Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly‐horseradish peroxidase (PolyHRP)

Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin‐containing poly‐horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP‐secondary antibody instead of HRP‐secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin‐conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin‐conjugated PolyHRP resulted in as much as a 110‐fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin‐conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin‐conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.     

Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.     

STRUCTURAL STUDIES ON THE PHOTORECEPTOR PHYTOCHROME: REEVALUATION OF THE EPITOPE FOR MONOCLONAL ANTIBODY Z-3B1

The photoreceptor phytochrome is widely distributed in the plant kingdom from angiosperms to ferns, mosses and algae. The epitope for the monoclonal antibody Z-3B1 which exhibits wide-ranging cross-reactivity with phytochromes from higher and lower plants was mapped by the combination of several methods: by Western blot with proteolytic fragments of known localization, by sequence comparison of phytochromes from various plants, and by production of overlapping fusion proteins. The only sequence which is common to all positively-reacting fusion proteins is the sequence A-830 to R-859. This sequence must contain the Z-3B1 epitope. The best candidate is suggested to be the T-cell antigenic sequence K-Y-V/I-E-A/C-L-L-T (= K-848 to T-855). The significance of the highly conserved epitope in all phytochromes is discussed.     

The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry

In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus.     

Serum protein immunogenicity: implications for liver xenografting

The objectives of this study were threefold: (i) assess immunogenicity of donor plasma proteins following hepatic xenotransplantation, (ii) identify potential immunogens, and (iii) consider the implications of antibody formation against these plasma proteins in xenograft survival. We studied liver and heart xenografts in a concordant combination, hamster to rat. All grafts were examined at necropsy for evidence of rat immunoglobulin G (IgG) deposition. Cardiac xenografts were placed in recipients who had, or had not, been sensitized with hamster serum. Hepatic xenografts were placed in naive recipients to see if antibodies to hamster serum proteins could be eluted from the rejecting organ. Sera of immunized rats were examined for the presence of anti-hamster antibodies by immunoelectrophoresis and by Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of hamster serum. Antibodies in sera of immunized rats were compared with those eluted from rejecting livers. Candidate antigens were identified by tandem mass spectrometry, sequence analysis, and reference to protein databases. Results showed that sera of immunized rats recognized a minimum of four different antigens in hamster serum by immunoelectrophoresis, and a minimum of seven by the more sensitive SDS-PAGE Western blot. IgG eluted from rejecting livers bound three of seven candidate antigens recognized by sera of the immunized animals. Sequence analysis searches revealed proteinase inhibitors in each of the three SDS-PAGE bands common to the above samples. All of these candidate proteinase inhibitor immunogens share a common catabolic fate, uptake via the lipoprotein-related protein (LRP/alpha 2-macroglobulin receptor (CD91). Sensitization to hamster serum proteins hastened cardiac xenograft rejection in 30-50% of recipients (depending on sensitization protocol). Vascular deposition of rat IgG occurred in all rejecting xenografts. Antibody binding to proteinase inhibitors could disturb their functional activity and contribute to the pathogenesis of delayed xenograft rejection.     

Reversed-phase high-performance liquid chromatographic tryptic digest peptide map comparisons of monoclonal antibodies to human tumor necrosis factor.

An automatic computer technique was used to compare the retention times and ultraviolet spectra of sixty-two peaks in peptide maps of three monoclonal antibodies against human tumor necrosis factor (TNF) and one monoclonal antibody against recombinant factor VIII. The anti-TNF monoclonal, B6, which has an overlapping epitope with the anti-TNF monoclonal, A10G10, had a 90% peak match with A10G10. The anti-TNF monoclonal, A6, with a different epitope to TNF than A10G10, had only a 60% peak match. The A6 match to A10G10 was similar to the 50% peak match of the anti-factor VIII monoclonal with A10G10. The results of this study suggest that peptide mapping can be used as a quantitative characterization technique for comparing monoclonal antibodies.     

