硝酸纤维素膜应用

1、YYYYYYLFIA Lateral Flow Immunochromatographic Assay strip横向流动的免疫诊断试剂条LFIA strip 免疫层析诊断试剂条Plastic backing塑料衬垫Cellulose nitrate membrane硝酸纤维素膜Conjugate pad结合垫Sample prefilter pad样品垫Absorbent pad吸水垫YYYTest line检测线YYYControl line对照线YYYYYYYYYYYYYYYYYYConjugate pad containing gold or latex particles with

2、conjugate antibody结合垫包含了底物颗粒如金标结合物颗粒或乳胶结合物颗粒YGold particles around 40nm胶体金颗粒约40nmLatex particles from 0.2m to 0.5m乳胶颗粒约0.2m - 0.5mYConjugate particles 底物颗粒YYYYSample flow into the conjugate pad样品流经结合垫YYYYYYYYYYYYYYYYYThe cellulose nitrate membrane is at the heart of the LFIA硝酸纤维素膜位于试剂条的心脏部位YCapture

3、antibodyYYYYYYYYYYYYYYYYYYYYYYYYYYYUniSart Cellulose Nitrate MembraneConsistency by Design !UniSart 硝酸纤维素膜均一品质源自完美设计Membrane role is critical, it : 膜的作用非常关键：binds antibodies on capture and control lines 吸附抗体：将抗体吸附在检测线和对照线上Drives the test by capillary force 免疫反响的载体：在毛细管力作用下，液体在膜上爬行，从而带动一系列反响的发生Allows

4、 to read on its surface a clear signal 提供清晰的背景：抗体线的结果信号，是以膜的外表为背景来判读的Cellulose nitrate is the polymer of choice硝酸纤维素是首选材质Cellulose nitrate membrane have unique properties :硝酸纤维素的特性 High protein binding capacity, due to high internal surface 蛋白吸附能力强，因其内部外表积大 ( 30g to 50g per cm) High and rapid bindin

5、g force through electrostatic and hydrophobic interactions ( protein binding is immediate) 吸附力来自静电和疏水性反响，吸附力强并且速度快蛋白的吸附是瞬间 完成的Fast capillary action through large pore size structure ( 8 to 10m wide open cell) 大孔径结构，毛细反响快 8 to 10m 开放式大孔结构Long history of preferred substrate for protein binding and imm

6、uno reaction ( western blot, dot blot)应用于蛋白吸附和免疫反响方面，已具有悠久历史Membrane most common parameters 膜的根本参数: Capillary speed 爬速 Thickness 厚度 Surfactant 外表活性剂 Protein binding 蛋白吸附能力 Surface characteristics 外表特性Intra and inter lot reproducibility批次内和批次间的可重复性Consistent capillary time爬速的一致性YYYYYYYYYYYYYYYYFast M

7、embrane lot爬速较快的膜Slow membrane lot爬速较慢的膜Capillary time controls reaction rate on the capture line爬速控制了反响线上的反响程度 Membrane thickness 膜的厚度Different membrane thickness 不同厚度的膜Line width线宽度For the same dispensed volume of antibody solution ,different membrane thickness , along or across the master roll ,w

8、ill give different line width !抗体的喷涂量相同时，膜的厚度不同，所得到的抗体线的宽度也不同10l10 l Membrane thickness and surface quality膜的厚度以及外表质量Uniform thickness and clean surface will give you uniform line厚度均匀、外表干净的膜，才能得到均匀的划线When developing new LFIA the question of the consistency of all the components, in particular the me

9、mbrane is the most important.当开发新的横向流免疫诊断产品时，所有组成材料的均一性，尤其是膜的均一性，是非常重要的Follow the consistency line at Sartorius遵循Sartorius的均一性原那么How to make a good and consistent membrane ?如何制造优质的膜？Cellulose nitrate membrane manufacturing 硝酸纤维素膜的生产From wood to high tech membrane 从木材到高科技的膜Homogeneous polymer solutio

