Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose

Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.     

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.     

General detection of proteins after electroblotting by trinitrobenzene sulphonic acid derivatization and immunochemical staining with a monoclonal antibody against the trinitrophenyl group

A sensitive method for the general detection of proteins electroblotted onto nitrocellulose sheets after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. The proteins on the blots were reacted with 2,4,6-trinitrobenzene sulphonic acid. The resulting trinitrophenyl groups on the proteins were rendered visible by immunochemical staining with a monoclonal anti-trinitrophenyl antibody, and a peroxidase-conjugated second antibody. Using various proteins, the method was compared to the amidoblack method for staining of protein blots. The method was 10-100-fold more sensitive than the amidoblack method. Amounts as low as 1 ng of human serum albumin could be detected.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.     

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Summary Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAF-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.Abbreviations MT Metallothionein - PBS Phosphate Buffered Saline - SDS Sodium Dodecyl Sulfat - PAGE Polyacrylamide Gel Electrophoresis Part of the work was reported in Proceedings of 54th Annual General Meeting of the Society of Biological Chemists, India, 1985.     

Factors affecting the assay of histone H1 and polylysine by binding of Coomassie blue G

Rabbit hepatic microsomal suspensions were bound directly to nitrocellulose sheets using a "Hybridot" apparatus to ensure uniformity. Cytochrome P-450, form 2, was then detected by a modified immunochemical method wherein the nitrocellulose paper was incubated sequentially with antibody to form 2 for 1 h at 25 degrees C, rabbit anti-goat immunoglobulin G (IgG) at a 1:100 dilution for 15 min at 25 degrees C, goat peroxidase-antiperoxidase at a 1:2000 dilution for 15 min at 25 degrees C, and 3,3'-diaminobenzidine at 0.3 mg/ml plus 0.002% hydrogen peroxide for 30 min at 25 degrees C. These conditions, as opposed to those previously published, yielded less background staining. The density of the stain, scanned with a soft laser (Zeineh), increased linearly from 2 to 100 fmol for purified form 2. Cytochrome P-450, form 2, was detected and quantitated in microsomal samples containing 0.1 to 0.5 and 0.02 to 0.05 micrograms protein for preparations from untreated and phenobarbital-treated rabbits, respectively. The results agreed with those obtained by Western blotting and single radial immunodiffusion. This assay is more sensitive than either Western blotting or radial immunodiffusion and has significant advantages such as ease of operation, increased sample numbers, and reduced interference from extraneous proteins.     

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.     

Analysis of platelet glycoproteins in thrombocytopathias using the lectin-avidin-biotin-peroxidase (LABP) technique

The application of the lectin-avidin-biotin-peroxidase (LABP) technique for detecting platelet glycoprotein abnormalities in thrombocytopathias is described. Platelet proteins from patients with Glanzmann's thrombasthenia or Bernard-Soulier syndrome were separated by two-dimensional O'Farrell gel electrophoresis, stained with silver or electroblotted onto nitrocellulose sheets. Nitro-cellulose blots were stained utilizing the LABP technique. The absence or severe reduction of glyco-proteins IIb and IIIa and fibrinogen in the platelet protein pattern of patients with thrombasthenia as well as the absence or marked reduction of glycoproteins Ib and V in the platelet protein pattern of a patient with Bernard-Soulier syndrome were clearly demonstrated.     

Binding of a 30-kDa protein to the pyruvate kinase gene of Neurospora crassa

Extracts of a wild-type strain of Neurospora crassa, electrophoresed on SDS-polyacrylamide gels and electroblotted onto nitrocellulose sheets, were hybridized to an end-labelled pyruvate kinase (PK) gene fragment containing the 5' noncoding sequence and a large part of the coding region. A 30-kDa protein was found to bind strongly to the PK gene DNA, while binding weakly to plasmid pUC12 DNA and to total N. crassa DNA. Probing of blots with individual restriction fragments derived from the PK gene showed that the protein binding occurred primarily to the 5' noncoding region. Nonspecific DNA from pUC12, PK gene DNA from the recombinant plasmid pNP460 (pUC12 containing a 1.8-kilobase EcoRI insert of the PK gene DNA), along with a 0.7-kilobase EcoRI-AccI restriction fragment containing the 5' flanking region, were used in filter-binding experiments to analyze the kinetics of binding. Formation of protein-DNA complexes was demonstrated by monitoring the electrophoretic mobility of this fragment on nondenaturing gels.     

