Theoretical considerations of the ability of monoclonal antibodies to detect antigenic differences between closely related variants, with particular reference to heterospecific reactions

In theory monoclonal antibodies can be used to analyse antigenic determinants in great detail by correlating differences in antibody affinity for variant antigens with their amino acid differences. In particular, heteroclitic antibodies should be detected, which would normally be masked in a polyclonal antiserum. Recognition of such antibodies may be important for our understanding of the scope of antibody repertoires particularly when the immunogen is closely related to a component of the immunised animal. In practice the immunoassays commonly used to measure affinity differences between different antigens fall short of these capabilities. Mathematical studies were carried out to identify factors controlling the sensitivity of 4 types of assay to differences in affinities for different antigens. The most important factors controlling assay sensitivity were found to be the ratio of antibody affinity (K) to epitope density in direct binding assays, the ratio of K to antibody concentration in liquid phase competition assays, and the ratio of solid phase to liquid phase values of K for solid-phase competition assays. It is predicted that a combination of solid-phase competition assay with high epitope density and direct binding assay with low epitope density would result in optimal detection of heteroclitic antibodies and small differences in antibody affinity for cross-reactive antigens.     

Glycation of monoclonal antibodies impairs their ability to bind antigen.

As elevated levels of glycated IgG have been detected in the plasma of patients with diabetes mellitus, a disease associated with increased susceptibility to infection, we have investigated whether glycation of MoAbs affects the kinetics and/or affinity of antigen binding. Three mouse MoAbs were incubated with 0.5 M glucose at pH 7.4 for 14-21 days at 37 degrees C. Control MoAbs were incubated using identical conditions but with no added glucose. Using a surface plasmon resonance technique we found that glycation significantly increased the rate of dissociation (kdiss) of the antigen-antibody complex for all three MoAbs (P < 0.05, n = 4), but had no significant effect on the rate of association (kass). For one of the MoAbs, against human IgG (Fab), we also measured kdiss by an alternative method utilizing radiolabelled antigen, which confirmed that glycation of the antibody significantly increases kdiss (P < 0.001, n = 8). We also found using an ELISA-based method that glycation of the same MoAb significantly increased the equilibrium dissociation constant (Kd) (P < 0.05, n = 6). A significant increase in kd was observed after glycation using glucose concentrations consistent with those found in poorly controlled diabetics (P < 0.02, n = 5). We conclude that in vitro glycation can significantly lower the affinity of an antibody for its antigen, and significantly increases the rate of dissociation of the antigen-antibody complex.     

Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus

An enzyme-linked immunosorbent assay (ELISA) in the form of a blocking test is described for the detection of group specific antibodies to bluetongue virus (BTV). The test relies upon interruption of the reaction between BTV antigen and a group specific murine monoclonal antibody against BTV by addition of serial dilutions of bovine or ovine test sera containing specific antibodies to BTV which inhibit binding of the monoclonal antibody to the BTV antigen. This is detected as a reduction in the optical density (O.D.) reading obtained with the monoclonal antibody alone. The test is capable of specific detection of antibodies to all 22 serotypes of BTV but, unlike the agar gel precipitin (AGP) test, does not show cross-reactions with antibodies to epizootic haemorrhagic disease of deer (EHD) viruses. Furthermore, antibodies to cellular proteins which complicate interpretation of the AGP test and the indirect ELISA are not detected in the blocking ELISA. The high sensitivity and specificity of the blocking ELISA make it an ideal alternative to the AGP test. The use of a monoclonal antibody would facilitate standardisation of diagnostic testing between laboratories.     

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.     

禽流感病毒核蛋白单克隆抗体的制备及部分特性鉴定

目的：制备抗禽流感病毒（AIV）核蛋白（NP）的单克隆抗体（mAb）并进行特性鉴定。方法：分别用甲醛灭活的和TritonX-100裂解的AIV H9N2及AIVNP基因的原核表达产物免疫BALB／c小鼠。经细胞融合、间接ELISA筛选及克隆化，建立能稳定分泌抗AIV NP mAb的杂交瘤细胞株。mAb的效价采用间接ELISA测定，用交义反应试验及间接免疫荧光染色法检测mAb的特异性。结果：经细胞融合、筛选及克隆化，间接ELISA法测定，mAb共得到6株能稳定分泌抗禽流感病毒NPmAb的杂交瘤细胞株，分别命名为4F4、1C3、1G11、1C2、1D10及2E7。1G11、1D10腹水的ELISA效价最高，分别为2^-13和2^-14。交叉反应试验及间接免疫荧光染色检测表明，两株mAb的特异性良好。结论：其获得6株抗AIVNP的mAb，其中2株mAb1C11和1D10的效价最高，特异性良好，为AIV的研究及快速诊断方法的建立奠定了基础。     

