Production and Analytical Properties of Antibodies with High Specificity to Zearalenone

The time course of production, specificity, and analytical potential of antizearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; -zearalenole, 0.15%; and -zearalenole, <0.02%. With synthetic analogues: zearalanone, 31.7% and -zearalanone, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Radioimmunoassay of Human Serum Thyrotrophin

The double antibody radioimmunoassay of serum thyroid-stimulating hormone (TSH) allows measurement of circulating levels of the hormone in most normal subjects. The serum TSH level in normal subjects is 1·6 ± 0·8μU/ml. Patients with non-toxic goitre and acromegaly have normal TSH levels. Values are always raised in hypothyroid patients (with primary thyroid disease) and are significantly lowered in those with hyperthyroidism. Of the many stimuli used in an attempt to raise TSH levels in normal adult subjects only three—synthetic thyrotrophin-releasing hormone, ethinyloestradiol, and carbimazole plus iodides—have been effective. The major clinical application of the TSH immunoassay lies in the diagnosis of minor degrees of hypothyroidism. An impaired response of serum TSH to synthetic thyrotrophin-releasing hormone should also help in the diagnosis of hypopituitarism affecting TSH production.     

Radioimmunoassay of Free Genistein in Human Serum

Two radioimmunoassay (RIA) systems for genistein have been established, based on polyclonal antibodies against genistein-4′-O-(carboxymethyl)ether-bovine serum albumin and genistein-7-O-(carboxymethyl)ether-bovine serum albumin conjugates. The sensitivities of assays were 4.44 and 10.4 fmol (1.2 and 2.8 pg)/tube, respectively, the intraassay coefficients of variation ranged from 3.54 to 9.30%, the interassay C.V. varied from 6.72 to 19.7%, depending on the type of method and on genistein concentration. The cross-reactivities with other chemically related compounds (with exception of genistein derivatives at the position used for construction of the immunogen) were 5.5 and 6.1% for daidzein and 3.9 and 0.04% for formononetin in RIAs using reagents prepared through positions 4′- and 7- of genistein, respectively. The method was used for measurement of genistein levels in 26 omnivore subjects and in three volunteers after consumption of a meal prepared from 125 g of cooked whole soybeans. The values obtained in ether extracts from human sera were almost identical for both RIA systems, indicating that both RIAs measure the same entity.     

Radioimmunoassay of Triiodothyronine in Unextracted Human Serum

Serum triiodothyronine (T-3) concentrations have been estimated by radioimmunoassay using unextracted serum. The serum T-3 concentrations have been shown to be similar in two separate European populations (0·76-1·67 ng/ml). Raised T-3 values have been observed in all subjects with hyperthyroidism. Low values are seen in hypothyroidism although there is some overlap with the normal range. There is a good correlation between serum T-3 and serum thyroxine (T-4) concentrations, and estimation of T-3 seems likely to prove a practical and reliable test of thyroid function.     

Radioimmunoassay of Bovine,Ovine and Porcine Luteinizing Hormone with a Monoclonal Antibody and a Human Tracer

A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human 125 ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH and oLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 µg/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar.     

Production, Characterization, and Use of Monoclonal Antibodies Against Acremonium coenophialum

Monoclonal antibodies were produced using intact mycelium of the fungus Acremonium coenopbialum as the immunogen. During the initial stages of characterization, the antibodies were found to react to A. coenophialum, A. loliae, Epichloe typhina , and also to cross-react with some other fungi normally associated with tall fescue. Careful selection of the ELISA system in which the antibodies were used eliminated reactions to all but the two Acremonium spp. and E. typhina. Asa result it was possible to detect Acremonium spp. (presumably A. coenophialum ) sensitively and unambiguously in leaf tissue of tall fescue.     

Characterization of Sulfhydryl Heterogeneity in Human Serum Albumin and Recombinant Human Serum Albumin for Clinical Use

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.     

