表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)单链抗体亲和力检测的间接ELISA的建立及优化

目的建立检测表皮生长因子受体变体Ⅲ(EGFRvⅢ)单链抗体PD0721亲和力检测的ELISA,并且对实验条件进行优化。方法利用本课题组制备的单链抗体PD0721,建立检测PD0721单链抗体亲和力的间接ELISA,并采用棋盘滴定法对实验条件中的抗体抗原浓度、二抗浓度、包被条件等方面进行优化,对本方法的灵敏度及精密度进行考察。结果使用PBS稀释抗原至1.25 mg/L于4℃包被12 h,加入120 ng/mL PD0721单链抗体以及1∶8000的酶标抗体为最佳检测结果。优化后的结果,在PD0721单链抗体浓度为15 ng/mL~480 ng/mL范围内回归模型拟合效果良好,最低检测限为7.5 ng/mL。组内差异系数为0.11%~0.99%,组间差异系数为0.68%~3.15%。结论建立了能准确、稳定检测PD0721单链抗体亲和力的间接ELISA。     

一种新型的酶免疫技术—TNP/抗TNP免疫组化法

目前广泛应用的酶免疫技术有过氧化酶—抗过氧化酶(PAP)法和生物素—亲和素(ABC)法。我们以牛血清白蛋白为载体,联结三硝基苯(TNP)半抗原,以此免疫新西兰兔,产生了抗TNP的特异性高效价抗体,再以三硝基苯磺酸(TNBS)修饰铁传     

间接ELISA法检测轮状病毒

本文报道检测急性小儿腹泻患者粪便中轮状病毒的间接 ELISA 法。分别用兔抗 SA_(11)轮状病毒血清和正常兔血清的粗提 IgG 包板,检测抗体为豚鼠抗 SA_(11)抗体。酶标抗体为辣根过氧化物酶标记的抗豚鼠 IgG 抗体。根据正常人标本结果为阴性,阻断试验成立,以及阳性标本用正常豚鼠血清检测为阴性,说明本试验具特异性。分析比较50份标本的电镜检查结果和间接 ELISA 试验结果,说明本试验的敏感性好。初步比较了抗牛轮状病毒(NCDV)抗体和抗 SA_(11)抗体检测的结果,两者基本一致。用硫酸铵粗提的 IgG 代替兔全血清,可减少非特异性显色。     

甲状腺过氧化物酶抗体光激化学发光间接检测法的建立

目的:利用抗免疫复合物IgG多抗体在光激化学发光(Lica)平台上建立间接法检测甲状腺过氧化物酶抗体(Anti-TPO)。方法:以人体IgG对兔子进行免疫耐受,以兔红细胞和人体血清构建免疫复合物IgG并免疫兔子。用纯化的抗免疫复合物IgG多抗和甲状腺过氧化物酶组成一步间接法,检测甲状腺过氧化物酶抗体浓度,并评估其最低检测限、回收率和批内精密度,与传统两步化学发光法(贝克曼)进行比较。结果:甲状腺过氧化物酶的检测方法的最低检测限为0.09 U/ml,平均回收率为96.26%,重复性变异系数(CV)为1.58%～1.97%,与贝克曼方法的结果的相关性r=0.989。结论:成功建立了光激化学发光定量检测Anti-TPO的方法(间接法)。该方法与传统两步间接化学发光法的相关性较好,弥补了光激化学发光平台无法使用间接法进行检测的缺陷。     

镉离子单克隆抗体的鉴定及竞争ELISA的建立

目的 制备Cd2+单克隆抗体(McAbs,单抗),建立Cd2+ 间接竞争酶免疫学检测方法 (EHSA).方法 利用双功能螯合剂iEDTA将Cd2+分别与血蓝蛋白(KLH)和牛血清白蛋白(BSA)偶联,制备免疫抗原和检测抗原.免疫Balb/c小鼠,应用杂交瘤技术制备抗Cd2+的单抗.鉴定单抗的效价、特异性、亚类及亲和力.以该抗体为基础建立检测镉离子的间接竞争ELISA方法 ,分析该方法 的灵敏度、检测限、工作范围.结果 成功制备出Cd-iEDTA-KLH和Cd-iEDTA-BSA偶联物;筛选出一株稳定分泌抗Cd-iEDTA单抗的杂交瘤细胞株C2G5该细胞株分泌的抗体亚类为IgG1,腹水抗体效价为1×106,相对亲和力结合常数Ka为1.6×109 L/mol.特异性结果 显示,除Hg2+外,该抗体与Ph2+、Fe3+、Mn2+、Mg2+、Zn2+、Al3+、Ni2+、Ca2+、Cu2+等金属离子的交叉反应低.建立了检测镉离子的间接竞争ELISA方法 ,该方法 的检测限为10 ng/mL,IC50为250 ng/mL.结论 建立了镉离子的竞争ELISA检测方法 ,检测限达到无公害水产品的国家标准(100 ng/g).     