人脱嘌呤脱嘧啶核酸内切酶单克隆抗体抗原表位的鉴定及应用

目的:研究鉴定人脱嘌呤脱嘧啶核酸内切酶单克隆抗体(hAPE1 mAb)的抗原表位,并建立定量检测hAPE1的ELISA一步法。方法:设计并合成APE1-15肽阵列,鉴定hAPE1 mAb 2-G1和4-F6的抗原表位,应用三维立体结构观察软件Molsoft.ICM-Pro模拟hAPE1 mAb抗原表位的立体结构;采用改良的过碘酸钠法标记抗体,以hAPE1 mAb为捕获抗体和酶标抗体,建立hAPE1的ELISA一步检测法。结果:APE1-15肽阵列检测结果和抗原表位三维结构显示,2-G1mAb的抗原表位对应为hAPE1天然蛋白氨基酸残基序列的76-90位和109-123位,位于氧化还原区域,为构象型抗原表位;4-F6 mAb的抗原表位对应为hAPE1天然蛋白氨基酸序列的109-147位,位于DNA修复内切酶活性区。ELISA一步法检测hAPE1蛋白的线性范围为8.0~200μg/L,最低检测限为2.0μg/L。平均批内变异系数为8.67%,平均批间变异系数为12.45%,平均回收率为105.47%。结论:hAPE1 2-G1 mAb和4-F6 mAb具有不同的抗原表位,成功建立的hAPE1 ELISA一步法为简便、快速、准确检测血清中hAPE1的含量奠定了基础。     

A simple method for ranking the affinities of monoclonal antibodies

Measurement of the binding of constant trace amounts of labelled antigen by increasing dilutions of culture supernatant allows the ranking of monoclonal antibodies in the order of their affinity for the antigen. The theoretical basis for this method is discussed and it is illustrated with data from a set of anti-alphafoetoprotein monoclonal antibodies. Hybridomas secreting antibodies of desired affinity for immunoassay, histochemistry or antigen purification can thus be selected at an early stage after fusion.     

Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure

Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 X 10(10) M-1 while the monoclonal antibodies possessed an affinity to LDL of 5 X 10(8) M-1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.     

抗H9亚型禽流感病毒血凝素单克隆抗体的制备及初步鉴定

目的 :制备抗禽流感病毒 (AIV)H9亚型血凝素蛋白的单克隆抗体 (mAb)。方法 :以AIVH9亚型油乳剂灭活疫苗作为免疫原 ,免疫 8wk龄BALB/c小鼠。采用淋巴细胞杂交瘤技术制备抗AIVH9亚型血凝素蛋白的mAb ;采用ELISA和血凝抑制试验(HI)检测腹水mAb的效价 ;采用ELISA、HI、免疫荧光染色 (IF)及Westernblot鉴定mAb的特异性。结果 :获得 3株可稳定分泌特异性mAb的杂交瘤细胞株 2A3、2H1和 1C8,其腹水mAb的ELISA效价依次为 1× 10 7、1× 10 5和 5× 10 6,血凝抑制效价为 1× 2 8～ 1× 2 13 ;3株mAb的Ig亚类均为IgG1。以mAb 2H1进行Westernblot的结果显示 ,该mAb能与AIV的Mr 为 75 0 0 0的蛋白条带起反应 ,表明其是针对AIVH9亚型血凝素蛋白的mAb。与 32株AIVH9亚型国内分离株进行血凝抑制试验表明 ,mAb 2H1具有良好的广谱性。结论 :成功地制备了抗AIVH9亚型血凝素蛋白的mAb ,为AIV的抗原性分析、血清学诊断、疫苗质量的监测及流行病学调查等奠定了基础     

抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

Antigenic variation of the Epstein–Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

The Epstein–Barr virus (EBV) nuclear antigen EBNA1 plays an essential role in the replication of EBV episomes in latently infected cells and is the only viral protein that is consistently expressed in all programs of latent EBV gene expression. In this study, four monoclonal antibodies (MoAbs) directed to a region (amino acid residues 442–530) of EBNA1 were generated. Competitive enzyme-linked immunosorbent assay (ELISA) experiments using biotinylated MoAbs showed that they recognized distinct epitopes. Reactivity of these MoAbs with various laboratory EBV strains and field EBV isolates was shown to be heterogeneous in that EBNA1 from certain strains (isolates) was recognized and that from others was not. All four MoAbs showed such heterogeneous reactivity, and moreover, each MoAb showed a distinct spectrum of reactivity with these EBV strains (isolates). These results demonstrate an extensive structural variation in this region of EBNA1 as predicted by previous sequencing studies. These MoAbs will be useful as probes to dissect this structural heterogeneity of EBNA1.     

