Resin embedment of organs and postembedment localization of antigens by immunoperoxidase methods

Various procedures for nonpolar and polar resin embedment were applied to mouse and rat livers for the study of postembedment immunolocalization of alpha 1-fetoprotein, albumin and the microsomal enzyme epoxide hydrolase. Fixations with formaldehyde and with formaldehyde-glutaraldehyde mixtures were used for tissue stabilization. Both fixation schedules did not abolish immunoreactivity. Treatment of liver with inert compounds such as polyvinylpyrrolidones or chemical modification of antigens with ethyl acetimidate prior to embedment improved immuno-staining. Either the low-polarity solvent ethanol or the highly polar ethylene glycol could be employed as dehydrating agents. Antigens were readily localized in sections from Epon 812 embedded livers. For this purpose, polymerized resin had to be partially removed. On the other hand, immunoreactivity of antigens was only faint after embedment in an epoxy-resin based on diepoxide octane. Also, antigens reacted faintly in sections from livers which were embedded at 0 degrees C in the polar acrylate-methacrylate based Lowicryl K4M resin. The indirect peroxidase labelled antibody method was as specific and sensitive as the PAP technique. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. Apart from purified immunological reagents, the addition of high molarity sodium chloride and bovine serum albumin to the wash solutions enhanced immunohistological specificity.     

Antibodies against p-aminophenyl-beta-D-galactopyranoside-containing proteins

The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG.     

Resin embedment of organs and postembedment localization of antigens by immunoperoxidase methods

Summary Various procedures for nonpolar and polar resin embedment were applied to mouse and rat livers for the study of postembedment immunolocalization of alpha₁-fetoprotein, albumin and the microsomal enzyme epoxide hydrolase. Fixations with formaldehyde and with formaldehyde-glutaraldehyde mixtures were used for tissue stabilization. Both fixation schedules did not abolish immunoreactivity. Treatment of liver with inert compounds such as polyvinylpyrrolidones or chemical modification of antigens with ethyl acetimidate prior to embedment improved immuno-staining. Either the low-polarity solvent ethanol or the highly polar ethylene glycol could be employed as dehydrating agents. Antigens were readily localized in sections from Epon 812 embedded livers. For this purpose, polymerized resin had to be partially removed. On the other hand, immunoreactivity of antigens was only faint after embedment in an epoxy resin based on diepoxide octane. Also, antigens reacted faintly in sections from livers which were embedded at 0° C in the polar acrylate-methacrylate based Lowicryl K4M resin. The indirect peroxidase labelled antibody method was as specific and sensitive as the PAP technique. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. Apart from purified immunological reagents, the addition of high molarity sodium chloride and bovine serum albumin to the wash solutions enhanced immunohistological specificity.Supported by the Deutsche Forschungsgemeinschaft (Ku 257/3) Bonn, Federal Republic of Germany     

Characterization of Mono- and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.     

口蹄疫病毒非结构蛋白3AB 的原核表达及应用

旨在建立一种检测口蹄疫病毒非结构蛋白抗体的敏感、特异的ELISA方法。克隆、表达了口蹄疫病毒非结构蛋白3AB基因,原核表达的重组蛋白经亲和层析法纯化及Western blotting鉴定后作为包被抗原,建立检测口蹄疫病毒非结构蛋白抗体的3AB间接ELISA方法,通过与商品化试剂盒3ABC-ELISA的比对试验对其进行评价。结果显示,重组蛋白3AB以包涵体形式表达;能与口蹄疫病毒感染血清发生特异性反应,而不能与疫苗免疫动物血清发生反应;在检测田间样品时,与3ABC-ELISA具有同样的特异性和敏感性 (P＞     

乳胶凝集试验检测H5亚型禽流感病毒血凝素抗体的研究

利用原核表达并经过纯化的H5亚型禽流感病毒的血凝素蛋白做为抗原,经碳二亚胺（EDAC）将表达产物和羧基化的乳胶共价偶联,并对偶联乳胶的蛋白量和偶联时间进行合理选择,建立H5亚型禽流感病毒血凝素蛋白乳胶凝集试验的方法。临床检测H5亚型禽流感病毒实验免疫鸡血清126份,同血凝抑制试验相比,其敏感性为87.03％,特异性为88.8％,两者的符合率为87.30%。结果证明建立的方法可用于H5亚型禽流感抗体水平监测和流行病学调查。     