Development of DNA-Designed Avian IgY Antibodies for Detection of Mycobacterium avium subsp. paratuberculosis Heat Shock Protein 70 (Hsp70) and Anti-Hsp70 Antibodies in the Serum of Normal Cattle

Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (MAPHsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAPHsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1-MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1-MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAPHsp70, and in a sandwich ELISA to assess the prevalence of anti-MAPHsp70 antibodies in cattle serum. Western blotting results indicate the expression of rMAP hsp70 in COS-7 cells and sandwich ELISA could detect anti-MAPHsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-MAPHsp70 could demonstrate the effective production of anti-MAPHsp70 IgY antibodies. Monospecific anti-MAPHsp70 antibody generated in chickens is useful for detection of MAPHsp70 peptide in cell culture and MAP lysate.     

MIMOTOPES,CONTINUOUS PARATOPES AND HYDROPATHIC COMPLEMENTARITY: NOVEL APPROXIMATIONS IN THE DESCRIPTION OF IMMUNOCHEMICAL SPECIFICITY

Most antigenic sites of proteins, known as discontinuous epitopes, are made up of residues on different loops that are brought together by the folding of the polypeptide chain. The individual loops are sometimes able, on their own, to bind to the antibody and they are then known as continuous epitopes. The binding sites of antibodies, known as paratopes, are built up from residues on six hypervariable loops known as complementarity determining regions (CDRs). Peptides corresponding to individual CDR loops are often able to bind the antigen and such peptides may be viewed as continuous paratopes. Using random combinatorial peptide libraries, it is possible to obtain peptides that bind to an antiprotein antibody without showing any sequence similarity with any part of the protein. Such epitope mimics are called mimotopes provided they are able also to elicit antibodies that react with the original antigen. The binding activity of mimotopes may partly be due to the phenomenon of hydropathic complementarity between epitope and paratope peptides. Although these concepts are vague in their structural connotation, they are useful for describing the immunological activity of linear peptides.     

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.     

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.     

Covalent display of oligosaccharide arrays in microtiter plates

A covalent array for the display of complex oligosaccharides in microtiter plates has been developed. This strategy is conducive to the display of carbohydrates to proteins of interest such as lectins and antibodies, including the broadly neutralizing antibody 2G12 against HIV envelope oligomannose and can be cleaved from the surface for further characterization by mass spectrometry. The system was used to probe the multivalent interaction of 2G12 with an optimal epitope (Kd 0.1 muM).     

Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes

A method for the immunodetection of several natural complex polysaccharides (glycosaminoglycans) after their separation by conventional agarose gel electrophoresis, blotting and immobilizing on nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride (CPC), and direct and specific immunodetection by antibodies is described. This new approach is based on the principles that were used to develop the Western blot, and is applied to the separation of the glycosaminoglycans purified from normal human urine. After migration in agarose gel electrophoresis, chondroitin sulfate samples of different origin were blotted and transferred onto nitrocellulose membranes treated with CPC. Immunodetection was performed using the anti-chondroitin-6-sulfate antibody that specifically recognizes intact chondroitin-6-sulfate. By calculating the ratio between the antibody staining (epitope) and alcian blue staining (mass), the epitope density expressed as a percentage, i.e., the number of repetitive epitopes per mass, was obtained. These values were in agreement with the quantitation of 6-sulfated groups of chondroitin sulfate performed by the evaluation of unsatured disaccharide-6-sulfate (DeltaDi6S) produced after treatment with chondroitinase ABC and separated by high-performance liquid chromatography (HPLC). Furthermore, immunodetection of heparan sulfate was performed using the anti-heparan sulfate antibody.     

Application of recombinant phage display antibody system in study of Codakia orbicularis gill proteins

We used the recombinant phage display antibody system (RPAS) to obtain chimeric single-chain fragment variable (ScFv) antibodies to gill proteins of the white clam Codakia orbicularis (Linné, 1758). After three rounds of selection on immunotubes loaded with total gill protein extract, recombinant phages exhibiting antibodies to gill proteins were isolated and tested by enzyme-linked immunosorbent assay (ELISA). Clones exhibiting a high affinity for the mollusk proteins were selected for production of soluble ScFv antibodies, which were purified for subsequent analysis. ScFv antibodies exhibited a reaction specific for a protein whose molecular mass was about 15,000 Daltons and that was detected by the antigen capture technique followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.     