Renaturation and ligand blotting of the major subunit of the rat asialoglycoprotein receptor after denaturing polyacrylamide gel electrophoresis

Rat hepatic asialoglycoprotein receptors (ASGP-Rs) bind terminalclustered galactosyl or N-acetylgalactosaminyl residues withhigh affinity. The affinity-purified ASGP-R consists of threesubunits designated RHL1, RHL2, and RHL3. The ligand-bindingactivity of individual subunits was investigated by ligand blotting,after separation of subunits by SDS-PAGE under nonreducing conditions,electrotransfer to nitrocellulose, and incubation with ¹²⁵I-asialo-orosomucoid(ASOR). No ligand-binding to any subunits could be detectedwhen proteins such as BSA, casein, gelatin, or fat-free drymilk were used as blocking agents. However, subsequent incubationof BSA-blocked nitrocellulose blots with some nonionic detergentsresulted in renaturation of RHL1. ¹²⁵I-ASOR-binding to RHL2or RHL3 was weaker and could be detected only after longer exposure.Similarly, direct use of detergents such as Tween 20, NonidetP-40, or Triton X-100 as blocking agents also preserved theASOR-binding activity of RHL1. Ionic detergents tested did notshow any ability to renature the ligand-binding activity ofRHL subunits. Among nonionic detergents tested, Tween 20, Tween85, Lubrol PX, Nonidet P-40, and Triton X-100 were more effectivethan Tween 40, Tween 65, Tween 80, or Brij 35, whereas SPAN,digito-nin, or octyl-glucoside showed no effect. Weak ¹²⁵I-ASORbinding to RHL2 or RHL3 could be detected only when the Tweenseries or Lubrol PX were used. Incubation of blots with dithiothreitolcaused a dose-dependent loss of binding activity. The carbohydraterecognition domain (CRD) of RHL1, isolated after subtilisindigestion of ASGP-R bound to ASOR-Sepharose, retained ligand-bindingactivity as assessed by its binding to ASOR-Sepharose and byligand blotting. ¹²⁵I-ASOR binding to electroblotted CRD afterSDS-PAGE was also dependent on the presence of nonionic detergents.We conclude that restoration of ligand-binding activity of RHL1after SDS-PAGE by some nonionic detergents is not dependenton the presence of the cytoplasmic, transmembrane, or stalkdomains of this subunit. asialoglycoprotein receptor Ligand blotting detergent renaturation RHL1     

Detection of low-molecular-weight polypeptides on nitrocellulose with monoclonal antibodies

An immunoblotting method to detect low-molecular-weight peptides with monoclonal antibodies that normally fail to demonstrate immunoreactivity using conventional blotting techniques is described. Detection of neurophysin, insulin, calcitonin, vasopressin, and beta-endorphin electroblotted on nitrocellulose membranes was optimized after introducing four modifications into the conventional procedure. These include renaturing the gels after sodium dodecyl sulfate electrophoresis, electroblotting the renatured gels in basic transfer buffer, fixing and/or heating the blots, and using avidin/alkaline phosphatase conjugates for antigen/antibody detection. This technique likely enables the denatured peptides to regain their native conformation and, therefore, restores antigenicity and recognition by highly structural specific monoclonal antibodies. Although the most dramatic improvement with this technique is with monoclonal antibodies, a modest improvement in sensitivity can be obtained when immunoblots are probed with polyclonal antibodies. The high resolution of this system will be useful in probing blots of partial proteolytic digests of proteins with both monoclonal and polyclonal antibodies.     

人肝金属硫蛋白-I_A基因在鱼腥藻中的克隆与表达

将人工合成的人肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,简称ＭＴ）－ＩＡ基因插入至中间载体ｐＲＬ－４３９上强启动子ｐｓｂＡ后,再将其与穿梭载体ｐＫＴ－２１０相连,得到大肠杆菌－蓝藻穿梭表达载体ｐＫＴ－ＭＴ,用三亲接合转移法将ｐＫＴ－ＭＴ转入丝状体蓝藻－鱼腥藻７１２０,经链霉素筛选,得到了稳定的转人肝ＭＴ－ＩＡ基因鱼腥藻．纯化单藻落,液体扩大培养．从鱼腥藻中提取的质粒经Ｓｏｕｔｈｅｒｎ印迹分析,确定人肝ＭＴ－ＩＡ基因已转入鱼腥藻７１２０中,Ｗｅｓｔｅｒｎ印迹分析表明,金属硫蛋白在转人肝ＭＴ－ＩＡ基因鱼腥藻中得到了表达．经原子吸收光谱法测定表达量约为７００μｇＭＴ／ｇ鲜藻,重金属耐受性实验表明,得到了能耐受重金属－镉的转人肝ＭＴ－ＩＡ基因鱼腥藻,它将在清除水域中重金属污染和医药研究方面发挥重要作用．     

Identification and characterization of taglin, a mannose 6-phosphate binding, trypsin-activated lectin from Giardia lamblia

