ssDNA-碳化树脂类风显关H免疫吸附剂性能研究

研究了以火棉胶作为包埋材料将DNA配基固定于碳化树脂表面制备的类风湿关节炎免疫吸附剂的吸附性能,静态吸附条件下,当ssDNA的固定量在0.4mg/mL树脂时吸附性能最佳,对3种类风湿因子(RF)的最佳饱和吸附量分别为IgMRF:2458IU/mL、IgGRF:2877IU/mL、IgARF:1100IU/mL.吸附120分钟后达到吸附平衡,对血浆中白蛋白及总蛋白的清除率分别低于8%和6%,表现出较好的吸附选择性.吸附剂经毒理实验证明,使用安全性较高.对活犬进行体外血液灌流实验表明该吸附剂具有良好的血液相容性.     

免疫吸附剂吸附类风湿因子（RF）

以聚乙烯醇水凝胶为载体，氨基酸为选择性配基，制备亲合性免疫吸附剂，用于清除类风湿性关节炎患者体内的类风湿因子（ＲＦ），实验中对水凝胶载体交联度、选择性配基种类、手臂效应等与吸附及选择性能的关系进行了研究。实验数据表明：当水凝胶交联度为４０％时，联接Ｌ－丝氨酸对ＲＦ具有最好的吸附选择性能。对患者血浆中ＲＦ－ＩｇＭ、ＲＦ－ＩｇＧ、ＲＦ－ＩＧＡ的吸附率分别达到６２．８％、４７．２％、和６０．０％，而对血浆蛋白无吸附。     

双亲型吸附剂对HDL的吸附动力学研究

设计合成了以葡聚糖为载体，同时具有亲水性磺酸基和疏水性胆固醇两类配基的新型吸附剂。通过对高密度脂蛋白（HDL）纯溶液中吸附等温线的测试，比较了以Dextran G-75为以亲型吸附剂与单一亲水型磺酸基配基、单一疏水型胆固醇配基吸附剂吸附量和亲和吸附系数的关系，对双亲型吸附剂的吸附动力学进行了初步研究。在HDL溶液中，亲水型磺酸基、疏水型胆固醇配基、双亲型吸附剂对HDL的吸附曲线基本符合Langmuir吸附方程。     

葡聚糖为载体的双亲型LDL吸附剂吸附动力学研究

合成了以葡聚为载体，同时具有亲水性磺酸基和疏水性胆固醇两类配基的新型低密度脂蛋白（LDL）吸附剂。通过对LDL纯溶液中吸附等温线的测试，比较了以Dextran G-75为载体的双亲型LDL吸附剂与单一亲水型磺酸基配基，单一疏水型胆固醇基吸附剂吸附量和亲和吸附系数的关系。对双亲型LDL吸附剂的吸附动力学进行了初步研究，在LDL溶液中，亲水型磺酸基，疏水型胆固醇配基，双亲型LDL吸附剂对LDL的吸附曲线基本上符合Langmuir吸附方程，另外通过高离子强度NaCl洗脱实验，测定了双亲型LDL吸附剂上具有的磺酸基与胆固醇两类基在对低密脂蛋白吸附过程中所起的配合效果，为下一步作用力机制研究提供了参考依据。     

以琼脂凝胶固定热聚IgG制备类风湿关节炎免疫吸附剂的研究

以环氧氯丙烷对琼脂凝胶珠进行活化反应后键联热聚ＩｇＧ,制成一种新型类风湿关节炎免疫吸附剂。确定了最佳制备条件,使凝胶上环氧基的含量达１１０μｍｏｌ／ｇ,对热苯ＩｇＧ的固定量达６ｍｇ／ｇ。在体外条件下吸附剂对三种类风湿因子ＩｇＭＲＦ,ＩｇＧＲＦ及ＩｇＡＲＦ的吸附量分别达３４００,２２５０和２４００ＩＵ／ｇ,具有良好的应用前景。     

亲和吸附剂对细菌内毒素吸附性能的研究

制备了以球形纤维素为载体、8种氨基酸和1种聚赖氨酸为配基的吸附剂,对质量浓度为100.0pg/mL的内毒素水溶液进行了吸附研究,绘制了吸附等温线,并初步探讨了吸附机理.结果表明,精氨酸和赖氨酸配基具有良好的吸附能力,在1.5mL100.0pg/mL内毒素溶液中吸附量分别达到182.0和160.0pg/mL;吸附等温线显示,以赖氨酸为配基的吸附剂其吸附量随溶液内毒素浓度增加而线性增加,符合Langmuir吸附方程,吸附能力强,具有一定的临床应用前景.     

