Pea lectin as a universal cell binding agent for ELISA (screening) tests

A cell binding assay (CBA) was developed in which plant lectins are used as binding agents between microtiter plates and human cells. After binding, the cells were fixed by mild glutaraldehyde treatment. Their antigenic activity was investigated by enzyme-linked immunosorbent assay (ELISA) with several monoclonal antibodies. The binding method resulted in cell layers that remained firmly attached to the plates during the washing and incubation procedures of the ELISA. A comparative phenotype analysis, performed by indirect membrane fluorescence, showed that cells bound by this method, do not lose their antigenic activity. This binding assay can be used as a rapid, large scale screening test for monoclonal antibodies to membrane antigens of malignant and normal cells.     

An enzyme immunofiltration assay useful for detecting human monoclonal antibody

A microenzyme-linked immunoassay (EIA) utilizing an immunofiltration manifold has been developed which provides a rapid, simple, and sensitive method of detecting human monoclonal antibody class, concentration, and specificity. In this assay either whole cells or soluble antigens were immobilized on glass fiber filters followed by incubating with the test human hybridoma supernatant with subsequent analysis by EIA. A specially designed 96-chamber immunofiltration plate is employed which serves as both an incubation chamber and as a filtration manifold. The assay described is unique in that small volumes of human hybridoma supernatant are required, crude preparation of only a few target cells are needed, labile cell surface antigens are preserved and it can be completed in 3 h. This assay is well suited for the rapid screening of large numbers of human hybridoma supernatants.     

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.     

Rapid screening of monoclonal antibodies by spin adherence double immunosorbent test (SADIST)

The spin adherence double immunosorbent test (SADIST) is a simple, rapid immunoassay with sensitivity similar to the enzyme-linked immunosorbent assay (ELISA). A 1-step SADIST has been found suitable for rapid screening of hybridomas for antigen-specific monoclonal antibodies (MAb). In this procedure hybridoma supernatants are added to antigen coated microplates followed by commercially available antiglobulin beads. The microplate is immediately centrifuged. Wells containing antigen-specific MAb produce a mat of beads whilst wells without antigen-specific MAb produce a button of beads. No washing or incubation steps are necessary and results are read within minutes of adding beads to test supernatants. By comparison, ELISA tests require several hours to perform with multiple wash steps and further reagent additions. A 2-step SADIST was also assessed. Supernatants are incubated in the microplate as for an ELISA and a wash step precedes the addition of antiglobulin beads. A panel of 117 hybridoma supernatants was selected to assess the suitability of the SADIST techniques for hybridoma screening. The supernatants were added to antigen-coated microplates and SADIST and ELISA tests performed. The SADIST correctly discriminated most hybridoma supernatants that were clearly positive or negative by ELISA. It was also found possible to perform SADIST followed by ELISA tests on the same microplate well without significantly affecting ELISA values.     

Immunodetection of cell-bound antigens using both mouse and human monoclonal antibodies

A micro enzyme-linked immunoassay (EIA) has been developed for the rapid and sensitive detection of either human or mouse monoclonal antibodies reactive with cell bound antigens. Whole intact cells are immobilized onto 96-well flat bottom microtiter plates by drying in an oven at 37 degrees C overnight prior to the start of the assay. This method of attachment was suitable for all cell types tested, regardless of origin, size and chromosomal content. The dried cells were then rehydrated, incubated with the appropriate test hybridoma supernatant, followed with subsequent analysis by EIA. The plates can be stored at 4 degrees C up to 1 month for future EIA analysis. This assay offers high sensitivity, requires only small amounts of target cells and test hybridoma supernatant, and can be completed within 3 h. This EIA is well suited for the rapid screening of large numbers of hybridoma supernatants and can also be adapted to include cells of any species, providing the appropriate antibody reagents are available.     

