Use Of Antigen Research Articles

Saline extract of Taenia saginata metacestodes as an alternative antigen for the immunodiagnosis of neurocysticercosis in human cerebrospinal fluid

The aim of the present research was to test the application of Taenia saginata metacestodes as an alternative antigen for use in enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) tests compared with the use of metacestodes antigen of Taenia solium in cerebrospinal fluid (CSF) samples. The samples were obtained from 35 patients with definitive neurocysticercosis (NCC)-group 1-and 44 patients with other neurological disorders (control)-group 2. The sensitivity and specificity of ELISA, using antigen obtained from T. solium, applied to the patients of group 1 yielded results of 100%. When the tests were conducted using T. saginata metacestodes, results were 100% and 93.2%, respectively. The 47-52-, 64-68-, and 70-kDa antigens showed high frequencies in CSF samples from patients with NCC when WB was conducted with both antigens. The results indicate that T. saginata metacestodes can be used as an alternative antigen for the diagnosis of human NCC in CSF samples.

Recombinant Mycobacterium smegmatis mc2155 vaccine expressing outer membrane protein 26 kDa antigen affords therapeutic protection against Helicobacter pylori infection

Orally administered recombinant Mycobacterium smegmatis (rM. smegmatis) vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, in the present study, we evaluated the capacity of the outer membrane protein 26kDa antigen (Omp26) of Helicobacter pylori (H. pylori) to induce therapeutic protection against H. pylori infection in mice. Omp26 was cloned and expressed in M. smegmatis mc2155 as a fusion with the Mycobacterium fortuitum β-lactamase protein under the control of the up-regulated M. fortuitum β-lactamase promoter, pBlaF*. The rM. smegmatis strain was shown to be relatively stable in vitro in terms of plasmid stability and bacterial persistence. We found that oral immunization of H. pylori-infected mice with rM. smegmatis-Omp26 induced protection, i.e., significant reduction in bacterial colonization in the stomach. The protection was strongly related to serum specific antibodies with a Th1 and Th2 profile as well as to local cytokines in the stomach and spleen. These findings suggest that Omp26 is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori. The rM. smegmatis expressing Omp26 antigen could constitute an effective, low-cost combined vaccine against H. pylori.

Peptide mimics selected from immune sera using phage display technology can replace native antigens in the diagnosis of Epstein-Barr virus infection

There is an expanding area of small molecule discovery, especially in the area of peptide mimetics. Peptide sequences can be used to substitute for the entire native antigen for use in diagnostic assays. Our approach is to select peptides that mimic epitopes of the natural immune response to Epstein–Barr virus (EBV) that may be recognised by antibodies typically produced after infection with EBV. We screened a random peptide library on sera from rabbits immunised with a crude preparation of EBV and serum antibodies from a patient with a high titer of EBV antibodies. We selected four peptides (Eb1–4) with the highest relative binding affinity with immune rabbit sera and a single peptide with high affinity to human serum antibodies. The peptides were coupled to the carrier molecule BSA and the recognition of the peptides by IgM antibodies in clinical samples after infection with EBV was measured. The sensitivities were Eb1 94%, Eb2, 3, 4 88%, H1 81% and all had 100% specificity. This study illustrates that the phage display approach to select epitope mimics can be applied to polyclonal antibodies and peptides that represent several diagnostically important epitopes can be selected simultaneously. This panel of EBV peptides representing a wide coverage of immunodominant epitopes could replace crude antigen preparations currently used for capture in commercial diagnostic tests for EBV.