IgA肾病免疫吸附剂吸附性能及血液相容性研究

针对IgA肾病研究制备用于IgA吸附的免疫吸附剂．采用不同活化方法、不同配基制备吸附剂，测定吸附效率并进行比较．体外吸附实验表明吸附剂对IgA肾病病人血清中的IgA具有较强的吸附性能；对正常人血液进行血液相容性实验及对健康小鼠的急性毒性实验证明该吸附剂具有良好的血液相容性且安全、无毒．该吸附剂可作为IgA肾病的免疫吸附剂．     

低密度脂蛋白吸附剂的研究.Ⅰ.氨基酸残基为配基的吸附剂

本文研究了以羧甲基纤维素和氯甲基化交联聚苯乙烯树脂为载体,以α—氨基酸为配基的吸附剂对血清中低密度脂蛋白(LDL)的选择性吸附。实验结果表明:载体上偶联赖氨酸和亮氨酸的吸附剂有较高的吸附率和选择性。     

IgA肾病免疫吸附剂的研究(Ⅱ)

通过1,4-丁二醇-二缩水甘油醚法活化琼脂糖载体,既起到了活化载体的作用,同时又由于该活化剂分子是长链结构而给载体提供了一定长度的手臂,有利于人免疫球蛋白G(IgG)大分子配基与载体的连接,用该方法制成的免疫吸附剂对高IgA肾病病人血清中IgA进行了吸附研究.通过体外实验条件的优化发现,最高可吸附60%的IgA,同CDI法比较(吸附率可达35%左右),该吸附剂具有较高的吸附效率.     

重症肌无力免疫吸附剂的制备及性能研究

重症肌无力患者体内致病毒素是乙酰胆碱的受体抗体.通过对载体材料的优化及对配基的筛选发现,使用质量分数为6%的纤维素球形载体,以环氧氯丙烷法活化并固定上L-色氨酸作为配基制备的血液灌流材料在静态吸附实验(1g吸附剂加入3mL病人血清,室温,3h)中可使抗体水平下降25%以上,吸附量达3.669×10^-3nmol/g,选择性较高,具有应用前景.     

A Rheumatoid arthritis study using Raman spectroscopy

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints and can lead to a progressive destruction of articular cartilage and bone. In this study, the specificity and sensitivity of the RA diagnostic methods based on the receiver-operating characteristic curves for monitoring C-reactive protein (CRP) and rheumatoid factor (RF) were compared with the Raman spectroscopic diagnostic method developed in this work. Sera from 24 patients with rheumatoid arthritis and from 16 healthy individuals were analyzed to assess the biochemical composition and presence of inflammatory activity by the aforementioned methods. By comparing with the clinical results for specificity and sensitivity from the RF and CRP tests, we show that the overall results from the newly developed Raman method were significantly better, with a specificity of 96%, a sensitivity of 88%, and correctly identifying 92% of the RA and healthy individuals, while the RF test gave a specificity of 100% and a sensitivity of 54%, and the CRP test gave a specificity of 87% and a sensitivity of 58%, respectively.     

Recombinant OmpL1 Protein as a Diagnostic Antigen for the Detection of Canine Leptospirosis

Microscopic agglutination test (MAT) is the standard method for the diagnosis of leptospirosis, which is laborious and the interpretation of the results is subjective. The present work describes the use of recombinant-based IgG ELISA for the serodiagnosis of leptospirosis. We used recombinant outer membrane protein OmpL1 as an antigen for conducting IgG enzyme-linked immunosorbent assay (ELISA). A total of 475 canine serum samples were subjected to IgG ELISA; 294 sera were positive to ELISA, while 283 were positive to MAT. All samples that were positive to MAT were positive to ELISA also, however, few samples which were negative to MAT were positive to ELISA, which suggested that recombinant-based IgG ELISA showed 100 % sensitivity when compared to MAT. Thus, this present study showed that recombinant OmpL1-based IgG ELISA appears to be a better alternative to MAT for the diagnosis of leptospirosis and rOmpL1 protein could be used as a potential diagnostic antigen in different assay formats for leptospirosis.     

邻苯二胺-过氧化氢-辣根过氧化物酶酶联免疫示差脉冲伏安法测定人血清类风湿因子抗体

建立了邻苯二胺（ＯＰＤ）－Ｈ２Ｏ２－辣根过氧化物酶（ＨＲＰ）酶联示差脉冲伏安分析体系并用于测定人血清中类风湿因子（ＲＦ），ＨＲＰ催化Ｈ２Ｏ２氧化ＯＰＤ所形成酶催化产物在ｐＨ２．０磷酸盐－枸橼酸缓冲溶液中于－０．１８ Ｖ左右产生一灵敏示差脉冲伏安峰，ＲＦ浓度在１．２５～２０．０ Ｕ／ｍＬ之间与峰电流呈线性关系，应用此峰检测人血清ＲＦ的检测限低至 ０．２８Ｕ／ｍＬ。该法较相同条件下ＥＬＩＳＡ显色光度测定法的灵敏度增加５倍，且受干扰较少。     

