Protein array of Coxiella burnetii probed with Q fever sera

Coxiella burnetii is the etiological agent of Q fever.To identify its major seroreactive proteins,a subgenomic protein array was developed.A total of 101 assumed virulence-associated recombinant proteins of C.burnetii were probed with sera from mice experimentally infected with C.burnetii and sera from Q fever patients.Sixteen proteins were recognized as major seroreactive antigens by the mouse sera.Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera.Notably,HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera.These results suggest that these seven major seroreactive proteins,particularly HspB,are potential serodiagnostic and subunit vaccine antigens of Q fever.     

Identification of centromere proteins in different mammalian cells

The characterization of centromeric proteins is facilitated using anti-centromere antibodies present in the sera of patients with the CREST variant of scleroderma. We have employed these sera to determine whether or not those proteins are present in different mammalian species, as well as to study their tissue distribution. Here, we describe the immunofluorescent pattern and the proteins recognized by CREST sera in dividing and resting cells from mouse, rat, swine, hamster, rabbit, and man. In nuclear preparations from cultured cells, thymocytes and spermatozoa from these species, the antigens recognized by CREST sera are proteins of 18 to 20 kDa in all species tested, except in rat. Additionally, two peptides of 80 and 140 kDa were observed in human preparations. In contrast, a 50 kDa peptide is the primary protein detected by the sera in rat nuclei.     

Antibodies to meningococcal antibodies in the sera of patients and donors

The comparative study of sera taken from healthy persons (pooled sera of 100 donors, 6 individual serum specimens) and sera taken from patients with meningococcal meningitis (pooled sera of 10 patients with meningococcal infection, group A, and 6 individual serum specimens from patients with meningococcal infection, groups A, B, C) was carried out by the method of immunoblotting. All proteins from healthy donors were found to contain antibodies to meningococcal iron-regulated protein (IRP) of 85 kD, designated as TbpB. In 30% of donor sera the presence of antibodies to meningococcal IRP of 34 kD (FbpA) was registered. Moreover, donor sera were found to contain antibodies to meningococcal IRP of 45 kD. The sera taken from convalescents were found to have the increased content of antibodies to IRP of 70 and 85 kD and somewhat lesser content of antibodies to proteins of 98, 44 and 34 kD. As regards other (non iron-regulated) proteins, in the process of convalescence the most intensive antibody production was observed with respect to minor protein with a molecular weight of 50 kD, as well as proteins of class 5, characterized by molecular weights of 30 kD and less.     

Characterization of new 18-kDa IgE-binding proteins in beer

The IgE-binding proteins in beer were examined by immunoblotting analysis with sera of patients sensitive to beer. Several proteins were immunoblotted with the sera, and among these, 18-kDa proteins were identified as new IgE-binding proteins in beer. Perhaps they originated from barley as a raw material.     

The serological specificities of Salmonella typhi antigens.

Sera prepared with two different strains of Salmonella typhi were analysed against all the soluble antigens isolated from S. typhi 0901, S. typhi Ty2 and S. typhi Vi. Agar-gel diffusion against individual sera showed that, in all the sera, antibodies were induced against somatic antigens and free proteins. Absorptions of the sera with polysaccharides, split from the somatic antigens, removed the antibodies induced against the polysaccharide and its proteinic carrier in most of the somatic antigens of S. typhi 0901. The antibodies left in the absorbed sera reacted against the proteinic moieties of more complex somatic antigens of S. typhi and against free proteins from all the analysed strains. Only the absorption with proteins removed all the precipitating antibodies from the sera. Moreover, in incomplete absorptions with proteins, the first antibodies removed are the antipolysaccharides, since antibodies are never induced against the haptenic polysaccharide but against somatic conjugates; in these the proteinic moiety eventually varies with every batch of bacteria. The sera exhausted of precipitins still agglutinate the bacteria, thus confirming the assumption that agglutinins and precipitins may be different antibodies.     

