DNA免疫吸附剂血液灌流治疗系统性红斑狼疮临床应用研究

目的 :应用DNA免疫吸附剂 ,对系统性红斑狼疮 (SLE)患者进行血液灌流治疗 ,以清除致病抗体及其免疫复合物 ,使血液得到净化以治疗疾病。方法 :将患者动脉血液流经血液灌流仪 ,以保温 (38℃± )和保持流速(2 0 0ml/min)进入DNA免疫吸附罐 ,在罐内经吸附后由静脉回输入体内。吸附 1次需 1.5～ 2h ,使患者体内血液流经 3～ 4遍。结果 :32例SLE患者吸附后 1～ 2天发热、乏力、关节痛消失 ,2周皮损 87.5 %消失 ,1周各项检测异常指标皆达正常参考值内。而血中有形成份红细胞、白细胞、血小板则无影响。所治疗 32例SLE患者已随访 12～ 4 4个月 ,与未用此法治疗的对比组 34例SLE患者相比 ,生活质量明显提高 ,病死率显著下降 (P <0 .0 1)。结论 :用DNA免疫吸附剂进行血液灌流的净化疗法是特异性强、疗效高、无菌、无毒性、无致热源、无溶血反应、便于应用的一种新疗法     

大分子吸附树脂清除血清中免疫复合物的实验研究

目的：采用血液灌流吸附方法清除血液中病理性循环免疫复合物(CIC),寻找安全有效的吸附剂。方法：从系列亲合吸附剂中筛选出对CIC具有良好吸附性能的AA3吸附剂,测定了该AA3的比表面积、孔容积和平均孔径等参数,分析了吸附剂用量、吸附速率、温度和血中CIC浓度等条件对吸附率、吸附量的影响。结果：AA3可有效地降低血液中CIC的浓度,其吸附率可达40％以上,为治疗多种免疫复合物疾病开辟了一条途径。     

重组质粒pcDNA3.1IL-2/Fc对HBV preS2S DNA疫苗免疫作用的影响

目的 探讨IL-2/Fc融合表达后对HBVpreS2S基因疫苗诱导免疫反应的佐剂效应.方法 采用HBV preS2S DNA疫苗作为基础免疫,重组质粒pcDNA3.1IL-2/Fc作为佐剂加强免疫BALB/c小鼠,采用0、2、4周的方案接种,检测各次接种后抗体水平.初次免疫后7周,测定免疫脾细胞的杀伤活性、增殖活性和细胞因子的分泌水平.结果 pcDNA3.1IL-2/Fc作为佐剂在HBV preS2S注射3 d后免疫组小鼠抗体滴度、免疫脾细胞的杀伤活性和增殖活性、TH1型细胞因子的分泌水平,均比各对照组明显增强.结论 IL-2/Fc是有效的HBV preS2S DNA疫苗佐剂之一.     

儿童散发性登革热免疫分析

自 1995年 10月～ 1996年 1月广州经济开发区医院收治 8例儿童登革热 ,年龄 10月～ 10岁 ,经实验室检查符合登革热诊热。1　方法　对拟诊登革热患儿采血作血凝抑制试验检测抗体 ,病人血清用高领土吸附、然后用鹅血球加到血清中混合后 ,经离心用血清进行抗体测定 ,单份血清滴度≥ 1∶2 5 0 0为阳性 ,同时检测病人恢复期血清。如滴度比急性期增加 4倍亦考虑为本病 ,同时在急性期用ＥＩＩＳＡ法测定血免疫循环复合物 (ＣＩＣ)2　结果　临床与实验室结果分析可见不论患儿年龄大小 ,全部病例均有发热 ,皮疹阳性率 10 0 % ,肌痛阳性率 6 2 5 % ,…     