抗精脒单克隆抗体的制备及其特性鉴定

本文用鼠×鼠细胞杂交瘤技术首次建立了抗多胺（精脒）单克隆抗体的杂交瘤细胞系。以此杂交瘤经体外组织培养扩大，并注入同系小鼠腹腔，产生的腹水中抗体滴度达１：１０６。单抗的类别是ＩｇＧ１。单价抗体是由分子量５２ｋＤ和２７ｋＤ组成的复合体。这个单克隆抗体可用于癌症的快速诊断和预后随访。     

人白细胞介素15单克隆抗体的制备及鉴定

目的 :用rhIL 15与沙门氏菌裸菌相偶联制备IL 15单克隆抗体 (monoclonalantibody ,McAb) ,以便为疾病的诊断、治疗和发病机制的研究提供可靠的生物制剂。方法 :将纯化的rhIL 15蛋白与沙门氏菌裸菌相偶联制成免疫原 ,用脾内、腹腔、静脉三种途径相结合免疫BALB c小鼠 ,以PEG为促融剂 ,将脾细胞与SP2 0细胞进行融合 ,HAT选择培养 ,间接ELISA筛选阳性克隆 ,通过多次克隆化 ,获得稳定分泌特异性McAb的杂交瘤细胞株 ,并对所获得的细胞株及分泌的McAb特性进行了分析。结果 :获得 4株能稳定分泌特异性McAb的杂交瘤细胞系 (hybridomacell,Hc) ,ELISA法检测其效价分别为 1:10 6 、1:10 7、1:10 7、1:10 7。Dotblot检测IL 15的灵敏度为 1 5ng ,其中一株 (1 7E4 )尚可用于Westernblot检测。结论 :成功制备了 4株IL 15McAb杂交瘤细胞系。     

H5N1流感病毒M1蛋白免疫制备的单克隆抗体的研究

目的 制备抗人流感病毒H5N1株M1蛋白的单克隆抗体,为流感的快速诊断和研究提供新的工具.方法 应用在大肠埃希菌中表达的人H5N1亚型禽流感病毒(A/Anhui/1/2005)株M1蛋白,以纯化的表达产物免疫BALB/c小鼠,取脾细胞与sp2/0细胞系作细胞融合后,间接ELISA法筛选阳性的杂交瘤细胞,并应用间接免疫荧光法对抗体的特异性进行鉴定.结果 获得3株能稳定分泌抗禽流感病毒M1抗原的McAb杂交瘤细胞株,交叉反应试验及间接免疫荧光检测表明,三株McAb具有型特异性.结论 用H5N1禽流感病毒M1蛋白免疫制备的单克隆抗体,具有一定的交叉反应性,可用于多种亚型甲型流感病毒的检测.     

Monoclonal Sperm Antibodies: Their Potential for Investigation of Sperms as Target of Immunological Contraception

ABSTRACT: We generated 149 hybridoma cell lines secreting antibodies against human spermatozoal antigens of which antibodies from 136 hybridoma lines also reacted with seminal plasma constituents. The occurrence of common antigeneic determinants on spermatozoa and seminal plasma was further demonstrated by competitive inhibition ELISA tests. We found that seven hybridoma clones secreted antibodies reactive with spermatozoa but nonreactive with seminal plasma. Antibodies from 5 clones were sperm-agglutinating and from 15 clones sperm-immobilizing. Localization of sperm antigens reactive with the monoclonal antibodies was demonstrated by indirect immunoperoxidase staining. A synthetic decapeptide, earlier shown to be reactive with naturally occurring human iso- and autosperm antibodies, was shown to be reactive with the monoclonal antibody VII-5 in ELISA tests.     

Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.     

[125I]protein a solid-phase assay for detection of monoclonal antibody

Summary The production of monoclonal antibodies involves testing hundreds of hybridoma supernatant samples for antibody content. This necessitates a rapid detection assay which can separate antibody of interest from antibodies that react nonspecifically. Solid-phase radioimmunoassay is one such method.     

