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1.
Food Chem ; 400: 134012, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36055143

RESUMO

Exploring a novel strategy for strengthening the catalytic activity of enzyme facilitates the development of a sensitive enzyme-linked immunosorbent assay (ELISA). Herein, a chemical staining (CS) strategy was firstly discovered to possess the ability to directly improve the catalytic activity of horseradish peroxidase. Based on this discovery, coomassie brilliant blue was introduced into ELISA to establish a CS enhanced ELISA (CS-ELISA) to detect clenbuterol (CL) by simply staining monoclonal antibodies. Satisfactorily, the most important analytical parameters of CS-ELISA, including sensitivity (0.074 ng mL-1) and linear range (0.2-2 ng mL-1) were all improving 2-folds compared with conventional ELISA. Moreover, the CS-ELISA shows good applicability in the detection of CL in pork tenderloin samples. The proposed CS-ELISA shows various advantages, such as cost-effective, easily accessible, enhanced catalytic activity of enzyme, higher sensitivity, and broader linear range, providing a new insight into enhanced ELISA for food safety.


Assuntos
Clembuterol , Anticorpos Monoclonais , Clembuterol/análise , Ensaio de Imunoadsorção Enzimática , Peroxidase do Rábano Silvestre , Coloração e Rotulagem
2.
Food Chem ; 400: 134067, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36084594

RESUMO

To determine gentamicin residues in animal tissues, a monoclonal antibody (Mab) was produced and a sensitive indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. At first, gentamicin was conjugated with bovine serum albumin as immunogens which were used to immunize BALB/c mice. Then, an anti-gentamicin Mab was prepared by hybridoma technology. Finally, a sensitive icCLEIA was developed with an 50% inhibition concentration (IC50) of 0.067 ng/mL for gentamicin. The limit of detection of the icCLEIA was 0.002 ng/mL. The cross reactivity of the Mab with structural analogues were<0.01%. The recoveries of gentamicin ranged from 80 to 101% and coefficient of variation was <6.4% in pork and fish samples. Samples were detected by UPLC-MS/MS for evaluating reliability of the icCLEIA. The results suggested that the prepared anti-gentamicin Mab can be used for rapid and convenient immunoassays to detect gentamicin residues in animal tissues.


Assuntos
Anticorpos Monoclonais , Gentamicinas , Animais , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , Técnicas Imunoenzimáticas , Luminescência , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Soroalbumina Bovina , Espectrometria de Massas em Tandem
3.
Food Chem ; 399: 134013, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36037695

RESUMO

Ovalbumin (OVA)-glucose mixture was treated with Co-60 irradiation at 0-25 kGy, and effects of irradiation on the glycation and allergenicity of OVA were investigated. Irradiation induced glycation between OVA and glucose, reflected in the significant increase of glycation sites from 3 to 14. Interestingly, OVA irradiated at 25 kGy had three new glycated peptides (568.782+, 739.382+ and 509.752+). The degree of substitution per peptide molecule (DSP) of glycated peptides exhibited different trends with increasing irradiation dose. Particularly, glycated peptides 17-26, 55-60, 263-267 and 368-375 showed markedly decreased DSP values after irradiation at 20 and 25 kGy, which could be caused by the generation of Maillard reaction products (MRPs). MS/MS spectra suggested that neutral loss occurred in glycated arginine, whose structure was similar to MRPs. The IgG- and IgE-binding abilities of OVA significantly decreased with increasing irradiation dose, indicating that the protein allergenicity was reduced.


Assuntos
Alérgenos , Radioisótopos de Cobalto , Alérgenos/química , Ensaio de Imunoadsorção Enzimática , Glucose , Ovalbumina/química , Peptídeos/química , Espectrometria de Massas em Tandem
4.
Shanghai Kou Qiang Yi Xue ; 31(2): 178-183, 2022 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-36110076