Immunization with Recombinant Murine Cytomegalovirus Expressing Murine Zona Pellucida 3 Causes Permanent Infertility in BALB/c Mice Due to Follicle Depletion and Ovulation Failure1

Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive vaccines because of their crucial role in mammalian fertilization. A single intraperitoneal immunization with recombinant murine cytomegalovirus engineered to express murine ZP3 (rMCMV-mZP3) induces permanent infertility with no evident systemic illness in female BALB/c mice. To investigate the mechanisms underpinning reproductive failure elicited by rMCMV-mZP3, ovarian parameters and reproductive function were evaluated at time points spanning 10 days to 5 wk after virus inoculation. Fertility was substantially impaired by 14 days after inoculation with rMCMV-mZP3 and was fully ablated by 21 days. Pregnancies established after inoculation but before complete infertility showed no adverse effects on fetal viability assessed at Day 17.5 post coitum (pc). Infertile mice retained estrous cycling activity and remained receptive to mating; however, at Day 3.5 pc there were fewer developing embryos and corpora lutea, plasma progesterone content was reduced, and there was no evidence of excess unfertilized oocytes. Consistent with this, profound ovarian pathology was evident from 10 days after rMCMV-mZP3 inoculation, with a decline first in mature ovarian follicles and then in immature ovarian follicles and with diminished expression of genes regulating follicle development, including Nobox, Gdf9, and Gja1 (connexin43). Follicle loss was associated with mild focal oophoritis and with recruitment of inflammatory leukocytes, predominantly CD4(+) and CD8(+) T cells evident from 10 days after virus inoculation. These data indicate that vaccination with rMCMV-mZP3 causes permanent infertility in BALB/c mice principally due to induction of ovarian autoimmune pathology leading to progressive oocyte depletion and eventual ovulation failure.

S-Arylcysteine−Keratin Adducts as Biomarkers of Human Dermal Exposure to Aromatic Hydrocarbons

To measure biomarkers of skin exposure to ubiquitous industrial and environmental aromatic hydrocarbons, we sought to develop an ELISA to quantitate protein adducts of metabolites of benzene and naphthalene in the skin of exposed individuals. We hypothesized that electrophilic arene oxides formed by CYP isoforms expressed in the human skin react with nucleophilic sites on keratin, the most abundant protein in the stratum corneum that is synthesized de novo during keratinocyte maturation and differentiation. The sulfhydryl groups of cysteines in the head region of the keratin proteins 1 (K1) and 10 (K10) are likely targets. The following synthetic S-arylcysteines were incorporated into 10-mer head sequences of K1 [GGGRFSS( S-aryl-C)GG] and K10 [GGGG( S-aryl-C)GGGGG] to form the predicted immunogenic epitopes for antibody production for ELISA: S-phenylcysteine-K1 (SPK1), S-phenylcysteine-K10 (SPK10), S-(1-naphthyl)cysteine-K1 (1NK1), S-(1-naphthyl)cysteine-K10 (1NK10), S-(2-naphthyl)cysteine-K1 (2NK1), and S-(2-naphthyl)cysteine-K10 (2NK10). Analysis by ELISA was chosen based on its high throughput and sensitivity, and low cost. The synthetic modified oligopeptides, available in quantity, served both as immunogens and as chemical standards for quantitative ELISA. Polyclonal rabbit antibodies produced against the naphthyl-modified keratins reacted with their respective antigens with threshold sensitivities of 15-31 ng/mL and high specificity over a linear range up to 500 ng/mL. Anti- S-phenylcysteine antibodies were not sufficiently specific or sensitive toward the target antigens for use in ELISA under our experimental conditions. In dermal tape-strip samples collected from 13 individuals exposed to naphthalene-containing jet fuel, naphthyl-conjugated peptides were detected at levels from 0.343 +/- 0.274 to 2.34 +/- 1.61 pmol adduct/microg keratin but were undetectable in unexposed volunteers. This is the first report of adducts of naphthalene (or of any polycyclic aromatic hydrocarbon) detected in the exposed intact human skin. Quantitation of naphthyl-keratin adducts in the skin of exposed individuals will allow us to investigate the importance of dermal penetration, metabolism, and adduction to keratin and to predict more accurately the contribution of dermal exposure to systemic dose for use in exposure and risk-assessment models.