重组人源细胞珠蛋白单抗制备及检测方法建立

目的制备重组人源细胞珠蛋白（recombinanthumanCytoglobin,rhCygb）单克隆抗体,并建立检测该蛋白双抗体夹心ELISA法,为下一步研究rhCygb药代动力学做准备。方法用纯化的rhCygb免疫BALB/c小鼠,采用甲基纤维素半固体培养基法获得抗rhCygb的单克隆抗体杂交瘤细胞,间接ELISA法筛选制备单克隆抗体,建立双抗体夹心ELISA法。结果筛选获取了稳定分泌单克隆抗体的杂交瘤细胞株,通过抗原表位相加法实验获得5株表位不同的细胞株,Western-blotting验证能与rhCygb特异性结合,间接ELISA法验证其不与本实验室制备的其它PET28a-BL21蛋白及BL21裂解液发生交叉反应。本方法灵敏度为1.25ng/ml,在浓度为10～1250ng/ml时,线性关系良好,相关性达0.9931,实验内和实验间平均变异系数分别为6.2%和10.92%。结论成功建立了灵敏度好、特异性高的双抗体夹心法,为下一步研究rhCygb药代动力学奠定了基础。     

McAb夹心ELISA测定人IL-2及其影响因素的探讨

用抗人白细胞间素-2(IL-2)单克隆抗体(McAb)及多克隆抗体建立了敏感的测定IL-2的夹心ELISA。测定范围为1.5u～1000u/ml,与生物学方法有较好的可比性。十二烷基磺酸钠、盐酸胍、尿素及二巯基乙醇等重组IL-2纯化制备中常用的变性剂对本法有不同程度的干扰,而血清则无。用戌二醛预处理包被板可明显减弱这些试剂的干扰作用,并可将测定的敏感性提高2倍。本法也可用以快速确定亲和层析纯化IL-2的条件及抗IL-2McAb的构相表位相关性。     

抗人白细胞介素15单克隆抗体的制备及特性鉴定

目的 :制备鼠抗人白细胞介素 15(hIL 15)单克隆抗体 (mAb) ,并鉴定其特性。方法 :自重组人白细胞介素 15(rhIL 15)基因工程菌中 ,提取融合蛋白GST IL 15,以 12 0g/LSDS PAGE分离鉴定 ,切取含有目的条带的凝胶 ,免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2 / 0骨髓瘤细胞常规融合 ,依次进行HAT选择培养 ,间接ELISA法筛选抗体阳性的杂交瘤细胞及克隆化。对杂交瘤细胞株的稳定性及其分泌的mAb的特性进行鉴定。另外 ,以rhIL 15包涵体蛋白 (rhIL 15IBP)免疫新西兰白兔 ,制备抗hIL 15的多克隆抗体 (多抗 )。用抗hIL 15的mAb与多抗建立双抗体夹心间接ELISA。结果 :获得 1株可稳定分泌特异性抗hIL 15mAb的杂交瘤细胞。建立了双抗体夹心间接ELISA ,检测rhIL 15蛋白的敏感性达 10 μg/L。结论 :成功地制备抗hIL 15mAb ,并建立了一种可用于hIL 15检测的双抗体夹心间接ELISA。     

Characterization of a method using viable human target cells as the solid phase in a cell concentration fluorescence immunoassay (CCFIA) for screening of monoclonal antibodies and hybridoma supernatants

A new method has been characterized for the use of viable target cells as the solid phase for screening of hybridoma supernatants in a cell concentration fluorescence immunoassay (CCFIA). Briefly, the specific target antigen on the cells is bound by the monoclonal antibodies and revealed by use of a fluoresceinated second antibody. Separation of free from bound antibody is accomplished by filtration in the 0.2 micron filter-bottom wells of specialized assay plates. Processing is automated in a Pandex screen machine, resulting in numerical fluorescence values for each well. This method is rapid (under 1 h per 96-well plate), highly sensitive (down to 0.2 ng/ml) and sparing of target cells (0.3-2.5 X 10(4) cells per assay well). It has been applied to 37 different varieties of human solid tumor cells, as well as to human peripheral blood mononuclear cells. The cells used as targets for the characterization of this method were still capable of attachment and growth when recovered post-assay. This method was compared with a viable cell enzyme-linked immunosorbent assay (ELISA) method, showing similar sensitivity and greatly shortened assay time. Comparison of the results from this method with those obtained from flow cytometric analysis performed on viable cells showed close correlation, whereas a lower correlation was seen with immunohistochemical methods using acetone-fixed cells. Development of this method made it possible to rapidly screen many thousands of hybridoma supernatants and successfully select those which were specific for surface antigens on viable cells.     

