Nitrocellulose Strips Research Articles

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens

AbstractSera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

A rapid and reproducible method for the analysis of immune complexes using affinity chromatography and Western blotting

A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human C1q or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared.

Isolation and Partial Characterization of Shared Antigens of Biomphalaria glabrata and Schistosoma mansoni and Their Evaluation by the ELISA and the EITB

Serum from humans infected with Schistosoma mansoni, when reacted with a Biomphalaria glabrata soluble hepatopancreas antigen extract (BgSHA) yields 2 lines of precipitation by gel diffusion and 1 by immunoelectrophoresis. The IgG from the serum of a human infected with S. mansoni was coupled to CNBr-activated Sepharose 4B. BgSHA (8.0 mg) was then filtered through the gel and the bound antigens, denoted BgSm, eluted with HCl-glycine, pH 2.6. These bound antigens comprised 2.8% of the total BgSHA. BgSm was then applied to an anti-Fasciola hepatica column as above. The drop through in PBS (38% yield) and containing the BgSm antigens depleted of cross-reactivity with F. hepatica was then tested by the ELISA to evaluate its serodiagnostic potential. These antigens detected a primary S. mansoni infection by 4 wk but were less sensitive than SmSEA in the detection of a primary infection with S. mansoni. However, the BgSm-specific antigens were more specific than SmSEA and showed less cross-reactivity with the serum of mice infected with F. hepatica. At least 16 peptides were seen by silver staining following SDS-PAGE with 5-20% gradient gels. The 2 more prominent bands obtained were estimated to have molecular weights of 62 and 66 kd. Nitrocellulose strips blotted with BgSHA were incubated with the serum of mice infected with S. mansoni for 12 wk and developed 6 bands with molecular weights of 66, 57, 55, 50, 48 and 32 kd.(ABSTRACT TRUNCATED AT 250 WORDS)

A dot enzyme-linked immunosorbent assay for detection of antibodies against Entamoeba histolytica

A visual micro-dot enzyme-linked immunosorbent assay (ELISA) based on detection of antibodies against soluble antigens of axenically grown cultures of Entamoeba histolytica is described. The antigen was spotted on a nitrocellulose sheet, the unsaturated sites blocked with bovine serum albumin (BSA) and incubated with 3-fold dilutions of patient sera followed by incubation with protein A conjugated to peroxidase. Enzymic activity was evidenced using the substrate 4-chloro-1-naphthol. A positive reaction produced a blue spot. The sensitivity of the assay was better and comparable to the indirect haemag-glutination assay (IHA) and plate ELISA. The entire assay could be completed within 3 h. Antigen-loaded and pre-blocked nitrocellulose strips could be stored up to 3 months at room temperature and 37°C.

Mouse sperm antigens that participate in fertilization. I. Inhibition of sperm fusion with the egg plasma membrane using monoclonal antibodies.

Monoclonal antibodies (mAbs) have been generated to determine the sperm components responsible for interaction with an egg that results in fertilization. Here, we report upon a group of six different mAbs, all of which localize to a restricted region of the sperm head, the equatorial segment. Several of these mAbs demonstrated cross-reactivity with sperm from the other species tested (human, hamster, rabbit); when cross-reaction occurred, the mAb distribution was restricted to the equatorial segment despite the various configurations that this homologous region assumes in different species. When tested for an effect upon the fertilization process in vitro, ascites fluids containing two of the six mAbs, M29 and M37, displayed significant inhibition. The concentration dependency of this inhibition was observed using purified M29 immunoglobulin M, over a range of 0 to 0.2 mg/ml. The mAb inhibition of fertilization was independent of the presence of either the cellular (the cumulus) or acellular (the zona pellucida) layers surrounding the egg, indicating that the specific locus of inhibition for both of these antisperm mAbs was the egg plasma membrane. Immunologic detection of sperm components separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose sheets was used to identify the sperm components recognized by two of the mAbs in this group: M29, which inhibited fertilization, and M2, which did not inhibit fertilization. Using M29 mAb, a single sperm component with an apparent subunit molecular weight of approximately 40,000 was detected, whereas in the nitrocellulose strips incubated with M2 mAb two components displayed reactivity, a very prominent band at approximately 44,000 and a tight cluster of bands at approximately 36,000. Parallel nitrocellulose strips of mouse liver did not display these reactivities, consistent with indirect immunofluorescence data in which only testis and sperm, and not liver, kidney, ovary, and epididymal epithelium, demonstrated positive reactivity. These results indicate that the use of mAbs permits identification of sperm components that participate, putatively, in individual events of the fertilization process. Furthermore, using this strategy, we have identified a specific sperm component that appears to be a candidate for a role in sperm fusion with the egg plasma membrane.