RESUMO

PURPOSE: To detect the level of B cell activating factor (B - cell activating factor of the TNF family, BAFF) in the serum of patientes suffering from systemic lupus erythematosus (SLE) with periodontitis, and analyze the relationship between the expression of BAFF with periodontitis and SLE. METHODS: According to the inclusion criteria, patients visiting the Department of Stomatology and Rheumatology, Shengjing Hospital Affiliated to China Medical University were selected, including 19 patients in the periodontitis group(P group), 22 in the systemic lupus erythematosus group (SLE group), 24 in the systemic lupus erythematosus combined with periodontitis group(SLE+P group), and 20 in the healthy control group(H group). The general information, periodontal probing depth (PD), clinical attachment loss (CAL), gingival sulcus bleeding index(SBI) were collected. Serum samples of patients in each group were collected, and BAFF content was determined by Elisa. Rheumatic and immunological indexes of subjects in SLE group and SLE+P group were determined, and the correlation between BAFF content and periodontal indexes was analyzed. SPSS 20.0 software package was used for statistical analysis. RESULTS: CAL in P+SLE group was significantly higher than that in P group(P<0.05). Serum BAFF concentrations in SLE+P group, SLE group and P group were significantly higher than those in the healthy control group (P<0.05). Serum BAFF concentration in SLE+P group was significantly higher than that in SLE group(P<0.05). ESR, SLEDAI and disease duration in SLE+P group were significantly higher than those in SLE group (P<0.05). The expression level of BAFF in serum was positively correlated with CAL and SBI(P<0.01). The expression level of BAFF in serum was positively correlated with PD(P<0.05). There was significant positive correlation between serum BAFF level and duration of disease and hormone use(P<0.01). Serum BAFF level was positively correlated with C3 (P<0.05). CONCLUSIONS: SLE has certain correlation with periodontitis, and serum BAFF in SLE patients with periodontitis is significantly increased.BAFF may be associated with the development of SLE and periodontitis.


Assuntos
Lúpus Eritematoso Sistêmico , Periodontite , Fator Ativador de Células B , Ensaio de Imunoadsorção Enzimática , Hormônios , Humanos
5.
Vopr Virusol ; 67(4): 290-303, 2022 Sep 11.
Artigo em Russo | MEDLINE | ID: mdl-36097710

RESUMO

INTRODUCTION: Prevention and control of African swine fever (ASF) transmission on the territory of the Russian Federation requires monitoring based on testing of samples from pigs and wild boars. Specific anti-ASFV antibodies are rarely detected in samples during routine serological diagnostics. Although, ASF isolates with weakened virulence were confirmed in Russia and neighboring countries.The aim of this work was to determine the possibility of using alternative samples for ASF diagnosis and evaluate the effectiveness of the diagnostic methods used on the territory of Russia. MATERIALS AND METHODS: Biological materials obtained from experimentally infected animals and samples collected in the "field" conditions were used in this study. RESULTS: Complex testing (RT-PCR and ELISA) is a more effective approach to diagnose chronic and asymptomatic forms of ASF compared to the separate use of these techniques. The possibility and efficiency of using alternative samples in diagnostics are demonstrated. It was confirmed that IPT method overcomes ELISA by high diagnostic sensitivity and detection of antibodies on earlier stages in extended range of samples. Anti-ASFV antibodies were detected in domestic and wild pigs in five regions of Russia. Samples from infected pigs that are negative in RT-PCR can be positive for anti-ASFV antibodies. The detection of antibodies in samples from shot wild boars (negative or uncertain in RT-PCR test) suggests the existence of animals surviving ASF infection. CONCLUSION: The data obtained suggest a revision of the ASF surveillance strategy, by introducing complex diagnostic methods aimed at detection of both the virus genome and anti-ASFV antibodies simultaneously.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Febre Suína Africana/diagnóstico , Febre Suína Africana/epidemiologia , Vírus da Febre Suína Africana/genética , Animais , Ensaio de Imunoadsorção Enzimática , Federação Russa/epidemiologia , Sus scrofa , Suínos
6.
Vopr Virusol ; 67(4): 331-340, 2022 Sep 12.
Artigo em Russo | MEDLINE | ID: mdl-36097714

RESUMO

INTRODUCTION: The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV). MATERIALS AND METHODS: The development of the cELISA was carried out following the OIE recommendations. RESULTS: The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95-13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001. DISCUSSION: Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%). CONCLUSION: The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.


