神经纤毛蛋白-1的原核表达及兔抗小鼠神经纤毛蛋白-1抗体免疫学活性的研究

目的:在原核系统中分段表达神经纤毛蛋白-1(Nrp1);将纯化的蛋白免疫家兔后获得特异的抗体,并将其应用于检测组织和细胞中Nrp1分子的表达。方法:提取BALB/c胎鼠脑组织总RNA,通过RT-PCR分段扩增获得Nrp1基因片段,将PCR产物插入表达载体pET28a( ),获得含5个Nrp1基因片段的重组质粒,在大肠杆菌BL21(DE3)中诱导蛋白表达并纯化;将纯化的重组蛋白免疫新西兰大白兔获得针对目的蛋白的特异性多克隆抗体;利用Nrp1特异性多抗检测胎鼠脑组织和HeLa细胞中Nrp1的表达。结果:在原核系统中分段表达了Nrp1蛋白,通过Ni-NTA纯化了Nrp1蛋白片段;纯化的Nrp1蛋白免疫新西兰大白兔获得了具有免疫活性的多抗;兔抗小鼠Nrp1特异性多抗可用于检测组织、真核细胞中Nrp1的表达。结论:应用原核系统成功地表达了Nrp1蛋白,兔抗小鼠Nrp1特异性多抗可用于免疫学检测Nrp1分子的表达。     

The reaction of SLE antibodies with native, single stranded RNA: radioassay and binding specificities.

Escherichia coli 3H-tRNA and MS2 phage 125I-RNA were prepared and used in a sensitive nitrocellulose filter assay. Antibodies that bound these RNA ligands occurred in the sera of several patients with SLE, but not in sera of patients with other connective tissue diseases. The antibody populations that bound polyribonucleotides (largely IgG) were distinct from antibody populations that bound polydeoxyribonucleotides. Competition experiments showed that the anti-RNA antibodies preferentially bound native ssRNA as compared with synthetic single and double stranded polyribonucleotides. There was increasing affinity with increasing m.w. of the ssRNA. The anti-tRNA population was of restricted heterogeneity (Sips index 0.83) and bound tRNA with an average association constant (Ko) of 9 x 10(6) l/mole at 4 degrees C. The anti-MS2 RNA population was much more heterogeneous (Sips index 0.67) and bound MS2 RNA with a Ko of about 3 x 10(9) l/mole at 4 degrees C. Whereas NZB/NZW mice spontaneously produce RNA reactive antibodies with conformation specificity for native tRNA, human SLE anti-RNA antibodies appear to have very little of this type of conformation specificity.     

Monoclonal antibodies can discriminate between some active and inactive forms of leukocyte interferon

Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification.     

A high-throughput hybridoma selection method using fluorometric microvolume assay technology

Monoclonal antibodies (mAb) are not only useful reagents but also represent a promising type of therapeutics due to their high affinity and exquisite specificity for their antigens. A critical step in mAb generation is to identify antigen-specific antibodies. Although enzyme-linked immunosorbent assay (ELISA) has been broadly applied for antibody selection against secreted antigens, an inherent disadvantage for ELISA is the difficulty in identifying antibodies that recognize the native conformation of cell surface antigens. To overcome this drawback, the authors have developed a high-throughput cell-based antibody binding assay using fluorometric microvolume assay technology (FMAT). This method offers a homogeneous assay for detection of antibody binding to its antigen on the cell surface. To distinguish antibodies that bind to antigen on the cell surface from those that bind nonspecifically to cells, the binding is assessed using both antigen-expressing cells and related cells devoid of the antigen expression. This assay can detect antibodies at a concentration as low as 5 ng/mL and cell surface antigen as low as 9000 copies per cell. Results demonstrate that the FMAT method provides a sensitive and homogeneous assay to detect antibody binding to cell surface antigens and is amenable for high-throughput hybridoma selection.     

Effects of sleep deprivation on the development of autoimmune disease in an experimental model of systemic lupus erythematosus

Sleep is hypothesized to play a restorative role on immune system. In addition, disturbed sleep is thought to impair host defense mechanisms. Chronic sleep deprivation is a common occurrence in modern society and has been observed in a number of chronic inflammatory conditions, such as systemic lupus erythematosus (SLE). New Zealand Black/New Zealand White (NZB/NZW) F1 mice develop an autoimmune disease that strongly resembles SLE in humans, exhibiting high titers of antinuclear antibodies associated with the development of rapidly progressive and lethal glomerulonephritis. On the basis of this evidence, the present study examined the onset and progress of lupus in as-yet healthy female mice submitted to sleep deprivation. Sleep deprivation was accomplished by two 96-h periods in the multiple-platform method when mice were 10 wk old, and they were observed until 28 wk of age. Blood samples were collected from the orbital plexus fortnightly to evaluate serum antinuclear antibodies and anti-double-stranded DNA. Proteinuria and longevity as well as body weight were also assessed. The results indicated that mice submitted to sleep deprivation exhibited an earlier onset of the disease, as reflected by the increased number of antinuclear antibodies. However, no statistical difference was found in the other parameters analyzed. According to these results, sleep deprivation could be considered as a risk factor for the onset but not for the evolution of the disease.     