Identification of a high density lipoprotein binding protein from adrenocortical membranes

A protein band with an apparent molecular weight of 78,000 daltons has been identified in the solubilised plasma membrane extract of sheep adrenal cortex which binds HDL 3 devoid of E apolipoprotein. Following ‘Western’ blotting, and development of the nitrocellulose strips with appropriate antisera and color reagent, the same band, unlike other cortical membrane proteins or albumin, bound AI and AII apolipoproteins. Human LDL bound weakly to the same band but more strongly to another two proteins of higher molecular weight. These studies confirm the same degree of specificity of HDL 3 binding found with cultured adrenal cells and strengthen the suggested existence of a specific HDL receptor.

Use of purified antipeptide sera of selected specificity for the detection of the simian virus 40 large T antigen by Western blotting.

Western blotting was used as a powerful alternative to immunoprecipitation for the detection of the simian virus 40 (SV40) large tumor (T) antigen. After resolution by electrophoresis on a SDS-polyacrylamide gel of a [35S]methionine labeled crude extract from SV40 infected monkey kidney cells, the separated proteins were transferred electrophoretically on nitrocellulose paper. T antigen was detected on nitrocellulose strips by using for the first time, specific, purified antipeptide monoclonal antibodies directed against the N- and C-terminal portions of the molecule, and 125I-labeled Protein A.

Characterization of allergens and patient sera by a nitrocellulose immunoprint technique.

A rapid and convenient protein-gel blot technique for qualitative detection of antigens/allergens in pollen allergen extracts and IgE/IgG antibodies in patient sera has been developed. The antigens were separated by isoelectric focusing in agarose gel and transferred to nitrocellulose by capillary migration. After incubation of the nitrocellulose strips with serum from allergic patients, the binding of the patient's specific IgE or IgG antibodies was analyzed by using isotope-labelled or enzyme-labelled anti-IgE or anti-IgG. The time needed for detection with isotope-labelled antibody was approximately 20 h and with enzyme-labelled antibody 2 h. The immunoprint technique is easy to use, which renders it suitable for routine use in allergy research and quality control.

Humoral antibody response to individual viral proteins after murine cytomegalovirus infection.

The purpose of this study was to identify viral proteins that played an important role in the humoral immune response to murine cytomegalovirus (MCMV). Viral proteins were separated from a purified virus preparation on polyacrylamide gels, were blotted onto nitrocellulose strips, and were reacted with antisera collected from mice on various days post infection. No antibody response was detected in serum obtained 5 days post infection, but by 10 days there was a faint response to five different proteins. Thereafter, the number of proteins eliciting an antibody response, as well as the intensity of the response, increased with time so that by 42 days post infection a response to 13 major antigens was detected. This method provides a means of separating out important immunogens from the more than 30 different MCMV proteins originally identified by polyacrylamide gel electrophoresis. Such information may improve our understanding of the pathogenesis of MCMV infection as well as host immune responses to the virus.

Human upper epidermal cytoplasmic antibodies are directed against keratin intermediate filament proteins.

