Antigenic Bands Research Articles

Evaluation of human IgG class and subclass antibodies to a 24 kDa antigenic component of Gnathostoma spinigerum for the serodiagnosis of gnathostomiasis

The immunoglobulin G class (total IgG) and subclass (IgG1, IgG2, IgG3, and IgG4) antibody responses to the spirurid worm Gnathostoma spinigerum were analyzed by immunoblotting technique for the antibodies' potential use in the serodiagnosis of human gnathostomiasis. Serum samples from patients with proven gnathostomiasis and from clinically suspected cases of gnathostomiasis with migratory swelling were tested. Sera from patients with other parasitic illnesses and from healthy volunteers were also analyzed. The total IgG antibody to an antigenic band of approximately 24 kDa from a somatic extract of G. spinigerum advanced third-stage larvae (GS24) gave the highest sensitivity (91.6%) and 87.8% specificity. Individual IgG subclass detection had a lower sensitivity than the detection of specific total IgG antibody, but IgG4 had a slightly higher specificity (93.9%). However, for cost effectiveness, we suggest that anti GS24 total IgG is sufficient for the routine serodiagnosis of human gnathostomiasis as well as for seroepidemiological studies in developing countries.

Characterization of the Western blotting IgG reactivity patterns in the clinical phases of acquired syphilis

We standardized the Western blotting (WB) method for detecting Treponema pallidum IgG (Tp-IgG) antibodies in sera samples of patients with syphilis and correlated the reactivity profile of bands with the clinical phases of the disease. The WB Tp-IgG has 100% sensitivity and 99.5% specificity. The clinical phases of the disease were associated with the reactive bands from TpN15 to TpN47. Quantitative Venereal Disease Research Laboratories was used to assist the WB Tp-IgG analysis. In primary syphilis, the reaction intensity for the antigenic band TpN47 was usually more intense when compared with other clinical phase. In secondary and sometimes in early latent syphilis, antibodies reacted with high numbers of antigenic proteins of T. pallidum. In late latent syphilis, various bands became negative, but the TpN15 and TpN47 were reactive. In tertiary syphilis, we observed reactivity with the TpN15 band and low reactivity with the TpN47. We concluded that WB Tp-IgG could be used to confirm serologic tests and characterize clinical phases of syphilis.

Immobilization antibodies of tiger puffer Takifugu rubripes induced by i.p. injection against monogenean Heterobothrium okamotoi oncomiracidia do not prevent the infection

We examined whether infection by the monogenean Heterobothrium okamotoi induces production of specific antibodies against oncomiracidia and their cilia, larvae on the gills, and adults on the branchial cavity wall of tiger puffer Takifugu rubripes. We also investigated whether specific antibody production participates in acquired protection against H. okamotoi. Sera from persistently infected fish immobilized H. okamotoi oncomiracidia 89 days after exposure and antibody levels (measured by enzyme-linked immunosorbent assays) in the sera against oncomiracidia and their cilia increased compared with sera from control (naïve) fish. Antibody levels in these sera against the larvae and adult stages did not increase. The number of H. okamotoi on persistently infected fish was significantly lower than for control fish (P<0.05) when persistently infected fish and control fish were exposed to oncomiracidia in the same tank. Thus tiger puffer produced specific antibodies against oncomiracidia and their cilia, and acquired partial protection against H. okamotoi. Intraperitoneal injection of proteins of sonicated oncomiracidia or their cilia with an adjuvant also produced oncomiracidium agglutination antibodies in sera from tiger puffer; the antibody levels in these sera against oncomiracidia and their cilia increased compared with sera from control fish (injection of BSA with an adjuvant) at 14, 44, and 75 days after the booster immunization. However, in a parasite challenge at 54-58 days after the booster immunization, the infection levels of fish immunized with parasites of sonicated oncomiracidia or their cilia were the same as the control fish. Western blot showed that sera from persistently infected fish and fish immunized with sonicated oncomiracidia or their cilia recognized similar antigenic bands, suggesting that tiger puffer tends to react against these antigens compared with other antigens. These results indicated that specific antibodies against these cilia and oncomiracidia induced by i.p. injection do not prevent H. okamotoi infection.