重组戊型肝炎氢氧化铝佐剂疫苗的磷含量与免疫效力关系研究

目的探索氢氧化铝佐剂戊肝疫苗的免疫原性与疫苗中PO_4~(3-)浓度的关系,并分析抗体滴度与抗原在羊淋巴液中吸附率有无相关性。方法用不同浓度PO_4~(3-)处理氢氧化铝佐剂制备试验疫苗,腹腔免疫Balb/c小鼠,ELISA法定量检测免疫后不同时间血清抗体滴度,同时采用钼酸铵还原法检测试验疫苗离心沉淀中的磷含量,根据兰格缪尔方程计算抗原在含不同浓度PO_4~(3-)的佐剂表面的最大吸附量(Γm)。试验疫苗在羊淋巴液中的抗原吸附率采用ELISA法检测。结果随疫苗中PO_4~(3-)浓度增加,抗原最大吸附量(Γm)减小,抗原在羊淋巴液中的吸附率下降,免疫血清抗体滴度渐次增加,直至达到平稳状态。结论在一定范围内增加疫苗磷酸根含量可减小抗原在羊淋巴液中吸附率,降低佐剂对抗原的吸附程度,提高疫苗免疫抗体滴度。     

血管紧张素Ⅰ化学发光免疫分析方法的建立

目的:建立人血管紧张素Ⅰ(AngⅠ)化学发光免疫分析方法,通过测定血浆中血管紧张素Ⅰ的含量计算肾素活性。方法:用生物素标记AngⅠ抗原,荧光素标记AngⅠ抗体,将抗荧光素抗体包被于化学发光板,以链亲和素酶为催化剂,采用竞争法建立AngⅠ化学发光免疫分析方法。结果:本方法的测量范围为100~7 800 pg/ml,灵敏度为24.3 mU/ml,批内变异为5.6%~8.7%,批间变异为6.8%~10.4%,回收率为95.4%~105.3%,稀释实验测定值与理论值呈线性相关,相关系数为r=0.999。与放射免疫分析试剂盒的相关性方程分别为y=0.95x+235.70,相关系数r=0.973。包被板、生物素标记AngⅠ抗原以及荧光素标记AngⅠ抗体在37℃存放一周稳定性良好。结论:方法学鉴定结果符合免疫分析的基本要求。     

电脉冲法增强CZP3 DNA 疫苗免疫不育效果的研究

目的:研究电脉冲法对CZP3 DNA 疫苗免疫不育效果的影响。方法:在小鼠腿部肌肉分别注射5、10、20 和50 μg的pcDNA3.0-CZP3 质粒,经电脉冲刺激,ELISA 法检测免疫小鼠的CZP3 抗体水平及最高抗体滴度。与雄鼠合笼三周后,计算各免疫组的不孕率及平均产仔数,统计学分析。结果:相比单独肌肉注射免疫,电脉冲法能极显著提高DNA 疫苗的免疫水平,电脉冲50 μg 组的抗体效价可以达到1 ∶4 000。此外随着血清中CZP3 抗体水平的增高,小鼠不孕率明显上升,平均产仔数显著减少。统计分析显示,免疫小鼠的CZP3 抗体水平与产仔数呈负相关关系。结论:电脉冲法能够显著提高CZP3 DNA疫苗的免疫水平,降低小鼠生育能力,这为CZP3 DNA 疫苗应用于流浪犬避孕奠定了基础。     

类风湿因子免疫吸附剂的制备及性能研究

目的制备一种类风湿因子（RF）免疫吸附剂并研究其性能。方法利用悬浮聚合法制备出大孔聚乙烯醇（PVA）树脂,以此为载体接枝苯丙氨酸,制备出可用于血液灌流吸附的RF免疫吸附剂（PVA-P树脂）,通过体外吸附实验观察不同处理方法对RF吸附性能的影响。通过体外血液学实验和溶血实验观察该吸附剂的血液相容性。结果免疫吸附剂的体外吸附实验表明,免疫吸附剂对RF的吸附清除率达到60%以上,处理工艺对RF吸附性能无明显影响。体外血液相容性实验结果表明：免疫吸附剂对红细胞、血小板、总蛋白的影响差异有统计学意义（P〈0.05）,其他血液指标差异无统计学意义（P〉0.05）,与日本同类吸附RF产品平行比较,对血细胞的影响差异无统计学意义（P〉0.05）。结论 PVA-P树脂作为RF免疫吸附剂具有良好的临床应用前景。     

