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.     

Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies

A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.     

Enzyme immunofiltration technique for rapid diagnosis of herpes simplex virus eye infections in a rabbit model.

A rapid enzyme immunofiltration assay for herpes simplex virus (HSV) has been developed which is sensitive enough to detect viral antigens in eye swabs from rabbits with primary herpes keratitis. This assay employs a specially designed filter manifold to immobilize whole cells and cell debris dissociated from the swabs. Viral antigens trapped on the filters are then detected in an indirect immunoassay utilizing staphylococcal protein A conjugated with horseradish peroxidase. The assay required only 2.5 h to perform and could be read visually. Reconstruction experiments indicated that antigen from as few as 49 HSV-infected cells could be detected. Calcium alginate swabs were shown to recover more viral antigen than dacron swabs. The enzyme immunofiltration assay detected HSV antigens on 95% of the eye swabs from which infectious virus was recovered. In addition, HSV antigen was also detected in several swabs from infected eyes which did not yield infectious virus, presumably because the virus was neutralized by native antibody present in the lacrimal fluid. This enzyme immunofiltration assay technique lends itself to the elution of native antibody bound to the viral antigens, and this may be especially applicable in the diagnosis of recurrent HSV keratitis, where antiviral antibody in the lacrimal fluid may interfere with virus isolation and fluorescent-antibody or other virus detection assays.     

Immobilization of viral antigens on filter paper for a [125I]staphylococcal protein A immunoassay: a rapid and sensitive technique for detection of herpes simplex virus antigens and antiviral antibodies.

A new technique is described for the rapid detection and quantitation of herpes simplex virus (HSV) antigens and antiviral antibodies. It involves immobilization of HSV antigens on filter paper discs and subsequent analysis by 125I-labeled staphylococcal protein A (SPA) radioimmunoassay. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. It is rapid, simple, sensitive and specific, and requires only small volumes of antiserum and few target cells. The results may be readily and objectively quantitated. This technique permits the simultaneous assay of a large number of specimens in less than 1 h. Its sensitivity is considerably greater than that of other currently used immunologic techniques, and it is amenable to automation. These characteristics suggest that this [125I]SPA immunofiltration technique may be applicable to the rapid diagnosis of viral infections.     

Rapid serological technique for typing herpes simplex viruses.

A rapid technique is described which can accurately identify a herpes simplex virus (HSV) isolate as type 1 or type 2. Filter paper disks were used to immobilize viral antigens, which were then identified by means of an (125)I-labeled staphylococcal protein A immunoassay. The assay was performed in a specially designed 96-well filtration device which served as both an incubation chamber and a filter manifold. By using this system and cross-absorbed antisera to HSV types 1 and 2, 69 coded clinical isolates of HSV were correctly and unequivocally typed. HSV was also clearly distinguished from varicella-zoster virus and cytomegalovirus. This assay can be rapidly executed (less than 2 h) and yielded an objective endpoint; it required only minute quantities of typing sera and can be easily performed with the cells from a single infected roller tube culture. Thus, it can be used to type initial clinical isolates of HSV, yielding results within hours after the first appearance of cytopathic effects in the culture used for primary virus isolation. Moreover, it is particularly well suited to the simultaneous analysis of many specimens and is amenable to automation. These characteristics suggest that this (125)I-labeled staphylococcal protein A immunofiltration technique will be applicable to the rapid identification of other herpesviruses, as well as other clinical isolates.     

A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens

This paper describes the adaptation of a cellular enzyme-linked immunosorbent assay (CELISA) for the detection of antibodies to cell-surface antigens. This CELISA has the advantages of convenience and rapidity and is therefore ideally suited for the screening of a large number of hybridoma culture supernatants. The basic procedure involves the direct drying of cell suspensions onto the wells of enzyme immunoassay (EIA) plates and a subsequent EIA with appropriate blocking reagents. In order to overcome high background binding of primary antibodies to Fc receptors and of secondary antibodies to surface Ig (sIg), this method involves a blocking step consisting of unlabelled secondary antibodies. Once CELISA plates are prepared, they can be stored for a period of at least 6 months and hence this assay does not rely on the availability of fresh, viable cells for each assay. This assay is simple, reproducible and sensitive. The results can be assessed in an objective manner and can also be adapted for the detection of cellular antigens. This paper describes a CELISA for the detection of antibodies to blood group antigens and human leukocyte (HLA) antigens.     

