中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2010年
3期
250-254
,共5页
韩纪举%潘少东%吴亚平%de GROOT Evelyn%de GROOT Philip G%王云%蔡洪信%赵晓民%夏作理
韓紀舉%潘少東%吳亞平%de GROOT Evelyn%de GROOT Philip G%王雲%蔡洪信%趙曉民%夏作理
한기거%반소동%오아평%de GROOT Evelyn%de GROOT Philip G%왕운%채홍신%조효민%하작리
von%Willebrand因子%酶联免疫吸附测定%紫癜,血小板减少性
von%Willebrand因子%酶聯免疫吸附測定%紫癜,血小闆減少性
von%Willebrand인자%매련면역흡부측정%자전,혈소판감소성
von Willebrand factor%Enzyme-linkedimmunesorbentassay%Purpura,thrombocytopenic
目的 建立一种测定血浆活化vWF的双抗体夹心方法 .方法 利用骆马来源的抗-活化vWF抗体(一抗),辣根过氧化物酶标记兔抗人vWF多克隆抗体(二抗)建立双抗体夹心方法 .随机选取健康人66名,糖尿病患者30例、vWD-2B 10例、先天性血栓性TTP5例、后天性血栓性血小板减少性紫癜13例、疟疾13例进行血浆活化vWF的测定.以基于AU/vWFa-11抗体酶标方法 测得的代表活化vWF浓度的A值与vWF抗原浓度的比值来表示活化vWF.同时在62、125、250μg/L 3个不同的vWF抗原浓度情况下测定代表活化vWF浓度的A值并使用GraphPad Prism 4软件处理数据,在达到良好线性回归的前提下计算样本斜率/标准血浆斜率比值即血浆活化vWF.根据该数值的大小表示血浆vWF的活化程度.结果 用双抗体夹心测定血浆活化vWF法检测高、中、低浓度3份不同标准品的批内CV分别为4.21%、4.98%、6.32%;批间CV分别为6.34%、7.02%、7.69%.健康对照组血浆活化血友病因子测定值为0.74±0.35,健康对照组中男性与女性的血浆vWF抗原浓度分别为(7.29±5.15)×10~3μg/L和(9.42±4.67)×10~3μg/L,活化vWF分别为0.74±0.34和0.73±0.35,两组的差异均无统计学意义(t分别为1.352和0.04,P均＞0.05).在不同年龄组间,≥40岁组血浆vWF抗原浓度[(10.64±5.39)×10~3μg/L]高于＜40岁组[(6.11±2.84)×10~3μg/L],差异有统计学意义(t=8.24,P＜0.01);而血浆活化vWF在两年龄组间差异无统计学意义[≥40岁组0.70±0.38,＜40岁组0.78±0.29,t:1.02,P＞0.05].而糖尿病组、vWD-2B、先天性TTP、后天性TTP、疟疾等各疾病组血浆活化vWF水平比健康对照组明显增高(1.07±0.66、6.52±1.30、5.82±1.47、1.53±0.11、2.17±0.53),差异有统计学意义(F=10.23,P＜0.01).结论 成功地建立了一种测定血浆活化vWF的双抗体夹心方法 ,可以用来表示疾病状态时vWF的真实活化状态,为分析引起血浆活化vWF升高的机制提供重要依据.
目的 建立一種測定血漿活化vWF的雙抗體夾心方法 .方法 利用駱馬來源的抗-活化vWF抗體(一抗),辣根過氧化物酶標記兔抗人vWF多剋隆抗體(二抗)建立雙抗體夾心方法 .隨機選取健康人66名,糖尿病患者30例、vWD-2B 10例、先天性血栓性TTP5例、後天性血栓性血小闆減少性紫癜13例、瘧疾13例進行血漿活化vWF的測定.以基于AU/vWFa-11抗體酶標方法 測得的代錶活化vWF濃度的A值與vWF抗原濃度的比值來錶示活化vWF.同時在62、125、250μg/L 3箇不同的vWF抗原濃度情況下測定代錶活化vWF濃度的A值併使用GraphPad Prism 4軟件處理數據,在達到良好線性迴歸的前提下計算樣本斜率/標準血漿斜率比值即血漿活化vWF.根據該數值的大小錶示血漿vWF的活化程度.結果 用雙抗體夾心測定血漿活化vWF法檢測高、中、低濃度3份不同標準品的批內CV分彆為4.21%、4.98%、6.32%;批間CV分彆為6.34%、7.02%、7.69%.健康對照組血漿活化血友病因子測定值為0.74±0.35,健康對照組中男性與女性的血漿vWF抗原濃度分彆為(7.29±5.15)×10~3μg/L和(9.42±4.67)×10~3μg/L,活化vWF分彆為0.74±0.34和0.73±0.35,兩組的差異均無統計學意義(t分彆為1.352和0.04,P均＞0.05).在不同年齡組間,≥40歲組血漿vWF抗原濃度[(10.64±5.39)×10~3μg/L]高于＜40歲組[(6.11±2.84)×10~3μg/L],差異有統計學意義(t=8.24,P＜0.01);而血漿活化vWF在兩年齡組間差異無統計學意義[≥40歲組0.70±0.38,＜40歲組0.78±0.29,t:1.02,P＞0.05].而糖尿病組、vWD-2B、先天性TTP、後天性TTP、瘧疾等各疾病組血漿活化vWF水平比健康對照組明顯增高(1.07±0.66、6.52±1.30、5.82±1.47、1.53±0.11、2.17±0.53),差異有統計學意義(F=10.23,P＜0.01).結論 成功地建立瞭一種測定血漿活化vWF的雙抗體夾心方法 ,可以用來錶示疾病狀態時vWF的真實活化狀態,為分析引起血漿活化vWF升高的機製提供重要依據.