Immunocytochemical localization of the Ca2+-ATPase polypeptide in human platelets

Specific polyclonal antibodies raised against purified human platelet Ca2+-ATPase were used with protein A-gold immunocytochemistry to localize this protein in human platelets. Immunolabeling specifically detected Ca2+-ATPase over the surface connected membrane system (SCS) in sections of paraformaldehyde-fixed, Lowicryl-embedded platelets. The maximum density of label, determined by quantitative morphometric techniques, was observed over electron-dense regions within the SCS which may represent specialized structures for uptake and release of Ca2+. Less intense immunolabeling was observed over cytosol and may represent localization over the dense tubular system (DTS) which was not readily visualized under the processing procedures employed.     

A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin

Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 +/- 5.5 microg/ml (mean +/- SD) in healthy women and 6.2 +/- 3.6 microg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).     

Isolation of anti-carcinoembryonic antigen (CEA), specific immunoglobulins and their use in immunocytochemical and enzyme-immunoassays (ELISA)

Anti-carcinoembryonic (CEA) polyclonal antibodies in sheep and rabbits were raised using purified CEA from acid extracts of human colon adenocarcinoma. CEA was purified by gel filtration on Sepharose 4B CL and chromatography on DEAE-Sephadex A50. The antiserum was adsorbed with human serum and perchloric acid extract from normal colon. Anti-CEA IgG was purified from monospecific antiserum by ion-exchange chromatography and its specificity was tested on cryostat sections from colon adenocarcinoma by the indirect immunoperoxidase technique. The specific reaction was compared with that obtained by using a similar technique and two CEA specific monoclonal antibodies. An anti-CEA IgG peroxidase conjugate was obtained allowing to establish a "sandwich" ELISA-CEA system with two antibodies. CEA determinations were made in a group of 15 normal controls (mean value 4.8 +/- 0.4 ng/ml) and in 30 colorectal tumor patients (mean value 26.6 +/- 2.15 ng/ml). The anti-CEA antibodies are proven useful in immunocytochemical and ELISA techniques and may be further used in radioimaging of tumors.     

Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum.     

Antigenic structure of double-stranded RNA analogues having varying activity in interferon induction.

Antibodies were induced by immunization of rabbits with methylated bovine serum albumin complexes of: poly(I).poly(BC), an effective interferon inducer; poly(c7A).poly(rT), a noninducer that can block induction by active poly(A).poly(rT); and poly(A).poly(Um), which has neither inducing nor blocking activity. Similar complexes of f2 phage RNA or tRNA did not induce anti-nucleic acid antibodies. Each anti-polynucleotide serum contained some antibodies specific for double-stranded structure. Antibodies were immunospecifically purified from precipitates made with each serum and homologous or cross-reacting double-stranded polynucleotides. The purified antibodies distinguished among varying helices bearing base or ribose modifications. Antipoly(I).poly(BC) specificity paralleled that of the interferon induction system. Anti-poly(A).poly(Um) specificity favored the 2'-modified polymers. Anti-poly(c7A).poly(rT) antibodies were the least discriminating. Cross-reaction results indicated that some antibodies reacted with determinants that included both sugar-phosphate backbones. In far antibody excess, antigen:antibody ratios in precipitating complexes reached a minimum of 7 to 12 base pairs per bivalent IgG molecule. Single antigenic determinants may span about 4 base pairs, with primary contact sites including the phosphate groups and the furanose.     

A nonredundant human protein chip for antibody screening and serum profiling

There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm(2) on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-microl sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis(6), anti-GAPDH, and anti-HSP90beta antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.     