抗乙肝病毒前S(2)蛋白单克隆抗体杂交瘤细胞系的建立及初步应用

本文用聚合人工血清白蛋白亲和层析柱纯化的含前S(2)-HBsAg,脾内接种BALB/c小鼠,取其脾细胞与Sp2/o-Ag骨髓瘤细胞进行常规方法融合,以四种酶联法进行筛选、并经多次有限稀释亚克隆培养,最终选出3林为分泌抗前S(2)单克隆抗体的细胞株。再经亚克隆培养和多次传代与冻存,并用抑制酶联法检测其抗体,均为抗前S(2)抗体阳性。接种BALB/c小鼠腹腔后,腹水均为抗前S(2)阳性反应,且效价可高达8万。用免疫双扩散法检测免疫球蛋白类型。均为鼠IgG1型。用经硫酸铵沉淀等方法纯化的IgG酶标物,采用双抗体夹心,检测了乙肝病人血清中的前S(2)蛋白,得到了初步结果,与现有检测受体的方法相比,具有敏感,特异,重复性好等优点,结果可靠。这一方法值得在临床上推广使用。     

姜黄素联合抗TfR抗体体外抗胶质瘤效应研究

目的探讨抗转铁蛋白受体(Tf R)抗体联合化疗药物姜黄素的抗瘤效应。方法利用MTT方法检测Tf R抗体与姜黄素单独及联合作用对胶质瘤细胞生存率的影响;利用流式细胞仪检测Tf R抗体与姜黄素单独及联合作用对胶质瘤细胞凋亡、细胞增殖和细胞周期的影响。结果 Tf R抗体与姜黄素联合作用可增强对胶质瘤细胞增殖的抑制作用,促进肿瘤细胞坏死,并将肿瘤细胞有效阻滞在S期。结论抗Tf R抗体可协同增强姜黄素对胶质瘤细胞的生长抑制效应。     

用酶标Ig交叉反应性单抗作间接ELISA检测肾综合征出血热(HFRS)病毒抗体

<正> 自Tkachenko等和Gavrilovskaya等应用酶联免疫吸附试验(ELISA)检测了HFRS病毒抗体以来,ELISA在HFRS的诊断与血清流行病学调查上的应用得到不断的发展。1986年,柴瑞珍等建立了ELISA抗IgM固相法检测人血清中的IgM抗体,但若用于不同种属血清的检测则需选用不同的固相包被抗体。本文在制备了Ig交叉反应性单抗(McAb)的基础上,制备了可用于人、猪、兔血清IgG检测的酶标McAb,建立了检测人、猪、兔血清中HFRS病毒抗体的ELISA间接法。     

小鼠抗KLH多克隆抗体的制备及TDAR实验间接ELISA定量检测方法的建立北大核心CSCD

目的:通过制备小鼠抗KLH多克隆抗体,优化反应条件建立KLH特异性抗体ELISA定量分析方法,以期提供一种特异性好、灵敏度高及误差低的TDAR实验检测方法。方法:利用盐析沉淀和亲和层析纯化技术分离和纯化小鼠抗KLH特异性多克隆IgG抗体;以纯化后的抗体作为标准品,建立TDAR实验间接ELISA定量检测方法,并对该方法进行线性范围、特异性、变异系数(CV)及检测限验证。结果:以KLH包被浓度为80μg/ml,山羊抗小鼠IgG/HRP酶标二抗的稀释倍率为1∶20000条件进行间接ELISA定量分析的方法学验证,检测线性范围为390.63~25000 ng/ml,R^(2)=0.9848,线性良好;批内和批间CV均<10%;检测限为194.83 ng/ml。结论:通过制备抗KLH多克隆抗体,并优化实验反应条件来建立间接ELISA定量分析方法,其线性范围、特异性及批内、批间CV均满足实验要求,是一种高灵敏度的TDAR实验检测方法。     

用HRP——抗IgM法快速诊断流行性出血热

本文报道用流行性出血热(EHF)病毒抗原片,以酶标抗人IgMu链单克隆抗体染色法(简称HRP-抗IgM)检测患者血清中的IgM抗体,从而对EHF进行早期诊断。为确证本方法的可靠性,同时与间接免疫荧光IgG法(IFA-IgG)进行了比较.经阻断试验和2-巯基乙醇耐性试验证明,HRP-抗IgM检出的抗体系EHF特异性抗体.用双盲法检测了51份血清,结果全部符合。实验结果表明,HRP-抗IgM特异性强.敏感性高,用普通显微镜便可观察,结果清晰.4小时内可获得诊断结果,可以用作早期诊断.     

