C1q binding by a high affinity anti-fluorescein murine monoclonal IgM antibody and monomeric subunits

Comparative interactions of purified rabbit C1q with 18-2-3, a high affinity (2-3 X 10(10) M-1) anti-fluorescein (anti-F1) murine monoclonal IgM antibody (pentamer) and constitutive monomeric subunits (IgMs) were studied. Using a solid phase radioimmunoassay (SPRIA), based on immobilized polyvalent antigen, it was shown that the mechanism of C1q binding to IgM was characteristically multiphasic while IgMs yielded monophasic binding curves. The latter compared qualitatively and quantitatively with a monoclonal IgG2a anti-fluorescein antibody with the same intrinsic affinity of 2-3 X 10(10) M-1. C1q binding efficiency to antibodies was significantly enhanced when the immunoglobulins interacted with immobilized multivalent antigen. Monoclonal IgM antibody bind identically to six F1-carrier protein conjugates independent of epitope (F1) density. In contrast, the C1q-antibody interaction binding was dependent upon epitope density. An average distance between F1 epitopes of 80 A was optimal for C1q binding by IgM. At low concn of IgM, when fluorescein was bound by antigen-binding sites on adjacent subunits of an intact pentamer, C1q appeared to bind IgM intramolecularly.     

A rat anti-mouse kappa chain specific monoclonal antibody, 187.1.10: purification, immunochemical properties and its utility as a general second-antibody reagent

A rat IgG1 monoclonal antibody, produced by hybridoma 187.1.10, exhibits specificity for mouse immunoglobulins containing kappa light chains (Yelton et al., 1981). The 187.1.10 hybridoma cell line secreted upwards of 200 micrograms/ml of monoclonal antibody in tissue culture and the secreted product was purified in a single step by antigen-immunoadsorbent affinity chromatography. The homogeneity of the purified 187.1.10 protein was determined by isoelectrofocusing and SDS gel electrophoresis. Equilibrium binding analyses of the radioiodinated 187.1.10 antibody indicated a strong interaction with its antigen of KA = 2 X 10(9) l/mole. The 187.1.10 antibody did not readily bind to Staph. aureus protein A unless it was complexed with antigen. The binding of immune complexes of 187.1.10 to protein A was shown to be dependent on the Fc region of the antigen. The utility of the 187.1.10 monoclonal antibody as a general second antibody reagent for studying mouse immunoglobulins was demonstrated in a rapid solid phase immunoprecipitation assay to detect and analyze radioiodinated membrane proteins of a human cytotoxic T cell line.     

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.     

Quantitative assessment of human leukocyte antigen-G protein in amniotic fluid by a double-determinant enzyme-linked immunosorbent assay using anti-human leukocyte antigen-G-specific antibody '87G'.

PROBLEM: The objective of this study was to establish an enzyme-linked immunosorbent assay (ELISA) system, in an attempt to quantify the amount of human leukocyte antigen (HLA)-G protein in amniotic fluid. METHOD OF STUDY: We established a double-determinant ELISA system using the anti-HLA-G specific mouse monoclonal antibody '87G' as a capture antibody and the horseradish-peroxidase labeled rabbit anti-human beta2-microglobulin antibody as a detection antibody. We then measured the concentration of HLA-G protein in amniotic fluid samples from nine normal second-trimester pregnant women and in serum samples from eight normal males. RESULTS AND CONCLUSION: HLA-G protein was detected in amniotic fluid at a concentration of 275 ng/ml (197-343 ng/ml) (median value and 95% confident range), whereas the concentration of HLA-G protein in male serum was below the minimum detection level.     

Measurement of IgG-blocking antibodies by ELISA using monoclonal antibody and fluorogenic substrate

An enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate has been developed for quantitation of IgG-blocking antibodies in human serum. Generation of fluorescent product was linear with time to 60 min. Using optimal conditions the ELISA was sensitive to less than 1 ng/ml of specific IgG to short ragweed pollen. The assay demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation = 8.8%). Reproducibility was determined by constructing precision profiles for intra and interassay variation for the entire working range of the assay. Intraassay CVs ranged from a mean of 13% at threshold to less than 5% at higher antibody concentration. Interassay reproducibility similarly ranged from 18 to 10%. In this assay the effect of serum dilution on nonspecific binding was minimal and specific binding of 4-10 ng IgG antibody to the antigen-adsorbed wells was largely complete (75.8 +/- 4.8%) and highly specific (greater than 98%). This application of ELISA for ragweed IgG antibody measurement has performance specifications equal or superior to previously developed radioimmunoassay and ELISA systems.     