计算机模式识别法在类风湿中医证侯的研究

根据计算机模式识别的马氏(Mahalanobis)距离判别法，用BASIC等语言缩制的IBM-PC等徽机适用的多元统计判别程序将健康人分别与类风湿性关节炎的寒热错杂型、寒湿阻络型及肝肾两虚型的发样徽量元素Zn、Fe、Cu、Cd及Zn／Cu五种因素进行分类判别．结果表明以Zn、Ft、Zn／Cu为特征参量．健康人与寒热情杂型类风温性关节炎患者分类准确亭为95．6%，而分别以Zn、Cu和Zn，Fe、Cu为特征参量．健康人与寒湿阻络型和肝肾两虚型患者分类准确率分别为100％和90．2％．说明以Fe、Cu、Zn(或Zn／Cu)组成的微量元素谱含量，在不同中医证型的类风温性关节炎和健康中是有差异的。     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

Purification of ascitic fluid-derived murine monoclonal antibodies by anion-exchange and size-exclusion high-performance liquid chromatography

Ascitic fluid-derived murine monoclonal antibodies (MoAbs) of immunoglobulin (Ig) M and IgG isotypes (IgG1 and IgG2a subisotypes) were previously prepared against an isolate of Actinobacillus sp (CAs8C) for the purpose of identifying and characterizing outer membrane antigens on this bacterium. An attempt was made to purify these MoAbs by anion-exchange and size exclusion high-performance liquid chromatography (HPLC). Hybridomas producing the IgG1 and IgG2a MoAbs posed unique difficulties in that they also secreted irrelevant IgG2b MoAbs that were present in the ascitic fluids. Anion-exchange chromatography (Protein-Pak DEAE-5PW column), with a simultaneous change in gradients of pH and ionic strength, was used to purify IgG and as a first step in the purification of IgM. There was good separation of IgG2b from IgG2a, but not from IgG1. Size-exclusion chromatography (Protein-Pak 300 SW column) was required to complete the purification of IgM. The presence of MoAbs in the HPLC fractions was confirmed by discontinuous gradient polyacrylamide gel electrophoresis (denatured and either reduced or non-reduced conditions) and the enzyme-linked immunosorbent assay. HPLC-purified MoAbs were free from transferrin and albumin and retained their specificity for As8C.     

Label-free detection of rheumatoid factor using YbYxOy electrolyte–insulator–semiconductor devices

In this study, we investigated the effect of yttrium content on the structural properties and sensing characteristics of YbY_xO_y sensing membranes for electrolyte–insulator–semiconductor (EIS) sensors to detect the rheumatoid factor (RF). The YbY_xO_y EIS device prepared at the 60 W plasma condition exhibited a higher sensitivity of 65.77 mV/pH, a lower hysteresis voltage of ∼1 mV, and a smaller drift rate of 0.14 mV/h than did those prepared at the other conditions. We attribute this behavior to the optimal yttrium content in the YbY_xO_y film forming a smooth surface. Furthermore, we used a novel YbTi_xO_y EIS biosensor to measure the RF antigen in human serum because of its rapid and label-free detection. Two different techniques were used for the immobilization of RF antibody onto the surface of an YbTi_xO_y EIS sensor. The RF antibody was directly immobilized on the EIS surface modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA). In contrast, a mixture of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution was used to functionalize the carboxyl groups at the tail of RF antibodies. RF antibodies functionalized with the active NHS esters were covalently immobilized on the APTES-modified YbTi_xO_y surface. The immobilized RF antibodies on the EIS that are functionalized with the EDC and NHS exhibit higher (41.11 mV/pC_RF) for detection of serum RF antigen in the range 10⁻⁷ to 10⁻³ M, compared to traditional antibody immobilization technique via APTES and GA linkage. The YbTi_xO_y EIS biosensor is a promising analytical tool for RF antigen monitoring due to its good sensitivity, stability and repeatability.     

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC₅₀ measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC₅₀ 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.     

Isolation of human serum immunoglobulins with a new salt-promoted adsorbent

In this work a highly acetylated-ethylenediamine-Novarose (HA-EDA-Novarose) gel was synthesized and used as a new agarose-based salt-promoted adsorption chromatography (SPAC) matrix to effectively isolate serum immunoglobulins without the need of denaturing conditions. Samples of human serum in 0.5 M Na2SO4, 10 mM 3-(N-morpholino)-propane-sulfonic acid (MOPS), pH 7.6 were applied to a chromatographic column packed with the SPAC gel. Immunoglobulins (Igs) with affinity for the HA-EDA ligands were specifically adsorbed to the matrix, non-bound serum proteins were readily removed by washing the column with the same feed solution buffer. Bound Igs were effectively and very gently eluted by simply removing the salt from the feed solution buffer. The elution buffer consisted thus of only 10 mM MOPS, at pH 7.6 and no salt. The salt-dependent adsorption capacity of this system was estimated to be 7.3 mg/ml with protein recovery of about 93%. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis analysis, radial immunodiffusion and enzyme-linked immunosorbent assays showed that immunoglobulins G, M and A (IgG, IgM and IgA) were the main components present in the elution fraction. The new SPAC adsorbent was used to purify Igs from human serum and IgG and IgA from non-pure commercially available Igs preparations in a very gentle single step.     

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′)₂ anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.