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.     

用硝酸纤维素膜亲和法纯化抗体

介绍一种改进的纯化抗体方法。将抗原蛋白固定在硝酸纤维素膜上,然后对抗体进行亲和纯化,能快速有效获得高特异性抗体。     

Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for detection of monoclonal antibodies and for studies of the properties of viral proteins

We report the use of herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell polypeptides (ICPs) separated by electrophoresis in polyacrylamide gels and transferred to nitrocellulose to (i) detect monoclonal antibodies to viral polypeptides and to (ii) study the properties of the proteins with the monoclonal antibodies. Our results were as follows. (i) When the antigens were electrophoretically separated in denaturing gels and then immobilized on nitrocellulose strips, we detected a greater diversity of monoclonal antibodies to viral proteins than when we used the technique of immune precipitation of soluble, nondenatured viral antigens. The primary advantage of the technique is in the detection of nonprecipitating antibody and of antibody to poorly soluble antigens not available for reaction in preparations cleared by high-speed centrifugation before immune reaction. (ii) Studies of the viral polypeptides reactive with three monoclonal antibodies indicated that the technique can be used to investigate several properties of the antigens. Specifically, monoclonal antibody to ICP 4 confirmed the accumulation of viral protein in the nucleus and the mapping of the gene in the S component. The results showed, however, that HSV-1 and HSV-2 ICP 4 do have common antigenic determinants. The reaction of a nonprecipitating monoclonal antibody with electrophoretically separated, immobilized polypeptides contained in cytoplasmic and nuclear fractions, those chemically deglycosylated, or those specified by specific HSV-1 x HSV-2 intertypic recombinants identified the antigens reactive with the second monoclonal antibody as various forms of glycoprotein gC. Of particular interest was a set of four antigens, 39,000 to 46,500 in apparent molecular weight, reactive with each of several monoclonal antibodies. These studies showed that two polypeptides partition in the cytoplasm and two in the nucleus and that all comap with the previously mapped ICPs 35 and 37 in the region of the genome defined by the viral thymidine kinase gene on the left and the glycoprotein gA/B gene on the right. Unlike ICP 4 and gC, the four polypeptides are linked by intermolecular bisulfide bonds, inasmuch as the polypeptides were not at the expected locations upon denaturation and electrophoresis in the absence of reducing agents.     

Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the alpha and beta subunits of F1-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with 125I-labelled purified monoclonal antibodies specific to various peptides. The 125I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H2O2 and alpha-naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F1.     

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.     

Determination of membrane antigens by a covalent crosslinking method with monoclonal antibodies

Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins. However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult. This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane. We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines. However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods. We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells. By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.     

Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the and β subunits of F₁-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with ¹²⁵I-labelled purified monoclonal antibodies specific to various peptides. The ¹²⁵I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H₂O₂ and -naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F₁.     

A dot-immunobinding assay for monoclonal and other antibodies

A procedure for the assay of antibodies in sera based on the application of the antigen as a spot to nitrocellulose filters is described. The method has the merit of being simpler in operation and more sensitive than comparable existing procedures. Applications for screening supernatants of hybridomas making monoclonal antibodies, and the use of such antibodies in the determination of the tissue distribution of the corresponding antigens, are described. An application for the screening of human pathological sera for multiple antibodies in one operation is also described.     

Transblot studies with biotin-labeled proteins: electrophoretic mobilities and detection limits

Molecular weight standard proteins, mouse IgG as well as several antigens cross-reacting with the carcinoembryonic antigen (CEA), were biotin labeled, submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The bound proteins were revealed by the use of avidin-peroxidase conjugates and a suitable substrate. The ratio of N-hydrosuccinimido biotin (NHSB) to protein yielding the lowest detection limit was determined. At an optimal NHSB/protein ratio, 0.33 ng of IgG heavy chains and 0.17 ng of IgG light chains could be visualized. With the exception of human albumin and ovalbumin, the increase in apparent molecular weight after biotin labeling was less than 10% for the proteins tested. The method has proven to be a valuable addition to Western blots performed with CEA-related antigens and monoclonal anti-CEA antibodies.     

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.     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

Identification and characterization of the high affinity [3H]ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel

The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.     

Glycosphingolipid immunostaining: detection of antibody binding with an avidin-biotin enzyme system

Glycosphingolipids carrying carbohydrate sequences recognized by antibodies and lectins can be detected on thin layer chromatograms using an avidin-biotin enzyme system (ABC reagents). This same method can be used to detect glycosphingolipids blot-transferred from thin layer chromatograms to nitrocellulose. This method has certain advantages over the original radioimmunoassay method, including development of positive bands in minutes after incubation with the substrate, avoidance of handling hazardous radioactive materials and stability of reagents. We have demonstrated the usefulness of this method for immunostaining glycosphingolipids with both monoclonal and polyclonal anti-carbohydrate antibodies. These reagents have previously been used to detect carbohydrate antigens in tissues and isolated cells and now it is possible to use the same reagents for the detection of glycosphingolipid antigens on chromatograms.     