Enzyme-linked coagulation assay. III. Sensitive immunoassays for clotting factors II, VII, and X

The solid-phase clotting assay utilizing fibrinogen coated on the wells of a microtiter plate and peroxidase-fibrinogen in solution as a substrate for thrombin (enzyme-linked coagulation assay, ELCA) has been modified for use as an immunoassay. Direct inhibition of factors II, VII, and X by polyclonal (rabbit) antibodies and of factor X by monoclonal antibodies has been demonstrated at high dilution of these antibodies and detection of the specific factors using ELCA. Using plates coated with a second antibody (goat anti-mouse IgG) as well as fibrinogen, monoclonal antibodies to factors X and VII were measured by binding the active factor to the plate and detection of the bound factor using ELCA. The assay was very sensitive, permitting the detection of as little as 0.2 ng/ml (30 pg/assay) of monoclonal antibody, or less than 0.4 ng/ml (60 pg/assay) of factor Xa. When plates were coated with monoclonal antibody to factor X and fibrinogen, the assay permitted the identification of distinct epitope specificities for two monoclonal antibodies to factor X by distinct competition of the monoclonal antibodies added in the solution phase for binding of factor Xa to the plate. This assay could be applied generally for immunoassay of clotting factors, and could have application in general as an immunoassay amplification system.     

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.     

Binding to native proteins by antipeptide monoclonal antibodies

mAb raised against synthetic peptides derived from cholera toxin, myohemerythrin, and sickle hemoglobin were analyzed by both solid-phase and solution-phase methods. Antipeptide mAb against cholera toxin (mAb TE32 and TE33), against myohemerythrin (mAb B13I2, B13C2, and B13F2), and against sickle hemoglobin (mAb HuS-1 and HuS-2), had been previously described and used for vaccine development, structural characterization, or identification of a specific antigenic determinant, and each was apparently capable of binding both peptide and native Ag. In this study, all were found to bind whole protein when tested against immobilized Ag in a standard solid-phase assay (ELISA), yet none of the antibodies recognized the Ag in its true native form, failing to bind when tested in several solution-phase assay systems, including size exclusion HPLC. This discrepancy may be the result of modifications of the epitope created by interaction and possible denaturation of the protein on the solid-phase matrix. As a consequence, binding of these antibodies to peptides, either immobilized or in solution, or to immobilized protein, cannot be used to infer that the peptide has assumed a conformation that corresponds to that of the cognate sequence in the native protein. A re-evaluation of binding data that relates antipeptide mAb to native structural characteristics may be necessary.     

Type-specific immunodetection of human heart fatty acid-binding protein with polyclonal anti-peptide antibodies

Summary In order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (HFABP_c), four oligo-peptides of 15–20 amino-acids each and corresponding with different antigenic parts of the human H-FABP_c molecule, were synthesized. Polyclonal antibodies against these synthetic peptides were raised in mice (Balb/C) and rabbits (Flemish giant). When tested in enzyme linked immunosorbent assays (ELISA, antibody-capture assay), antisera against three of the four peptides showed a high immunoreactivity with the synthetic peptide selected for immunization as well as with the native human H-FABP_c. Some cross-reactivity with the other synthetic peptides was observed for the rabbit antisera but not for those from mice. Polyclonal antibodies against synthetic peptides can be applied for the specific detection of the native protein in biological preparations containing proteins that show a high degree of homology with the protein to be assayed.     