人心肌肌钙蛋白Ⅰ单克隆抗体及多克隆抗体的制备

目的:以重组人心肌肌钙蛋白Ⅰ(cTnⅠ)为抗原制备鼠源单克隆抗体(McAb)及兔源多克隆抗体,并鉴定抗体的特性。方法:以纯化的重组人cTnⅠ为抗原免疫BALB/c小鼠,取鼠脾细胞同Sp2/0骨髓瘤细胞融合,利用选择培养基筛选融合的杂交瘤细胞,用有限稀释法分离获得能够稳定分泌抗cTnⅠ的McAb阳性克隆,并利用体内诱生法大规模制备McAb,用辛酸-硫酸铵沉淀法纯化抗体;兔多抗制备以cTnⅠ为抗原常规免疫后取其血清;用间接ELISA和Western印迹鉴定抗体的性质。结果:经ELISA鉴定,筛选出5株能分泌cTnⅠMcAb的杂交瘤细胞株,即C5B2、C5B3、C5B4、C5B1、B1A6,效价最高的B1A6株分泌的McAb为IgG3型,纯化后效价为1∶10000,亲和常数为1.08×10-9mol/L,Western印迹鉴定表明cTnⅠMcAb有良好的特异性;兔多抗纯化后的效价为1∶8000。结论:制备了具有良好特性的cTnⅠMcAb和多克隆抗体。     

Single-Batch Production of Recombinant Human Polyclonal Antibodies

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the Sympress^TM I technology. The Sympress^TM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the Sympress^TM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the Sympress^TM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the Sympress^TM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.     

Production and Use of Monoclonal Antibodies to Pseudomonas andropogonis

Pseudomonas andropogonis is an important pathogen of worldwide distribution in ornamental and other plant species from 15 families. This paper reports the production and characterization of monoclonal antibodies (MAbs) to P. andropogonis and evaluation of their use in the detection of the pathogen in carnation cuttings. Ten stable hybridoma cell lines were produced. Results of indirect ELISA and indirect immuno-fluorescence showed that MAb 6B3 was specific for P. andropogonis; MAb 3D5W1 reacted with both P. andropogonis and P. caryophylli; six other MAbs reacted with all strains of seven species of rRNA group II pseudomonads tested except P. solanacearum and P. pickettii. Eight of the ten MAbs failed to cross-react with other non-fluorescent or fluorescent pseudomonads, xanthomonads and other bacteria tested. P. andropogonis was similar in protein profile to other rRNA group II pseudomonads tested except P. solanacearum and P. pickettii. Epitopes were clearly located within the cell by immunogold labelling. Of four MAbs that were isotyped, two possessed IgGl and two the IgM heavy chain. P. andropogonis was readily detected by combining immunofluorescence and detached carnation leaf assay using an initial inoculum of 4 × 10° colony forming units (cfu) ml^?1 after enrichment at room temperature for 4 days.     

Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial β-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UAS_E) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.     

Monoclonal Antibodies to Substance P: Production, Characterization of Their Fine Specificities, and Use in Immunocytochemistry

Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholinesterase as label. All five monoclonal antibodies were directed to the C-terminal pentapeptide of substance P, especially to the Phe7 residue. They cross-reacted with neurokinin A and to some extent with neurokinin B but not with other nontachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin-like immunoreactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP 14 monoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.     

Production and Use of Monoclonal Antibodies for Identification of Strains of Rhizobium trifolii

We produced a monoclonal antibody against Rhizobium trifolii 162×95. This antibody in cell culture supernatant was used in an indirect enzyme-linked immunosorbent assay to differentiate strain 162×95 from naturalized strains in the Appalachian region. Nodules crushed in 0.1 to 0.2 ml of phosphate-buffered saline and used to charge enzyme-linked immunosorbent assay plates gave strong absorbance readings. Heat-inactivated and noninactivated portions of 162×95 cultures were strongly reactive, indicating that the antigen is probably a carbohydrate. Of 10 strains from California, where 162×95 was isolated, 6 strongly cross-reacted with the antibody. The cellular protein patterns in a sodium dodecyl sulfate-polyacrylamide gradient gel of cross-reactive strains were essentially identical. A Western blot analysis indicated that the antibody was against a 19.8-kilodalton band. The Western blot analysis also revealed that the polyvalent antiserum contained other strongly reacting antibodies with molecular weights of approximately 20,000, indicating the possibility that other monoclonal antibodies to detect strain 162×95 may be produced. However, the available antibody has been shown to be useful for short-term experiments. Based upon protein profiles and immunological reactions, there are 4 or 5 California strains rather than 10.     