Characterization and Scale-Up of Cyanogen Bromide Chemical Activation of Sepharose CL-4B in a Stirred Tank Reactor to Purify the rHBsAg

The aim of this paper was to characterize and to scale-up the CNBr chemical activation of Sepharose CL-4B in a stirred tank reactor to purify the recombinant Hepatitis B surface antigen for use in vaccines. The activation quality was assessed measuring the concentration of the cyanate esters and the percentage of nitrogen in the activated matrix. The Sepharose CL-4B incorporated less than 50% of the initial CNBr amount added to the matrix, but the monoclonal antibody CB.Hep-1, used as immunoligand to purify the rHBsAg, was efficiently immobilized in a weakly activated matrix (<8 ?mol/mL of matrix). The impellent type has a significant influence on activated matrix quality. The constant power per volume unit was a proper criterion to scale-up the Sepharose CL-4B March's modified chemical activation procedure with CNBr.

Identification of prostate cancer antigens by automated high-throughput filter immunoscreening

There is a need for earlier and more accurate cancer diagnostics as well as new targets for cancer immunotherapy. To this end, it is important to identify sets of tumour antigens specific for different cancer forms. Several methods that identify potential tumour antigens in an arrayed and high-throughput format have been developed during the last years of SEREX (serological identification of antigens by recombinant expression cloning) related research. Such techniques may hold the potential to describe the complete immunogenic part of the cancer proteome, also called the cancer immunoproteome. We have developed a powerful platform for automated serological high-throughput filter screening of tumour cDNA libraries. The screening format of this method is 18,000 single cDNAs clones, which is superior to other high-throughput methods described. The output is antigens, which are potential diagnostic cancer markers and vaccine targets. We present here the results from the screening of a prostate tumour cDNA library with autologous patient antibodies. We first demonstrated the feasibility of the automated high-throughput filter immunoscreening method by use of the NY-ESO-1sv (NY-ESO-1 splice variant) antigen. We then screened 18,000 cDNA clones from a phage display selected prostate tumour cDNA library with autologous patient antibodies and identified several relevant antigens such as NY-ESO-1, XAGE-1, DJ-1 and transcription factor 25 (TCF25). The present high-throughput immunoscreening method has the potential to identify both patient-specific and disease-specific antigens for use in diagnostics and therapy.

Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge

The bacillus Calmette--Guerin (BCG), a live attenuated Mycobacterium bovis strain is considered a promising candidate as a vector system for delivery of foreign antigens to the immune system. The gene coding for the Leptospira interrogans external membrane protein LipL32, a highly immunogenic antigen found in all pathogenic leptospira, was cloned into several mycobacterial vectors for expression in BCG. Hamsters immunized with recombinant BCG (rBCG) expressing LipL32 were protected against mortality (P<or=0.05) upon challenge with a lethal inoculum of L. interrogans serovar Copenhageni. Autopsy examination did not reveal macroscopic or histological evidence of disease in rBCG immunized hamsters that survived lethal challenge. The data presented here further enhance the status of LipL32 as a promising candidate antigen for use in the control of leptospirosis, when presented to the immune system by an appropriate delivery system.

Induction of Cross-Serovar Protection against Genital Chlamydial Infection by a Targeted Multisubunit Vaccination Approach

An important consideration for antichlamydial vaccine development is the induction of cross-serovar protection, since multiple serovars (D to L) of Chlamydia trachomatis cause genital infections. We have shown previously that vaccination with C. trachomatis-derived recombinant chlamydial protease-like activity factor (rCPAF) induced significant earlier resolution of Chlamydia muridarum infection and reduced oviduct pathology. However, the vaccinated mice continued to shed chlamydiae for up to 2 weeks after challenge. In this study, C. trachomatis serovar D recombinant proteins, such as recombinant major outer membrane protein (rMOMP), recombinant inclusion membrane protein A (rIncA), and rCPAF were administered intranasally, individually or in combinations, with murine interleukin-12 (IL-12) as an adjuvant, and cross-species immunity against intravaginal C. muridarum infection was examined. Immunization with rCPAF plus IL-12 (rCPAF+IL-12), compared to immunization with rIncA+IL-12 or rMOMP+IL-12, induced the greatest antigen-specific gamma interferon production from purified CD4(+) T cells and concurrently enhanced serum antibody production. All (100%) the animals vaccinated with rCPAF+IL-12 alone or in any combination completely resolved the infection by day 18 after challenge compared to animals vaccinated with rIncA+IL-12 (50%), rMOMP+IL-12 (33%), or phosphate-buffered saline (mock vaccinated; 0%). Moreover, oviduct pathology in mice vaccinated by any regimen that included rCPAF, but not rMOMP+IL-12 or rIncA+IL-12 alone, was markedly reduced compared to mock-immunized animals. The addition of rMOMP and/or rIncA did not significantly enhance the rCPAF+IL-12-induced effect on bacterial clearance or oviduct pathology. These results suggest a greater conservation of protective linear antigenic epitopes within CPAF than MOMP or IncA across the examined serovars and the need to identify other highly conserved antigens for use with rCPAF in a multisubunit recombinant vaccine.

