time-saving because it did not include the preincubation protocol of samples. Additionally, the intra- and inter-assay coefficients of variations in the sandwich ELISA were 2.1%～5.7% and 3.0%～7.3%, respectively, which were lower than the values of competitive ELISA, revealing that the sandwich format had a higher precision. This study revealed that the simple and highly sensitive sandwich ELISA was suitable for the accurate detection of VTG inductions in zebrafish exposed to environmental estrogens.

zebrafish; vitellogenin; polyclonal antibody; competitive ELISA; sandwich ELISA

山東省自然科學基金重點項目(ZR2014DZ001)資助 Supported by the Shandong Province Natural Science Foundation of China (ZR2014DZ001)

2016-06-23；

2016-08-26

邴 欣(1977-)，男，高級工程師，博士，研究方向為生態(tài)毒理學。E-mail: bingxinhaida@163.com

?? 通訊作者：E-mail: rusg@ouc.edu.cn

X835

A

1672-5174(2017)07-056-07

10.16441/j.cnki.hdxb.20160223

邴欣， 李鵬， 王軍, 等. 斑馬魚卵黃原蛋白的純化鑒定及其2種ELISA的開發(fā)[J]. 中國海洋大學學報(自然科學版), 2017, 47(7): 56-62.

BING Xin, LI Peng, WANG Jun, et al. Vitellogenin inDaniorerio: Purification, characterization, and development of competitive ELISA and sandwich ELISA[J]. Periodical of Ocean University of China, 2017, 47(7): 56-62.