Multiplex localization of sequential peptide epitopes by use of a planar microbead chip

Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.     

Matrix-assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody

In this study we have applied epitope excision and epitope extraction strategies, combined with matrix assisted laser desorption/ionization mass spectrometry, to determine the fine structure of epitopes recognized by a polyclonal antibody to human immunodeficiency virus envelope glycoprotein gp120. This is the first application of this approach to epitope mapping on a large, heavily glycosylated protein. In the epitope excision method, gp120 in the native form is first bound to the antibody immobilized on sepharose beads and cleaved with endoproteinase enzymes. In the epitope extraction method, the gp120 was first proteolytically cleaved and then allowed to react with the immobilized antibody. The fragments that remain bound to the antibody, after repeated washing to remove the unbound peptides, contain the antigenic region that is recognized by the antibody, and the bound peptides in both methods can be characterized by direct analysis of the immobilized antibody by matrix assisted laser desorption ionization/mass spectrometry. In this study we have carried out epitope excision and extraction experiments with three different enzymes and have identified residues 472–478 as a major epitope. In addition, antigenic regions containing minor epitopes have also been identified.     

Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three Gram-negative bacteria: Proteus morganii O34, Escherichia coli O111 and Salmonella Adelaide O35

Bacterial strains have complex and individual antigenic structure, which provides basis for their serological identification. However, serological cross-reaction may occur when antibodies against a certain strain recognize other strains too. The molecular basis of this phenomenon is the expression of similar or identical antigenic epitopes on the surface of different bacterial cells. Such cross-reactions might harden the serological diagnosis of pathogenic bacteria. But it can be also advantageous, when antigens of non-pathogenic strains can be used in the serological examinations. Serological cross-reaction between three taxonomically different strains--Proteus morganii O34 (8662/64), Escherichia coli O111 and Salmonella Adelaide O35--have been described. It has been proven that it is based partially on the similar lipopolysaccharide structures of these pathogens. In this study the involvement of the outer membrane proteins of these strains in the serological cross-reaction is presented. Microfluidic chip technology was applied for the detection of common proteins, which provided fast and quantitative data about the proteins that might be responsible for serological cross-reaction. Two outer membrane proteins with apparent molecular mass of 36 and 41 kDa, respectively, could be detected in the profile of each strain, while individual dominating protein peaks have also appeared in the protein profiles. The presence of common protein antigens was proven by Western blotting.     

STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light-harvesting antenna of photosystem I or II, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c-binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.     

Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis

The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate proteins and analyze results. We demonstrate the overall capabilities of such a method combination in a complex cell lysate background, while comparing the specific results obtained using a biarsenical fluorescein-derivative and tetracysteine epitope-tagged proteins with total protein staining using a fluorescent gel stain and with Western blotting where an anti-oligohistidine (His) tag antibody has been employed. When applied on purified target proteins without extraneous protein background, the demonstrated sensitivity of the assay on the extended-run 96-array precast SDS-PAGE system allows detection of quantities of tagged protein as low as 1 pmol per band.     

The characterization and use of different antibodies against the hsp70 major heat shock protein family for the development of an immunoassay.

The hsp70 family of major stress proteins is composed of several different members exhibiting similar structural and functional properties. In order to obtain an antiserum with wide epitope reactivity, rabbits were immunized with a mixture of native and denatured hsp70 purified from bovine muscle by ATP-affinity chromatography. Screening for antibody specificity was performed by a "sandwich" enzyme linked immunosorbent assay (ELISA). Immunoprecipitation and immunoblotting analyses demonstrated that the polyclonal antiserum obtained by us and a monoclonal antibody raised against a different preparation of antigen recognized the same determinant on the native hsp70 molecule (inducible form). With a different specificity the polyclonal antiserum recognized only the denatured monomers of the other members of the hsp70 family. These results are discussed in relation to the immunological features of the hsp70 molecule and to the development of an immunoassay for the detection of hsp70 in cell and tissue extracts.