10、n均匀的聚合物溶液phase inversion 相转化:porous Structure多孔结构polymer-rich phase富含聚合物的相polymer-lean phase不含聚合物的相Liquid-Liquid Demixing 相转化法nitrateRaw material原材料:Polymers聚合物 & solvents溶剂Mixing tankUniSart Membrane manufacturing- casting processN2 N2Evaporation and solvent recycling溶剂蒸发和再循环N2 flow氮气Casting machin

11、e 制膜机Post treatment bath and final drying后处理槽和最后烘干Mixing tank混合罐Casting solution recipe新制膜溶液配方analytical selection of raw material原材料的分析选择Define physical key parameters that influence casting solution 对影响制膜溶液的主要物理参数进行明确定义Choose and audit our strategic suppliers regularly有方案地对供给商进行选择和审核Proceed to rig

12、orous incoming quality control steps严格执行进货过程的质控 Clean and open structure from the new casting solution 用新的制膜溶液制造的新型膜，具有干净和开放的结构 Able to generate high intra and inter lot consistency 批次间和批次内都能保证均一性Consistent structure 均一的结构inhomogenous structures 不均一的结构Lower consistency 可重复性差Globular 球状结构Dust 布满灰尘的结构

13、Large scale production on casting equipment大规模生产时的制膜设备Carefully slit the membrane 切膜步骤QQQQQQQLot approval批次放行Delivery 出厂Solvent tanks溶剂罐Semi finished area半成品区域Raw material原材料Mixing control混合步骤Casting line制膜机Slitting 切膜Consistency by design !There are many ways to adjust and tune reagents in order to

14、 get the right line shape and intensity 还有一些方法和试剂可以调节显色线形状和强度But before starting the development of new LFIA, it is important to be sure of the consistency of the different solid components 各组分质量的均一性是非常重要的Application Lab in Goettingen德国哥廷根应用实验室LFIA strip层析条Plastic backing塑料板Cellulose nitrate membran

15、e硝酸纤维素膜Conjugate pad结合垫Sample prefilter pad样品垫Absorbent pad吸附垫YYYTest line测试线YYYControl line控制线Manufacturing Lateral flow immunochromatographic assay strip:Prepare all your component准备层析条原材料Pre treat the prefilter pad ( sample pad)预处理样品垫Pre treat the conjugate pad 预处理结合垫Prepare the conjugate particl

16、es准备结合垫颗粒Stripe the capture antibodies and the control antibodies onto the membrane喷测试线和控制线抗体Block or not the membrane 封闭(或者不封闭)膜Dry the membrane膜的枯燥Stripe the particles onto the conjugate pad结合垫喷显色颗粒Assemble components装配Cut strips切割PlasticBacking card塑料板Prefilter sample 样品垫Conjugate pad结合垫Membrane膜

17、Absorbent pad吸附垫Prefilter sample padPrefilter sample padPaper or non woven, for particles clarification or blood separation无纺布或纸, 颗粒澄清或血液别离Conjugate padConjugate padUsually a glass fiber or polyester non woven玻璃纤维或者聚酯无纺布The cellulose nitrate membrane is the best substrate for LFIA硝酸纤维素膜A clean and o

18、pen structure is important.干净和开放结构,非常重要Pre treat sample padPre treatment is needed to adjust Ph or viscosity of the sample. It will greatly differs according to the type of fluid being tested: Whole blood or urine or else预处理调节PH和黏度, 对于不同样品,处理方法差异很大Typical blocking solution for urine will be :典型对于尿液样

19、品的处理方法0.5% to 1% BSA or casein 0.25% to 0.5% Tween 200.1M Tris Buffer ( MW 121.14 ) adjust ph 8,0 with HCLPre treat conjugate padPre treatment is needed so the conjugate particles can be stored on the pad and then can redisolve easily end be completely released目的是结合颗粒能够固定在结合垫上.并且容易别离.Typical blockin

20、g protocole is :典型封闭工艺0.5% to 1% BSA or casein 0.25% to 0.5% Tween 200.1M Tris Buffer ( MW 121.14 ) 1% to 3% sucroseadjust ph 8,0 with HCLConjugate particles结合颗粒Gold 金or Latex乳胶20nm to 50nm0.1m to 0.5mConjugate particles结合颗粒Check for :检查No free antibodies无剩余抗体No aggregation无凝聚Right concentration浓度适宜

