Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies

A very sensitive method for the detection of antigen-antibody complexes on nitrocellulose paper immunoblots is described. The protein antigens are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by their electrophoretic transfer onto a nitrocellulose sheet (“Western blot”). The protein antigens bound to the nitrocellulose paper are exposed to the monoclonal antibody and the antibody-antigen complexes are detected on the paper by an immunoenzymatic reaction. The improved sensitivity of this method is the result of (i) the use of the detergent Tween 20 in blocking the nonspecific binding of the antibodies to the nitrocellulose paper, (ii) the use of a peroxidase-antiperoxidase (PAP) reaction, and (iii) the intensification of the diaminobenzidine reaction product with nickel and cobalt ions in phosphate buffer.     

Visualization of antigen on nitrocellulose membrane by the oxidative coupling reaction of N,N'-dimethyl-p-phenylenediamine and 4-chloro-1-naphthol

A method which allows the highly sensitive and simple immunodetection of antigen-antibody complexes on nitrocellulose papers has been developed. The method is a modification of the procedure known as the "Nadi reaction" (oxidative coupling of 1-naphthol and N,N'-dimethyl-p-phenylendiamine) for histochemical purposes. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. Bound proteins were first reacted with a primary antibody and then with a horseradish peroxidase-labeled second antibody. The antigen-antibody complexes on the membranes were visualized with the oxidative coupling solution containing N,N'-dimethyl-p-phenylendiamine and 4-chloro-1-naphthol. Sensitivity was enhanced 4 to 16 times by the new method relative to that of the 4-chloro-1-naphthol method or the 3,3'-diaminobenzidine method.     

The use of a dot immunoenzyme method for detection of viral antigens

The technique of dot immunoenzyme detection has been elaborated for viral antigens identification. The investigated samples (extracts of infected cells, fractions obtained during viruses and viral proteins purification) are placed in nitrocellulose filters, the free binding sites are blocked and then treated with specific immune serum. The formed antigen-antibody complexes are detected using antispecies immunoglobulins or protein A from Staphylococcus aureus, conjugated with horseradish peroxidase. The brown dots appear at samples location containing the viral antigens. Sensitivity of the technique is 1 ng of protein per sample as tested using adenoviral antigens.     

Rapid quantitation of uncoupling protein in brown adipose tissue mitochondria by a dot immunobinding ("dot blot") procedure: application to the measurement of uncoupling protein in Richardson's ground squirrel, rats, and mice

A dot immunobinding ("dot blot") method for measuring uncoupling protein in brown adipose tissue mitochondria is described. Mitochondrial proteins were solubilized in sodium dodecyl sulfate and applied directly to a nitrocellulose membrane housed in a 96-well microfiltration manifold. Spare binding sites on the nitrocellulose membrane were blocked with bovine serum albumin and then anti-(uncoupling protein) serum was applied. The antigen-antibody complex was detected by the addition of 125I-labelled protein A. Each nitrocellulose "dot" was cut out and its radioactivity was counted. A calibration curve was constructed from purified uncoupling protein standards, taken through the entire procedure. The dot immunobinding method is sensitive (nanogram quantities of uncoupling protein), and in contrast to conventional radioimmunoassay and enzyme-linked immunosorbent assay procedures, it is also rapid and appears to be very robust. The method has been successfully applied to the measurement of uncoupling protein in brown adipose tissue mitochondria of Richardson's ground squirrel, rats, and mice.     

检测烟草中烟草花叶病毒的RNA斑点杂交法

用普通烟草花叶病毒OM株3′-端约2kb的cDNA为探针,探索了用RNA斑点杂交法对烟草组织中烟草花叶病毒RNA进行检测的条件。这些条件包括用分子杂交法观察云南烟区和上海烟草上分到的烟草花叶病毒与OM株的同源性,从烟草组织中提取烟草花叶病毒的几种方法的比较,使RNA有效地固定在硝酸纤维素滤膜上的方法,烟草组织中是否有干扰RNA固定和杂交的物质,斑点杂交方法检测烟草花叶病毒的特异性、灵敏度等。     

The isolation of tobacco mosaic virus RNA fragments containing the origin for viral assembly