Assuntos
Lyssavirus , Vírus da Raiva , Rhabdoviridae , Animais , Anticorpos Antivirais , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária
7.
Vopr Virusol ; 67(4): 341-450, 2022 Sep 12.
Artigo em Russo | MEDLINE | ID: mdl-36097715

RESUMO

INTRODUCTION: Yellow fever (YF) remains one of the most common natural focal infectious diseases in the world. In connection with the increasing tourist flow to countries endemic for YF, the discovery of stable populations of Aedes aegypti and Ae. albopictus which are the main vectors of the yellow fever virus (YFV), in the southern regions of Russia, and the fact that in medical institutions in our country it is possible to obtain a live attenuated vaccine against YF, but there is no way to evaluate the effectiveness of vaccination, the question arises of the development and implementation of diagnostic kits for detecting antibodies (AB) to the pathogen by enzyme immunoassay (ELISA).The aim of this study was to develop a method for detecting specific IgG antibodies to the E protein of YFV by ELISA and assessing its diagnostic characteristics. MATERIALS AND METHODS: A specific cDNA was synthesized by reverse transcription on an RNA template of YFV isolated on a cell culture of Aedes albopictus clone C6/36, and a fragment of the genome coding the YFV E protein was amplified and subsequently cloned into the plasmid pET160 (Thermo Fisher Scientific, USA). The resulting gene fragment was used as a DNA template to obtain a recombinant analog of the third domain of the YFV E protein in Escherichia coli cells (BL-21(DE3)). Next, the immunogenicity of the obtained antigen was evaluated and the analysis conditions were optimized. RESULTS: The optimal conditions for the production of the obtained recombinant E protein of YFV were determined, its specificity was confirmed by immunological methods (Western blot and ELISA), sorption buffers and blocking solutions were selected, and sensitivity and specificity of detection of antibodies to YFV using the recombinant antigen were assessed. CONCLUSION: A method for the detection of specific IgG antibodies to the YFV E protein by ELISA was developed. This diagnostic kit can be used both to study the protective properties of the YF vaccine and to detect imported cases of infection in non-endemic areas.


Assuntos
Aedes , Flaviviridae , Flavivirus , Febre Amarela , Animais , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G , Mosquitos Vetores , Vacinas Atenuadas , Febre Amarela/diagnóstico , Vírus da Febre Amarela/genética
8.
J Pharm Biomed Anal ; 220: 115020, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36049377

RESUMO

Artemether, an artemisinin derivative, is a component of the commonly used artemisinin-based combination therapy, artemether-lumefantrine. In this study, we cloned the VH and VL genes of a cell line (mAb 2G12E1) producing a monoclonal antibody specific to artemether, and used to construct a recombinant DNA of single-chain variable fragment (scFv). The scFv was constructed into prokaryotic expression vectors pET32a (+), pET22b (+), pGEX-2T, and pMAL-p5x, respectively. However, only the pMAL-p5x/scFv could be induced to express soluble scFv with comparable sensitivity and specificity to that of mAb 2G12E1. Based on the anti-artemether scFv, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The 50% of inhibition concentration (IC50) value and the working range based on IC20 to IC80 were 4.33 ng mL-1 and 1.05-22.65 ng mL-1, respectively. The artemether content in different drugs were determined by the developed icELISA, and the results were consistent to those determined by ultra performance liquid chromatography (UPLC). The anti-artemether scFv prepared in the current study could be a valuable genetically engineered antibody applied for artemether monitoring and specific binding mechanism studying.


Assuntos
Antimaláricos , Artemisininas , Anticorpos de Cadeia Única , Anticorpos Monoclonais , Artemeter , Combinação Arteméter e Lumefantrina , Artemisininas/análise , DNA Recombinante , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
9.
Acta Vet Scand ; 64(1): 22, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064726

RESUMO

BACKGROUND: In human and murine obesity, adipose tissue dwelling macrophages and adipocytes produce monocyte chemoattractant protein-1 (MCP-1) leading to systemic low-grade inflammation. The aim of the study was to validate a canine MCP-1 ELISA assay for use in cats and to investigate whether a difference in MCP-1 concentrations could be detected between: a) cats having normal or elevated circulating serum amyloid A (SAA) levels and b) normal weight and obese cats. Serum obtained from 36 client-owned cats of various breed, age and sex with normal (n = 20) to elevated SAA (n = 16) was used for the validation of the canine MCP-1 ELISA assay. As no golden standard exists for measurement of inflammation, circulating MCP-1 concentrations were compared to SAA measurements, as an indicator of systemic inflammation. Analytical precision, dilution recovery and detection limit were calculated. A possible correlation between MCP-1 concentrations and obesity related measures (body fat percentage (BF%), insulin sensitivity and cytokine expression) were investigated in another population of 73 healthy, lean to obese, neutered domestic short-haired cats. RESULTS: Intra- (2.7-4.1%) and inter-assay (2.2-3.6%) coefficient of variation and dilution recovery were acceptable, and the detection limit was 27.1 pg/mL. MCP-1 did not correlate with SAA, and there was no difference between the inflammatory (SAA > 20 mg/L) and non-inflammatory group, due to a marked overlap in MCP-1 concentrations. Circulating MCP-1 concentrations were unaffected by BF% (r2 = 2.7 × 10-6, P = 0.21) and other obesity-related markers. CONCLUSIONS: The present canine ELISA assay seems to be able to measure circulating feline MCP-1. However, further studies are needed to determine its possible use for detecting inflammation in relation to disease processes or obesity-related low-grade inflammation in cats.