Nucleic acid-based analyses of non-group A rotaviruses.

Simple genome profile studies on polyacrylamide gels allow all non-group A rotaviruses isolated so far to be recognized by the absence of the tight triplet (7-9) of RNA segments seen in all group A viruses. However, reliance solely on genome profile studies for rotavirus grouping can be misleading and, for virus group definition, additional corroborating nucleic acid and serological studies are essential. Terminal fingerprint analysis was the first generation of nucleic acid-based assays that allowed discrimination between the various rotavirus groups. By means of this technique the clear definition of five rotavirus groups (A-E), correlating exactly with those found by a serological assay, has been possible, with preliminary evidence for at least two additional groups. The technical sophistication of fingerprinting techniques prevents their widespread use in epidemiological studies; the development of a second generation of nucleic acid-based assays is therefore under way. These employ molecularly cloned cDNA probes to the genomes of non-group A viruses which can be widely distributed for use in 'dot-blot' screening of faecal samples and, if expressed as protein in Escherichia coli, should provide a ready source of viral antigen for use in surveying viral prevalence through the screening of serum antibody levels.

Discovery and analysis of Bartonella henselae antigens for use in clinical serologic assays

The antibody response to Bartonella henselae has been studied in a number of mammals; however, the human response needs to be further studied. After natural infection, humans have antibody reactivity to a large number of B. henselae proteins. We used a proteomic approach to identify antigenic proteins of B. henselae to determine their capacity to elicit a human antibody response. Comparing patient sera by Western blot analysis demonstrated significant amounts of reactivity to B. henselae. The immunofluorescence assay (IFA)-positive sera identified several protein spots of interest. However, a consistent reactivity to a single spot by all sera was not observed. Three of these spots demonstrated reactivity in 71%, 64%, and 64% of positive sera tested with negligible reactivity to the negative sera. These proteins were identified as GroES, BepA, and GroEL. Most IFA-positive sera demonstrated reactivity to GroES, GroEL, and BepA. The usefulness of these proteins for a clinical serologic assay is discussed.

Immunogenicity of porcine circovirus type 2 capsid protein targeting to different subcellular compartments

Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging disease in swine. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS, and a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, recent studies have revealed that interferons (IFNs), especially IFN-γ, can enhance the replication of PCV2, indicating that the high levels of IFN-γ induced by DNA vaccination seem to have potential deleterious effect on protective immunity. Strategies to improve the neutralizing antibody response and simultaneously decrease the IFN-γ response will facilitate the clinical application of DNA vaccines against PCV2. In the present study, four different DNA vaccine constructs encoding cytoplasmic (Cy-ORF2), secreted (Sc-ORF2), membrane-anchored (M-ORF2) or authentic nuclear-targeted (pc-ORF2) Cap protein were generated to evaluate the neutralizing antibody and IFN-γ responses in a mouse model. Although all four DNA constructs could elicit PCV2-specific humoral immune responses, mice inoculated with Sc-ORF2 developed a significantly higher level of neutralizing antibodies than those that received M-ORF2, pc-ORF2 or Cy-ORF2. Furthermore, mice immunized with Sc-ORF2 or M-ORF2 showed a significantly decreased or enhanced IFN-γ level, respectively, compared with those inoculated with pc-ORF2. With respect to neutralizing antibody and IFN-γ levels, Sc-ORF2 is a good candidate for DNA vaccination, and the secreted Cap protein appears to be an ideal antigen for use in development of vaccines against PCV2.