Identification of antigenic proteins encoded by human herpesvirus 8 and seroprevalence in the general population and among patients with and without Kaposi's sarcoma

To establish a sensitive and specific antibody assay, potent antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied. Fifteen recombinant HHV8-encoded proteins were produced as glutathione S-transferase fusion proteins. The sera from AIDS-associated Kaposi's sarcoma (KS) patients reacted with four proteins encoded by open reading frames (ORFs) K8.1, 59, 65, and 73 in a Western blot assay. An enzyme-linked immunosorbent assay (ELISA) using these four proteins as antigens (mixed-antigen ELISA) revealed that all 26 sera derived from KS patients (24 with and 2 without human immunodeficiency virus infection) became positive for anti-HHV8 antibodies. The presence of HHV8 was demonstrated in 14 (1. 4%) of 1,004 sera from the Japanese general population and 10 (1.9%) of 527 sera from patients without HHV8-associated diseases. The presence of immunoglobulin G (IgG) and IgM antibodies against HHV8 examined further by the mixed-antigen ELISA and Western blotting revealed IgG antibody in all ELISA-positive sera, while IgM antibody against ORF K8.1 was absent. These data suggest that the ORF 73 and 65 proteins are potent antigens for a sensitive serological assay.     

Identification of the secreted macromolecular immunogens of Staphylococcus aureus by analysis of serum

The ability of sera to recognise secreted macromolecules of Staphylococcus aureus was examined by ELISA and Western immunoblotting. Individual secreted proteins were also studied using both human sera and sera from rabbits immunised with secreted macromolecules. Patients sera showed a wide range of IgG antibody titres to secreted macromolecules and whole bacteria. Controls showed a significantly lower IgG response. Western immunoblotting revealed that a significant number of secreted proteins were recognised by circulating IgG antibodies. Surprisingly, both the sera from controls and from patients recognised similar macromolecules including a number of potential virulence factors. The major difference was in the IgG binding to a 16-kDa component, which was recognised by the majority of the sera from infected individuals, but only by a small number of sera from healthy controls. The higher incidence of antibodies recognising the 16 kDa component may be related to our earlier finding that the major bone resorbing component of S. aureus is a heterodimeric protein containing a 16-kDa subunit, the activity of which could be blocked by sera.     

Autoantibodies as potential biomarkers for nasopharyngeal carcinoma

Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma (NPC). We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor-associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage-displayed proteins, 22 have sequence identity with known or putative tumor-associated proteins. The results of immunochemical reactivity of patients' sera with phage-expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors. The autoantibody against phage-expressed protein MAGE, HSP70, Fibronectin, and CD44 measured by ELISA had greater predictive value than that against EBNA-1, respectively. The antibody levels against MAGE in sera positively correlated with the clinical stages of NPC, and the antibody levels against other three proteins partly correlated with the clinical stages of NPC. Our studies suggested that the autoantibodies against tumor-associated antigens in the sera of NPC patients could be used as a screening test for NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy.     

Protein profiling in human sera for identification of potential lung cancer biomarkers using antibody microarray

To identify potential biomarkers of lung cancer (LC), profiling of proteins in sera obtained from healthy and LC patients was determined using an antibody microarray. Based on our previous study on mRNA expression profiles between patients with LC and healthy persons, 19 proteins of interest were selected as targets for fabrication of an antibody microarray. Antibody to each protein and five nonspecific control antibodies were spotted onto a hydrogel‐coated glass slide and used for profiling of proteins in sera of LC patients in a two‐color fluorescence assay. Forty‐eight human sera samples were analyzed, and expression profiling of proteins were represented by the internally normalized ratio method. Six proteins were distinctly down‐regulated in sera of LC patients; this observation was validated by Wilcoxon test, false discovery rate, and Western blotting. Blind test of other 32 human sera using the antibody microarray followed by hierarchical clustering analysis revealed an approximate sensitivity of 88%, specificity of 80%, and an accuracy of 84%, respectively, in classifying the sera, which supports the potential of the six identified proteins as biomarkers for the prognosis of lung cancer.     

Echinococcus multilocularis: identification of proteins inducing antibody formation in metacestode infection

An approach to the identification of parasite proteins which are immunogenic in natural infections is described, using the infection with the larval stage of Echinococcus multilocularis as a parasite model. Metacestode proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose sheets (Western blotting). Subsequently, immune recognition of the proteins was performed with various host sera and antigen-antibody complexes were detected enzymatically. Using homologous antisera, different patterns of immunogenic bands were revealed by sera of different host species. Cross-reactions with sera from individuals infected with unrelated helminths were analysed. Four proteins of E. multilocularis which failed to show any cross-reaction were identified.     

Salmonella choleraesuis proteins and their relation to proteins from bacteria of heterologous sero-groups.