Enzyme immunofiltration staining assay for immediate diagnosis of herpes simplex virus and varicella-zoster virus directly from clinical specimens.

A simple, sensitive, 30-min assay for the detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) antigens in clinical specimens is described. The assay utilizes a filter manifold, biotinylated monoclonal antibodies, streptavidin-horseradish peroxidase conjugate, and the substrate aminoethylcarbazole. The method stains infected cells and cell debris a bright red and is easily interpreted with a dissecting microscope. Reconstruction experiments indicated that as few as two virus-infected cells per swab could be detected. This was equivalent to an infectivity titer of 29 to 160 50% tissue culture infective doses per infected cell and 250 times more sensitive than an enzyme immunofiltration assay which detects a soluble reaction product. Clinical swab specimens collected from ocular, oral, skin, and genital lesions were cultured for virus, and the remaining cell debris was immobilized on glass fiber filters and stained for the presence of virus antigens. Of 71 HSV culture-positive specimens, 66 (93%) were positive for HSV antigens. All of nine VZV culture-positive specimens were positive for VZV antigens. Of 98 culture-negative specimens, 7 were positive for either HSV (n = 3) or VZV (n = 4) antigens.     

Detection of monoclonal influenza antibodies synthesized in culture by hybridoma cells with a solid-phase indirect immunofluorometric assay

A solid-phase indirect immunofluorometric assay for measuring reactions of mouse monoclonal antibodies with antigen has been developed, with influenza virus as a model. Purified IgG from hyperimmune rabbit sera is covalently linked to polyaminostyrene beads, to which influenza viruses are then bound immunologically to make solid-phase antigens. Alternatively, the virus is covalently coupled directly to the beads. Mouse antibodies, produced by hybridoma cells in culture, are reacted with constant amounts of solid-phase antigens, and then indirectly quantitated by adding FITC-labeled antimouse Ig and measuring the flourescent intensity with a filter-fluorometer. The assay system permits rapid screening for low levels of antibodies synthesized by hybridoma cells in culture. It is about 25- to 150-fold more sensitive than hemagglutination inhibition tests in detecting monoclonal antibodies reactive with influenza virion HA protein.     

Screening Monoclonal Antibodies against Cell-Surface Antigens High Frequency of Natural Antibodies

Two commonly used methods for screening hybridoma supernatants against cell-surface antigens were performed simultaneously on the supernatant culture fluids of different hybridizations, and compared with the detection of Ig secretion. Supernatants reacting with glutaraldehyde-fixed cells (ELISA (Enzyme-linked immunosorbent assay) on fixed cells) are mostly non-specific for the immunogen; in addition, with this method, only half of the antibodies detected by immunofluorescence are identified. These results can be explained by the frequent occurrence of hybridomas secreting antibodies displaying 'natural antibody' properties, which are strongly reactive with intracellular antigens, and apparently made accessible by the fixation procedure. Since artefacts impair the interpretation of results with ELISA on fixed cells, and complement-mediated cytotoxicity usually as a low yield, membrane immunofluorescence remains the best method for screening hybridoma antibodies.     

The Rose Bengal assay for monoclonal antibodies to cell surface antigens: comparisons with common hybridoma screening methods

An automated colorimetric procedure for detection of antibodies specific for cell surface antigens (1) has been compared for specificity and sensitivity to other methods of hybridoma supernatant screening. The Rose Bengal colorimetric assay (RBA) compared favourably in these respects with whole cell radioimmunoassay and indirect immunofluorescence with manual or flow cytometric analysis (FACS). A major advantage of the method is that it allows a large number of samples to be screened in a comparatively short time. Unlike other semi-automated colorimetric assays, such as ELISA, the procedure does not require cell fixation, which can destroy some antigenic determinants. The original assay of O'Neill and Parish (1) has been modified to give increased sensitivity and also to enable the detection of erythrocyte specific antibodies by elimination of the dye staining step and direct measurement of haemoglobin by spectrophotometry. The RBA allows detection of monoclonal antibodies (MoAb's) binding to only a proportion of the cells in a sample, which is an important feature when hybridoma supernatants are screened for reactivity to a minor cell population, for example against leukaemic cell samples with low percentages of blast cells.     