A Solid-Phase Assay for the Detection of Anti-Sperm Antibodies

PROBLEM: ELISA is an ideal assay method for a large-scale screening of anti-sperm antibodies among a large number of infertile males. However, conventional ELISA with whole spermatozoa needs time-consuming steps of centrifugation. METHOD: A solid-phase assay used for detecting anti-sperm antibodies was established. This assay is suitable not only for detecting circulating anti-sperm antibodies of IgG, IgM, and IgA subclass simultaneously but also for screening hybridomas secreting anti-sperm monoclonal antibodies (mAbs). The microtiter plates, on which solubilized sperm antigens are fixed, can be stored at ? 80°C for up to six months without losing reactivity with anti-sperm antibodies. RESULTS: Using this assay, 53 sera (13 were proven positive and 40 were proven negative for sperm agglutination antibody) were tested. Although the false-negative rate was 0%, the false-positive rate was 32%. One thousand one hundred sixty-five supernatants from hybridomas constructed with splenocytes of mice who were hyperimmunized with human sperm and nonsecreting myeloma cells were tested by this solid-phase assay and two anti-sperm mAb secreting clones were selected and established. CONCLUSIONS: It is recommended that for research work this assay could be used for the first screening of the hybridoma secreting anti-sperm mAb, and for clinical use this assay might be suitable for the first screening of sera of infertile patients. However, conventional bioassays should follow to confirm the biological meaning of the positivity.     

生物素化抗细胞因子单克隆抗体的制备

目的 探讨生物素化单克隆抗体的制备方法。方法 应用经超滤、硫酸铵沉淀、ProteinG亲和层析纯化的来源于杂交瘤细胞培养上清中的大鼠抗小鼠IL-5、IL-4和IFN-γ单克隆抗体，以每mg抗体加入生物素制剂80ug室温反应60min，其反应液经SephadexG-25层析纯化，然后用生物素-亲和素ELISA法测定所制备的生物素化抗体活性。结果 该法制备的生物素化抗体活性高，用于检测感染巴西钩虫的IL-5转基因小鼠和非转基因小鼠的血清标本敏感性好。结论 该生物素化抗体的抗备方法具有制备简单、稳定性好等优点。     

杀虫剂西维因单克隆抗体的研制及鉴定

目的 制备特异性的西维因单克隆抗体。方法 合成了西维因的半抗原并对其进行了结构鉴定，将半抗原与牛血清白蛋白和卵清蛋白共价偶联分别制备免疫抗原和包被抗原。再将免疫的Balb／c小鼠脾脏细胞与SP2／0小鼠骨髓瘤细胞融合，建立一株分泌西维因单克隆抗体的杂交瘤细胞。分析该杂交瘤细胞的染色体，并以间接酶联免疫吸附分析法(ELISA)检测了单克隆抗体免疫球蛋白的类型及其与西维因的亲和性和特异性。结果 杂交瘤细胞的平均染色体数目为98条，它分泌IgG1亚类的单克隆抗体；该单克隆抗体与西维因的亲和性较高(IC50=52ng，mL)而与西维因结构类似物的交叉反应很小。结论 本实验成功的制备了西维因特异性的单克隆抗体，为其ELISA方法的建立提供了条件。     

基于基因免疫制备抗人绒毛膜促性腺激素β亚基单克隆抗体及其特性研究

用真核表达人绒毛膜促性腺激素β亚基((human chorionic gonadotrophinβ,hCGβ)的重组质TR421-hCGβ免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,ELISA法筛选阳性细胞株,经过多次的克隆化培养,最终获得10株持续、稳定分泌抗hCGβ单克隆抗体的杂交瘤细胞株。随机抽取6H1杂交瘤细胞进行抗体的制备、纯化及免疫学特性的分析。间接ELISA法证明6H1单抗属于IgG2a亚类。Western blot证明6H1单克隆抗体可以特异性结合hCGβ,间接免疫荧光和流式细胞仪等结果表明,6H1单克隆抗体能够不同程度的结合不同来源的肿瘤细胞膜上表达的hCGβ分子,为进一步研究其生物学功能奠定了物质基础。     

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.