A diversity of proteins was identified in the material isolated from S. choleraesuis with the help of sera prepared in rabbits with this material. The sera displayed, in agar-gel diffusions, numerous superimposed precipitation lines against proteins from: Salmonellae, Shigellae and E. coli. In contrast to proteins from S. paratyphi C, sharing identical identical 'O' 'factors, the serological activity of the S. choleraesuis proteins was impaired by heating. The immunochemical analysis of the sera before and after exhaustive absorptions with heterologous proteins exhibited a stronger relation of S. choleraesuis with S. thyphimiurium and S. Newport than with S. paratyphi C. The antibodies induced against free proteins with S. paratyphi C specificity, present in the mosaic of proteins isolated from S. choleraesuis, were removed by the respective absorption without substantial modifications of the homologous precipitation. In contrast, the absorption of the serum with proteins from either S. newport or S. typhimurium removed almost all the homologous induced antibodies. The strong relations found among species belonging to different serogroups underline the non-conformity of the empirical established serofactors.     

Molecular identities of human sperm proteins reactive with antibodies in sera of immunoinfertile women

Antisperm antibodies (ASA) can cause infertility in both men and women. It is important to delineate the sperm antigens against which these ASA are directed. Sperm proteins were separated by 2D gel electrophoresis and transferred to nitrocellulose membrane and incubated with sera from fertile women or immunoinfertile women having ASA. The corresponding immunoreactive peptide spots were cored from the gel and analyzed by the two-dimensional (2D) gel electrophoresis/matrix-assisted laser desoprtion ionization-time of flight-mass spectrometry and liquid chromatography-mass spectrometry (MALDI-TOF-MS/LC-MS). A total of 68 spots belonging to 38 different proteins and their isomers were identified. Fourteen of these proteins and their isomers reacted with both the fertile and immunoinfertile sera. Twenty-four of these proteins reacted specifically only with the immunoinfertile sera and not with the fertile sera. Among them was a novel protein designated as a hypothetical protein FLJ32704 (accession # Q96MA6). An immunodominant sequence (amino acid 151-159) of this protein was identified and a nonamer peptide based upon this sequence (IQTLG1TPR) was synthesized and examined for its immunoreactivity. This synthetic peptide reacted with 90% (36/40) of immunoinfertile sera and not with any of the fertile sera (0/40) in the enzyme-linked immnosorbent assay (ELISA). In conclusion, using the 2D gel electrophoresis/MALDI-TOF-MS/LC-MS procedure, we have identified several known and at least one novel antigen against which the antibodies are present in sera of immunoinfertile but not fertile women. Some of these antigens may find applications in specific diagonsis and treatment of infertility/immunoinfertility, and in the development of new generation of contraceptive modalities including contraceptive vaccines.     

Blood serum as a factor of chromatin condensation in trisomy-21 (Down's syndrome)]

The influence of blood sera from patients with Down's syndrome and healthy ones, and different serum fractions on the structure of the deoxyribonucleoprotein systems (DNP-system) has been studied. It was demonstrated that non-fractionated sera of the patients produce a condensation effect on the DNP-system in contradistinction to the sera from healthy people. The analysis of the action of single serum fractions showed that different condensation effect results from the activity of high molecular nondialysable thermosensitive components whose action disappears at gel-filtration of serum proteins. A possibility of the humoral control over chromatin structural organization in vivo is discussed in terms of the evidence on the similarity of serum proteins and chromosomal nonhistone proteins.     

Efficient isolation of cDNA clones encoding rheumatoid arthritis autoantigens by lambda phage surface display

Bacteriophage lambda surface display was used to isolate cDNA clones encoding autoantigens recognized by synovial fluid (SF) or sera from patients with rheumatoid arthritis (RA). We constructed cDNA libraries from human synovial sarcoma cells and synovial tissue, using the surface display vector lambdafoo. The cDNA libraries were screened by affinity selection using 40 SF and 44 sera as probes separately immobilized in microtiter wells. Phage clones isolated encode 13 different autoantigens; an unknown protein, two proteins previously unanalyzed as autoimmune antigens, three proteins previously unknown to be recognized by RA sera, and seven known RA antigens. When analyzed their sensitivity and specificity for RA by phage enzyme-linked immunosorbent assay, frequencies of sera that recognize the newly-isolated autoantigens ranged from 20.5 to 6.8% of a panel of RA sera, and 13.6-0% of other autoimmune disease sera. These results indicate that the lambda phage surface display may be powerful for the isolation of cDNA clones encoding autoantigens recognized by SF or sera from patients with not only RA but also other autoimmune diseases.     

