Direct Binding Enzyme-linked Immunosorbent Assay Research Articles

Nonenzymatic glycosylation of human serum albumin and its effect on antibodies profile in patients with diabetes mellitus.

BackgroundAlbumin glycation and subsequent formation of advanced glycation end products (AGEs) correlate with diabetes and associated complications.MethodsHuman Serum Albumin (HSA) was modified with D-glucose for a 40 day period under sterile conditions at 37°C. Modified samples along with native HSA (unmodified) were analyzed for structural modifications by UV and fluorescence, FTIR, Liquid chromatography mass spectrometry (LCMS) and X–ray crystallography. New-Zealand white female rabbits immunized with AGEs, represent auto-antibodies formation as assessed by competitive and direct binding enzyme-linked immunosorbent assay (ELISA). Neo-epitopesagainst In-vitro formed AGEs were characterized in patients with diabetes mellitus type 2 (n = 50), type 1 (n = 50), gestational diabetes (n = 50) and type 2 with chronic kidney disease (CKD) with eGFR level 60–89 mL/min (n = 50) from serum direct binding ELISA.ResultsGlycated-HSA showed amarked increase in hyperchromicity of 65.82%,71.98%, 73.62% and 76.63% at λ280 nm along with anincreasein fluorescence intensity of 65.82%, 71.98%, 73.62% and 76.63% in glycated-HSA compared to native. FTIR results showed theshifting of Amide I peak from 1656 cm_1 to 1659 cm_1 and Amide II peak from 1554 cm_1 to 1564 cm_1 in glycated-HSA, with anew peak appearance of carbonyl group at 1737 cm-1. LCMS chromatogram of glycated-HSA showed thepresence of carboxymethyl lysine (CML) at 279.1 m/z. Immunological analysis showed high antibody titre>1:12,800 in theserum of rabbits immunized with glycated-HSA (modified with 400 mg/dL glucose) and inhibition of 84.65% at anantigen concentration of 20μg/mL. Maximum serum auto-antibody titre was found in T2DM (0.517±0.086), T1DM (0.108±0.092), GDM (0.611±0.041) and T2DM+CKD (0.096±0.25) patients immunized with glycated-HSA (modified with 400 mg/dL glucose).ConclusionsNon-enzymatic glycosylation of HSA manifests immunological complications in diabetes mellitus due to change in its structure that enhances neo-epitopes generation.

Lytic Inactivation of Human Immunodeficiency Virus by Dual Engagement of gp120 and gp41 Domains in the Virus Env Protein Trimer.

We recently reported the discovery of a recombinant chimera, denoted DAVEI (dual-acting virucidal entry inhibitor), which is able to selectively cause specific and potent lytic inactivation of both pseudotyped and fully infectious human immunodeficiency virus (HIV-1) virions. The chimera is composed of the lectin cyanovirin-N (CVN) fused to the 20-residue membrane-proximal external region (MPER) of HIV-1 gp41. Because the Env gp120-binding CVN domain on its own is not lytic, we sought here to determine how the MPER(DAVEI) domain is able to endow the chimera with virolytic activity. We used a protein engineering strategy to identify molecular determinants of MPER(DAVEI) that are important for function. Recombinant mutagenesis and truncation demonstrated that the MPER(DAVEI) domain could be significantly minimized without loss of function. The dependence of lysis on specific MPER sequences of DAVEI, determination of minimal linker length, and competition by a simplified MPER surrogate peptide suggested that the MPER domain of DAVEI interacts with the Env spike trimer, likely with the gp41 region. This conclusion was further supported by observations from binding of the biotinylated MPER surrogate peptide to Env protein expressed on cells, monoclonal antibody competition, a direct binding enzyme-linked immunosorbent assay on viruses with varying numbers of trimeric spikes on their surfaces, and comparison of maximal interdomain spacing in DAVEI to that in high-resolution structures of Env. The finding that MPER(DAVEI) in CVN-MPER linker sequences can be minimized without loss of virolytic function provides an improved experimental path for constructing size-minimized DAVEI chimeras and molecular tools for determining how simultaneous engagement of gp120 and gp41 by these chimeras can disrupt the metastable virus Env spike.