Assuntos
Doenças do Gato , Doenças do Cão , Inflamação , Resistência à Insulina , Doenças dos Roedores , Animais , Gatos , Quimiocina CCL2 , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Humanos , Inflamação/veterinária , Camundongos , Obesidade/veterinária
11.
Turkiye Parazitol Derg ; 46(3): 195-200, 2022 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-36094120

RESUMO

Objective: Cystic echinococcosis (CE) is one of the most common zoonotic diseases worldwide. Diagnosis of CE is predominantly based on imaging techniques and serological tests are used in cases of non-characteristic imaging findings as diagnostic reference. However, serological test results cannot be completely reliable as they are affected by multi-factors. P-selectin and resistin are inflammatory markers that are altered during the acute stages of infection. In this purpose, inflammatory markers as P-selectin and resistin have been investigated for a potential diagnostic reference for CE diagnosis. Methods: A total of 60 patients who were diagnosed with CE and twenty-five healthy individuals were included in this study. Blood samples were obtained from all participants. Obtained sera were evaluated using the P-selectin and resistin ELISA kits for protein levels. Additionally, the relative expression of SELP (P-selectin) and RETN (resistin) genes were determined using the comparative CT (ΔΔCT) method between groups as CE patients with active and inactive cysts, CE patients and healthy controls. Results: SELP (13.9-fold change, p<0.05) and RETN (8.1-fold change, p<0.05) were differentially expressed in CE patients compared in the control group. Whereas resistin protein levels were significantly higher in CE patients than the healthy controls (p<0.001), the difference in P-selectin protein levels was not significant (p>0.05). There was no difference between active and inactive CE patients in terms of P-selectin and resistin in gene and protein levels (p>0.05). Conclusion: Although there was no difference between the active and inactive CE patients, the good differentiation between the healthy controls and the CE patients suggested that resistin is a potential inflammatory diagnostic reference.


Assuntos
Equinococose , Resistina , Equinococose/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Selectina-P , Resistina/genética , Resistina/metabolismo
12.
BMC Vet Res ; 18(1): 335, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36068568

RESUMO

BACKGROUND: Toxoplasma is an obligate intracellular protozoan that causes an important zoonotic disease with a worldwide distribution. Felids are the definitive hosts of this parasite, while virtually all warm-blooded animals, including birds, serve as intermediate hosts. Four ring-tailed lemurs (Lemur catta) in the Taipei Zoo died of acute Toxoplasma infection in June 2019. Since then, Toxoplasma has occasionally been identified in this Zoo during necropsy of dead animals and PCR of animal blood samples. Therefore, a general survey of Toxoplasma infection in animals in the Zoo seems to be needed. METHODS AND RESULTS: An indirect multispecies ELISA was used for the first time to screen for Toxoplasma infection in 326 serum samples collected from 75 species of animals. The infection rate of Toxoplasma was 27% (88/326). A commercial latex agglutination (LAT) assay was used to re-examine the samples with doubtful and uncertain ELISA results (151 samples from 42 species). The infection rate increased to 36.2% (118/326), and the indirect multispecies ELISA appeared to be applicable to 31 of 75 species animals included in this study. Nested PCR assays targeting the dense granule protein 7 (GRA7) gene and B1 gene were also used to detect Toxoplasma in DNA samples extracted from 10 liver or blood specimens from 8 animals. GRA7 gene fragments were amplified from 8 samples from 7 animals, while B1 gene fragments were amplified from only 4 samples from 4 animals. From the B1 nested PCR and the sequence data of GRA7 fragments amplified from infectious specimens, the animals in the Zoo were speculated to have been infected by at least three different Toxoplasma variants. CONCLUSIONS: According to the serological investigation, we speculated that over one-third (36.2%) of animals in Taipei Zoo presented the infection of Toxoplasma, and the indirect multispecies ELISA we used can be applied to detect Toxoplasma infection in 31 animal species included in this study. Sequence analysis revealed that at least three Toxoplasma variants were infecting the animals of Taipei Zoo.