Monoclonal antibodies specific for apoproteins of lipophorins from the migratory locust

Spleen lymphocytes from mice immunized with locust native low-density lipophorin A⁺ (LDLp) were fused with nonproducing myeloma cells, strain Sp 2/0. Hybridomas that were isolated from the fused cells produced antibodies specific for LDLp and the high-density lipophorin A_yellow (HDLp). Monoclonal strains were generated through cloning by limiting dilution from those hybridomas synthesizing antibodies specific for apolipophorins (apoLp)-I, -II, and -III of LDLp. Additionally, a hybridoma strain that was obtained after fusion of lymphocytes from mice immunized with apoLp-III produced antibodies that bind to apoLp-III and native LDLp. Some features of LDLp and HDLp were studied using these antibodies. It could be demonstrated that apoLp-I and apoLp-II are not immunochemically identical and are exposed in the native particle of both LDLp and HDLp. It was also shown that in both lipophorins apoLp-II is less exposed than apoLp-I, whereas in LDLp apoLp-III is mainly exposed; some apoLp-III could also be detected in HDLp. Tween-20, a nonionic detergent, appears to affect the binding of anti-apoLp-I, -II, and -III to both LDLp and HDLp. The monoclonal antibodies specific for locust apolipophorins do not bind to the respective apoproteins of lipophorins from other insects.     

Production and characterization of anti-bifidobacteria monoclonal antibodies and their application in the development of an immuno-culture detection method

An immuno-culture method has been developed by combination of specific monoclonal antibodies and plate culture to allow detection of viable bifidobacteria. Cell wall proteins were selected as surface antigen to produce antibodies against bifidobacteria. The cell wall proteins were extracted and purified from six ATCC strains of bifidobacteria grown in MRS broth using an anaerobic system. To compare the profile of the protein extracts, all the protein solutions obtained were analyzed by SDS-PAGE. Similar bands corresponding to the major proteins of each species of bifidobacteria were observed. The proteins were tested for their immunogenicity in Balb/c mice after immunization and subsequent analysis using ELISA procedures. High immune responses were generated in mice immunized by proteins from Bifidobacterium bifidum and Bifidobacterium longum. Monoclonal antibodies were produced against B. longum and tested for their specificity, sensitivity and cross reactivity with other bifidobacteria species. All the hybridoma cells selected produced anti-B. longum antibodies cross-reacting with native and purified proteins from five other bifidobacteria species. An epitope supported by a cross-reacting protein of 58 kDa shared by bifidobacteria was revealed by western blot. This was confirmed by immune-transmission electron microscopy observations which showed the specific interaction of these antibodies with bifidobacterial cell wall proteins. Also, the antibody obtained was found to be specific for the genus Bifidobacterium and sensitive, allowing the detection of at least 10(5) target cells/ml. An immuno-culture detection approach was then developed using the selected anti-B. longum antibodies. This method was shown to be very efficient for the detection of viable cells of bifidobacteria suggesting the possibility of its use to quantify these bacteria in various food matrices.     

The beta subunit of E. coli glycyl-tRNA synthetase plays a major role in tRNA recognition.

The contributions made by the alpha and beta subunits of E. coli glycyl-tRNA synthetase to the recognition of tRNA have been investigated via binding and immunological methods. Using the nitrocellulose filter assay, we have shown that isolated beta subunit, but not the alpha subunit, binds [14C]glycyl-tRNA with an affinity comparable to that of the native enzyme. Further, the data indicate that the beta subunit possesses one binding site for glycyl-tRNA while the native or reconstituted enzyme (alpha 2 beta 2) has two sites. Rabbit antibodies directed at the beta subunit or the holoenzyme inhibit efficiently the ability of the enzyme to aminoacylate tRNA while alpha-subunit antibodies have a smaller effect. Since none of the antisera have an appreciable effect on the ATP-PPi exchange activity of the enzyme under these conditions, the beta-subunit (and holoenzyme) antisera evidently interfere with productive tRNA binding. Taken together, the data indicate that the larger, beta subunit of glycyl-tRNA synthetase plays a major role in tRNA recognition.     

Monoclonal antibodies against distinct determinants of histone H5 bind to chromatin

A series of monoclonal antibodies specific for distinguishable epitopes in chromosomal protein histone H5 were obtained from mice immunized with either free H5 or H5 . RNA complexes. The antibodies elicited by H5 could be distinguished from those elicited by H5 . RNA by their binding to native or acid-denatured H5, by their interaction with the globular region of H5, and by their cross-reactivity with H1o. The specificity of the antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting experiments. The antibodies could distinguish between H5 and the closely related histones H1 and H1o. The binding of some of the antibodies to the antigens was dependent on the type of assay used, suggesting nonrandom binding of the antigen to the solid supports used in ELISA and immunoblotting. Competitive ELISA experiments indicate that 8 of the 11 antibodies characterized bind to distinct epitopes. Three monoclonal antibodies bind to epitopes which are in close spatial proximity, causing mutual steric hindrance. The monoclonal antibodies bind to nuclei of fixed cells and to isolated chromatin, indicating that the epitopes are present both in the purified protein and in chromatin-complexed H5. These monoclonal antibodies can be used to study the organization of distinct regions of histones H5 and H1o in chromatin and chromosomes.     