Augmentation by 2‐Mercaptoethanol of In Vitro Anti‐TNP Antibody Production Induced by Butyrate Plus IL‐2 in Murine Splenic B Cells

Abstract

We previously reported that anti‐trinitrophenyl (TNP) antibody production in murine splenic B cells stimulated with TNP‐lipopolysaccharide in vitro was promoted by sodium butyrate (NaBu) in an IL‐2‐dependent manner. In the present study, we found that the effect of NaBu plus IL‐2 was markedly augmented by 2‐mercaptoethanol (2‐ME), which showed a slight or null effect on the response of untreated, IL‐2‐treated or NaBu‐treated B cells, as assessed by both anti‐TNP plaque‐forming cell assay and anti‐TNP IgM ELISA. Other thiol compounds such as dithiothreitol, cysteamine and reduced glutathione (GSH) also had this activity. 2‐ME enhanced the anti‐TNP antibody production induced by other short‐chain fatty acids with three to five carbon atoms plus IL‐2. The proliferation of B cells was significantly inhibited by NaBu or NaBu plus IL‐2, and the proliferation was completely restored by the simultaneous addition of 2‐ME. These results demonstrate that 2‐ME markedly enhanced anti‐TNP antibody production in murine B cells induced by NaBu plus IL‐2 and suggest that the effect of 2‐ME is at least partly due to its blocking activity of the growth‐inhibitory action of NaBu.     

IgM-Induced Specific Antibody Responses: Direct Correlation between Responsiveness and Natural or Induced Recurrence of the Idiotype

The idiotype (Id) expressed on antibodies of the trinitrophenol (TNP)-spccific BALB/c hybridoma TNP.11 is defined as a 'nonrecurrent' or private Id in BALB/c mice, whereas the analysis of splenic plaque-forming cells (PFC) and circulating antibody in DBA/2 mice immunized with TNP antigens revealed that TNP.11 is a 'recurrent' Id in this strain. TNP.11 antibodies were characterized by their ability to induce an antigen-independent anti-TNP response in these two strains. Whereas this antibody was negative in BALB/c mice [18], a clearcut increase in splenic anti-TNP PFC, preferentially expressing the TNP.11 Id, was observed in DBA/2 mice. This correlation between Id recurrency and ability to induce antigen-independent responses was further probed in BALB/c mice repeatedly immunized with anti-TNP.11 Id antibodies (Ab3). Such BALB/c mice express the TNP.11 Id in detactable amounts even without antigen challenge and respond with increased numbers of splenic anti-TNP PFC to the injection of low amounts of TNP.11 antibody. These findings suggest that the ability of an antibody to induce antigen-independent PFC responses is associated with the'recurrency', either natural or induced, of the antibody Id.     

Different effect of LPS-induced macrophage factor on antibody responses to TI-1 and TI-2 antigens.

Effect of macrophage culture fluid (MF) on thymic independent (TI) antibody responses was examined. MF potentiated antibody responses of spleen cells to dinitrophenyl (DNP)-Ficoll and DNP-liposome, TI-2 antigens, but not to trinitrophenyl (TNP)-BA and TNP-LPS, TI-1 antigens. The enhancing effect of MF on the anti-DNP-Ficoll response was dose-dependent. Neither T cells nor macrophages were required for MF to exert the effect, suggesting that MF works on B cells directly. B cells modulated by MF in their antibody responses were indicated to be in mature B-cell subset for the following reasons: (i) the cells were in (CBA/N x BALB/c) F1 female but not in F1 male mice; (ii) the cells bore the receptors for C3 on their surface. MF was indicated to exert the enhancing effect on the antibody response by modulating the proliferation and/or early events in differentiation of B cells and not by promoting antibody secretion. The active component of the MF was indicated to be Interleukin 1.     