목적 건립일충측정혈장활화vWF적쌍항체협심방법 .방법 이용락마래원적항-활화vWF항체(일항),랄근과양화물매표기토항인vWF다극륭항체(이항)건립쌍항체협심방법 .수궤선취건강인66명,당뇨병환자30례、vWD-2B 10례、선천성혈전성TTP5례、후천성혈전성혈소판감소성자전13례、학질13례진행혈장활화vWF적측정.이기우AU/vWFa-11항체매표방법 측득적대표활화vWF농도적A치여vWF항원농도적비치래표시활화vWF.동시재62、125、250μg/L 3개불동적vWF항원농도정황하측정대표활화vWF농도적A치병사용GraphPad Prism 4연건처리수거,재체도량호선성회귀적전제하계산양본사솔/표준혈장사솔비치즉혈장활화vWF.근거해수치적대소표시혈장vWF적활화정도.결과 용쌍항체협심측정혈장활화vWF법검측고、중、저농도3빈불동표준품적비내CV분별위4.21%、4.98%、6.32%;비간CV분별위6.34%、7.02%、7.69%.건강대조조혈장활화혈우병인자측정치위0.74±0.35,건강대조조중남성여녀성적혈장vWF항원농도분별위(7.29±5.15)×10~3μg/L화(9.42±4.67)×10~3μg/L,활화vWF분별위0.74±0.34화0.73±0.35,량조적차이균무통계학의의(t분별위1.352화0.04,P균＞0.05).재불동년령조간,≥40세조혈장vWF항원농도[(10.64±5.39)×10~3μg/L]고우＜40세조[(6.11±2.84)×10~3μg/L],차이유통계학의의(t=8.24,P＜0.01);이혈장활화vWF재량년령조간차이무통계학의의[≥40세조0.70±0.38,＜40세조0.78±0.29,t:1.02,P＞0.05].이당뇨병조、vWD-2B、선천성TTP、후천성TTP、학질등각질병조혈장활화vWF수평비건강대조조명현증고(1.07±0.66、6.52±1.30、5.82±1.47、1.53±0.11、2.17±0.53),차이유통계학의의(F=10.23,P＜0.01).결론 성공지건립료일충측정혈장활화vWF적쌍항체협심방법 ,가이용래표시질병상태시vWF적진실활화상태,위분석인기혈장활화vWF승고적궤제제공중요의거.
Objective To establish an double-antibody sandwich method to detect the amount of active vWF in the plasma of healthy people and diabetic patients.Methods A double-antibody sandwich method has been established using llama-derived anti-active vWF antibody and horseradish peroxidaselabelled rabbit anti-human vWF polyclonal antibodies.The study randomly selected 66 healthy people,30 diabetic patients,10 cases of von Willebrand disease 2B(vWD-2B),5 cases of congenital thrombotic thrombocytopenic purpura(TIP),13 cases of acquired TIPs and 13 cases of malaria.The activated vWF level were described as the ratio of absorbance value(A)representing activated vWF value using AU/vWFa-11 antibody-based ELISA method to the concentrations of vWF antigen.At the same time,the absorbance values representing the concentrations of active vWF were measured when the concentrations of vWF antigen were 62,125,250μg/L respectively.The data were analyzed with GraphPad Prism 4 software.Under the premise of reaching a good linear regression,plasma active vWF values were calculated as the slope ratio of the sample plasma to the standard plasma.Accordingly to the ratio evaluated the levels of active vWF in plasma.Results The coefficients of variation(CVs)for intra assays were 4.21%,4.98% and 6.32% respectively.The CVs for interassays were 6.34%,7.02% and 7.69% respectively.In healthy adults,the level of active vWF in plasma was 0.74±0.35.There was no significant difference of the vWF levels[male (7.29±5.15)×10~3μg/L,female(9.42±4.67)×10~3μg/L]and active vWF levels(male 0.74±0.34,female 0.73±0.35)between male and female in healthy people(t=1.35,0.04,P＞0.05).The vWF levels of the group over 40 years old[(10.64±5.39)×10~3μg/L]were higher than these below 40 years old[(6.11 ±2.84)×10~3 μL)(t=8.24,P ＜ 0.01)],but there was no significant difference of the active vWF levels difference between these groups(over 40 years old 0.70±0.38,under 40 years old 0.78 ± 0.29,t=1.02,P＞0.05).The plasma levels of active vWF in vWD-2B type,congenital TIP,acquired TIP,malaria and diabetic groups(1.07±0.66,6.52±1.30,5.82±1.47,1.53±0.11,2.17±0.53)were higher than those in the control group(F=10.23,P ＜ 0.01).Conclusions Double-antibody sandwich method for detection of active vWF levels in plasma had good reproducibihty and specificity,which could be used to indicate the true active vWF levels under disease state.It could also provide an important contribution to analyze the mechanism for the active vWF in plasma.