用新型免疫吸附剂治疗系统性红斑狼疮

本文介绍特异性DNA免疫吸附剂制备方法及用血浆灌流治疗系统性红斑狼疮。DNA免疫吸附剂是以碳化树脂为载体,将小牛胸腺DNA与蓝四唑阳离子络合后同火棉胶混合固定于载体表面而制成。6例SLE女性患者,年龄22～43岁,免疫复合物高于正常值,有不同程度贫血。其中,关节疼痛5例,水肿3例,皮肤损伤2例。用血浆灌流进行治疗。结果:5例关节疼痛全部缓解,3例水肿中2例减轻,2例皮肤损伤中1例好转。DNA抗体平均减少了37.5％。BUN平均减少40.6％。而血小板及血红蛋白变化不明显。此方法是较理想的血液净化形式,为难治性免疫性疾病提供了新治疗方法。     

CA242免疫放射分析的稳定性研究

本文报道了以单克隆抗体C241作为捕捉抗体, 以C242为标记抗体的碘标固相双位点夹心式CA242免疫放射分析(IRMA)的稳定性研究. 实验结果表明, 包被珠、标记抗体、加入稳定剂后的标准及缓冲液在4℃存放8周仍可获得理想的实验结果.而未加入稳定剂的标准, 当存放条件为4℃、室温和37℃时, 分别在第7周、第2周第第1周失活.37℃存放包被珠时, 各标准点的结合率降低, 并且非特异性吸附升高. 37℃存放标记抗体到第3周时仍很稳定, 到第4周时略有下降.     

Purification of DNP specific antibodies using sepharose bound DNP-para-aminobenzoylglutamate.

An immunoabsorbent column employing a DNP derivative having restricted molecular freedom (dinitrophenylated-para-aminobenzoylglutamate--Sepharose 4B) was prepared in order to isolate and purify DNP specific antibodies in high yield. The antibodies were recovered from the immunoabsorbent column in yields from 80 to 90% using 3 M sodium thiocyanate, and these antibodies retained their immunologic activity. There appeared to be limited selection of antibodies with specific affinities as was noted in a parallel experiment comparing a second antibody purification procedure.     

In situ immune complex formation in the mouse glomerulus: reactivity with human IgM rheumatoid factor and the effect on subsequent immune complex deposition.

An in situ model of immune complex formation in the mouse was utilized to study the interaction of human IgM rheumatoid factor (RF) with glomerular bound immune complexes (IC). Prior intravenous injection of RF did not interfere with in situ IC formation, but reacted with complexes once formed. RF injected after formation of in situ IC was deposited in the same distribution as the complexes and was capable both in vivo and in vitro of acting as an immunoabsorbent and binding IC with the same antibody species as the in situ IC, but which had a different antigen component. The potential for this in situ immunoabsorbent activity of RF in the pathogenesis of the chronicity of the lesion in glomerulonephritis is discussed.     

Measurement of antibodies to pneumococcal, meningococcal and haemophilus polysaccharides, and tetanus and diphtheria toxoids using a 19-plexed assay