Genetic structure of an introduced paper wasp,Polistes chinensis antennalis (Hymenoptera,Vespidae) in New Zealand

Several eusocial wasps are prominent invaders to remote islands. The paper wasp Polistes chinensis antennalis is native to East Asia, was introduced to New Zealand in 1979 and has expanded its distribution there. This provides an excellent opportunity to examine the impacts of an initial bottleneck and subsequent expansion on genetic structure. We analysed and compared the genetic population structures of the native (Japan and South Korea) and invasive New Zealand populations. Although 94% of individuals had shared haplotypes detected across both populations, the remaining 6% had private haplotypes identified in only one of the three countries. The genetic variation at microsatellite loci was lower in New Zealand than in native countries, and the genetic structure in New Zealand was clearly distinct from that in its native range. Higher frequencies of diploid‐male‐ and triploid‐female‐producing colonies were detected in New Zealand than in the native countries, showing the reduction in genetic variation via a genetic bottleneck. At least two independent introductions were suggested, and the putative source regions for New Zealand were assigned as Kanto (central island) and Kyushu (south island) in Japan. Serial founder events following the initial introduction were also indicated. The estimated dispersal distance between mother and daughter in New Zealand was twice that in Japan. Thus, the introduction history of P. chinensis antennalis in New Zealand is probably the result of at least two independent introductions, passing through a bottleneck during introduction, followed by population expansion from the point of introduction.     

Structural studies on human glutathione S-transferase pi. Family of native-specific monoclonal antibodies used to block catalysis.

The glutathione S-transferases are a family of related detoxification enzymes that have been shown to conjugate numerous electrophiles to the common cellular thiol glutathione. We have generated a panel of monoclonal antibodies against the human pi class isozyme of this enzyme, and, in this report, we characterize the binding of these antibodies to the glutathione S-transferase antigen. Of the 10 monoclonal antibodies that we have isolated, 7 are able to recognize the native form of the enzyme while the remaining 3 are only able to bind to glutathione S-transferase pi in assays that partially denature the antigen, such as an enzyme-linked immunosorbent assay or a Western blot. We synthesized seven partial protein fragments and asked whether the monoclonal antibodies could bind to these fragments in an immunoprecipitation reaction. The antibodies that can bind the native form of the enzyme all bind to the carboxyl-terminal domain of the protein. Two antibodies are able to inhibit the glutathione S-transferase-catalyzed reaction noncompetitively against glutathione. Incubation of a 10-fold molar excess of either antibody over enzyme can inhibit the reaction by 50%. We have also used the same protein fragments of glutathione S-transferase pi to show that amino acids 1-77 retain the capacity to bind glutathione in a glutathione-agarose binding assay.     

Serologic evaluation of New Zealand sea lions for exposure to Brucella and Leptospira spp

A serologic survey of anti-Brucella and antileptospiral antibodies was conducted on 147 adult, female New Zealand sea lions (Phocarctos hookeri). Most sea lions (n=138) were sampled at Sandy Bay, Enderby Island, Auckland Islands (50°30'S, 166°17'E), January 2000-March 2005. Nine were sampled at Otago, New Zealand (46°0'S, 170°40'E); four in April 2008 and five in March 2009. Serum from one of the Enderby Island females was weakly positive for antibodies to Brucella abortus using the competitive enzyme-linked immunosorbent assay, and one female had a low titer for Leptospira interrogans serovar pomona using the microscope agglutination test. All serum samples from Otago animals were negative. Brucellosis and leptospirosis are therefore considered unlikely to play a major role in population dynamics of these populations, and the low antibody prevalence of these agents suggests that they are an unlikely source of infection for humans, wildlife, or domestic species on mainland New Zealand.     

On the coordination properties of Eu3+ bound to tRNA

The luminescence properties of Eu3+ have been used to investigate the binding and coordination properties of the ion with tRNA, as an attempt to resolve the discussion of whether metal ions bind to tRNA in solution only by Debye-Hückel screening, or whether direct coordination to specific sites may occur. Binding studies with Escherichia coli tRNAmet/f (taking advantage of 4-thiouracil-sensitized Eu3+ emission) distinguish three classes of binding affinities. Two of these are single sites with affinities approx. 10(4) and approx. 10(3) tighter than the nonspecific affinity of Eu3+ for native DNA. Mg2+ competes for binding at both these sites. Measurement of the lifetime and excitation spectrum of Eu3+ bound to the highest affinity site shows that the ion has two to five non-phosphate ligands in its inner coordination sphere. The existence of this coordinated site demonstrates that electrostatic screening is not the only mechanism for metal ion interaction with tRNA. The coordination properties of the high-affinity Eu3+ site do not agree with the properties of any of the metal ion sites found in the two tRNAphe crystal forms. Possible reasons for this discrepancy are discussed; it may be that ions bind differently to isolated molecules in solution than to molecules packed in a crystal lattice.     