Immune carrier properties of acid-treated Salmonella minnesota R595 bacteria. The immune response to TNP-bacterial conjugates in rabbits and mice

Salmonella minnesota R595 bacteria from which the core region of the lipopolysaccharide on the cell wall had previously been removed by mild acid treatment were trinitrophenylated. Differing amounts of these trinitrophenyl naked bacterial conjugates (TNP-NB), covering a range of epitope densities, were used for immunising mice and rabbits via the intraperitoneal or intravenous routes without adjuvants. It was found that such acid-treated, naked bacteria were effective carriers for the covalently linked hapten, TNP, with an optimum epitope density of 15 micrograms TNP/mg NB. Significant immune responses were obtained with dose levels as low as 50 ng TNP. The possible applications of acid-treated, naked bacteria as universal carriers having inherent adjuvant activity are discussed.     

In Vitro TNP-Specific Antibody Formation by Peripheral Lymphocytes from Patients with Systemic Lupus Erythematosus

Immunological reactivity in patients with systemic lupus erythermatosus (SLE) was assessed by investigating in vitro trinitrophenyl (TNP)-specific antibody formation by peripheral lymphocytes. Peripheral lymphocytes from 16 patients with SLE were cultured with TNP conjugated with horse erythrocytes (TNP-HRBC) in the presence of 2-mercaptoethanol. The hemolytic plaque assay was used to detect hapten (TNP)-specific antibody-forming cells. Peripheral lymphocytes from normal individuals failed to produce antibody to TNP, whereas SLE lymphocytes produced a significant number of plaque-forming cells. Co-culture experiments with SLE and normal lymphocytes suggested that patients with SLE have a defect in T lymphocytes, leading to abnormal antibody production.     

Rat basophil leukaemia (RBL) cells sensitized with low affinity IgE respond to high valency antigen

Background The very low concentrations of IgE antibodies in serum tnake investigations of the affinity of allergen-specific antibodies extremely difficult. In the absence of such studies, the fact that low IgE concentrations are capable of inducing powerful effector function has encouraged the view that IgE antibodies are typically high affinity antibodies. Yet the phenomenon of allergic cross-reactivity suggests that lower affinity IgE antibodies may sometitnes be of clinical significance. Objectives To investigate the effect of antibody affinity upon mast cell sensitivity in an in vitro model. Methods Rat basophil leukaemia (RBL) cells were sensitized with one of three monoclonal IgE antibodies which bind to trinitrophenyiated proteins with varying affinity. Serotonin release was measured after challenge of sensitized cells with trinitrophenyiated human serum albumin (TNP-HSA). Results Low valency TNP₃-HSA failed to stimulate degranulation of RBL cells sensitized with SPE-7 anti-DNP IgE, which binds TNP with low affinity. However, upon challenge with high concentrations (1250 ng/mL) of TNP₈-HSA, or as little as 10 ng/mL of highly substituted TNP₂₃-HSA, low levels of degranulation were seen. A similar relationship between antigen valency and cell sensitivity was seen with cells sensitized with the H-le-DNP anti-DNP IgE. which binds with moderate affinity to TNP proteins. Conclusion High valency antigen is capable of activating RBL cells sensitized with low affinity antibody. This has important implications for our understanding of allergic sensitization. It also suggests that the long-rccognized relationship between antigen valency and RBL cell sensitivity may partly reflect the high functional affinity of cell-bound IgE when directed against multivalent antigen.     

Antibody responses of cattle immunized with the Tf190 adhesin of Tritrichomonas foetus.

The antibody response patterns of cattle after subcutaneous and intranasal immunizations with adhesin Tf190 of Tritrichomonas foetus were investigated. Reactions of antibody from cattle parenterally immunized with Tf190 revealed antigen specificity and Tf190 sensitization in the majority of the animals, as determined by Western blotting. The results also demonstrated strong preimmune immunoglobulin G2 (IgG2) binding to T. foetus antigens not seen in IgG1 profiles. Subcutaneous injections of Tf190 resulted in significant (P < 0.05) increases in serum IgG1 and IgG2 titers over time, as determined by parasite specific enzyme-linked immunosorbent assay. Immune sera also significantly inhibited parasite adhesion to mammalian cell lines compared to the level of inhibition obtained with preimmune sera (P < 0.05). Intranasal immunization with Tf190 failed to produce measurable parasite-specific antibody in serum; however, this immunization route did result in significant (P < 0.05) increases in parasite-specific IgA titers in cervical mucus secretions from immunized animals that were more resistant to intravaginal challenge with T. foetus than controls. These results suggest that systemic immunization with Tf190 results in serum antibody production and antiparasitic adhesin antibodies. Additionally, the results of challenge experiments with intranasally immunized animals suggests that Tf190 primes protective immune responses that lead to lower rates of infection among these animals.     

