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.     

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.     

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.     

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.     

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.     

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.     

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.     

Production of Monoclonal Antibodies to Barley Mild Mosaic Virus and Their Use for Strain Differentiation

A panel of five stable hybridoma cell lines secreting mono- clonal antibodies (mAbs) were produced using a French mechanically transmitted isolate of barley mild mosaic virus (BaMMV-MF) as antigen. All mAbs reacted with BaMMV-MF in two enzyme-linked immunosorbent assay (ELISA) formats: triple antibody sandwich (TAS)-ELISA and antigen-coated plate (ACP)-ELISA. These mAbs recognized epitopes, present on both degraded virions and intact particles. Four mAbs (5C8, 1D5, 1B12, 1A12) belong to the immunoglobulin (Ig)G class and one mAb (3A9) represents an IgM. The five mAbs were compared in TAS- and ACP-ELISA for reactivity with numerous French isolates. These isolates were detected in TAS- and ACP-ELISA with four mAbs (5C8, 1D5, 1B12, 3A9). In both ELISA systems the mAb 1A12 recognized only an epitope specific for BaMMV-MF. All mAbs, except 1A12 recognized also the German (BaMMV-MG), Italian (BaMMV-I) and Japanese (BaMMV-Ka1) isolates in both TAS- and ACP-ELISA. The Japanese isolate (BaMMV-Na1) only reacted with two mAbs (1D5, 5C8) in TAS-ELISA. Only one mAb (3A9) reacted with BaMMV-MF, BaMMV-PF, BaMMV-I,BaMMV-MG and BaMMV-Ka1 in Western blot. These mAbs make it possible to distingish between the three BaMMV serotypes.     

Human CD59 Incorporation into Porcine Endogenous Retrovirus Particles: Implications for the Use of Transgenic Pigs for Xenotransplantation

Transgenic pigs have been engineered to express human CD59 (hCD59) in order to suppress hyperacute rejection of xenotransplants in human recipients. In this study, porcine endogenous retrovirus (PERV) was produced in a porcine cell line expressing hCD59 in order to examine the effect of this complement control protein on PERV neutralization by human sera. hCD59 was found to be incorporated into PERV particles produced from engineered ST-IOWA cells. PERV incorporation of hCD59 resulted in a dramatic inhibition of complement-mediated virolysis by human serum. However, incorporation of hCD59 had no effect on neutralization of PERV by human serum, as measured in infectivity assays. Our results suggest that the use of organs from hCD59 transgenic pigs will inhibit complement-mediated virolysis, but will not compromise the protective effects of human sera on the neutralization of PERV particles.     

Antigen-Binding Properties of Monoclonal Antibodies Reactive with EBNA1 and Use in Immunoaffinity Chromatography

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG₁) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.     

Variation in Deoxynivalenol, 15-Acetyldeoxynivalenol, 3-Acetyldeoxynivalenol, and Zearalenone Production by Fusarium graminearum Isolates

Of 88 isolates of Fusarium graminearum collected from soil or cereals in the United States, 49 produced 15-acetyldeoxynivalenol (15-ADON) as the major isomer; one produced 3-acetyldeoxynivalenol (3-ADON). A total of 26 isolates collected from cereals or soil in Australia, New Zealand, Norway, China, and Poland were used for comparison. Of these, 15 produced 3-ADON as the major isomer and 2 produced 15-ADON.     

Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B

Human FAM76B (hFAM76B) is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s) of FAM76B, murine monoclonal antibodies (MAbs) against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B^-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B) specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s) of FAM76B.