Polyaniline-dacron composite as solid phase in enzyme linked immunosorbent assay for Yersinia pestis antibody detection

Polyaniline (PANI) was chemically synthesized on a dacron disk surface and an antigen (F1 fraction) obtained from Yersinia pestis was covalently fixed onto this composite via glutaraldehyde. The enzyme linked immunosorbent assay (ELISA) or rapid ELISA procedure detected immunoglobulin G (IgG) anti-F1 fraction in human serum employing this derivative. The appropriate conditions for carrying out the test were established as an antigen concentration of 2 microg/PANI-dacron disk, peroxidase labeled goat anti-human IgG conjugate diluted 4000 times, and a serum dilution of 1:100. The PANI-dacron disks showed greater antigen retention than conventional poly(vinyl chloride) plates and less antibody unspecific adsorption.     

Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma.     

A cell ELISA for the quantitation of leukocyte antigens. Requirements for calibration

A cell ELISA for human leukocytes has been established and standardized. The assay was based on the use of air-dried and methanol-fixed cells which were attached to microplate wells. Cellular antigens were measured through the subsequent binding of monoclonal antibody (MoAb), biotinylated sheep Ig against mouse IgG and streptavidin-peroxidase conjugate. The test was standardized by measuring both the specific antigen and the amount of cellular protein in each single sample and was calibrated either with intact cells or isolated plasma membranes prepared from the cells under study. The detection of the pan T-lymphocyte-specific 3A1 antigen (CD7, P40) in fewer than 5000 cells or less than 50 ng membrane protein was possible.     

人可溶性B7-2分子的定量检测及应用

选取识别不同抗原位点的鼠抗人B7-2单抗1F9和6C8分别作为包被和检测抗体,用生物素(Biotin)标记检测抗体,建立双单抗夹心的sB7-2检测方法。在对其特异性、稳定性和准确性进行分析的基础上,对20例系统性红斑狼疮(SLE)患者及30名健康献血者血清中sB7-2的含量进行了测定。建立的sB7-2检测方法的灵敏度为0.15 ng/ml,具有良好线性关系的检测范围0.31~20.00 ng/ml。单抗1F9包被的测定板,4℃放置30 d内,工作曲线中各测定点的变异系数<5.9%。SLE患者血清中sB7-2的含量为(2.207±0.517)ng/ml,与健康献血者的(1.275±0.263)ng/ml比较具有统计学的显著差异(p<0.01)。本研究中建立的人sB7-2检测方法,能够对血清中sB7-2进行定量分析,可为该分子的基础研究及临床相关疾病的辅助诊断与判断预后提供参数。     

Quantitation of mouse monoclonal antibody and human anti-mouse antibody response in serum of patients

The assays required to study pharmacokinetics of monoclonal antibodies and human anti-mouse antibody response in man, utilizing enzyme linked immunosorbent assay, have been at best semi-quantitative. We have developed and well characterized a quantitative assay for measurement of murine monoclonal antibodies and human anti-mouse antibody in circulation of patients injected with murine monoclonal antibodies. The sensitivity of mouse IgG assay is approximately 1 ng/ml and cross reactivity of IgG's from other species were less than 0.01%. The pre-therapy samples from 30 patients treated with monoclonal antibody CO17-1A as well as serum from 54 individuals had no detectable human anti-mouse antibody indicating specificity. The sensitivity of this assay was approximately 10 mg/ml. The utility of these assays was demonstrated in a group of patients receiving 400 mg of monoclonal antibody CO17-1A. The half-life and the degree of immune response can be measured using these assays.     

Detection of circulating excretory secretory antigens in human fascioliasis by sandwich enzyme-linked immunosorbent assay.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigen in humans with fascioliasis. The assay uses antibodies against Fasciola hepatica excretory secretory (ES) antigens. A monoclonal antibody was used to capture circulating ES antigens, and a human polyclonal antibody peroxidase conjugate was used to identify circulating ES antigens. Optimal dilutions of all reagents were determined by block titration. The antigen concentration in sera from patients was estimated by comparing the optical density at 492 nm of test sera with a standard curve. All of the serum samples from 25 patients with parasitological evidence of fascioliasis had a detectable antigen concentration (more than 10 ng/ml). None of the serum samples from 80 patients infected with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay.     