The effect of epitope density and antibody affinity on the ELISA as analysed by monoclonal antibodies

The antibody titre and the optical density values in an ELISA are influenced by the epitope density of the antigen and the affinity of the antibody tested. This has major implications in the interpretation of ELISA results.     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.     

H1亚型流感病毒HA蛋白单克隆抗体的制备及部分特性鉴定

目的:制备针对H1亚型流感病毒HA蛋白的单克隆抗体(mAb),并分析其反应特性。方法:分别以2009年甲型H1N1、季节性A1流感病毒裂解疫苗为免疫原,常规法免疫、融合、克隆化,获得各抗原特异性mAb。应用ELISA、HI试验和Western blot等技术研究mAb的反应性和特异性。结果:获得稳定分泌抗H1亚型流感病毒HA蛋白的杂交瘤细胞97株。其中株特异性mAb39株,29株具有HI活性;亚型特异性mAb7株,5株具有HI活性;2009年流行株与季节性A1、A3流行株共同抗原的mAb16株,9株具有HI活性;针对流感病毒共同抗原mAb35株,22株具有HI活性。结论:两种疫苗均具有较好的免疫原性和免疫保护活性,这些mAb的获得为流感病毒株特异、亚型特异性诊断试剂盒及流感病毒通用诊断试剂盒的制备提供了实验资料,为进一步研究H1N1流感病毒HA的抗原表位奠定了基础。     

促肾上腺皮质激素单克隆抗体的筛选和鉴定

目的制备促肾上腺皮质激素 ( ACTH)单克隆抗体 ,拟建立高灵敏度 ACTH- IRMA或 EL ISA。方法 ACTH1 - 1 3、ACTH1 - 2 8、ACTH 1 - 39和 ACTH1 8- 39分别与牛血清白蛋白偶联制备免疫原 ,在背部皮下和腹腔注射免疫 BAL B/ c鼠。按常规方法进行细胞融合 ,以 RIA法检测培养上清液 ,阳性孔经亚克隆 ,获得 2株分泌特异性抗体的杂交瘤细胞株 ;瘤细胞种于预致敏的BAL B/ c鼠腹腔 ,批量生产单抗性腹水 ;琼脂扩散法鉴定抗体类型 ;利用标准曲线的参数计算单抗的亲合常数 ;亲合纯化单抗并进行抗体固化试验。结果从 ACTH1 - 2 8免疫鼠获得单抗株为 5 G2 ,从 ACTH1 - 1 3免疫鼠获得单抗株为 D3 ;单抗腹水 5 0 %结合率时腹水最终稀释度分别为 5× 10 3和 2× 10 3;抗体类型均为 Ig G1/λ;5 G2和 D3亲合常数分别为 5 .1× 10 9L M- 1 和 4 .9× 10 7L M- 1 ;抗体包被实验显示 5 G2和 D3的固化抗体量分别为 1.4μg和 8.8μg时结合率达到饱和。结论 5 G2和 D3可以用于构建灵敏的 ACTH- IRMA或 EL ISA。     

生物传感器对抗戊型肝炎病毒单克隆抗体部分特性的研究

目的　应用生物传感器对新制备的抗戊型肝炎病毒单克隆抗体 (mAb)的亲和力、Ig亚类 (型 )及抗原结合的动力学进行研究。方法　用HEVORF2区基因工程重组蛋白NE2 ,免疫BALB/c小鼠 ,经杂交瘤技术制备mAb ,采用ELISA、Westernblot和生物传感器鉴定其有关特性。结果获得 12株可稳定分泌抗NE2mAb的杂交瘤细胞系。用ELISA及生物传感器等鉴定各株mAb ,分别为IgM和IgG1、IgG2a,轻链均为κ型。其中在用ELISA法对mAb3F5亚类鉴定过程中 ,发现其与HRP GAMIgG2a和HRP GAMIgM均有反应 ,生物传感器鉴定其为IgM。Westernblot验证各株mAb的特异性 ,同时各株mAb对于不同聚合形式的NE2蛋白的反应性有一定的差别。应用生物传感器测定了 4株mAb的KD 值 ,mAb 8C11为 4 .36× 10 7,mAb8H3为 1.5 3× 10 5,mAb 13D8为 1.2 1× 10 6,mAb 16D7为8.0 3× 10 7。从生物传感器测得的各株mAb与NE2的结合、解离过程中发现 ,mAb 8H3与NE2可快速结合、快速解离 ,其它 3株mAb结合稳定。结论　经多种方法鉴定 ,所获得的 12株杂交瘤细胞分泌的抗体 ,为抗HEVORF2区段的特异性抗体     