Upon mixing purified TMV RNA with limited amounts of viral coat protein in the form of the disk aggregate, a unique region of the whole RNA becomes protected from nuclease digestion. The protected RNA consists of fragments up to 500 nucleotides long in varying yields, which are found in nucleoprotein particles having a protein-nucleic acid ratio similar to the mature virus. The protected RNA, when reextracted, is able to rebind to coat protein disks rapidly, quantitatively and with high affinity, becoming once more RNAase-resistant in the process. Small aggregates of TMV protein (A protein) are inactive in formation of the nuclease-resistant complexes. On the basis of this evidence, we identify the isolated RNA fragments as portions of TMV RNA containing the origin or initiation site for in vitro reassembly, which have been protected from digestion by incorporation into assembly nucleation complexes.The yield, but not the length distribution, of the protected RNA pieces is found to double upon increasing the protein added from 1–2 disk-equivalents of protein per RNA molecule. This implies that the formation of the nucleation complexes may involve a highly cooperative initial addition of protein.     

A new method for induction of specific polyclonal antibodies using immunostained proteins on nitrocellulose membrane, and HIV 1 core antigen and Escherichia coli verotoxin as examples

Abstract Two polyclonal antibodies against verotoxin 1 and the core protein p24 of HIV 1 were raised in mice by a new immunization procedure. Both proteins were transferred to nitrocellulose, reacted with polyspecific antisera and the antigen-antibody complexes were then visualized by immunostaining. For preparation of antisera the stained protein bands were cut from the nitrocellulose sheets and implanted subcutaneously into the backs of BALB/c mice, without any adjuvant. A single booster was given 4 weeks later by implanting a second strip. All mice produced high titers of antibody directed against the antigen used for immunization. Thus, antibodies of high specificity can be elicited against protein bands in stained immunoblots.     

A new method for induction of specific polyclonal antibodies using immunostained proteins on nitrocellulose membrane, and HIV 1 core antigen and Escherichia coli verotoxin as examples

Two polyclonal antibodies against verotoxin 1 and the core protein p24 of HIV 1 were raised in mice by a new immunization procedure. Both proteins were transferred to nitrocellulose, reacted with polyspecific antisera and the antigen-antibody complexes were then visualized by immunostaining. For preparation of antisera the stained protein bands were cut from the nitrocellulose sheets and implanted subcutaneously into the backs of BALB/c mice, without any adjuvant. A single booster was given 4 weeks later by implanting a second strip. All mice produced high titers of antibody directed against the antigen used for immunization. Thus, antibodies of high specificity can be elicited against protein bands in stained immunoblots.     

Multiple immunoreplica Technique: screening for specific proteins with a series of different antibodies using one polyacrylamide gel

A simple method for immunological identification of proteins resolved electrophoretically is presented. Proteins from one polyacrylamide gel can be subjected to a series of electrophoretic transfers to nitrocellulose paper (partial “western-blots”), providing several replicas of the gel. Each replica can be reacted with a series of different antisera (at least three), where the preceding antibody is removed by treatment with pH 2.2. The antigen-antibody complexes are visualized using ¹²⁵I-Protein A. Reactivity and antigenic specificity of proteins immobilized on nitrocellulose paper is not affected by repeated incubations and low pH treatments. Identical size of the replicas and superimposable profiles of proteins detected by antibodies allow a precise localization of particular polypeptides in the original gel.     

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture

A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.     

Intracellular signalling of initiation of DNA synthesis: effect of cell-free extracts from anti-receptor-stimulated B lymphocytes on spleen cell nuclei

To study the relatively late intracellular signals involved in the proliferative response of B lymphocytes to antibodies specific for surface membrane immunoglobulins, extracts from antibody activated cells were mixed with Xenopus laevis splenic nuclei, and the incorporation of thymidine 5'-triphosphate into DNA was assessed. The slight incorporation observed with either nuclei or extract alone was markedly enhanced upon mixing the two entities when the extract was derived from cells cultured with but not without anti-receptor antibody. The appearance of active extract correlated well with the culture requirements necessary for the induction of B lymphocyte proliferation and, as revealed by time course studies, the active component arises relatively late in the activation process. Moreover, the appearance of active extracts is independent of DNA synthesis but is dependent on protein synthesis as judged from studies with metabolic inhibitors. Appropriate homogenization of activated cells yielded nuclei and cytoplasm with 85% of the activity confined to nuclei. In addition, purified active extracts exhibited DNA binding although the active component was readily distinguishable from polymerase alpha by chromatographic techniques. It is tentatively concluded that the active component represents either some replication protein other than polymerase or some earlier signal necessary to induce the formation or utilization of replicating proteins.     