Immunoreactive proteins of Campylobacter concisus, an emergent intestinal pathogen

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Recently, a significantly higher prevalence of C.?concisus DNA and higher levels of antibodies specific to C.?concisus was detected in children with Crohn's disease when compared with controls. The aim of this study was to identify C.?concisus immunoreactive antigens. Proteins from C.?concisus were separated using two-dimensional gel electrophoresis, and sera from 10 C.?concisus-positive children with Crohn's disease were employed for immunoprobing. The patients' sera reacted with 69 spots, which corresponded to 31 proteins identified by mass spectrometry. The proteins were functionally classified as involved in chemotaxis, signal transduction, flagellar motility, surface binding and membrane protein assembly. Although the individual patients' sera reacted to different sets of proteins, common antigens that were recognized by all patients were flagellin B, ATP synthase F1 alpha subunit, and outer membrane protein 18. Cross-reactivity between proteins of the Campylobacter genus was tested using patients' sera absorbed with Campylobacter showae, Campylobacter jejuni and Campylobacter ureolyticus. Most of the C.?concisus immunoreactive proteins identified in this study showed cross-reactivity with other species except for three antigens. In conclusion, this study has identified C.?concisus proteins that are immunoreactive within patients with Crohn's disease.     

Immune response in subacute sclerosing panencephalitis: reduced antibody response to the matrix protein of measles virus.

Immune precipitation was used to study the humoral immune response of patients with subacute sclerosing panencephalitis (SSPE). Patients with SSPE have a progressive infection of the CNS by measles or a measles variant despite high serum antibody levels to measles virus as measured by standard serologic techniques. However, when the antibody response to individual measles virus proteins was measured, we found a striking reduction in the ability of sera from patients with SSPE to precipitate the matrix (M) protein as compared to the precipitation of the M protein by sera from normal adults who had natural measles infection in childhood, or by convalescent sera obtained 3 to 5 weeks after a naturally occurring measles infection. The decreased antibody response to the M protein in sera from patients with SSPE occurred despite a vigorous antibody response to the other viral proteins, suggesting a selective defect in the production of antibody to a single viral protein. The reduced anti-M antibody in sera from patients with SSPE was demonstrated whether immune precipitation was performed with wild-type measles virus or SSPE virus proteins. These results suggest that in SSPE only small amounts of the M protein are produced. This result may help explain how measles virus persists in the central nervous system of patients with SSPE.     

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.     

Identification of a novel potential antigen for early-phase serodiagnosis of leptospirosis

This study examined four genes encoding for predicted membrane proteins selected from the genome sequences of Leptospira interrogans. Genes were cloned and the proteins expressed in E. coli. Immunoblotting analysis of the recombinants with sera from early and convalescent phases of a leptospirosis patient showed that two proteins, namely Lp29 and Lp49, were reactive with serum from both phases of the illness. These data were further confirmed in enzyme-linked immunosorbent assay using sera from both phases of seventeen confirmed leptospirosis specimens, suggesting that these proteins are presented to the host immune system during infection. In the early phase, anti-Lp29 IgM was detected in all sera when microscopic agglutination tests (MAT), the reference method for diagnosis of leptospirosis, were negative. The gene encoding Lp49 is conserved among five tested leptospiral pathogenic serovars, while Lp29 is present in serovars that are predominant in urban settings. These recombinant antigens might be valuable for serodiagnosis of both phases of leptospirosis.     

Autoantigenic properties of some protein subunits of catalytically active complexes of human ribonuclease P.

At least six proteins co-purify with human ribonuclease P (RNase P), a tRNA processing ribonucleoprotein. Two of these proteins, Rpp30 and Rpp38, are Th autoantigens. Recombinant Rpp30 and Rpp38 are also recognized by Th sera from systemic sclerosis patients. Two of the other proteins associated with RNase P, Rpp20 and Rpp40, do not cross-react with Th sera. Polyclonal antibodies raised against all four recombinant proteins recognize the corresponding proteins associated with RNase P and precipitate active holoenzyme. Catalytically active RNase P holoenzyme can be separated from the nucleolar and mitochondrial RNA processing endoribonuclease, RNase MRP, even though these two enzymes may share some subunits.