21、Make sure all reagents are filtered before being dispensed onto the membrane在喷膜之前,试剂需要过滤Test and control linesDispense the antibodies with non contact or contact systems.喷抗体可使用接触式或者非接触式喷膜系统Typical is :Volume : 1l/cmSpeed: 50 to 90mm/secConc.: 1to 4mg/mlBuffer : 10mM phosphateEthanol: up to 5%For the

22、 test line, we use :测试线推荐配方Antibody:polyclonal Anti-hCG from goat (Arista Biologicals, USA)Concentration:4 mg.ml-1Buffer:5 mM borat w. 1% (w/v) Saccharose, pH 8Striping:1 l -1Striping speed:50 mm.s-1 For the control line, we use:控制线推荐配方Antibody:Rabbit Anti-mouse IgG (Alchemy, GB) Concentration:1 mg.

23、ml-1Buffer: 5 mM borate w. 1% (w/v) Saccharose, pH 8 Striping:1 l -1Striping speed:50 mm.s-1 Immediately check the quality of the wet line在喷膜未干时检查喷膜质量Block or not the membrane膜的封闭与否Dry the membrane膜的枯燥1 hour at 40COr 30min at 60COr overnight room TValidation is necessary需要验证Spray the conjugate parti

24、cles喷结合颗粒For example :3.5l gold at 10 O.D. or 3l latex at 10ODWe use :推荐Particles: 40 nm gold particles (Alchemy, GB)Antibody:monoclonal Anti-hCG from goatOr dip the pad 或者浸渍结合垫, It is important to have always the same amount of conjugate per strip控制一致数量的结合物And it is essential to check for complete

25、release of the particlesPrepare backing 准备底版Laminate the membrane onto the plastic backing将膜贴在底版上.Laminate the other components贴其他组分Never store the membrane directly in contact with plastic or other material. Always use paper to protect it不能将膜和塑料及其他材料直接接触,膜须放在保护纸上保存Make sure, there is a good contact

26、 between all components保证所有组分接触良好Cut strips.Non proper cut can alter flow front on the membrane切割:不良切割将影响膜上流动A top tape is useful to maintain a good contact between the pads and the membrane上部的胶带作用是保持膜和垫之间良好接触Reader for performance characterization 性能检测表征Reader for: quantitative reading of test- and

27、 control-line定量分析 characterization of line shape and quality 线质量表征 picture recording of magnified line图象记录Intensity a.u.Position mm Standard hCG test: 标准HCG测试= Evaluate line intensity with reader评估线强度How to get the right line shape and intensity 如何得到良好的线形和强度Some parameters influencing line intensity

28、 and shape影响参数YYYYYYLFIA important parametersAntibody buffer, volume & concentration 抗体缓冲液, 用量和浓度Distance from pad contact to test line距离Drying T and time枯燥温度和时间Distance from conjugate stripping to test line距离Conjugate amount and conditions of release 结合颗粒数量和释放条件Analyte concentration检测物浓度Blocking or

29、 not the membrane 封闭Test line concentration and printed volume浓度和用量Distance from release pad to test line 结合垫到T线距离 Dependence on applied conjugate volume 结合剂用量Influence of drying time on test line: t 4.5h T线枯燥时间Influence of drying time on control line: t 4.5hC线枯燥时间TLCLInfluence of relative humidity湿

30、度影响Test was equilibrated for a defined time at different relative humidities and run under the same conditions. 相对湿度和时间的平衡Gold conjugate test line on a CN-membrane:胶体金在包被线上状态Technique: vapor pressure SEM without sputtering * vp-SEM was performed by Schossig, GKSS, Geesthacht, GermanyYGold particles

31、around 40nmLatex particles from 0.2m to 0.5mYConjugate particles结合颗粒ultrasound treatment超声波处理A non-diluted and a 1:100 diluted conjugate solution was treated for 1 h with ultrasoundIntensities of selected areas are measured with Image J and the integrated values are displayed in a window of 90,000 pixelsRelease of conjugated latex beadsRelease of conjugated latex beadsi.e. drying treatment and perhaps drying temperature affects the release of conjugate枯燥时间和温度对于结合物释放的影响After application of th