磁免疫电化学发光检测肺癌血清p53抗体

肿瘤抑制基因——p53基因的突变可能产生p53抗体.p53抗体在肿瘤的诊断、预后及监测等方面具有重要的临床价值.目前检测p53抗体的方法,如酶联免疫分析方法,需要多个步骤,比较费时,且大部分检测指标只能是半定量,具有一定的局限性.提出了一种磁免疫电化学发光(IM-ECL)分析方法定量检测人血清p53抗体.这种新的分析方法检测人血清p53抗体的最低检测极限可达到10 ng/L,标准曲线的动力学范围和线性范围达到5个数量级（0.01～1 000 μg/L）.我们应用IM-ECL分析法检测肺癌病人血清,只需要50 μl的样品量,30 min的孵育时间和少于50 s的采集时间,得出肺癌血清中p53抗体的阳性率为28.6 ％,然后通过标准曲线定量阳性血清中p53抗体的浓度.从肺癌血清的结果中发现,随着临床分期的升高,p53抗体浓度增加.IM-ECL分析方法在检测限、线性范围、分析时间等方面都优于酶联免疫分析,是一种可行的快速、灵敏、定量检测人血清p53抗体的分析方法.     

Production and Use of Monoclonal Antibodies against Rice Embryo Lipoxygenase-3

A DNA fragment coding for a part of a putative phosphatidylinositol 3 kinase was cloned from Schizosaccharomyces pombe by cross-hybridization with Saccharomyces cerevisiae VPS34 gene, a yeast homologue of mammalian PI-3 kinase. The clone contained an open reading frame of 797 amino acids but lacked the initiation codon, ATG. The predicted amino acid sequence was homologous to those of S. cerevisiae VPS34 and mammalian PI-3 kinase genes. Disruption of the gene resulted in extremely low levels of PI-3-P and higher levels of PI-4-P, supporting the idea that the gene codes for the PI-3 kinase of S. pombe. The disruptants harbored large vacuoles and were sensitive to stresses such as high temperature or high concentration of monovalent and divalent cations.     

Substitution of Milk for Serum in the Production of Human Leukocyte Interferon

The presence of serum in suspensions of Sendai-induced human leukocytes is necessary for the synthesis of significant amounts of interferon. Very little interferon is obtained from serum-free suspensions. Cow's milk or milk casein can substitute for serum in the production of high yields of human leukocyte interferon.     

Accounting for Fluorine: Production,Use, and Loss

Fluorine is an essential element to human health and to the chemical industry. In spite of our dependence on fluorine and fluorine compounds, we have yet to learn to use them wisely. Our fluorine history, which spans about a hundred years, has had negative effects such as hydrofluoric acid pollution caused by aluminum smelters and ozone depletion due to chlorofluorocarbon (CFC) emissions. More recent concerns center on greenhouse effects from CFCs, hydrofluorocarbons (HFCs), and sulfur hexafluoride (SF6). In this article we note also that fluorine is a nonrenewable resource that is nonsubstitutable for many purposes. This article tracks fluorine from sources through conversion processes to end uses, most of which are dissipative. We present a stock‐flow model of the fluorine system. Based on this model we consider some possible measures that could be taken to increase the degree of recovery. To mention one example, a large percentage of the world demand for fluorspar could be supplied by the phosphate rock (fertilizer) industry, which currently dissipates a great deal of recoverable fluorine in waste phospho‐gypsum.     

Lecithin-Agar Assay for Lecithinase Antibodies in Serum

A technique for assay of lecithinase antibodies in serum was developed in this laboratory by using a lecithin-agar plate diffusion procedure based on a combination of described plate assays. Egg yolk lipoprotein composed primarily of lecithin was used as a substrate for reaction with free or non-neutralized lecithinase C after incubation of known amounts of lecithinase C with various dilutions of control and test sera. It was found that the size of the reaction zone was a function of enzyme concentration and inversely proportional to the antibody concentration. Accuracy and precision of the assay were determined. In addition, lecithinase antibody levels in sera from experimentally inoculated rats and rabbits and sera from randomly selected human patients were studied.