与TMV-RNA装配起始位点互补的cDNA片段对TMV体外装配的抑制

合成了与TMV-RNA病毒装配起始位点互补的、长度为二十个核苷酸的DNA片段。该片段用~(32)P标记后,代替反义RNA(antisense RNA)与TMV-RNA进行硝基纤维素膜点杂交和溶液杂交。结果表明,该cDNA片段在两种条件下均能与TMV-RNA进行杂交。将溶液杂交的RNA-cDNA复合体经酒精沉淀,再与TMV衣壳蛋白的20S聚合体制剂进行体外装配,用测定310nm吸收光谱变化和电子显微镜观察的方法鉴定装配结果。实验证明,该cDNA片段与TMV-RNA杂交后抑制了装配起始位点的活力,从而使TMV病毒颗粒的装配不能完成。这一结果提示,TMV基因装配起始位点顺宁的cDNA和反义RNA能够在体外抑制TMV病毒颗粒的装配。     

Solid-Phase Immunoassay of PO Glycoprotein of Peripheral Nerve Myelin

To explore the immunological properties of PO protein, antibodies were elicited in rabbits against the purified chick PO protein. Peripheral nervous system protein was fractionated on sodium dodecyl sulfate-polyacrylamide slab gels and then transferred electrophoretically ("blotted") onto nitrocellulose sheets. The PO protein was detected by its capacity to bind its specific antibody present in the rabbit serum. The PO-specific antibody complex was then exposed to goat anti-rabbit immunoglobulin G (IgG) coupled to peroxidase or labeled with 125I. The resulting PO antigen-antibody "sandwich" was visualized and quantitated by densitometry of the colored peroxidase reaction product or by autoradiography and gamma-radiation counting of the 125I-IgG complex. The methods permitted quantitation of the PO protein in various nerve extracts. The limit of detection of the PO antigen was about 1 ng of protein. The antibody was specific for the PO glycoprotein in the peripheral nerve extracts. The PO proteins from various species, including human, were also detected by the antibody to chick PO protein. Preliminary experiments indicate the solid-phase immunoassay is a useful method for monitoring PO protein levels in small quantities of tissue extracts under various physiological and pathological conditions.     

Proteinaceous complexes from mitochondrial contact sites

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.     

Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues

The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.     

Alu-family repeat binding protein from HeLa cells which interacts with regulatory region of SV40 virus genome

Using a gel retardation assay the protein which binds selectively to the Alu-family repeat (AFR) has been identified and partially purified from HeLa cell nuclear extract. The protein (AFR-binding protein, ABP) forms multiple discrete complexes with AFR even in the presence of 200 to 2000-fold excess of non-specific (E. coli) DNA. The most stable complex has a relative mobility in 4% polyacrylamide gel (as compared to the free Alu-fragment) of 0.54. Heterogeneity of protein-DNA bands seen in the polyacrylamide gel suggests that ABP is able to form multimeric complexes with AFR. Competition experiments show that ABP do not interact with the RNA polymerase III promoter and with the TGGCA-sequence, but a high affinity binding site for ABP was found within a 660 bp restriction fragment containing the SV40 virus promoter and replication origin.     

Rapid measurement of protein kinase and phosphatase activities by slot-filtration.

A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.     

Enhancement of immunoblot sensitivity by heating of hydrated filters

Immunoblots of either dot or Western type were exposed to heat before reaction with antibody. Dramatic increases in immunoblot sensitivity were seen for certain antigen-antibody pairs after heating of either dry or hydrated nitrocellulose filters at or above 100 degrees C. Heating of filters in the hydrated state improved the linearity of immunodetection and produced the highest signal-to-noise ratio. This treatment greatly increased immunoblot sensitivity with several peptide-generated antibodies, whereas decreased sensitivity was seen with antibodies against native proteins. Heating of hydrated filters after antigen immobilization is thus a potentially powerful way to increase the sensitivity of immunoblot analysis for antibodies that preferentially recognize epitopes in denatured proteins.