Assuntos
Felidae , Toxoplasma , Toxoplasmose Animal , Animais , Animais de Zoológico , Anticorpos Antiprotozoários , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Reação em Cadeia da Polimerase/veterinária , Proteínas de Protozoários/genética , Sensibilidade e Especificidade , Toxoplasma/genética , Toxoplasmose Animal/diagnóstico , Toxoplasmose Animal/epidemiologia
13.
J Dairy Sci ; 105(10): 8354-8363, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36055833

RESUMO

Johne's disease and bovine tuberculosis are diseases of economic, public health, and animal welfare importance. The single intradermal cervical comparative tuberculin (SICCT) test, which is used to determine bovine tuberculosis status as part of eradication schemes in the United Kingdom and some other countries, has been reported to interfere with the results of the widely used ELISA to detect antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) in milk. Better understanding of the relationship between SICCT and MAP tests can improve management and control of Johne's disease. The aim of this study was to characterize the relationship between SICCT testing and milk ELISA performance and to assess whether the immunological response to the SICCT test is different for MAP-infected cows and noninfected cows. We used repeated MAP milk ELISA test results of a cohort of 805,561 cows in the United Kingdom between 2010 and 2018 that had milk ELISA tests within 90 d of SICCT testing to identify cows likely to be infected. We then assessed, separately, for cows deemed to be MAP-infected and noninfected, the association between MAP test results and proximity to SICCT testing by means of survival analysis and generalized additive mixed models. The results were used to quantify the effect SICCT testing may have on performance of milk ELISA tests conducted soon after SICCT testing. At high prevalence levels (20%) of MAP in the infected herd, overall accuracy of the milk ELISA is not reduced when testing occurs within 14 d from SICCT testing. Milk ELISA values of cows deemed to be infected were highest when MAP testing was closer in time to SICCT testing, suggesting the SICCT test enhances antibody response for MAP in infected cows. This corresponds to higher sensitivity of the MAP milk ELISA when testing within 30 d of the SICCT test. For cows deemed to be noninfected, the effect of previous SICCT testing was delayed compared with infected cows, with MAP milk ELISA values peaking at around 15 d post-SICCT testing. For both, MAP-infected and noninfected cows, interference from SICCT test diminished 30 d after SICCT testing, suggesting post 30 d to be the most appropriate time for evaluating the milk ELISA for MAP after SICCT testing. Our results provide strong evidence that the effect of the SICCT test on serological response against MAP is different for MAP-infected versus noninfected cows and that, as a result of this distinct effect, it is possible to improve interpretation of MAP milk ELISA test results (higher accuracy) by taking into consideration time since SICCT testing.


Assuntos
Doenças dos Bovinos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Tuberculose Bovina , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Humanos , Leite/microbiologia , Paratuberculose/microbiologia , Tuberculina
14.
J Virol Methods ; 309: 114611, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36058340

RESUMO

African swine fever (ASF) is a highly fatal viral disease affecting pigs. It is caused by the ASF virus (ASFV), and causes serious economic losses to the swine industry worldwide, including in Korea. Commercially available enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-ASFV antibodies are used for the diagnosis and surveillance of ASF. In this study, an ELISA was developed to detect anti-ASFV antibodies using two recombinant proteins, p22 and p30, from genotype II ASFV. Recombinant transmembrane domain-deleted p22 (p22∆TM) and p30 were expressed in E.coli vector system pET32a and mixed for use as antigens in indirect ELISA. The p22∆TM/p30-based indirect ELISA was validated using 31 sera from genotype I ASFV-infected pigs and 1133 sera from uninfected pigs. Area under the curve of this test was 0.999 [95 % concentration interval 0.992-1.000], and sensitivity and specificity were 93.5 % and 99.8 %, respectively. The between run coefficient of variation for internal quality control serum was 6.61 %. In the seroconversion analysis, the p22∆TM/p30-based indirect ELISA showed equal or better ability to detect antibodies in pigs experimentally challenged with ASFV p72 genotypes I and II (p < 0.05). In conclusion, the p22∆TM/p30-based indirect ELISA is a reliable diagnostic method for detecting anti-ASFV antibodies.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Vírus da Febre Suína Africana/genética , Animais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Proteínas Recombinantes/genética , Suínos , Proteínas Virais/genética
15.
Methods Cell Biol ; 172: 163-178, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36064222