Population density and geographical range size in the introduced and native passerine faunas of New Zealand

The current avifauna of New Zealand comprises species with two distinct origins: those that evolved in New Zealand or colonized naturally from neighbouring landmasses, and those that were deliberately introduced to the islands by European settlers. Elsewhere, it has been shown that for species introduced to New Zealand from Britain there is a positive interspecific correlation between the geographical range sizes attained in both countries. Since positive relationships between abundance, measured either as population size or density, and geographical range size are a near ubiquitous feature of assemblages of closely related animal species, this suggests that species’ abundances may also be so correlated between the two countries. Here, data for 12 passerine bird species introduced to New Zealand from Britain are used to compare population densities and density–range size relationships in their native and alien ranges. In addition, the density–range size relationship for 12 passerine bird species that can be considered native to New Zealand is compared to that for the introduced species. The geographical range size and the mean and maximum densities of introduced species in New Zealand were significantly positively correlated with those values for the same species in Britain. However, in no case was the relationship between mean density and range size significant. While not statistically significant, density–range size relationships for introduced species are similar in New Zealand and Britain, but those for introduced and native species in New Zealand are quite different. Implications of these patterns are discussed.     

Autoantibodies to OxLDL fail to alter the clearance of injected OxLDL in apolipoprotein E-deficient mice

This study tests the hypothesis that autoantibodies to oxidation epitopes on oxidized LDL (OxLDL) promote the clearance of OxLDL from the plasma. Human LDL (hLDL) was injected into immune-competent apolipoprotein E-deficient (apoE(-/-)) mice and immune-deficient apoE(-/-)/recombination-activating gene-deficient mice that lack mature T and B cells and thus antibodies. There was a progressive decrease in human apoB-100 in the plasma in all mice, but the rate of clearance was not greater in the immune-competent mice than in the immune-deficient mice. Interestingly, oxidized phospholipid (OxPL) epitopes as detected by the EO6 antibody on the hLDL increased over time, suggesting de novo oxidation of the LDL or transfer of OxPL to the particles. Because the native LDL was not extensively modified, we also examined the clearance of copper OxLDL. Although the extensively OxLDL was cleared faster than the native LDL, there was no difference in the rate of clearance as a function of immune status. There appeared to be some transfer of OxPL to the endogenous murine LDL. Together, these results suggest that oxidation-specific antibodies do not participate to any great extent in the clearance of OxLDL from plasma. However, it is possible that such antibodies may bind to oxidation epitopes and modulate lesion formation within the vessel wall.     

Antibody-nucleic acid complexes. Conformational and base specificities associated with spontaneously occurring poly- and monoclonal anti-DNA antibodies from autoimmune mice

An enzyme-linked immunosorbent assay (ELISA) was developed to characterize spontaneously occurring, mono-and polyclonal anti-DNA antibodies. The assay consists of adsorbing single- (ss) and double-stranded (ds) DNA and various nucleoside-bovine serum albumin conjugates (e.g., A-, G-BSA, etc.) to microtiter wells and assesses the ability of various antibodies to bind to these immobilized antigens. The conformational and base specificity of two monoclonal antibodies (designated MRss-1 BWds-3) was examined in this manner. The exclusive binding of MRss-1 to ssDNA and guanosine-BSA (G-BSA) confirms our previous findings [Munns, T.W., Liszewski, M.K., Tellam, J.T., Ebling, F. M., & Hahn, B.H. (1982) Biochemistry 21,2929-2936] that this antibody recognizes single-stranded nucleic acids by virtue of their guanine content. The extensive binding of BWds-3 to dsDNA, its limited binding to ssDNA, and complete absence of binding to nucleoside-BSA antigens implied a double-stranded conformational specificity. Further, competitive studies with naturally occurring and synthetic alternating copolymers indicated that BWds-3 preferentially recognized the native dsDNA antigens. ELISA analysis of the spontaneously occurring, polyclonal anti-DNA antibodies from MRL/lpr and NZB/NZW-F1 mice revealed that the majority of anti-ssDNA antibodies bound to nucleoside-BSA conjugates. Anti-G antibodies were most prominent in both strains of mice, yet lesser and more variable quantities of anti-A, -C, -U, and -T antibodies were also detected. Preadsorption of serum with G-BSA/Sepharose resulted in the complete removal of anti-G antibodies and a 60% reduction in anti-ssDNA antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)