Expression of monoclonal-antibody-defined antigens in fractions isolated from human breast carcinomas and patients' serum

The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these smaples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.     

Grid-immunoblotting: a fast and simple technique to test multiple allergens with small amounts of antibody for cross-reactivity

Grid-immunoblotting is a procedure that allows the simultaneous testing of up to 20 different antibodies such as monoclonal antibody-containing hybridoma supernatants or human sera for specific antibodies to up to 20 different antigens or allergens on a single sheet of nitrocellulose membrane. Since only 150 to 200 μl of antibody-containing solution are required this technique is uniquely suited to test growing hybridomas and small amounts of sera (e.g. mouse and children’s sera). Compared to a standard ELISA, approximately ten times less antibody is needed to obtain the same information.     

Trypanosoma rhodesiense: antigenicity and immunogenicity of flagellar pocket membrane components

A low density membrane fraction, isolated from the bloodstream stage of Trypanosoma rhodesiense and enriched in flagellar pocket membrane, was characterized with regard to antigenicity using antibodies raised against purified flagellar pocket membrane. Mild trypsinolysis of flagellar pocket membrane released two small peptides (Mr = 13-16 X 10(3)) separated by chromatofocusing (pI = 6.8 and 5.8) that were antigenic as monitored by fused rocket immunoelectrophoresis. Both of these antigenic peptides were enriched in relative fluorescence when flagellar pocket membrane was prepared from surface labeled (fluorescamine-beta-cyclodextrin) trypanosomes, indicating that cleaved peptides were on the external (luminal) side of the flagellar pocket membrane. More extensive release of fluorescamine labeled flagellar pocket membrane components was affected using mild detergent treatment (0.15% Zwittergent 3-12/0.4% Triton X-100), crossed immunoelectrophoresis separating two prominent antigens was more pronounced after incubation of flagellar pocket membrane with either porcine pancreas phospholipase A2 or umbilical cord sphingomyelinase. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent electroblotting to nitrocellulose also revealed two principal flagellar pocket membrane antigens (Mr approximately 60 and 66 X 10(3)), the latter showing greater release after exposure to sphingomyelinase or phospholipase, compared to mild detergent or 50 mM acetate, pH 5.0. Both antigens were glycoprotein as judged by electroblotting and the use of concanavalin A conjugated horseradish peroxidase as probe. Neither flagellar pocket membrane antigen was found to react with monoclonal antibodies prepared against T. rhodesiense variable surface antigen. The use of flagellar pocket membrane in the presence of Freund's complete adjuvant was found to protect mice against challenge infections with either the CP344 clone or uncloned CT Well-come isolate of T. rhodesiense.     

P-epitope is characteristic for the neural tissue of vertebrates

In a search for antigens immunologically related to chordin, a notochord-specific glycoprotein of sturgeneous fishes, extracts from 55 samples of human and rabbit tissues were tested for inhibition of [125I]chordin binding to rabbit polyclonal antibodies. The strongest inhibition was observed with brain extracts of both species. Human, chicken, rabbit, and newt brain extracts also inhibited chordin binding in liquid phase to monoclonal antibodies (MAbs) against the P-epitope, the most immunogenic epitope of this glycoprotein. Immunohistochemical studies done on human and chicken embryos, newt, sterlet, and sturgeon embryos, larvae, and juveniles revealed a strong immunoreactivity of the brain, spinal cord, and tissue of the peripheral nervous system with an anti-P MAb. Other tissues, with several exceptions, showed a negative reaction in immunohistochemical experiments. The authors found that the P-epitope is ontogenetically expressed in the neural tissue of chicken, newt, and sterlet at the period of cytodifferentiation. Gel chromatography of human, chicken, and newt brain extracts showed that in each case the P-epitope was associated with a polydisperse macromolecular material of similar size. These antigens were designated as neurochordins. Prolonged pronase digestion of human and chicken brain extracts resulted in fragments with M about 3 kDa (presumably glycopeptides), which reacted with anti-P MAbs. These fragments were of the same size as corresponding glycopeptides of the pronase digest of chordin. Thus, in the present study, the P-epitope has been shown to be characteristic for the neural tissue of several vertebrate species; in the brain, it has been found in association with neurochordins, macromolecular antigens that are presumably protein conjugates with carbohydrates.     

The association on SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins of Pseudomonas aeruginosa as revealed by monoclonal antibodies and Western blotting

Abstract Monoclonal antibodies raised against single serotype components of a Pseudomonas aeruginosa vaccine have been shown to bind to the O antigen region of lipopolysaccharide (LPS). Outer membrane (OM) proteins, prepared by detergent treatment of envelope fractions and by EDTA/sonication treatment of whole cells, were separated on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretically transferred to nitrocellulose membrane and reacted with LPS-specific monoclonal antibodies. The patterns produced revealed that many of the protein bands were in fact protein-LPS complexes.     

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.