The Rose Bengal Assay for Monoclonal Antibodies to Cell Surface Antigens: Comparisons with Common Hybridoma Screening Methods

An automated colorimetric procedure for detection of antibodies specific for cell surface antigens (1) has been compared for specificity and sensitivity to other methods of hybridoma supernatant screening. The Rose Bengal colorimetric assay (RBA) compared favourably in these respects with whole cell radioimmunoassay and indirect immunofluorescence with manual or flow cytometric analysis (FACS). A major advantage of the method is that it allows a large number of samples to be screened in a comparatively short time. Unlike other semi-automated colorimetic assays, such as ELISA, the procedure does not require cell fixation, which can destroy some antigenic determinants. The original assay of O'Neill and Parish (1) has been modified to give increased sensitivity and also to enable the detection of erythrocyte specific antibodies by elimination of the dye staining step and direct measurement of haemoglobin by spectrophotometry. The RBA allows detection of monoclonal antibodies (MoAb's) binding to only a proportion of the cells in a sample, which is an important feature when hybridoma supernatants are screened for reactivity to a minor cell population, for example against leukaemic cell samples with low percentages of blast cells.     

Generation of human monoclonal antibodies to cancer-associated antigens using limited numbers of patient lymphocytes

A limiting dilution method for the efficient transformation by Epstein-Barr virus (EBV) of human B lymphocytes has been applied to the production of human monoclonal antibodies to ovarian cancer-associated antigens. Limited numbers (e.g., 2 X 10(5)) of EBV-infected B lymphocytes from ovarian cancer patient spleen, lymph node, tumor, ascites and blood were successfully transformed using this method. An immunofiltration assay system was employed to identify EBV transformants secreting IgM antibody which reacted selectively with ovarian cancer patient ascites tumor cells, but not with a mixture of normal cell types. A miniature Western blot assay was utilized to screen for IgG reactivity to protein species in detergent extracts of ovarian cancer tumor cells. EBV-transformed cells selected after screening were then fused with heteromyeloma fusion partner SHM-D33 resulting in efficient recovery of hybridomas secreting MAb of the desired specificity. Human MAbs which selectively react with antigens associated with ovarian cancer tumor cells were obtained.     

抗人红细胞膜抗原非凝集型单克隆抗体的研制及特性鉴定

目的:制备抗人红细胞膜抗原的非凝集型单克隆抗体(mAb)并鉴定其特性。方法:应用杂交瘤技术,以人O型红细胞膜抗原免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,用聚凝胺试管法筛选识别红细胞表面共同抗原的抗体,玻片凝集实验剔除凝集型抗体(完全抗体),再将分泌非凝集型mAb(不完全抗体)的杂交瘤细胞株用有限稀释法克隆化3次。对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果:获得1株可稳定分泌mAb的杂交瘤细胞2E8。mAb2E8为IgG1类,可特异性地识别红细胞膜上的H抗原,没有种属交叉血凝反应。杂交瘤细胞的培养上清与人的A、B、AB和O型红细胞均能产生强凝集,凝集效价为1∶1024,腹水mAb的凝集效价达到1∶64×106。mAb的亲和力用凝集试验检测,出现血凝的时间为7s,3min以内凝块>1mm。结论:成功地制备了针对红细胞膜H抗原的非凝集性mAb,此mAb的凝集效价、相对亲和力及特异性均较良好,为构建双特异性抗体奠定了基础。     

A simple immunofiltration assay for mucins secreted by a human colonic epithelial cell line

An enzyme-linked immunosorbent assay for the quantitative detection of mucins secreted in culture by a human colonic epithelial cell line has been developed. Dilutions of mucin standards and culture media were applied to nitrocellulose membranes by vacuum filtration through a specially designed immunofiltration manifold. Sequential incubation of the nitrocellulose with blocking buffer, anti-mucin polyclonal antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG permitted quantitation of mucins. Linear and reproducible standard curves were obtained with mucins purified by gel filtration chromatography using a gel of large pore size. The assay was found to be simple, sensitive, reproducible, rapid and not influenced by contaminating proteins up to a concentration of 500 micrograms/ml. This assay has been used to monitor column fractions obtained during the analysis of mucin preparations, and to study the effects of cholera toxin on the secretion of mucins by cultured cells.     

Indirect 125I-labeled protein A assay for monoclonal antibodies to cell surface antigens.