Upper cytoplasmic (U-Cyt) antibodies are directed against cytoplasmic antigens found in keratinocytes in the upper layers of the epidermis. Until now, they have been defined by indirect immunofluorescence and are known to occur in the sera of patients with cutaneous diseases such as bullous dermatoses, basal cell carcinomas, and melanomas. An increased incidence of U-Cyt antibodies has also been reported in the sera of patients with noncutaneous diseases, such as pulmonary neoplasms. They have been found in addition in the sera of some normal individuals. In this study we have identified keratin intermediate filaments (KIF) as antigens U-Cyt antibodies are directed against. KIF proteins were prepared, separated by polyacrylamide gel electrophoresis, transblotted to nitro-cellulose strips, and used as substrates for antibody binding. Sera containing U-Cyt antibodies by indirect immunofluorescence also had antibodies that were directed against high molecular weight (65,000, 63,000, 61,500) KIF proteins. When KIF proteins were separated according to their charge and their molecular weight by two-dimensional gel electrophoresis and transblotted, the anti-KIF protein antibodies bound to virtually all charge isomers of the KIF proteins at the respective molecular weight. The antibody titers measured using the transblotting technique were 10 to 160 times higher than those found by indirect immunofluorescence. To determine whether U-Cyt antibodies were directed against KIF, a series of absorption and elution experiments were performed. Absorption of test sera with purified KIF removed both U-Cyt antibodies and anti-KIF protein antibodies. Absorption with another type of intermediate filament derived from fibroblasts, vimentin, did not remove U-Cyt or anti-KIF protein antibodies. Absorbed U-Cyt and anti-KIF protein antibodies were both eluted from the same KIF preparation and shown to bind to U-Cyt antigens by indirect immunofluorescence and KIF proteins by transblotting. Absorption of a serum containing U-Cyt antibodies, anti-nuclear antibodies, and anti-basement membrane zone antibodies with purified KIF resulted in the removal of the U-Cyt antibodies but not the other types of antibody. In addition, all test sera, even those that lacked U-Cyt antibodies, were found to have low-titer antibodies against KIF proteins by the transblotting technique. These data indicate that KIF proteins bear antigens to which U-Cyt antibodies are directed and that low titer antibodies against KIF proteins may be much more common than previously appreciated.

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.

Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated anti-immunoglobulin for determination of the specificity of monoclonal antibodies to protein mixtures

The target antigens of a series of monoclonal antibodies raised against a complex mixture of proteins have been identified. The protein mixture was fractionated by polyacrylamide gel electrophoresis in SDS, and the fractionated proteins electrophoretically transferred to nitrocellulose sheets. Protein bands reacting with antibody were identified by incubating the nitrocellulose strips with the ‘hybridoma’ culture supernatants, followed by detection of bound antibody using alkaline phosphatase conjugated to rabbit anti-mouse F(ab′) 2 antibody, and histochemical substrates. With this technique, the antigens reacting with 4 of a series of 5 monoclonal antibodies have been identified.

Identification of immunogens of [formula omitted] by protein blotting

Proteins of Mycoplasma pneumoniae were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet by blotting. Sera obtained from infected hamsters and immunized rabbits were then incubated with the nitrocellulose strips. Proteins which are capable of eliciting antibodies were detected by indirect immunoradioautography using 125I-labeled antisera against hamster or rabbit IgG. Antibodies to seven immunogens were demonstrated in the sera of hamsters infected with M. pneumoniae by inhalation, while many more proteins were found to be capable of stimulating antibodies in rabbits immunized parenterally with mycoplasmas. This method should prove useful for identifying those components of a microorganism which elicit antibody responses and contribute to the protective state.

The Role of DNA Damage and Repair in the Function of Eukaryotic Genes: Radiation-Induced Single-Strand Breaks and Their Rejoining in Chromosomal and Extrachromosomal Ribosomal DNA of Tetrahymena

The production and rejoining of single-strand breaks (SSB) in chromosomal DNA and extrachromosomal ribosomal DNA (rDNA) were investigated after sublethal doses of ..gamma.. radiation to exponentially growing Tetrahymena. Hydrogen-3-labeled total nuclear DNA isolated from either control or irradiated cells was heat denatured and electrophoresed in agarose gels containing formaldehyde. Ribosomal DNA was identified by hybridization to (/sup 32/P)rRNA after transferring the DNA from the gels to nitrocellulose strips. It was found that (a) approximately 0.68 SSB is produced in each strand of rDNA exposed to 40 krad; (b) greater than 80% of SSB were rejoined within the first 20 min after irradiation in both chromosomal and rDNA; and (c) the rejoining process in both chromosomal and rDNA proceeded in the presence of inhibitors of protein synthesis, RNA synthesis, or oxidative metabolism. While the majority of SSB induced by 40 krad is rejoined within 20 min after irradiation, the resumption of rRNA synthesis does not occur until 30 min thereafter; it is concluded that the restoration of the normal size of the rDNA template is probably necessary but not sufficient for the resumption of rRNA synthesis.