Antibodies to α B-Crystallin, Vimentin, and Heat Shock Protein 70 in Aqueous Humor of Patients with Normal Tension Glaucoma and IgG Antibody Patterns Against Retinal Antigen in Aqueous Humor

Purpose: To show the existence of IgG antibodies against retinal antigens in aqueous humor of normal tension glaucoma patients. Methods: Forty-two patients were included in this study. Aqueous humor was collected from control subjects (CO; n = 21) and patients with normal tension glaucoma (NTG; n = 21). Western blot methods against bovine retinal antigens were used to detect the IgG antibody patterns. The complex antibody repertoires were analyzed by multivariate statistical techniques. Mass spectrometry was used to identify the most important antigens. Results: Very complex IgG antibody patterns against retinal antigens were found in all analyzed aqueous humor samples. Our multivariate approach could quantify differences in immunoreactivities, and including all peaks, the analysis of discriminance revealed a statistical significant difference between the patterns of the NTG and the CO group (p < 0.001). The antigen band at 21 kDa was identified as α B-crystallin, the 57-kDa antigen band as vimentin, and one at 70 kDa as heat shock protein 70. Conclusions: We could demonstrate that complex IgG antibody patterns against retina exist in aqueous humor. The significant differences in the antibody pattern of the glaucoma group compared with the nonglaucoma group in aqueous humor confirm the results of previous studies using sera of glaucoma patients. These differences in antibody patterns might be further evidence for an autoimmune involvement in the pathogenesis of some glaucoma patients.

Caracterización de un anticuerpo monoclonal dirigido contra Trypanosoma evansi y su aplicación en la detección de antígenos circulantes

Monoclonal antibodies were obtained against Trypanosoma evansi. The 2-4F6 IgM monoclonal antibody (Mab) was chosen for the study because of its ability to detect antigens and its specificity (as it did not recognise T. cruzi, T. equiperdum, Babesia equi or B. caballi). The immunoblot test revealed that the 2-4F6 IgM Mab recognises epitopes in two antigenic bands, one measuring 85 kDa and the other 122 kDa. An immunoassay for antigen detection in serum using polyclonal antibodies for capture, the Mab 2-4F6 as primary antibody and an antimouse IgM as secondary antibody gave positive results in 10 of the 11 equidae infected with T. evansi, whereas 20 controls gave negative results. These research results show that the Mab 2-4F6 and the antigen it recognises are useful in identifying equidae infected with T. evansi.

Analysis of IgG antibody patterns against retinal antigens and antibodies to α-crystallin, GFAP, and α-enolase in sera of patients with “wet” age-related macular degeneration

The aim of this study was to compare the IgG antibody patterns against retinal antigens in sera of patients with age-related macular degeneration (AMD) and healthy subjects to learn more about possible immunological aspects of this disease and to identify some of the most important antigens. Sera of 140 patients were analyzed: healthy volunteers (CO, n=101) and patients with "wet" age-related macular degeneration (AMD, n=39). The sera were tested against western blots of bovine retinal antigens. The IgG antibody patterns were analyzed by multivariate statistical techniques and some antigens were identified via LC-MS/MS. All patients showed complex patterns of IgG antibodies against retinal antigens. The discriminant analysis revealed a statistical significant difference between the antibody profiles of the AMD and the CO group (P=0.000023). Not only up-regulations of antigen-antibody-reactivities in the AMD group at some molecular weight ranges, e.g. at 46 and 52 kDa, could be seen, but also down-regulations, e.g. at 18 and 36 kDa. The 18 kDa antigen band was identified as alphaB-crystallin, the band at 46 kDa as alpha-enolase, and one at 52 kDa as glial fibrillary acidic protein. We could demonstrate that both groups (wet AMD and CO) show complex IgG antibody patterns against retinal antigens, which are highly specific for each group. This provides further hints for the immunological basis of the disease. These changes in the antibody profiles in "wet" AMD could represent a secondary response to retinal damage or can play a causative role in the disease.

Biochemical and genetic characterization of wheat ( Triticum spp.) kernel polyphenol oxidases

Genetic variability of polyphenol oxidase (PPO) activity was evaluated in hexaploid, tetraploid, and aneuploid wheat ( Triticum spp.). A DNA probe to kernel-specific PPO hybridized to PPO clones from an immature wheat kernel cDNA library but not to clones from a seedling cDNA library. Recombinant protein and antibody were developed from the kernel-specific PPO. PPO transcript and antigenic proteins were most abundant during kernel development and decreased rapidly at physiological maturity. PPO proteins were more abundant in immature kernels, but PPO activity was greater in mature kernels. Wheat kernel antigenic protein bands of ∼58, 60, 62, and 75 kD were identified; immunoprecipitated proteins of ∼60 and ∼62 kD were confirmed to be PPOs by LC-MS/MS. The 2D chromosome was implicated as a major locus of PPO activity in cultivar (cv.) ‘Chinese Spring’ based on antigenic staining intensity of ∼60–62 kD proteins and kernel PPO activity in aneuploid lines. The ∼58 kD protein was not well-correlated with kernel PPO activity, while the ∼75 kD protein was likely a PPO precursor. Tropolone completely inhibited PPO activity extracted from mature kernels but not from immature kernels. Total PPO activity in mature kernels is thus a function of specific PPO isoforms present, their abundance, and activation levels.