A comparison of concentration methods applied to non-infectious flavivirus recombinant antigens for use in diagnostic serological assays

Since the introduction of West Nile virus into the United States in 1999, there has been a greater awareness of arboviruses, consequently, diagnostic testing for West Nile virus and other arboviruses has increased both in U.S. and international public health laboratories. The Centers for Disease Control and Prevention/Division of Vector-Borne Infectious Diseases/Arbovirus Diagnostic and Reference Laboratory produces and provides the serodiagnostic reagents which are not available commercially. Reagents needed to conduct the enzyme-linked immunoassay (ELISA) include a virus-specific non-infectious antigen. Antigens for Japanese encephalitis and the four dengue virus serotypes have been developed from COS-1 transformed cells that secrete non-infectious, virus-like particles into the cell culture supernatant. Four methods for concentrating the supernatant are discussed here. The methods are ultracentrifugation, polyethylene glycol precipitation, and two ultrafiltration methods: the Stirred Cell (Millipore Corporation, Billerica, MA) and the Pellicon 2 (Millipore Corporation, Billerica, MA). Ultracentrifugation and the Pellicon 2 ultrafiltration system produced antigen at a sufficient concentration for use in the ELISA. Large volumes were concentrated in a shorter time in the Pellicon 2 ultrafiltration system. An additional filtration step was necessary to produce antigen of sufficient concentration for use in the microsphere-based immunoassay, which requires antigen concentrated an additional 10 times.

Minor histocompatibility antigens as targets for immunotherapy using allogeneic immune reactions

Minor histocompatibility antigens (mHag) were originally identified as antigens causing graft rejection or graft-versus-host disease in human leukocyte antigen (HLA)-matched allogeneic transplantation. Molecular identification has revealed most to be major histocompatibility complex (MHC)-bound short peptide fragments encoded by genes which are polymorphic due to single nucleotide polymorphisms (SNP). Genotypic disparity of SNP between transplantation donors and recipients gives rise to mHag as non-self antigens for both the donor and the recipient. Subsequently, mHag have been explored as immunotherapeutic antigens for use against recurring hematological malignancies after allogeneic hematopoietic cell transplantation (HCT), because mHag expressed only on hematopoietic cells are considered to augment graft-versus-leukemia/lymphoma (GVL) effects without increasing the risk of life-threatening graft-versus-host disease (GVHD). Accumulating evidence suggests that T-cell responses to mHag aberrantly expressed on solid tumor cells are also involved in the eradication of sensitive tumors such as renal cell carcinomas following HCT. Over the past decade, the number of putative GVL-directed mHag has increased to a level that covers more than 30% of the Japanese patient population, so that clinical trials may now be executed in the setting of either vaccination or adoptive immunotherapy. As it is expected that immune responses to alloantigens are more powerful than to tumor antigens mostly derived from overexpressed self-proteins, mHag-based immunotherapy may lead to a new treatment modality for high-risk malignancies following allogeneic HCT.

Identification by genomic immunization of a pool of DNA vaccine candidates that confer protective immunity in mice against Neisseria meningitidis serogroup B

We have shown previously that expression library immunization is viable alternative approach to induce protective immunity against Neisseria meningitidis serogroup B. In this study we report that few rounds of library screening allow identification of protective pools of defined antigens. A previously reported protective meningococcal library (L8, with 600 clones) was screened and two sub-libraries of 95 clones each were selected based on the induction of bactericidal and protective antibodies in BALB/c mice. After sequence analysis of each clone within these sub-libraries, we identified a pool of 20 individual antigens that induced protective immune responses in mice against N. meningitidis infection, and the observed protection was associated with the induction of bactericidal antibodies. Our studies demonstrate for the first time that ELI combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in a meningococcal vaccine.