4-Aminobiphenyl and Nitric Oxide Synergistically Modified Human DNA: Its Implication in Bladder Cancer

Bladder cancer is a disease of continuing public health significance. Some known risk factors for bladder cancer development include smoking, occupational exposure, genetic susceptibility, infectious diseases and radiation therapy. Smoking and occupational exposure have strongly implicated aromatic amines as being carcinogenic for the bladder, with 4-aminobiphenyl (4-ABP) being one of the most potent. 4-ABP is a major etiological agent of human bladder cancer, and its metabolites are able to form DNA adducts that may induce mutation and initiate bladder carcinogenesis. Binding characteristics and specificity of bladder cancer anti-DNA antibodies were analyzed by direct binding and inhibition enzyme-linked immunosorbent assay. The data show preferential binding of bladder cancer antibodies to 4-ABP-NOmodified DNA in comparison with native native DNA. A band shift assay further substantiated the enhanced recognition of 4-ABP-NO-modified DNA by anti-DNA antibodies. Cancer antibodies exhibited enhanced binding with the modified human DNA as compared to the native form. Lymphocyte DNA from cancer patients showed appreciable recognition of anti-4-ABP–NO-DNA IgG as compared to the DNA from healthy subjects. The 4-ABP-NO- modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in bladder cancer patients. The results suggest that 4-ABP-NO-modification of self-antigen(s) can generate neoepitopes capable of inducing bladder cancer characteristic autoantibodies. The preferential binding of 4-ABP-NO-modified DNA bladder cancer anti- DNA antibodies points out the likely role of oxidatively modified in the initiation/progression of bladder cancer. Moreover, oxidatively modified genomic DNA antigen, appear to be more suitable as a trigger for bladder cancer. The aim of this study was to evaluate whether biomarkers of environmental tobacco smoke exposure [i.e., 4-aminobiphenylDNA (4-ABP-DNA) adducts] were common pathway for the predictive of the risk of cancers.

Seroprevalence of Molluscum contagiosum Virus in German and UK Populations

Molluscum contagiosum virus (MCV) is a significant but underreported skin pathogen for children and adults. Seroprevalence studies can help establish burden of disease. Enzyme linked immunosorbent assay (ELISA) based studies have been published for Australian and Japanese populations and the results indicate seroprevalences between 6 and 22 percent in healthy individuals, respectively. To investigate seroprevalence in Europe, we have developed a recombinant ELISA using a truncated MCV virion surface protein MC084 (V123-R230) expressed in E. coli. The ELISA was found to be sensitive and specific, with low inter- and intra-assay variability. Sera from 289 German adults and children aged 0–40 years (median age 21 years) were analysed for antibodies against MC084 by direct binding ELISA. The overall seropositivity rate was found to be 14.8%. The seropositivity rate was low in children below the age of one (4.5%), peaked in children aged 2–10 years (25%), and fell again in older populations (11–40 years; 12.5%). Ten out of 33 healthy UK individuals (30.3%; median age 27 years) had detectable MC084 antibodies. MCV seroconversion was more common in dermatological and autoimmune disorders, than in immunocompromised patients or in patients with multiple sclerosis. Overall MCV seroprevalence is 2.1 fold higher in females than in males in a UK serum collection. German seroprevalences determined in the MC084 ELISA (14.8%) are at least three times higher than incidence of MC in a comparable Swiss population (4.9%). While results are not strictly comparable, this is lower than Australian seroprevalence in a virion based ELISA (n = 357; 23%; 1999), but higher than the seroprevalence reported in a Japanese study using an N-terminal truncation of MC133 (n = 108, 6%; 2000. We report the first large scale serological survey of MC in Europe (n = 393) and the first MCV ELISA based on viral antigen expressed in E. coli.

Binding of circulating autoantibodies in breast cancer to native and peroxynitrite-modified RNA