Diversified serum IgG response involving non-myelin CNS proteins during experimental autoimmune encephalomyelitis

We sequentially analyzed the serum IgG response against normal mouse brain during experimental autoimmune encephalomyelitis in SJL/J mice injected with CFA, Bordetella pertussis toxin (BPT) and proteolipid protein 139–151 peptide, compared with mice that received CFA and BPT or were uninjected. Dynamic changes were observed from day 0 to day 28 in the 3 groups. Six highly discriminant antigenic bands ( κ = 0.974) were identified. Three non-myelin proteins were characterized (mitochondrial aconitase hydratase 2, phosphoglycerate mutase 1, brain specific pyruvate deshydrogenase). The IgG response against two of them was less frequent in EAE whereas it was associated with multiple sclerosis in our previous work.

Western Blot Immunoassay as a Confirmatory Test for the Presence of Anti-Mycoplasma Hyopneumoniae Antibodies in Swine Serum

A Western blot immunoassay (WBI) was developed as a confirmatory test for 2 commercial Mycoplasma hyopneumoniae (Mhyo) ELISAs. The WBI detected at least 5 antigen bands (150, 130, 74, 70, and 30 kDa) from Mhyo whole membrane proteins that were not present in the antigens prepared from M. hyorhinis and M. hyosynoviae. Among discrepant sera from vaccinated pigs (n = 17) and field samples (n = 91) assayed by WBI: 1) 2 of the ELISA-positive samples reacted with all 5 antigens bands; 2) all blocking ELISA-positive samples (n = 53) bound 150-, 130-, 74-, and 70-kD antigen bands; and 3) all indirect ELISA-positive samples (n = 55) bound 150-, 130-, and 30-kD antigens. We conclude that the WBI targeting the top 4 antigen bands is a useful confirmatory test for samples initially screened using the commercial Mhyo ELISAs.

Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384–605 or aa 384–746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23 kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.

Kinetics and Diagnostic and Prognostic Potential of Quantitative Western Blot Analysis and Antigen-Specific Enzyme-Linked Immunosorbent Assay in Experimental Canine Leishmaniasis

Quantitative computerized Western blot analysis of antibody responses during experimental canine Leishmania infantum infection distinguished between immunodominant and nonimmunodominant protein bands. Six infected beagles, positive by both PCR and parasite culture, were monitored over 75 weeks postinfection and during a 12-week allopurinol treatment course. All dogs were symptomatic at the time of treatment. Of 12 antigenic bands examined, the immunodominant bands (12, 14, 24, 29, 48, and 68 kDa) showed significantly increased intensities (P<0.01) and higher frequencies of recognition than the nonimmunodominant bands at all time points. Detection of the former bands at 6 weeks postinfection preceded seroconversion by enzyme-linked immunosorbent assay (ELISA) both on crude Leishmania antigen or the recombinant proteins rK39 and HSP70. Reactivity with the 14-, 48-, and 68-kDa bands signified early infection, whereas increased reactivity with the 14-, 24-, and 29-kDa bands was associated with posttreatment parasite persistence and potential unfavorable prognosis. Total lane intensity (TLI) emerged as a sensitive marker for early infection and increased as early as 4 weeks postinfection. TLI had a significantly higher (P<0.01) relative increase rate than crude Leishmania antigen or HSP70 or rK39 ELISA at all time points. These immunodominant antigens and TLI, as determined by quantitative Western blotting, will be valuable for early detection and treatment evaluation of canine leishmaniasis.