Isolation and characterization of monoclonal antibodies specific for the cardiac muscarinic acetylcholine receptor

A number of monoclonal antibodies were raised against the purified porcine atrial muscarinic acetylcholine receptor. The antibodies were shown to exhibit a high degree of specificity for the receptor by their ability to recognize the purified receptor but not other porcine atrial glycoproteins in enzyme-linked solid-phase immunosorptive assays and by immunoblot analyses. Several of the antibodies were able to quantitatively precipitate the muscarinic receptor in both pig and rat heart and a portion of the receptor from rat cerebellum but little if any receptor from rat cerebral cortex. Thus, these monoclonal antibodies not only exhibit specificity for the muscarinic receptor but also are specific for the cardiac receptor subtype.     

Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)—based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galβ1-3GalNAcα- and Galβ1-3GalNAcβ-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcα-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC₅₀ values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 × 10⁻⁸ M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcβ and GalNAcβ1-3GalNAcβ ligands was found in human serum. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation and characterization of anti-paroxetine antibodies

6-nitroparoxetine was synthesized and reduced to 6-aminoparoxetine. After coupling to glutaraldehyde at the 6-position and to bovine serum albumin, the resulting Schiff's base was further reduced into an amino-derivative which served as the antigen. Anti-paroxetine antibodies were raised against this antigen in rabbits and the anti-paroxetine IgG purified by Protein A affinity chromatography. The anti-paroxetine IgG demonstrated high specificity towards paroxetine and 6-nitroparoxetine without significant cross-reactivity with other commonly used antidepressant and neuroleptic drugs. These antibodies may be useful for both plasma paroxetine level assays and uptake inhibitor binding site studies.     

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.     

Presence of natural anti-Galα1-4GalNAcβ1-3Gal (anti-NOR) antibodies in animal sera

Rare polyagglutinable NOR erythrocytes contain unusual globoside extention products terminating with a Galα1-4GalNAcβ1-3Gal- unit. This trisaccharide epitope is recognized by recently characterized antibodies naturally occurring in most human sera (Duk et al., Glycobiology, 15, 109, 2005). These antibodies represent two major types of fine specificity. All these antibodies are most strongly inhibited by Galα1-4GalNAcβ1-3Gal (NOR-tri), and weakly by Galα1-4Gal. However, the type 1 antibodies are strongly inhibited by Galα1-4Galβ1-3Gal-R and weakly by Galα1-4GalNAc, while the type 2 antibodies show the opposite reactivities with these two oligosaccharides. Similar antibodies have now been found in horse, rabbit and pig sera. The antibodies were purified from animal sera by affinity chromatography on Galα1-4GalNAcβ1-3Gal-human serum albumin(HSA)-Sepharose 4B conjugate. The specificity of the antibodies was determined by binding to ELISA plates coated with several α-galactosylated oligosaccharide-polyacrylamide (PAA) or -HSA conjugates and by inhibition with synthetic oligosaccharides. The purified antibodies bound specifically to conjugates containing NOR-tri. The inhibition of binding showed that the animal sera also contain two types of anti-NOR antibodies: type 2 was found in the horse serum, and a mixture of both types was present in rabbit and pig serum. These results indicate that anti-NOR, a new and distinct kind of anti-αGal antibody, are present in animal sera and show similar specificties and diversity as their counterparts found in human sera.     

Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver.

Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled.     

猿猴疱疹病毒B糖蛋白D的表达及抗原性的探讨

目的表达猴B病毒糖蛋白D(BVgD)并对其抗原性进行初步研究。方法应用体外原核表达BVgD,继而进行Ni离子亲和层析纯化。采用Western blotting对蛋白抗原性进行分析,评估其抗原潜力。结果完成了对BVgD的小量表达和纯化。Western blotting结果表明表达出的重组蛋白BVgD具有特异性结合猴B病毒抗体的能力。结论BVgD重组蛋白具有良好的抗原性,与HSV-1抗体无交叉反应。具有进一步开发的潜力,也为BVgD免疫原性等方面的进一步研究提供了条件。     

Immunological crossreactivity to the catabolite control protein CcpA from Bacillus megaterium is found in many Gram-positive bacteria

Abstract The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, streptococci, lactic acid bacteria, and some actinomycetes.