The induction and characterization of monoclonal antibodies specific to GP of Ebola virus

The Ebola virus is highly infectious and characterized by hemorrhagic fever, headache, and so on with a high mortality rate. Currently, there are neither therapeutic drugs or vaccines against the Ebola virus nor fast diagnostic methods for the detection of Ebola virus infection. This study reported the induction and isolation of two monoclonal antibodies that specifically recognized the glycoprotein (GP) and secreted glycoprotein (sGP) of the Ebola virus. Plasmids encoding either GP or sGP were constructed and immunized BALB/c mice, accordingly purified sGP was boosted. The antisera were analyzed for binding activity against sGP protein in enzyme-linked immunosorbent assay (ELISA) and neutralization activity in a pseudotyped virus neutralization assay. A number of reactive clones were isolated and two monoclonal antibodies T231 and T242 were identified to react with both GP and sGP. Western blot and ELISA assays showed that the monoclonal antibodies could react with GP and sGP, respectively. Moreover, they could recognize Ebola pseudovirus by cellular immunochemistry assay. We labeled the monoclonal antibody T231 with biotin and analyzed the competitiveness of the two antibodies by the ELISA test. The results showed that the binding epitopes of the two monoclonal antibodies to sGP were partially overlapped. In summary, two GP-specific mAbs were identified, which will be used to detect the Ebola virus or investigate GP.     

抗凋亡抑制蛋白——survivin单克隆抗体的制备

目的制备抗 survivin蛋白单克隆抗体。方法以大肠杆菌表达的 survivin融合蛋白为抗原 ,免疫 Balb/c小鼠 ,通过细胞融合 ,建立分泌单克隆抗体的杂交瘤细胞系。结果获得了 6株稳定分泌单克隆抗体的杂交瘤细胞系。 6株单克隆抗体均能特异识别和结合大肠杆菌表达的 survivin融合蛋白 ,但其中仅有 3株能够结合肿瘤细胞系表达的天然 survivin蛋白 ,并与某些肿瘤细胞系中 5 0 0 0 0 u±分子量的未知蛋白存在交叉反应 ,与正常人外周血淋巴细胞样本未见任何反应。结论通过单克隆抗体的免疫印迹反应 ,可以观察 survivin特定分子量蛋白的表达 ,为深入研究该蛋白的细胞凋亡调控功能提供了工具。此外 ,这些单克隆抗体对于肿瘤诊断也具有一定应用价值。     

Directed selection of influenza virus produces antigenic variants that match circulating human virus isolates and escape from vaccine‐mediated immune protection

Influenza vaccination does not provide 100% protection from infection, partly due to antigenic drift of the haemagglutinin (HA) protein. Low serum antibody titres increase the risk of infection. To determine whether there were additional correlates of risk, we examined the relationship between human serum immunity and antigenic variation in seasonal H3N2 influenza viruses. Seasonal H3N2 vaccine strains grown in the presence of heterogeneous human or mono‐specific ferret antisera selected variants with mutations in the HA antigenic sites. Surprisingly, circulating strains infecting human subjects in the same seasons displayed mutations in the same positions, although only in one case did the change correspond to the same amino acid. Serum antibody titres were lower against both the in vitro selected and clinical isolates compared with the vaccine strains, suggesting that the mutations are relevant to vaccine failure. Antibody titres were also significantly lower in sera from infected subjects than in non‐infected subjects, suggesting relatively poor responses to vaccination in the infected subjects. Collectively, the data suggest that risk from influenza infection is a result of poor response to vaccination, as well as encounter with drifted seasonal influenza virus antigenic variants. The results also show that directed selection under human immune pressure could reveal antigenic variants relevant to real‐world drifted viruses, helping in annual vaccine re‐formulation.