Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody,RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter

Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac(1) flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac(1) mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac(1) Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac(1) mutant. These results validate the Drosophila nac(1) mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency.     

Immunological analysis of chemoattractive proteins from earthworm to garter snakes.

1. Two sets of polyclonal antibodies to two highly purified prey-derived snake-attractive proteins, a low molecular weight (3000) protein and a 20,000 mol. wt protein, were generated in rabbits. 2. They are immunospecific for their respective purified immunogens and do not cross-react with each other. 3. Eight prey-derived proteins that elicit attack by garter snakes (Thamnophis sirtalis) from earthworms (Lumbricus terrestris) were analyzed with these antibodies, and can be assigned to three distinct groups on the basis of their antigenic properties. 4. Unfolding or denaturation of the low molecular weight protein did not alter its antigenic activity to its polyclonal antibodies, suggesting the antigenic epitopes contain contiguous amino acid sequences. 5. In contrast, unfolding of the 20,000 mol. wt protein resulted in a loss of its binding with antibodies, suggesting that the epitope of this protein contains noncontiguous amino acid sequences. 6. The snake-attractivity of the 20,000 mol. wt protein could not be neutralized by reacting it with its antiserum, suggesting that the antigenic determinant (the epitope) of the antigen is not an integral part of the attractive domain of the ES20 protein. 7. In contrast, the attractivity of the purified low molecular weight protein could be neutralized by the polyclonal antibodies.     

Identification and purification of vitamin K-dependent proteins and peptides with monoclonal antibodies specific for gamma -carboxyglutamyl (Gla) residues

Novel monoclonal antibodies that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins and peptides have been produced. As demonstrated by Western blot and time-resolved immunofluorescence assays the antibodies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, prothrombin, protein C, protein S, growth arrest-specific protein 6, bone Gla protein, conantokin G from a cone snail, and factor Xa-like proteins from snake venom). Only the Gla-containing light chain of the two-chain proteins was bound. Decarboxylation destroyed the epitope(s) on prothrombin fragment 1, and Ca(2+) strongly inhibited binding to prothrombin. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin that contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered. Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical methods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequences of the Gla domain and portions of the heavy chain of the snake protein were determined.     

Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

Stoichiometry of murine leukemia virus envelope protein-mediated fusion and its neutralization

Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.     

Antibody performance in western blot applications is context‐dependent

An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well‐designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application‐specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full‐length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full‐length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context‐ and application‐dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross‐reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.     

Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice

The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M(1)ASLIYRQLL(10), a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1-10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection.     

GM-CSF and IL-2 as adjuvant enhance the immune effect of protein vaccine against foot-and-mouth disease

Background

The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported.

Results

In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae.

Conclusion

The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.     

Expression of the B subunit of<Emphasis Type="Italic"> Escherichia coli</Emphasis> heat-labile enterotoxin as a fusion protein in transgenic tomato

Epitopes often require co-delivery with an adjuvant or targeting protein to enable recognition by the immune system. This paper reports the ability of transgenic tomato plants to express a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) and an immunocontraceptive epitope. The fusion protein was found to assemble into pentamers, as evidenced by its ability to bind to gangliosides, and had an average expression level of 37.8 microg g(-1) in freeze-dried transgenic tissues. Processing of selected transgenic fruit resulted in a 16-fold increase in concentration of the antigen with minimal loss in detectable antigen. The species-specific nature of this epitope was shown by the inability of antibodies raised against non-target species to detect the LTB fusion protein. The immunocontraceptive ability of this vaccine will be tested in future pilot mice studies.     

Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.     

Epitope Mapping by Label-Free Biomolecular Interaction Analysis

The diversity of B-cell response to a large immunogen gives rise to a series of antibodies that can be used for epitope mapping of an antigen. This is based on the relative reaction pattern for all antibodies in relation to each other and other ligands to the studied protein. With the introduction of an instrument system, BIAcore, label-free real-time biomolecular interaction analysis (BIA) was made possible. It is based on biosensor technology, with a carboxymethyl-dextran-coated gold surface and an integrated fluidics for transport of liquid. The basic idea is to measure label-free binding of an analyte from a continuous flow to an immobilized ligand in real time. With an automatic approach, quantitative analysis and sequential injection characteristic biospecific binding parameters such as affinity and kinetic constants can be measured. The instrument system was adopted at an early stage for epitope mapping. With label-free detection, antibodies from tissue culture media can be analyzed without purification. Binding of both antigen and a series of antibodies can be individually determined in molar ratio by sequential injections. The quantitative aspects of BIA offer the possibility of further refined epitope mapping. The relative binding pattern for 30 monoclonal antibodies against HIV-1 p24 core protein has been analyzed. Multideterminant analysis and peptide identification of binding sites were performed. Verification of the binding pattern has also been performed in relation to mapping with ELISA as well as the binding to peptides derived from the antigen sequence. Functional domains of proteins in relation to an epitope map have been identified forTaqpolymerase.     