Potential use of Trichinella spiralis antigen for serodiagnosis of human capillariasis philippinensis by immunoblot analysis

Intestinal capillariasis is an emerging helminthic zoonosis caused by Capillaria philippinensis and is frequently fatal if not diagnosed correctly. The present study demonstrates cross-reactivity between Trichinella spiralis larval antigens and C. philippinensis-infected human sera by immunoblotting. Sera from 16 proven intestinal capillariasis patients and 16 proven trichinosis patients were tested. The antigenic patterns recognized by intestinal capillariasis sera varied with the molecular masses, ranging from less than 20.1 to more than 94 kDa. The immunoblotting profiles of the trichinosis sera were similar to those of the intestinal capillariasis sera. The antigenic bands with 100% reactivity were located at 36.5, 40.5, and 54 kDa, respectively. Sera from patients with trichuriasis, strongyloidiasis, opisthorchiasis, and healthy controls differed clearly from the previous two and produced very faint patterns of reactivity and attenuated bands. This assay is potentially useful for large-scale screenings of persons at risk for C. philippinensis infection. Parasitological stool examinations of the positive cases are necessary as second-tier laboratory tests for confirming the diagnosis.

Diagnostic values of IgG4 in human gnathostomiasis

The diagnostic values of immunoglobulin G subclass antibodies from patients with gnathostomiasis were assessed by immunoblot technique. Antigen was prepared from crude extracts of Gnathostoma spinigerum advanced third-stage larvae obtained from naturally infected eels. The sera were obtained from 14 parasite-confirmed gnathostomiasis cases, 63 patients with other helminthic infections and 13 healthy controls. Nine prominent IgG4 reactive bands appeared with molecular weights of 94, 51, 47, 43, 38, 24, 21, 20 and 15 kDa. The diagnostic sensitivity of each of the nine reactive bands ranged from 100% to 64.3% in 14 parasite-confirmed gnathostomiasis cases. All (100%) confirmed cases recognized the 21 kDa antigenic band, but not other helminthic infections or parasite-free control. Recognition of 21 kDa antigen in G. spinigerum advanced third-stage larvae crude extracts is the most specific diagnostic marker for human gnathostomiasis, with 100% sensitivity and specificity. The 20 and 24 kDa protein bands were additional diagnostic bands for confirming diagnosis of infection where the 21 kDa band was faint. No specific binding of IgG1, IgG2, or IgG3 antibodies was observed in any sera from confirmed gnathostomiasis cases.

Immunoblot analysis of specific antigen bands predictable for Dirofilaria immitis infection in cats

Serial sera from four mongrel cats experimentally inoculated with infectious larvae of Dirofilaria immitis were analyzed by immunoblot patterns against a phosphate buffered saline-extract of D. immitis. Antigen-specific protein bands detected indicate that the low molecular weight bands of 36, 32, 22, 19 and 14 kDa, are predictable for positive adult worm infection, suggesting diagnostic usefulness for adult D. immitis infection in cats.

STRONGYLOIDES STERCORALIS RECOMBINANT NIE ANTIGEN SHARES EPITOPE WITH RECOMBINANT Ves v 5 AND Pol a 5 ALLERGENS OF INSECTS

A new recombinant protein (NIE) for immunodiagnosis of human Strongyloides infection has 13% to 18% amino acid identity with antigen 5 insect venom allergen, but the C-terminal segment of NIE showed highest identity with Ves v 5 (yellow jacket) and Pol a 5 (paper wasp). A rabbit polyclonal anti-NIE antibody identified a single band of NIE antigen as well as bands of Pol a 5 and Ves v 5 antigens, and mouse anti-Pol a 5 and anti-Ves v 5 sera reacted with recombinant NIE antigen by Western blot. A cyanogen bromide-digested C-terminal fragment of NIE was reactive with mouse anti-Ves v 5 and Pol a 5 antibodies as well as with rabbit anti-NIE serum. Although IgE and IgG antibodies from pooled sera from Strongyloides-infected patients reacted with Pol a 5 and Ves v 5 recombinant antigens on immunoblots, neither antigen inhibited human IgG reaction with NIE antigen in a competitive enzyme-linked immunosorbent assay.

Immunogenic cross-reaction among outer membrane proteins of Gram-negative bacteria

In the present study, antigenic cross-reactivity of OMPs was investigated in several species of bacterial pathogens. Heterogeneous mouse or fish antisera were used to ascertain OMPs with cross-reactivity and cluster analysis was performed to analyze the distribution of cross-antigenic OMPs in diverse bacterial strains. We interestingly found that eleven and seven bands could be reacted with four kinds of heterogeneous mouse and fish antisera, respectively, and the phenograms constructed could provide ideal targeted bacteria for candidate genes of polyvalent vaccines. Importantly, there were significant differences in reaction with bacteria between mouse and fish antisera, but commonly antigenic bands still existed between them. Our results suggest that the cross-reactivity of OMPs exists commonly in Gram-negative bacteria, which may be a promising choice for the development of polyvalent OMP vaccines. Meanwhile, cluster analysis will help to understand the relation of cross-antigenic OMPs among Gram-negative bacteria.