Peroxynitrite (ONOO(-)) is a powerful oxidant and nitrosative agent and has in vivo existence. The half life of ONOO(-) at physiological pH is less than 1 s. It can react with nucleic acids, proteins, lipoproteins, saccharides, cardiolipin, etc., and can modify their native structures. Action of ONOO(-), synthesized in the authors' laboratory by a rapid quenched flow process, on structural changes of commercially available RNA was studied by ultraviolet (UV), fluorescence, and agarose gel electrophoresis. Compared to native RNA, the ONOO(-)-modified RNA showed hyperchromicity at 260 nm. Furthermore, the ethidium bromide (EtBr) assisted emission intensities of ONOO(-)-modified RNA samples were found to be lower than the emission intensity of native RNA-EtBr complex. Agarose gel electrophoresis of ONOO(-)-modified RNA showed a gradual decrease in band intensities compared to native RNA, an observation clearly due to the poor intercalation of EtBr with ONOO(-)-modified RNA. Native and ONOO(-)-modified RNA samples were used as an antigen to detect autoantibodies in sera of patients with clinically defined breast cancer. Both direct binding and inhibition enzyme-linked immunosorbent assay (ELISA) confirmed the prevalence of native and 0.8 mmol/L ONOO(-)-modified RNA specific autoantibodies in breast cancer patients. Moreover, the progressive retardation in the mobility of immune complexes formed with native or 0.8 mmol/L ONOO(-)-modified RNA and affinity purified immunoglobulin G (IgG) from sera of breast cancer patients supports the findings of the direct binding and inhibition ELISAs. The peroxynitrite treatment to RNA at a higher concentration appears to have damaged or destroyed the typical epitopes on RNA and thus there was a sharp decrease in autoantibodies binding to 1.4 mmol/L ONOO(-)-modified RNA. It may be interpreted that cellular nitrosative stress can modify and confer immunogenicity on RNA molecules. Higher concentrations of nitrogen reactive species can be detrimental to RNA. However, the emergence of native as well as 0.8 mmol/L ONOO(-)-modified RNA as a novel antigen/substrate for autoantibodies in breast cancer patients indicates that, in future, these molecules might find a place on the panel of antigens for early diagnosis of breast cancer.

Peroxynitrite-induced modification of H2A histone presents epitopes which are strongly bound by human anti-DNA autoantibodies: Role of peroxynitrite-modified-H2A in SLE induction and progression

Peroxynitrite is a potent oxidant and nitrating agent and has in vivo existence. It is a powerful proinflammatory substance and may increase vascular permeability in inflamed tissues. Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease of unknown etiology. Since its discovery, numerous self- and non-self, nuclear, and cytoplasmic antigens have been suggested as stimuli for SLE initiation, but the exact trigger is yet to be identified. In this study, an attempt has been made to investigate the binding characteristics of SLE anti-DNA autoantibodies to native DNA and native and peroxynitrite-modified H2A histone to explore the possible role of modified protein antigen(s) in SLE initiation and progression. The nuclear protein (H2A histone) was modified by peroxynitrite synthesized in our laboratory. The peroxynitrite-modified H2A revealed generation of nitrotyrosine, dityrosine, and carbonyls when subjected to investigation by physicochemical methods. Binding characteristics and specificity of SLE anti-DNA antibodies were analyzed by direct binding and inhibition enzyme-linked immunosorbent assay. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H2A histone in comparison with native H2A histone or native DNA. A band shift assay further substantiated the enhanced recognition of peroxynitirite-modified H2A histone by anti-DNA autoantibodies. The results suggest that peroxynitrite modification of self-antigen(s) can generate neoepitopes capable of inducing SLE characteristic autoantibodies. The preferential binding of peroxynitrite-modified H2A histone by SLE anti-DNA antibodies points out the likely role of oxidatively modified and nitrated H2A histone in the initiation/progression of SLE. Moreover, oxidatively modified and nitrated nuclear protein antigen, rather than nucleic acid antigens, appear to be more suitable as a trigger for SLE.

Structure and immunological function of oxidised albumin in lung cancer: its potential role as a biomarker of elevated oxidative stress

The role of reactive oxygen species (ROS)-damaged human serum albumin (HSA) in lung cancer (LC) patients is investigated. The binding characteristics of LC antibodies with native and ROS-damaged HSA are assessed. Smoking and non-smoking LC patients (n=40) are examined by a direct binding enzyme-linked immunosorbent assay (ELISA) and the results are compared with healthy ageand gender-matched smoking and non-smoking subjects (n=38). A high degree of specific binding in approximately 70% of cancer immunoglobulin G (IgG) towards ROS-damaged HSA was observed, compared to results with its native analogue (P<0.05). Affinity purified IgG from those LC patients with a history of smoking showed substantially stronger binding to damaged HSA over native HSA. Competitive inhibition ELISA substantiated the enhanced recognition of ROS-HSA by circulating antibodies in LC patients. The increase in total serum protein carbonyl levels in the LC patients was largely due to an increase in oxidised albumin. Purified HSA from LC patients (LC-HSA) contained higher levels of carbonyls than did HSA from healthy subjects (normal-HSA; P<0.01). LC-HSA was conformationally altered and showed greater exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in LC patients.