Novel expression-linked copies of the genes for variant surface antigens in trypanosomes.

Pathogenic African trypanosomes evade the immune system of their mammalian hosts by the sequential expression of alternative cell-surface glycoproteins (reviewed in refs 1,2). Variant surface glycoproteins (VSGs) purified from cloned variants of Trypanosoma brucei have similar molecular weights (about 60,000), but differ in amino acid composition, N-terminal amino acid sequence and C-terminal structure. We have cloned DNA complementary to the messenger RNA's for four immunologically distinct VSGs and hybridised these complementary DNAs (cDNAs) with restriction digests of T. brucei nuclear DNA, fractionated by gel electrophoresis and transferred to nitrocellulose strips. Each cDNA recognises a unique set of fragments and this basic set is present unaltered in the nuclear DNAs from the four variants. In addition, each probe recognises an extra fragment only in nuclear DNA isolated from cells expressing the VSG corresponding to the cDNA probe. We infer that activation of a VSG gene involves the production of an expression-linked copy of that gene.

Characterization of DNA from Trypanosoma brucei and related trypanosomes by restriction endonuclease digestion

Abstract We have digested non-kinetoplast DNA from various Trypanosoma strains with restriction endonucleases and analysed the fragment distribution by one-dimensional agarose gel electrophoresis. Visual inspection of ethidium-stained gels shows differences in banding pattern between DNAs from Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum and even between different T. brucei strains, but not between three different antigenic variants derived from the same T. brucei cell. By blotting the DNA on to nitrocellulose strips the restriction endonuclease recognition sites around genes available in cloned form can be analysed by molecular hybridization and we demonstrate this for the gene which codes for one of the variant surface glycoproteins of T. brucei. The use of this method for strain classification is discussed. Renaturation analysis of T. brucei non-kinetoplast DNA shows that about 68% of this is present as single-copy DNA with a complexity of 2.5 × 107 base pairs, whereas the remainder consists of intermediate repetitive and highly repetitive DNA. The latter fraction contains a DNA which is cut by AluI into fragments of 180 base pairs, bands in CsCl at 1.690 g/cm3 and contains duplex circles heterogeneous in size and circles with duplex tails.

Anatomy of herpes simplex virus DNA. VI. Defective DNA originates from the S component

We previously reported that serial propagation of the Justin strain of herpes simplex virus 1 [HSV-1 (Justin)] results in the generation of defective DNA molecules consisting of tandem repetitions of sequences of limited complexity. In the present study, HSV-1 DNA was cleaved with the restriction endonucleases BglII and EcoRI. The fragments were electrophoretically separated on agarose gels, transferred to nitrocellulose strips, and then hybridized with 32P-labeled HSV-1 (Justin) defective DNA. The data allow us to conclude that DNA sequences contained in the repeat unit of defective DNA originate from the S segment of the wild-type viral DNA molecule.

Microchromatography, immunochromatography and detection of proteolytic activity on nitrocellulose membranes impregnated with proteins

Nitrocellulose membrane filters impregnated with proteins were found to be very suitable supporting media for microchromatography and detection of various substances, especially those interacting with the stationary protein layer. This was demonstrated in experiments with phenolsulphophthalein dyes and immunochemically active sera, as well as with proteolytic enzymes. A simple detection of specific antibodies was achieved within about 60 min by chromatographing o.1 μl aliquots of rabbit antibovine serum on membranes impregnated with the antigen, i.e. bovine serum or serum albumin. Nitrocellulose strips impregnated with heat denatured serum were used for the rapid detection of proteolytic activity. Picogram amounts of trypsin were thus detected within about 30 min.