Bioinformatic requirements for protein database searching using predicted epitopes from disease-associated antibodies

We describe a new approach to identify proteins involved in disease pathogenesis. The technology, Epitope-Mediated Antigen Prediction (E-MAP), leverages the specificity of patients' immune responses to disease-relevant targets and requires no prior knowledge about the protein. E-MAP links pathologic antibodies of unknown specificity, isolated from patient sera, to their cognate antigens in the protein database. The E-MAP process first involves reconstruction of a predicted epitope using a peptide combinatorial library. We then search the protein database for closely matching amino acid sequences. Previously published attempts to identify unknown antibody targets in this manner have largely been unsuccessful for two reasons: 1) short predicted epitopes yield too many irrelevant matches from a database search and 2) the epitopes may not accurately represent the native antigen with sufficient fidelity. Using an in silico model, we demonstrate the critical threshold requirements for epitope length and epitope fidelity. We find that epitopes generally need to have at least seven amino acids, with an overall accuracy of >70% to the native protein, in order to correctly identify the protein in a nonredundant protein database search. We then confirmed these findings experimentally, using the predicted epitopes for four monoclonal antibodies. Since many predicted epitopes often fail to achieve the seven amino acid threshold, we demonstrate the efficacy of paired epitope searches. This is the first systematic analysis of the computational framework to make this approach viable, coupled with experimental validation.     

Antitubulin antibodies. II. Natural autoantibodies and induced antibodies recognize different epitopes on the tubulin molecule

Natural and induced antitubulin antibodies were compared for their epitope recognition on alpha- and beta-tubulin subunits by immunoenzymatic assays and Western blot techniques on partially digested tubulin molecules. Our results indicated that natural autoantibodies recognized different epitopes from those recognized by induced antibodies, because: 1) all polyspecific natural autoantibodies tested so far recognized the same or very overlapping epitopes in the central part of both alpha- and beta-subunits (between positions 100 and 300 on the tubulin amino acid sequence) and that this epitope differed from the various epitopes recognized by induced antitubulin antibodies on the amino-terminal or carboxy-terminal parts of the tubulin subunits; 2) one human myeloma protein (monoclonal (m)IgA, kappa) with a monospecific antitubulin activity bound to an epitope around position 310 on both alpha- and beta-subunits and a second human mIg (mIgM, kappa) with a monospecific anti-beta activity bound to an epitope on the carboxy-terminal part of the subunit around amino acid position 350. Both epitopes differed from epitopes recognized by induced antitubulin antibodies. These results thus confirmed our previous findings indicating that natural and induced antitubulin antibodies do not share cross-reactive idiotopes.     

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.     

Epitopes on the major capsid protein of simian virus 40

Thirteen monoclonal antibodies which react with the major capsid protein (VP1) of simian virus 40 (SV40) have been isolated. Of these, five neutralized viral infectivity when added in sufficient concentration. Seven of the antibodies reacted with denatured VP1 and also recognized fragments generated by protease or cyanogen bromide cleavage. The region of VP1 recognized by all seven antibodies was mapped within a nine-amino-acid segment located in the carboxyl portion of the protein (from amino acid positions 312 to 321). This region is likely to protrude from the surface of the protein as judged by high hydrophilicity and low hydropathy predicted from the amino acid sequence and lack of secondary structure by contrast with the rest of the protein for which predominantly beta-sheet structure is predicted. Competition between these antibodies and synthetic peptides for binding to virus particles confirmed that the continuous epitope is contained within the nine-amino-acid sequence. Competition between the different monoclonal antibodies suggested that the continuous epitope was also part of more complex discontinuous epitopes recognized by some of the other antibodies. These results support a model in which a segment of the carboxyl-terminal portion of VP1 protrudes from the surface of the virus to form an antigenic structure.     

The major outer membrane protein of Chlamydia trachomatis: critical binding site and conformation determine the specificity of antibody binding to viable chlamydiae

The major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies-specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid-phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface-exposed, conformation-dependent, subspecies epitope and a buried, conformation-independent species epitope some 10 A distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species-specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the natural protein.     

Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system.

The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.     

Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots

Background

Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the “in-cell western” (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC₂₀) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.

Methodology/Principal Findings

ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey®) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC₂₀ phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.

Conclusion/Significance

ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required.