RESUMO

Cancer cell-intrinsic type I interferon (IFN-I) activation is required to initiate early innate immune responses and the subsequent radiation-induced anti-tumor immunity. Investigating the secretion of IFN-I cytokines in response to radiation therapy (RT) is therefore a critical readout for selecting the best immunogenic radiation dose-fractionation regimen. In this chapter, we present different ELISA-based quantification techniques that can be utilized to assess the secretion of tumor-derived IFN-I cytokines, namely IFN-α and IFN-ß.


Assuntos
Interferon Tipo I , Neoplasias , Citocinas , Ensaio de Imunoadsorção Enzimática , Imunidade Inata , Interferon Tipo I/farmacologia , Interferon-alfa , Neoplasias/radioterapia
16.
Vet Parasitol ; 310: 109789, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36063580

RESUMO

The aim of this study was to assess the suitability of PCR and ELISA as diagnostic method in young sheep naturally infested by Oestrus ovis larvae. The experiment was carried out from December 2020 to April 2021 with 39 lambs divided into two groups: infested (n = 26) and control treated group (n = 13). The infested group did not receive treatment against oestrosis, and the control group was treated with closantel (10 mg/kg orally) every 28 days in order to keep the animals as free as possible of O. ovis infestation. The clinical signs varied among animals regardless of the number of recovered larvae of each lamb, however, the thick mucus and mucopurulent nasal discharge scores were less frequent in lambs from treated group. There was no correlation between the nasal discharge score and the number of O. ovis recovered larvae (R² = 0.012, P = 0.165). Three control treated animals only presented first instar larvae (L1) (1 - 4 larvae/animal) which were smaller than L1 found in the lambs of the infested group. Ninety-two percent of the lambs from infested group (24/26) were parasitized by O. ovis with number ranging from 1 to 54 larvae per animal. A gradual increase in plasma IgG (anti-antigen of O. ovis larvae) levels of animals from infested group after the third week of the trial was observed, whereas the control lambs had low levels of IgG until the end of the experiment. The PCR had low sensitivity (26 %) and high specificity (100 %), and it presented poor agreement (k = 0.177) with the larvae detection after the lamb slaughter. The oestrosis clinical signs were not related to larvae infestation intensity and ELISA showed a greater advantage over the PCR technique in identifying animals that are carrying O. ovis.


Assuntos
Dípteros , Ectoparasitoses , Miíase , Doenças dos Ovinos , Animais , Ectoparasitoses/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Imunoglobulina G , Larva , Miíase/diagnóstico , Miíase/tratamento farmacológico , Miíase/veterinária , Reação em Cadeia da Polimerase/veterinária , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/tratamento farmacológico
17.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 38(9): 842-847, 2022 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-36082715

RESUMO

Objective Mice were immunized with purified virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) to prepare polyclonal antibody and identify specificity. Methods BALB/c mice were injected with duck tembusu virus to generate viperin in mouse brain by intracranial injection. Viperin gene, cloned from mouse brain tissue by reverse transcription PCR, was inserted into pGEX-6p-1 prokaryotic expression vector and transformed into E. coli Rosetta. The recombinant viperin protein was induced by isopropyl thiogalactoside (IPTG) and its solubility was analyzed. The protein was purified by potassium chloride (KCl) staining and gel cutting method. Polyclonal antibody was prepared by immunizing mice with purified recombinant viperin protein subcutaneously through abdomen, and the titer of polyclonal antibody was determined by indirect ELISA. Western blot analysis and indirect fluorescence assay (IFA) were used to detect the transient expression of viperin protein in BHK-21 cells to identify the specificity and sensitivity of the prepared polyclonal antibody against viperin protein. Results The mouse viperin gene was successfully cloned and the viperin protein was expressed. The titer of the prepared anti-viperin polyclonal antibody reached 1:25 600. The mouse anti-viperin polyclonal antibody could specifically recognize the transient expression of viperin protein in BHK-21 cells. Conclusion Mouse polyclonal antibody against viperin protein with high specificity and sensitivity was successfully prepared.