Application of Taenia saginata metacestodes as an alternative antigen for the serological diagnosis of human neurocysticercosis

Serological tests are an important tool for the diagnosis of neurocysticercosis (NCC), the disease caused by Taenia solium metacestodes. The aim of the present research was to test the application of Taenia saginata metacestodes as an alternative antigen for use in the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB) tests compared with the metacestodes antigen of T. solium in serum samples. The samples were obtained from 130 individuals: 20 from patients with definitive NCC, Group 1; 18 from individuals infected by Taenia sp., Group 2; 40 from individuals infected by various parasites, Group 3; and 40 from healthy individuals, Group 4. The sensitivity of IFAT, ELISA, and WB using antigen obtained from T. solium applied to the patients of Group 1 yielded results of 85, 95, and 95%, respectively, for the three tests. When the tests were conducted using T. saginata metacestodes, results were 75, 80, and 85%, respectively. The specificity of IFAT, ELISA, and WB using antigen obtained from T. solium yielded results of 94.9, 88.8, and 93.9%. When the tests were conducted using T. saginata metacestodes, results were 95.9, 88.8, and 93.6%, respectively. No statistical differences for sensitivity or specificity among the antigens were found. In conclusion, the results indicated that T. saginata metacestodes can be used as an alternative antigen for NCC diagnosis.

HAGE, a cancer/testis antigen with potential for melanoma immunotherapy: identification of several MHC class I/II HAGE-derived immunogenic peptides.

There remains a need to identify novel epitopes of potential tumour target antigens for use in immunotherapy of cancer. Here, several melanoma tissues and cell lines but not normal tissues were found to overexpress the cancer-testis antigen HAGE at the mRNA and protein level. We identified a HAGE-derived 15-mer peptide containing a shorter predicted MHC class I-binding sequence within a class II-binding sequence. However, only the longer peptide was found to be both endogenously processed and immunogenic for T cells in transgenic mice in vivo, as well as for human T cells in vitro. A different class I-binding peptide, not contained within a longer class II sequence, was subsequently found to be both immunogenic and endogenously processed in transgenic mice, as was a second class II epitope. These novel HAGE-derived epitopes may contribute to the range of immunotherapeutic targets for use in cancer vaccination programs.

Genetic and Antigenic Diversity of the Surface Protective Antigen Proteins of Erysipelothrix rhusiopathiae

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types ( approximately 60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.

Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines

AbstractThe unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli—based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Estabilidade do antígeno de célula total de Brucella abortus para uso no diagnóstico sorológico da brucelose bovina pela reação de fixação de complemento

A reação de fixação de complemento é um dos testes usados no diagnóstico confirmatório da brucelose bovina, e para sua realização emprega-se o mesmo antígeno usado na prova de soroaglutinação lenta, porém não foi possível encontrar na literatura estudos sobre a estabilidade desse antígeno para uso na prova de fixação de complemento, de modo a estabelecer um prazo de validade para o mesmo. Por isso, esta investigação teve por objetivo avaliar a estabilidade do antígeno de célula total de Brucella conservado sob refrigeração, para uso na reação de fixação de complemento. Analisaram-se 14 partidas de antígeno, preparado com Brucella abortus amostra 1119/3 e padronizado para uso na prova de soroaglutinação lenta, com tempo de fabricação variando de 9 meses a 23 anos e 11 meses. Testaram-se 167 soros bovinos com títulos variáveis de anticorpos contra Brucella, adotando-se a técnica com incubação a 37ºC nas duas fases da reação e 5 unidades hemolíticas 50% de complemento. Considerou-se como positivo o soro com pelo menos 25% de fixação de complemento na diluição 1:4. Compararam-se os resultados obtidos com as 13 partidas de antígeno com aqueles obtidos com a partida com 9 meses de fabricação, usando o teste de chi2 de McNemar e o coeficiente kappa. A grande maioria dos soros apresentou resultados muito próximos quando testados com as diversas partidas de antígeno, e não se observou relação entre tempo de fabricação do antígeno e diferenças nos resultados obtidos.