两株抗人CD40配体单克隆抗体的制备及生物学特性的研究

目的 :研制功能性抗CD4 0L单克隆抗体 ,探讨其生物学特性及其运用价值。方法 :采用B淋巴细胞融合、免疫荧光、免疫印迹和竞争抑制等方法获得鼠抗人CD4 0LmAb ,通过Daudi细胞生长抑制试验、混合淋巴细胞反应观察抗CD4 0LmAb的生物学功能。结果 :经表型分析、Westernblot和竞争抑制试验 ,证实mAbB1、mAb4F1是识别人CD4 0L分子的特异性单抗 ,且识别的位点不同 ;mAbB1、mAb4F1均能不同程度地拮抗CD4 0L TC介导的Daudi细胞生长抑制和抑制混合淋巴细胞反应。结论 :成功地获得两株能稳定分泌特异性抗人CD4 0L单克隆抗体的杂交瘤 (1B1、4F1) ,这两株单抗对CD4 0L的生物学功能具有明显的阻断作用 ,为阻断型单抗     

Selection of anti-F protein B-cell repertoires in normal mice

The hypothesis that self-tolerance to F protein antigen exclusively concerns T cells was tested by determining the frequencies of B lymphocytes producing anti-F antibodies in bone marrow (BM), spleen and peritoneal exudate (PEC) cells from normal, immune or tolerant animals, and in responder and non-responder mouse strains. Using an ELISA spot assay and lipopolysaccharide stimulation, we found that anti-F frequencies were highest in BM and “naturally activated” large spleen cells, followed by resting spleen and PEC cells. Anti-F specificities were also induced among “natural” Ig-secreting cells of normal individuals. Specific immunization of responder mice doubled the splenic frequencies, while tolerization had no effect. Similar results were obtained in BALB/c and A/J mice, while C57BL/6 contained fewer anti-F B cells in spleen, but not in BM. These results support the notion that self-tolerance to F antigen can primarily be ascribed to T cells, but they also show F-antigen-specific selection of B-cell repertoires.     

Characterization of the early antibody response in bovine tuberculosis: MPB83 is an early target with diagnostic potential

A 26-kDa antigen has been shown to be a dominant antibody target in Mycobacterium bovis-infected cattle. In this study, that antigen was used as an immunogen to raise a panel of mouse monoclonal antibodies. The majority of those bound to native protein with a molecular mass of 26 kDa and to recombinant MPB83, strongly suggesting that MPB83 is an important B-cell antigenic target in bovine tuberculosis. In order to provide assessment of the potential of measuring antibody responses to the native protein, one monoclonal antibody, 1F11, was incorporated into an enzyme-linked immunosorbant assay format to trap antigen from a crude bacterial extract. Despite some disadvantages of this format, serum samples from cattle which had been infected experimentally with M. bovis, and from tuberculin skin-test-negative and -positive field cattle were tested for the presence of antibodies. Data from the skin-test-negative cattle allowed an arbitrary cut-off value to be established and, under these conditions, test sensitivity and specificity were estimated at 37.5 and 89%, respectively. These results indicate potential for MPB83 in the development of assays for serological diagnosis of bovine tuberculosis.     

Selection of anti-F protein B-cell repertoires in normal mice

The hypothesis that self-tolerance to F protein antigen exclusively concerns T cells was tested by determining the frequencies of B lymphocytes producing anti-F antibodies in bone marrow (BM), spleen and peritoneal exudate (PEC) cells from normal, immune or tolerant animals, and in responder and non-responder mouse strains. Using an ELISA spot assay and lipopolysaccharide stimulation, we found that anti-F frequencies were highest in BM and "naturally activated" large spleen cells, followed by resting spleen and PEC cells. Anti-F specificities were also induced among "natural" Ig-secreting cells of normal individuals. Specific immunization of responder mice doubled the splenic frequencies, while tolerization had no effect. Similar results were obtained in BALB/c and A/J mice, while C57BL/6 contained fewer anti-F B cells in spleen, but not in BM. These results support the notion that self-tolerance to F antigen can primarily be ascribed to T cells, but they also show F-antigen-specific selection of B-cell repertoires.     

Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.     

A novel and effective intranasal immunization strategy for respiratory syncytial virus.

In designing subunit vaccination strategies for respiratory syncytial virus (RSV), immunization by mucosal routes may present a realistic alternative to parenteral administration for inducing protective immune responses. To this end, we have utilized the BALB/c mouse model and an adjuvant formulation containing caprylic/capric glycerides (CCG) and polyoxyethylene-20-sorbitan monolaurate (PS). The intranasal (i.n.) delivery of purified natural F protein (3 microg per vaccine) formulated with CCG-PS resulted in the generation of statistically heightened serum anti-F protein immunoglobulin G (IgG), IgG1, IgG2b, and IgA antibodies. In addition, the presence of locally produced anti-F protein IgA was demonstrated in both vaginal and nasal washes of vaccinated mice. That production of specific serum and mucosal immunoglobulins resulted in functional immune responses was shown in neutralizing antibody assays and protection of mouse lungs against subsequent live virus challenge. Consequently, we propose a novel vaccine formulation composed of purified natural RSV F protein in CCG-PS as a viable intranasal immunogen to stimulate anti-RSV immune responses in humans.     

Specificity of mouse hybridoma antibodies to DNA. II. Phospholipid reactivity and biological false positive serological test for syphilis

Using mouse hybridoma monoclonal antibodies to DNA from MRL/lpr and NZB X NZW (B/W F1) mice, the reactivity of anti-DNA antibodies to several phospholipids was analysed. The anti-DNA antibody which reacted with the common antigenic determinants on the phosphate-sugar backbone of nucleic acid could bind to the cardiolipin, but failed to bind to other phospholipids, including VDRL antigen. We tentatively conclude that the anti-cardiolipin antibody is identical with the anti-DNA antibody, but differs from the BFP reactor.     

Preparation and characterization of polyclonal and monoclonal antibodies against fusarenon‐X

Polyclonal and monoclonal antibodies against fusarenon‐X were prepared by using a fusarenon‐X oxime derivative coupled to human serum albumin as the antigen for the immunization of rabbits and BALB/c mice, respectively. The specificity and sensitivity of these antibodies were tested by using fusarenon‐X oxime coupled to horseradish peroxidase as an enzyme‐linked toxin in a competitive assay with a double antibody solid phase. The relative cross‐reactivities of the polyclonal antiserum with T‐2 toxin, diacetoxyscirpenol, fusarenon‐X and neosolaniol were 3.67, 2.75, 1.0 and 0.58, respectively. The monoclonal antibody, however, showed considerable cross‐reactivity only with neosolaniol (0.28). The detection limit for fusarenon‐X was 150 pg/ml (5 pg per assay) and 300 pg/ml (10 pg per assay), respectively, using the polyclonal and monoclonal antibodies.     

Purification and characterization of human liver specific F antigen.

This paper reports the properties of purified human F antigen (liver-specific antigen). Homogenates of liver in 0-25 M sucrose were centrifuged at 105,000 g. The supernatants were chromatographed on Sepharose 6-B and four major peaks were separated. The third peak proved to be predominantly F antigen. This fraction was subsequently subjected to DEAE-cellulose column chromatography and F antigen was eluted at a concentration less than 0-2 M NaCl in 0-01 M sodium phosphate buffer (pH 7-2). Finally, purified F antigen was obtained after preparative isoelectric focusing. Purified human F antigen was found to have a mol. wt between 40,000 and 80,000, a pI of 6-5-6-7 and a density of 1-26. It is a protein antigen and contains no detectable carbohydrate or lipid. No differences were found in purified F-antigen preparations from several species when tested by sodium dodecyl sulphate (SDS) disc gel electrophoresis. Immunofluorescent studies showed that F antigen was homogeneously distributed in the cytoplasm of liver cells but was not present on the cell surface. Immunization of guinea-pigs with purified human liver-specific protein did not induce antibody to the F antigenic determinant defined by mouse anti-F antiserum. It did, however, induce antibodies to two human liver antigens. One of these seems to be a human-specific determinant on the F antigen molecule and the other appears to be a separate molecule which is similar in molecular weight and electrophoretic mobility to the F-antigen molecule.