Active in situ immune complex glomerulonephritis using the hapten-carrier system: role of epitope density in cationic antigens.

The role of epitope density in cationic antigen was investigated in an active model of in situ immune complex glomerulonephritis (ICGN) using the hapten-carrier system. Trinitrophenol (TNP) was conjugated with variable density to cationic human immunoglobulin (C-HIgG) to yield TNP6.2-C-HIgG (low-valency antigen) and TNP31.3-C-HIgG (high-valency antigen). In rats preimmunized with TNP17.3-bovine serum albumin (BSA), endocapillary proliferative GN with proteinuria developed in rats receiving high-valency antigen. In contrast, no significant abnormalities in renal histology or urinalysis were observed when a low-valency antigen was injected. These results indicate that glomerular injury produced by hapten-specific immune reaction is affected by the number of haptenic groups conjugated to the carrier molecule (epitope density) in active in situ ICGN.     

The IE Allogeneic Response of T Cells from C57B1/6 Mice is Associated with Genes in the TCRa Locus

It has been demonstrated that induction of immune responses, infectious diseases and autoimmune manifestations can be associated with at least four gene loci: the major histocompatibility complex (MHC) locus; the immunoglobulin (Ig) heavy chain (Hc) locus; and the T–cell receptor (TCR) TCR–β or TCR–β chain loci. In the present study, we have analysed whether T–cell responses of IE–negative C57B1/6 (B6) mice to IE alloantigen (IEα transgenic B6 mice = B6. Eα 16) or to trinitrophenylated (TNP) syngeneic spleen cells were influenced by changes in the Ig–Hc locus or the TCRa locus. Whereas the fine specificity of T–cell responses to IE alloantigen was the same in B6 mice and in Ig–Hc congenic B6.26a or TCRa congenic B6.10TCa mice, the latter strain of mice demonstrated much higher IE–specific T–cell responses against B6. Eα16 spleen cells than B6 or B6.26a mice. This high responsiveness was a dominant feature and associated with the TCRa locus. In addition, the TCRVα or Vα repertoire of the congenic strains of mice was polyclonal and very similar. The TNP–specific T–cell responses of B6 and B6. 10TCa mice showed the same restricted TCRVα and Vα repertoire. It is concluded that in both an oligoclonal T–cell response (anti–TNP) and a polyclonal T–cell response (anti–IE), exchange of Ig–Hc or TCRa loci does not significantly influence the TCRVα or Vα repertoire in IE–negative C57B1/6 mice.     

Murine anti-TNP antibodies positive for either lambda 1 or lambda 2 light chains express common idiotypic determinants

Although the lambda-bearing antibodies represent only 5% of the total mouse serum immunoglobulins, some antigens such as B1355 dextran (alpha (1-3)Dex), the 4-hydroxy-3-nitrophenyl acetyl (NP) and 2,4-dinitro or 2,4,6-trinitrophenyl (DNP/TNP) antigens can induce lambda-positive immune responses. In contrast to the lambda antibody response against alpha (1-3)Dex and NP antigens which is restricted to the lambda 1 isotype it was shown that the response to the DNP (or TNP) antigen uses lambda 1 and lambda 2 and lambda 3 isotypes. The idiotypy of the alpha (1-3)Dex and NP systems has been well characterized contrary to that of the lambda-positive anti-TNP/DNP response which has been poorly studied. In this paper, we describe two idiotopes (Id C19-3 and Id D11-2) shared by two BALB/c monoclonal anti-TNP antibodies (TNP5 and TNP9) which, respectively, use the lambda 1 and lambda 2 light chains. These idiotopes were independently expressed on other monoclonal anti-TNP/DNP antibodies and appear to require the use of a unique VH gene associated with a particular V lambda region. After TNP-Ficoll immunization, BALB/c mice recurrently express both idiotopes on lambda 1 and (lambda 2 + lambda 3) anti-TNP antibodies. In addition, all the mouse strains immunized against TNP-Ficoll give a lambda 1- and (lambda 2 + lambda 3)-positive immune response with the exception of SJL and SJA strains which present a deficit for the expression of lambda 1 light chain. The expression of Id C19-3 was restricted to the strains with the Igh-Va allotypic haplotype (including SJA) whereas the Id D11-2 was extensively expressed in the various strains.