Comparative study of four different assays for the detection of binding antibodies against interferon-β

Background Binding antibodies (BAB) against interferon-β (IFNβ) are often determined as screening assays before performing an expensive and elaborate neutralizing antibody (NAB) test. Methods In this study, we compared four BAB tests, a western blot (WB), a direct binding enzyme-linked immunosorbent assay (ELISA) (dELISA), a capture ELISA (cELISA), and a commercial enzyme immuno-assay (EIA) in 325 multiple sclerosis patients with and without neutralizing antibodies to evaluate the sensitivity and specificity to detect NAB by receiver operating characteristics analysis. Results The area under the curve (AUC) values were 0.907 for the dELISA, 0.925 for the cELISA, and 0.776 for the EIA (P < 0.0001 for all). At a sensitivity of 95%, the specificity was approximately 30% in the dELISA, 55% in the cELISA, and 13% in the EIA. The WB as a qualitative BAB detection method had a given sensitivity of 97% and a specificity of 55%. There was a strong and significant correlation between high NAB titers (>500 neutralizing units [NU]) and titers obtained by all quantitative BAB assays. However, low to medium NAB titers (20–500 NU) did not significantly correlate with BAB titers. Conclusion We conclude that the cELISA seems to be most suitable for NAB screening, but BAB titers cannot reliably predict NAB titers.

Binding Characteristics of SLE Anti-DNA Autoantibodies to Catecholestrogen-modified DNA

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease in which anti-double-stranded DNA antibody is a classic autoantibody that characterizes SLE. A role for oestrogens in the pathogenesis of SLE has been suspected for many years but the exact patho-aetiology remains elusive. In this study, the binding of SLE autoantibodies with native and 4-OHE(2)-NO-modified plasmid DNA were assessed. Binding specificity of antibodies was analysed by direct binding and inhibition enzyme-linked immunosorbent assay, quantitative precipitin titration and gel retardation assay. Anti-DNA IgG from SLE sera, purified on Protein A-Agarose matrix, exhibited increased recognition of 4-OHE(2)-NO-DNA than native DNA (P < 0.001). Gel retardation assay further substantiated the enhanced recognition of modified DNA by anti-DNA autoantibodies. The affinity of anti-DNA antibodies for modified polymer was found to be high as calculated by using Langmuir plot. DNA modified by 4-OHE(2)-NO presents unique neo-epitopes that might be one of the factor in antigen-driven induction of SLE autoantibodies.

Measurement and Significance of 3‐Nitrotyrosine in Systemic Lupus Erythematosus

Nitration of free and protein associated tyrosine represents, in vivo, a mechanism that can severely compromise the cell function. The detection of 3-nitrotyrosine (3-NT) in pathological tissues is suggestive of the occurrence of nitrating pathways and has been identified as a marker of inflammation and a stable end product of increased reactive nitrogen intermediate production. Protein nitration occurs in many disease conditions including systemic lupus erythematosus (SLE). In this study we show that the level of both free and protein bound 3-NT, which is produced by reactive nitrogen species (RNS)-dependent oxidative damage, is elevated in patients with SLE and that there is a possible role of RNS-modified epitopes in the aetiology of the disease. Commercially available poly L-tyrosine was exposed to nitrating species, inducing nitration in tyrosine residues. Immunoglobulin-G (IgG) purified on Protein-A-Sepharose matrix from 24 SLE patients was studied for their recognition of native and nitrated poly L-tyrosine by direct binding and competition enzyme-linked immunosorbent assay (ELISA). The formation of immune complex between SLE IgG and nitrated poly L-tyrosine was visualized by gel retardation assay. Free 3-NT in patients' sera was detected and quantitated by high performance liquid chromatography whereas protein-bound 3-NT was analysed by Western blotting and the concentration was calculated by sandwich ELISA. The concentration of free 3-NT was found to be 1.4 +/- 0.09 microm whereas the concentration of protein bound 3-NT was 96.52 +/- 21.12 microm nitrated bovine serum albumin equivalents/mg protein, which was significantly higher when compared with healthy controls. Elevated level of 3-NT was observed in SLE patients using two different techniques, when compared with healthy subjects confirms the overproduction of RNS in the pathogenesis of human SLE.

Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates

Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product or the quantum yield of a fluorescent product, coupled with the efficiency of the immobilized enzyme, is the determining factor for the sensitivity and precision of a given ELISA. The enhancement of precision and sensitivity using fluorogenic substrates was demonstrated in a direct-binding ELISA in a low-analyte concentration range compared with commonly used chromogenic substrates. The enhancement in precision was demonstrated quantitatively with lower coefficients of variation in measurements of signal intensities, approximately a five- to six-fold enhancement in signal-to-noise ratio at a given analyte concentration with fluorogenic substrates. Similarly, the amplitude of the enhancement in sensitivity, as reflected by relative limits of detection or quantitation, is approximately two- to five-fold when compared with commonly used chromogenic substrates. Additional advantages of a fluorescence-based ELISA format include the continuous monitoring of initial rates of enzymatic reactions, the measurement of fluorescence changes in the presence of particulate materials, the absence of a quench step, and a larger quantifiable analyte range.

Anti-neutrophil cytoplasmatic antibodies and lung disease in cystic fibrosis

Background: Bactericidal-permeability-increasing protein (BPI) is a potent anti-microbial protein produced by neutrophil granulocytes. Anti-neutrophil cytoplasmatic antibodies (ANCA) directed against BPI have been detected in up to 91% in patients with cystic fibrosis (CF). We aimed to evaluate the prevalence of BPI-ANCA in our CF patients and to determine whether presence of BPI-ANCA is correlated with organ damage. Methods: Twenty-four patients performed respiratory function testing and pulmonary high-resolution computed tomography (HRCT). HRCT was scored by using a modified Bhalla method. Serum samples were analysed by direct binding enzyme-linked immunosorbent assay for BPI-ANCA. Results: The prevalence of anti-BPI-IgG was 71% and anti-BPI-IgA 33%. Twenty-nine percent of our patients were positive for both BPI-ANCA isotypes. Mean HRCT score was 8.0 ranging from 0 to 22, bronchiectasis presented the most common finding (79%). There was a significant correlation between BPI-ANCA and both HRCT score and FEV 1 ( p<0.01). High levels of BPI-ANCA were correlated to chronic Pseudomonas aeruginosa lung infection ( p<0.01). Conclusions: BPI-ANCA was common in our study group. Highly significant correlations between BPI-ANCA and parameters to evaluate lung disease in CF may be a consequence of the inflammation process, or it may indicate a pathogenic role of BPI-ANCA levels in the development of lung disease. More research is needed and the clinical significance of our findings needs further evaluation.

Monoclonal Antibody-Based Sensitive Enzyme-Linked Immunosorbent Assay for Murine Serum Amyloid A

Enzyme-linked immunosorbent assay (ELISA) methods for measuring murine serum amyloid A (SAA), a representative acute phase reactant, were developed utilizing a newly produced monoclonal antibody. Two site-ELISA, in which the monoclonal antibody was used as the captured antibody, was sensitive enough to determine the SAA concentration in mice at the steady state. Direct binding ELISA, in which the sample SAA bound to the plastic wells was detected by the antibody, was simple and suitable for measuring the elevated SAA, but could not analyze the resting level of SAA because of the need for high dilution in plasma samples. Plasma SAA concentrations were measured in ten ICR mice on the day of purchase and at the end of seven days of ordinary rearing. the SAA concentration of one animal decreased from 1.6 to 0.5 mg/1 during a week, while the others had no obvious changes. the plasma SAA of the ten animals after one week of rearing ranged from 0.3 to 0.8 mg/1 with a mean of 0.47. These mice, two days after 10 μ g lipopolysaccharide were given, had increased SAA values up to a mean of 300 mg/1, though with variations between animals.

Recombinant Phabs reactive with 7,8-dihydro-8-oxoguanine, a major oxidative DNA lesion.