Combined use of Western blot/ELISA to improve the serological diagnosis of human tuberculosis

Two recombinant antigens and a crude bacterial antigen of a wild M. tuberculosis strain were used to detect specific IgG antibodies in sera from 52 patients with pulmonary tuberculosis, confirmed by an acid-fast smear and serum culture of these patients and that of 25 contacts. The patients were not infected with HIV. We evaluated the sensitivity and specificity of ELISA, based on the recombinant TbF6 and TbF6/DPEP antigen and a search for reactivity patterns in the Western blot technique, using whole mycobacterium antigen. Serum samples from 22 healthy individuals and from 30 patients with lung diseases other than tuberculosis were used as controls. The best ELISA results were obtained with the TbF6/DPEP antigen combination, which gave 85% sensitivity and 91% specificity. ELISA sensitivity improved from 85% to 92% when the Western blot results were used. Western blot specificity was 100% when antibody reactivity with different antigenic bands was analyzed and associated. The association of TbF6/DPEP antigens used in ELISA with specific patterns of reactivity determined by Western blot can help make an identification when classic methods for the diagnosis of pulmonary tuberculosis are not sufficient.

Exploration of immunoblot profiles of Neospora caninum probed with different bovine immunoglobulin classes

The present study was attempted to compare the Neospora caninum (N. caninum) antigenic bands recognized by different bovine immunoglobulin classes. A total 10, 5, 2, and 6 antigenic bands were exhibited on immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. A 46 kDa band was probed as a common antigenic band except IgA; 69 kDa band was bovine IgM and IgE; 33, 37, 55, and 79 kDa bands were bovine IgM and IgG; 72 kDa band was found IgM and IgA profiles. Based on the analysis, it appeared that different immunoglobulin classes recognizing different antigenic molecules were cooperating to cope with neosporosis.

Characterizaation of a Mycoplasma agalactiae Strain, Candidate to an Attenuated Vaccine

Assunção, P., Fe, C. de la, Ramírez, A.S., Sarradell, J.E. and Poveda, J.B. 2004. Characterization of a Mycoplasma agalactiae strain, candidate to an attenuated vaccine. J. Appl. Anim. Res., 26: 1–5. The aim of this work was to study the stability of a M. agalactiae attenuated strain after lyophilization and its characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Immunoblot against homologous antisera showed that the attenuated M. agalactiae strain had 2 characteristic antigenic bands of 32 and 21 kDa. The assays related to strain lyophilization showed that the CFUs were decreased lesser than 2log10 after 1 year storage at 4C with the exception of one vial, which lost all its viability. The subcutaneous inoculation dose (105 CFU), did not produce any symptom or sign of disease during the 45 days of the experiment. However, a local reaction was observed on the day after the inoculation. At necropsy, no macroscopic lesions were found which related to contagious agalactia or other disease. These results suggest that this attenuated strain has good possibilities as a live attenuated vaccine.

New antigenic candidates in multiple sclerosis: identification by serological proteome analysis.

Myelin antigen targets that are clearly associated with pathogenic events in multiple sclerosis (M.S.) patients remain to be defined. We recently demonstrated that the analysis of global IgG antibody response against human brain antigens using one-dimensional (1-D) immunoblotting, allowed us to discriminate M.S. patients from controls (both healthy subjects and patients with Sjögren's syndrome). Additionally, this approach also differentiated the three clinical forms of M.S. Indeed, 42 brain antigenic bands (26 from healthy brain and 16 from the M.S. brain) showed the discriminant IgG immune responses. The aim of our study was to characterize the 26 discriminant antigenic bands detected in healthy brain. Protein identification was successively performed by 1-D and two-dimensional (2-D) immunoblottings using sera from 18 M.S. patients, followed by mass spectrometry (MS) analysis and a database search. One hundred and two antigenic spots were then detected on 2-D immunoblots, with M.S. sera against healthy brains. Sixty-four spots were successfully matched with 2-D Coomassie brillant blue stained gels, which were further selected for MS analysis and annotated leading to the identification of 14 of the 26 discriminant antigens. Thus, serological proteome analysis may provide a useful tool for the identification of potentially new M.S.-associated antigens, whose relevance to physiopathological events remains to be defined.