The measurement of antibody responses to vaccination is useful in the assessment of immune status in suspected immune deficiency. Previous reliance on enzyme-linked immunoabsorbent assays (ELISA) has been cumbersome, time-consuming and expensive. The availability of flow cytometry systems has led to the development of multiplexed assays enabling simultaneous measurement of antibodies to several antigens. We optimized a flow cytometric bead-based assay to measure IgG and IgM concentrations in serum to 19 antigens contained in groups of bacterial subunit vaccines: pneumococcal vaccines, meningococcal vaccines, Haemophilus influenzae b (Hib), and tetanus and diphtheria toxoid vaccines. 89-SF was employed as the standard serum. The assay was used to determine specific antibody levels in serum from 193 healthy adult donors. IgG and pneumococcal IgM antibody concentrations were measurable across 3 log10 ranges encompassing the threshold protective IgG antibody levels for each antigen. There was little interference between antibody measurements by the 19-plexed assay compared with monoplexed assays, and a lack of cross-reactive IgG antibody, but evidence for cross-reacting IgM antibody for 3/19 pneumococcal antigens. 90th centile values for 15/19 IgG concentrations and 12/12 IgM concentrations of the 193 adult sera were within these ranges and percentages of sera containing protective IgG antibody levels varied from 4% to 95% depending on antigen. This multiplexed assay can simultaneously measure antibody levels to 19 bacterial vaccine antigens. It is suitable for use in standard clinical practice to assess the in vivo immune response to test vaccinations and measure absolute antibody levels to these antigens.     

BSA-Y-DOTA免疫噬菌体Fab抗体库的构建及鉴定

目的 :构建钇 十二烷四乙酸 (Y DOTA)免疫噬菌体Fab抗体库。方法 :将牛血清白蛋白 (BSA)与DOTA交联 ,并与金属Y鳌合制备成BSA Y DOTA ,用其免疫BALB/c小鼠。检测抗血清的滴度后 ,分离抗体阳性的小鼠脾淋巴细胞 ,提取总RNA。利用RT PCR扩增全套重链Fd和轻链基因 ,依次插入经改造的噬菌体载体pComb3M的相应酶切位点 ,构建成Fab噬菌体抗体库。用酶切、序列测定及ELISA等方法 ,对重组率、多样性及Fab的展示情况进行鉴定。结果 :成功得制备了BSA Y DOTA交联物 ,并获得较好的免疫效果。免疫小鼠的全套重链Fd片段和轻链均得到正确扩增 ;Fd片断和轻链基因均插入到载体pComb3M中 ;Fab抗体库的库容量达 8× 10 7;重组率约为90 % ,且具有良好的抗体基因多样性。另外 ,Fab片段也被展示于噬菌体表面。结论 :成功地构建了半抗原Y DOTA的Fab噬菌体抗体库 ,为筛选Y DOTA特异性抗体奠定了基础     

Seroepidemiological study of measles after the 1992 nationwide MMR revaccination program in Taiwan

The incidence of measles declined rapidly in Taiwan after the introduction of the measles vaccine into the routine immunization schedule in 1978. However, an epidemic still occurred every 3–5 years until recently. A nationwide measles-mumps-rubella (MMR) revaccination program for school and preschool children has been in place since 1992 to control the indigenous transmission of measles. In order to understand the current immune status after this recent nationwide revaccination program, we determined the presence of measles IgG antibodies by enzyme-linked immunosorbent assay (ELISA) in 1,281 blood samples from healthy persons aged from 2 months to above 30 years collected between 1993 and 1995, and also in another batch of 90 sera samples from children aged 2 years collected before 1992. The results showed that 1) the measles antibody seropositive rate (36.4%) was lowest in children aged 5–7 months and rose to an unexpectedly high level of 85.8% at the age of 12–14 months, 2) the seropositive rate rose further to between 85.9% and 95.1% after 2 years of age and remained high in adults and pregnant women, and 3) the seropositive rate of the 2-year-old children collected before 1992 was 61.4%, which was significantly lower than the rate of the same age group collected after the nationwide MMR revaccination program. We conclude that the national revaccination program has promoted effectively measles immunity in Taiwan. This immunity explains the rarity of reported measles cases since the last epidemic in 1989. This revaccination program should continue and be extended to all preschool children and young adults so that indigenous measles can be eliminated by the year 2000. J Med Virol 51:32–35, 1997. © 1997 Wiley-Liss, Inc.     