Antibody Fabs that bind to DNA damages provide useful models for understanding DNA damage-specific protein interactions. BSA and RSA conjugates of the nucleoside and nucleotide derivatives of the oxidative DNA lesions, 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), were used to immunize mice. RNA from the responders was isolated and used to repertoire clone and phage display Fabs that bind to these haptens. Direct binding and competitive enzyme-linked immunosorbent assay (ELISA) demonstrated that phage Fabs (Phabs) specific for 8-oxopurine-BSA conjugates and 8-oxoguanine were produced although the Phabs did not react with 8-oxopurines in DNA. Amino acid sequence comparisons among clones having different binding properties suggested that a relatively small portion of the binding surfaces defined by the complementarity determining regions (CDR) accounted for hapten binding specificity, whereas other regions appeared to stabilize hapten binding by interacting with protein or DNA epitopes. Chain shuffling between 8-oxopurine-BSA binding Fabs and a DNA binding Fab showed that the heavy chain of the DNA binder conferred DNA binding capacity to the light chain of only one of the 8-oxopurine-BSA binders. Homology modeling of the 8-oxoG-specific clone g37 showed significant similarities to two previously isolated monoclonal antibodies specific for single-stranded nucleic acids. In the 8-oxoG Fab, which did not bind to DNA, the presumptive DNA binding canyon was blocked by heavy chain residues in the CDR2 region and appeared to lack part of the canyon wall due to the different placement of the light chain framework region.

Quantitative assessment of IgM antibodies towards an immunodominant B-cell epitope within the preS2 domain of HBV in the natural course and during combined prednisone/interferon alpha 2b treatment of chronic hepatitis B virus infection.

A direct binding enzyme-linked immunosorbent assay (ELISA) was established for quantitative determination of serum IgM antibodies towards a synthetic peptide corresponding to a selected segment (14-21) of the preS2-gene product containing an immunodominant linear B-cell epitope. The prevalence of IgM anti-preS2 (14-21) antibody titers > 1,000 for hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B virus (HBV) infection was 38% (22/58) and 10% (2/21) for HBeAg-negative subjects (P < 0.005). IgM anti-preS2 (14-21) reactivity was detected during the clinical course of chronic HBV infection and IgM anti-peptide antibody titers declined and disappeared before spontaneous HBe/anti-HBe seroconversion. Recombinant interferon (IFN)-alpha 2b with an antecedent short course of corticosteroids was administered to eight Chinese patients with chronic HBV infection. The IgM anti-preS2 (14-21) reactivity was monitored consecutively during treatment and patients were followed for more than 1 year. A close association between the presence of pretreatment IgM anti-preS2 (14-21) in serum and the capacity to respond favorably to the combined prednisone/IFN-alpha 2b therapy was detected. The IgM anti-preS2 (14-21) titers decreased during treatment with subsequent loss of detectable antibodies 8-16 weeks after the initiation of therapy. This decrease was concomitant with an alanine aminotransferase (ALT) augmentation preceding the disappearance of HBV-DNA and anti-HBe seroconversion. Long-term remission was not observed in treated patients who lacked detectable levels of pretreatment IgM anti-preS2 (14-21) in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a Receptor Peptide Antagonist to Human γ-Interferon and Characterization of Its Ligand-bound Conformation Using Transferred Nuclear Overhauser Effect Spectroscopy

Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human gamma-interferon (huIFN-gamma) sequence recognizes soluble human gamma-interferon receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363). We sought to use this reagent to identify a ligand-binding domain within IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic antibody was used to probe overlapping linear peptide octamers of the extracellular domain of the huIFN-gamma receptor. A 22-amino-acid residue receptor segment 120-141 identified by the antibody was synthesized. CD and NMR analysis indicates that peptide 120-141 has no apparent secondary structure in water or in water containing 50% trifluoroethanol. The synthetic receptor peptide inhibited huIFN-gamma induced expression of HLA/DR antigen on Colo 205 cells with an approximate IC50 of 35 microM. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating factor on a microtiter plate in a direct binding enzyme-linked immunosorbent assay. The binding results are supported by two-dimensional transferred nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the conformation of the bound peptide by TRNOE suggests that this peptide assumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a third region comprising a beta-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand-binding domain.