Assuntos
Escherichia coli , Proteína Viperina , Animais , Anticorpos , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Interferons , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética
18.
Int J Mol Sci ; 23(17)2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-36076966

RESUMO

Background. Monitoring of biological TNF inhibitors is a very important tool to guide clinical decisions using specialized algorithms, especially in gastroenterology. A new chemiluminescent instrument (i-TRACK10® from Theradiag) could replace ELISA techniques to calculate the dosage of drugs and anti-drug antibodies. In this bi-centric study, we explored the analytical performances of i-TRACK10® using manual or automated (DS2®) ELISA Lisa-Tracker® assays, and compared the results. Patients and methods. Intra- and inter-run performances were evaluated with i-TRACK10® in two different laboratories and for two different ranges of values for infliximab, adalimumab, and their respective antibodies. Patients' samples were used in the labs to compare the results obtained between the new instrument and either the manual Lisa-Tracker® or the automated DS2. Results. Intra- and inter-run performances were satisfactory, with values between 1.8% and 16.1% (for inter-run imprecision at low/medium values of infliximab). Results were generally comparable between assays. with the lowest value of correlation at 0.59 (anti-adalimumab dosage between i-TRACK10® and manual ELISA). Most often, values of drugs and anti-drug antibodies were higher with i-TRACK10® than with manual ELISA assay, and correlation values were better with automated ELISA. Agreements were globally acceptable, and the lowest coefficients of 0.7 was obtained for adalimumab values between i-TRACK10® and the two ELISA methods, and for anti-adalimumab values between i-TRACK10® and manual ELISA. The type of assay can potentially induce a change in the class of patients and lead to divergent therapeutic decisions. Conclusions. The new random-access instrument i-TRACK10® presents many advantages in a routine laboratory: rapidity, the possibility of standardization, usability, and expansion of the measurement range. Despite the relatively good agreement of results, it is preferable to use the same assay in longitudinal follow-up of a patient, because quantitative results were not completely equivalent especially for anti-drug antibodies.


Assuntos
Monitoramento de Medicamentos , Luminescência , Adalimumab/uso terapêutico , Anticorpos , Monitoramento de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Infliximab/uso terapêutico , Fator de Necrose Tumoral alfa
19.
Mem Inst Oswaldo Cruz ; 117: e220012, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36074421

RESUMO

BACKGROUND: Zika virus (ZIKV) was discovered in 1947 with the virus isolation from Rhesus monkey (Macaca mulatta) in Uganda forest, Africa. Old World Primates are involved in a sylvatic cycle of maintenance of this arbovirus, however a limited knowledge about the role of New World primates in ZIKV transmission cycles has been established. OBJECTIVE: This work aimed to investigate the presence of enzootic circulation of ZIKV in New World Primates from three Brazilian states: São Paulo, Paraíba, and Paraná. METHODS: We analyzed 100 non-human primate samples collected in 2018 and 2020 from free-ranging and captive environments from São Paulo (six municipalities belonging to Sorocaba region), Paraíba (João Pessoa municipality), and Paraná (Foz do Iguaçu municipality) using reverse transcriptase quantitative polymerase reaction (RT-qPCR) assays, indirect enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization test (PRNT). FINDINGS: All samples (n = 141) tested negative for the presence of ZIKV genome from tissue and blood samples. In addition, all sera (n = 58) from Foz do Iguaçu' non-human primates (NHPs) were negative in serological assays. MAIN CONCLUSION: No evidence of ZIKV circulation (molecular and serological) was found in neotropical primates. In addition, the absence of antibodies against ZIKV suggests the absence of previous viral exposure of NHPs from Foz do Iguaçu-PR.


Assuntos
Infecção por Zika virus , Zika virus , Animais , Anticorpos Antivirais , Brasil , Ensaio de Imunoadsorção Enzimática , Primatas , Zika virus/genética
20.
Nat Commun ; 13(1): 5359, 2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36097164

RESUMO

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.


Assuntos
Anticorpos , Proteínas , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , Sensibilidade e Especificidade
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