We have previously reported the presence of a cell surface associated lectin activity in Giardia lamblia, a human protozoan parasite that is a significant cause of diarrheal disease worldwide [Lev, B., Ward, H., Keusch, G. T., & Pereira, M. E. A. (1986) Science (Washington, D.C.) 232, 71-73]. This lectin is specifically activated in vitro by a host protease, trypsin, which is secreted in vivo at the site of infection. The activated lectin agglutinates cells to which the parasite adheres in vivo and binds specifically to isolated brush border membranes of these cells. These findings suggest that this lectin may be of importance in the host-parasite interaction. We now report the identification of this lectin, which we have named taglin (to denote trypsin-activated Giardia lectin), and describe some of its properties. A monoclonal antibody that inhibits the hemagglutinating activity of taglin recognizes a protein of 28,000/30,000 kdaltons in Western blots of Giardia lysates. This finding was confirmed by direct demonstration of lectin activity with the technique of erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody. This study also elucidates the binding of taglin to terminal phosphomannosyl residues. The involvement of cell surface phosphate in binding of taglin to erythrocytes is shown by the abolition of lectin activity by alkaline phosphatase treatment of the erythrocytes. Taglin also requires divalent cations, Ca2+ or Mn2+, for hemagglutinating activity and is active within a narrow pH range of 6-7.     

Suppression of nonspecific binding of avidin-biotin complex (ABC) to proteins electroblotted to nitrocellulose paper

Nitrocellulose blots of cell extracts reacted in sequence with biotinylated lectins and horseradish peroxidase-labeled avidin-biotin complex (ABC) often show considerable nonspecific staining of protein bands. Experiments were performed to determine which of the components of the ABC were responsible for this and whether or not the nature and ionic strength of the buffer used could alter this binding. Furthermore, as powdered non-fat milk has been proposed as a possible blocking agent for nonspecific binding of ABC, we sought to determine if it would adequately block that binding in our system. The initial experiments showed that nonspecific binding of ABC to proteins transferred to nitrocellulose membranes was due to the avidin component of the ABC; little, if any, binding was seen if biotin alone was incubated with these blots. The spurious binding was shown to be primarily due to the high affinity of avidin to proteins electroblotted to nitrocellulose, when incubated in low-salt buffers. Low-fat milk added to the buffer reduced overall nonspecific reactivity but produced additional artifacts in the form of bands that were not seen in other preparations. Nonspecific avidin binding to proteins transferred to nitrocellulose can therefore be effectively reduced by adding extra salt to buffers, whereas the addition of non-fat dry milk does not seem suitable for this purpose.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

Histochemical localization of mushroom tyrosinase in whole tissue sections on nitrocellulose

Mushrooms were cut into vertical and horizontal sections. These sections were blotted onto nitrocellulose sheets and the sheets were then stained for tyrosinase using L-dopa. Tyrosinase was localized throughout the mushroom tissues but more enzyme was located in the epidermis of the cap, the gill region, and the stipe. Preincubation of the nitrocellulose sheets in specific inhibitors of tyrosinase completely blocked enzyme staining, suggesting that the enzyme stained areas on the nitrocellulose blots were regions of tyrosinase activity. Immunochemical localization of tyrosinase was similar to that observed by histochemical staining. Nitrocellulose blotting of mushrooms allows localizations of enzyme at the whole tissue level and may be useful for other enzymes in mushrooms as well.     

Improvements of Western blotting to detect monoclonal antibodies

A comparison of the effects of different factors on the sensitivity of Western blotting technique to detect monoclonal antibodies is described. The major improvements were obtained by: A) renaturating the antigen in the gel before transferring it in carbonate buffer at pH 10 onto nitrocellulose and B) using alkaline-phosphatase-conjugated second antibody instead of peroxidase-conjugated second antibody.     

Monoclonal Antibodies Against Vasoactive Intestinal Polypeptide: Studies of Structure and Related Antigens

Abstract: Hybridomas secreting monoclonal anti-vaso-active intestinal polypeptide (VIP) antibodies were constructed from spleen cells sensitized to VIP in vitro . The secreted antibodies were characterized by binding to VIP in indirect radioimmunoassays and enzyme-linked immunosorbent assays. Two monoclonal antibodies, characterized for their binding activities with synthetic fragments of VIP, were found to bind different sites on the VIP molecule. These monoclonal antibodies may recognize tertiary structures of the VIP. A search was conducted for antigens recognized by the monoclonal antibodies in brain: brain proteins separated on polyacrylamide gels were electroblotted onto nitrocellulose filters and were reacted first with the mouse antibody and then with goat anti-mouse imunnoglobulin coupled to horseradish peroxidase as a means of detection. The monoclonal antibodies were found to react with a protein of molecular weight 60,000, which was also recognized by polyclonal antibodies, although the latter reacted with a number of additional proteins. The relationship of the protein of molecular weight 60,000 to VIP is discussed.     

Purification and partial characterization of rat ovarian lutropin receptor

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea and mouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.