Inter and intra laboratory concordance of HLA antibody results obtained by single antigen bead based assay

Single antigen bead based assays (SAB) to identify antibodies to HLA are marketed as a qualitative test, however often used as a quantitative test as the results provide mean fluorescence intensity (MFI) which is found to correlate with the strength/avidity of the antibody. We studied the between and within laboratory variability in performing the SAB from one manufacturer. Ten samples were tested at four laboratories according to the manufacturer’s suggested protocol. Additionally same samples were tested on four consecutive days in one laboratory. All tests were performed using the same lot of beads and secondary antibody. Results were classified as positive at four different MFI cutoffs: 1000, 5000, 8000 and 10,000. MFI values across and within the laboratory were compared in a pair-wise fashion with Pearson’s correlations. Overall concordance for Class-I was 97% between laboratories and 98% within laboratory at all cutoffs. Pair-wise Pearson correlation between laboratories was 0.989–0.99, while within laboratory it was 0.998–0.999. For Class-II, overall concordance between and within laboratory was 98%. Pair-wise Pearson correlation between laboratories was 0.991–0.997, while within laboratory it was 0.997–0.999. There is good correlation between laboratories and within laboratory using the same manufacturer, same lot and same protocol while performing SAB.     

Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine

Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine. DNA vaccination represents a new strategy for induction of humoral and cellular immune response. To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay. We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies. C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections. We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target. When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200-1:6400, average titre is 1:4580) and high CTL activity (67.4+/-8.4% (E:T = 25:1), 69.6+/-6.7% (E:T = 50:1) and 71.8+/-6.2% (E:T = 100:1)) were observed. In the case of two vaccine injections, CTL activity was also high (56.6+/-12.7% (E:T = 25:1), 57.4+/-11.7% (E:T = 50:1) and 63.0+/-6.3% (E:T = 100:1)) However, antibody titres were much lower (1:200-1:3200, average titre is 1:980). Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.     

Long-term preservation of transfecting activity of avian sarcoma proviral DNA

The integrated proviral DNA of avian sarcoma virus (ASV) in host cell chromosomes has been isolated and stored in saline sodium citrate (SSC) solution or in 70% ethanol at 4 degrees C in a refrigerator over 4 years. This DNA was assayed by transfection of chick embryo cells(CEC). The biological activity of cellular transformation by the stored DNA was compared with that of a fresh isolate of the proviral DNA. The efficiency of the transfection by each DNA was almost the same.     

Tissue distribution and quantitation of la-like antigens in the rat

The tissue distribution of a major histocompatibility complex (MHC)-linked alloantigen in the rat was studied using antiserum raised by alloimmunization in a MHC-congenic pair of rat strains. HO (Ag-B5) rats were immunized with HO.B2 (Ag-B2) skin grafts, and the tissue distribution of antigens recognized by the resultant antiserum was determined by cellular radioimmunoassay using different HO.B2 tissues as targets or immunoabsorbent. Linkage to the rat MHC was formally established by backcross analysis. The results obtained indicated that the tissue distribution of antigens recognized by this antiserum was closely similar to that reported for the distribution of Ia antigens in the mouse. Thus, T cells and thymocytes bound, respectively, 2% and 10% as much antibody as peripheral B cells while no binding to erythrocytes was detectable. Quantitative immunoabsorption studies revealed that thymocytes and kidney tissue had, respectively, 20% and 2% of the absorbing capacity of spleen cells. However, both tissues were capable of completely absorbing out the anti-B cell activity showing that they both possessed all the Ia-like specificities detectable by this antiserum on peripheral B cells. A variety of procedures aimed at reducing the passenger leukocyte content of kidneys had only a slight effect on the absorbing capacity. The cellular radioimmunoassay results indicated that on B cells, Ia-like molecules are a major membrane component, present in amounts comparable with surface immunoglobulin molecules. Studies at the single cell level, using a fluorescence-activated cell sorter, indicated that the expression of Ia-like alloantigens by rat thymocytes was very heterogeneous and that large thymocytes were the most heavily labeled. No Ia-like alloantigens were detected on alloantigen-activated T cells. The findings are compared with published data on the mouse.