Production of Monoclonal Antibodies to Potato Virus Y Helper Component-Protease and Their Use for Strain Differentiation

Ten monoclonal antibodies (MAbs) and a rabbit antiserum containing polyclonal antibodies (PAbs) were prepared against helper component-protease (HC-Pro) purified from plants infected with a non-aphid-transmissible strain of potato virus Y (PVY-O NAT). All MAbs reacted with PVY HC-Pro in two enzyme-linked immunosorbent assay (ELISA) protocols, and seven were useful for Western blot analysis. Six MAbs and the PAbs were compared in direct antigen binding ELISA for reactivity with 22 isolates of PVY, potato virus A (PVA), tobacco etch virus (TEV), and pepper mottle virus (PepMoV). None of the MAbs or the PAbs reacted with PVA or TEV HC-Pro. The immunoreactivity of one MAb was similar to the PAbs, reacting with all 22 PVY isolates and with PepMoV, but failing to react with PVA and TEV HC-Pro. Another MAb reacted with all of the common strains of PVY (PVY O and a stipplestreak strain of PVY (PVY C ) but not with any of the tobacco veinal necrosis strains (PVY N ). The other MAbs could be used to differentiate a subgroup within the PVY O and PVY N strain groups. These MAbs proved useful for identifying strains of PVY

Characterization of the humoral response induced by a synthetic peptide of the major outer membrane protein of Chlamydia trachomatis serovar B

The major outer membrane protein of Chlamydia trachomatis has been extensively studied and is still considered one of the most promising candidates for development of a synthetic vaccine. Neutralizing epitopes in variable domains I, II, and IV have already been reported. In variable domain I, residues 69 to 78 have been identified as a neutralizing epitope for some of the C- and C-related complex serovars (A, C, I, J, L3, and K). It is not known whether epitopes located at the same position in B-complex serovars are neutralizing. To clarify this point, rabbit polyclonal antibodies directed against the peptide 69TTTGNAVAPS78 from the B serovar were produced. Rabbit antisera were further rendered peptide specific by purification on a peptide-bovine serum albumin-Sepharose affinity column. Peptide-specific rabbit immunoglobulin reacted with five of the B-complex serovars (B, Ba, E, L1, and L2) by immunoblot and by direct-binding enzyme-linked immunosorbent assay. Furthermore, this peptide-specific rabbit immunoglobulin neutralized the chlamydial infectivity of both serovars B and E for HaK cells in a complement-independent in vitro assay. The importance of these results stems from the fact that peptide 69TTTGNAVAPS78 was able to induce an antibody response directed against B- and B-related complex serovars, including serovar E, which is responsible for a high proportion of genital infections. This peptide could therefore be considered for the construction of a multivalent synthetic vaccine.

The specificity of antiglobulin autoantibodies in patients with primary Sjögren's syndrome

Antiglobulin autoantibodies have already been demonstrated in the sera of patients with primary Sjögren's syndrome (primary SS). In our study, the specificity of primary SS antiglobulins for different regions of IgG molecules was examined by employing both direct binding and competitive inhibition enzyme-linked immunosorbent assay. We found that a considerable amount of total antiglobulins in primary SS was specific for the Fab portion, although the remainder was specific for the Fc portion, namely rheumatoid factor (RF). In contrast, most of the antiglobulins in RA were specific for the Fc portion of IgG. These results indicate that in primary SS, antiglobulins directed against epitopes different from those of RF are produced. These antiglobulins may prove to have a different role in primary SS than that ascribed to RF in RA.

High-level production of Escherichia coli STb heat-stable enterotoxin and quantification by a direct enzyme-linked immunosorbent assay

A convenient and sensitive enzyme-linked immunosorbent assay (ELISA) for the STb heat-stable enterotoxin of Escherichia coli was developed and used to quantify STb production by strains with a high level of expression. Based on an antigenic profile of the secreted form of STb, a synthetic peptide (STb3-27) spanning the major predicted epitope was synthesized, coupled to keyhole limpet hemocyanin, and used to immunize rabbits. Anti-STb3-27 antibodies were affinity purified on a synthetic peptide-Sepharose 4B column and used in a direct-binding STb ELISA. Based on a highly purified form of toxin as a standard, the ELISA detected as little as 1 to 2 ng of STb from crude culture filtrates. ELISA data revealed that natural STb-producing strains elaborate little STb in defined-medium cultures relative to that elaborated by a recombinant strain harboring a cloned copy of the estB gene. Replacement of the endogenous STb promoter with any of several highly active promoters, including a bacteriophage T7 promoter, a beta-galactosidase promoter, and a tryptophan-beta-galactosidase hybrid (tac) promoter, increased the yield of STb 10- to 20-fold over levels obtained by an E. coli strain harboring the recombinant estB gene. The high level of STb antigen detected by the ELISA correlated with intestinal secretory activity. The combination of a convenient assay and effective hyperproduction of STb will serve as a basis for a large-scale toxin purification strategy.