An assay for detection of monoclonal hybridoma antibodies against cell surface antigens is described. Samples of spent medium from the hybridoma cultures are incubated in microtest wells with cells, either as adherent monolayers or in suspension. Antibodies bound to surface antigens are detected by successive incubations with rabbit anti-immunoglobulin serum and 125I-labeled protein A from Staphylococcus aureus, followed by autoradiography of the microtest plate or scintillation counting of the individual wells. Particular advantages of this assay for screening hybridomas are: (1) commerically available reagents are used, (2) antibodies of any species and of any immunoglobulin class or subclass can be detected, and (3) large numbers of samples can be screened rapidly and inexpensively. We have used the assay to select hybridomas producing monoclonal antibodies to surface antigens of human melanomas and mouse sarcomas.     

人瘦素单克隆抗体的制备及初步应用

以亲和纯化的人重组瘦素作为免疫原，用经典的杂交瘤技术制备出抗人瘦素单克隆抗体。放免法（Ｌｉｎｃｏ ＲＩＡ ｋｉｔ）筛选阳性孔，进行三次克隆化培养。体内诱生法收集杂交瘤细胞腹水。经不同稀释度２Ｅ４单克隆腹水抗体与＾１２５Ｉ－瘦素高度结合呈量效关系。以辣根过氧化物酶标记瘦素单克隆抗体、荧光免疫组化技术，在原位检测人脂肪组织细胞内瘦素。结果显示，正常人脂肪组织细胞内含有大量棕褐色沉淀。表明人体内瘦素和共     

Detection of antibodies to cell surface antigens by a simplified cellular elisa (CELISA)

An improved method for screening human hybridoma antibodies to cell surface antigens is described. The following modifications have been developed: rapid expansion of desired screening cell types by EBV transformation; use of only 5 X 10(4) cells/well; elimination of the need for glutaraldehyde fixation; elimination of the requirement for PLL to attach cells to microplates; preparation of a large number of plates which can be stored at 4 degrees for 3 months; Protein A-peroxidase ELISA assay yielding excellent replicates, low background "noise", and high OD readings for positive wells. The techniques we have developed should greatly simplify and shorten the assay procedures for detecting human antibodies to a variety of cell surface antigens.     

B细胞生长因子活性上清对人—人杂交瘤细胞生长影响的研究

本研究用金黄色葡萄球菌(Cowan 1株)死菌制剂(SAC)刺激人B淋巴细胞建立了人B细胞生长因子(BCGF)的检测系统。发现经PHA刺激的人扁桃体细胞培养上清可以促进SAC活化的B细胞的生长,~3H-TdR掺入率提高了2～3倍。同时,用~3H-TdR掺入法和有限稀释法观察了该上清对人-人B细胞杂交瘤生长和克隆率的影响,发现其可以促进人-人B细胞杂交瘤的生长、~3H-TdR掺入率提高2～3倍,可提高克隆率3倍以上。但人-人B细胞杂交瘤可吸收掉上述BCGF活性上清中的促杂交瘤细胞生长因子(HGF)活性,而对BCGF活性无明显影响。提示该上清同时具有人BCGF和HGF活性,部分BCGF活性与HGF活性可能是由不问的分子所介导。     

Evaluation of two commercial tests for human immunodeficiency virus antigens in culture supernatant fluid

Two commercial enzyme-linked immunosorbent assays (EIAs) for human immunodeficiency virus (HIV) antigens were evaluated for sensitivity and reproducibility by use of supernatant fluid from cell cultures of peripheral mononuclear cells of 18 patients with acquired immunodeficiency syndrome (AIDS) and 12 asymptomatic HIV antibody-positive patients. Both commercial assays detected HIV antigen in all cultures by day 21; however, the Abbott HIV antigen EIA (Abbott Laboratories, North Chicago, IL) detected HIV antigen three to seven days earlier in 15 of 48 cultures (31%), on the same day in 32 cultures (67%), and three days later in 1 culture (2%) when compared with the DuPont HIV antigen EIA (DuPont Laboratories, Wilmington, DE). In serial twofold dilution experiments, the Abbott HIV Ag EIA was found to have at least a twofold to eightfold increased sensitivity over the DuPont assay. Repeat testing of 15 initially positive supernatant fluids by both assays revealed that 13 of 15 and 12 of 15 were consistently positive by the Abbott and DuPont assays, respectively. The authors conclude that the Abbott EIA demonstrated better performance in this study than the DuPont EIA for detection of HIV in cell culture because of its shorter time-to-positivity and greater sensitivity.