RESUMO
The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.
Assuntos
Anticorpos Monoclonais/química , Mapeamento de Epitopos , Proteínas do Nucleocapsídeo/imunologia , Síndrome Respiratória Aguda Grave/veterinária , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Especificidade de Anticorpos , Western Blotting/veterinária , Chlorocebus aethiops , Clonagem Molecular , Coronavirus Humano 229E , Proteínas do Nucleocapsídeo de Coronavírus , Reações Cruzadas , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Humanos , Camundongos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Síndrome Respiratória Aguda Grave/diagnóstico , Células VeroRESUMO
A novel severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been discovered. The detection of both antigens and antibodies in SARS-CoV from human specimens with suspected SARS plays an important role in preventing infection. We developed a novel rapid immunochromatographic test (RICT) based on the sandwich format enzyme immunoassay (EIA) with an all-in-one device for detecting the native nucleocapsid antigen (N-Ag) of SARS-CoV using monoclonal antibodies (MoAbs), which we produced by immunizing recombinant N-Ag to mice. RICT is a qualitative assay for respiratory aspirates and serum specimens. With this assay, a positive result can be judged subjectively by the appearance of a blue line on the device 15 min after the sample is applied. RICT with several pairs of MoAbs showed a high sensitivity for the detection of recombinant N-Ag as well as viral N-Ag of SARS-CoV. rSN122 and rSN21-2 were the best MoAbs for immobilized antibody and enzyme labeling, respectively. With regard to analytical sensitivity, RICT detected N-Ag at 31 pg/mL for recombinant N-Ag, and at 1.99 x 10(2) TCID(50)/mL for SARS-CoV. The specificity of RICT was 100% when 150 human sera and 50 nasopharyngeal aspirates (NSPs) were used. RICT based on an EIA using the rSN122/rSN21-2 pair is a sensitive, specific, and reliable rapid assay for detecting N-Ag in SARS-CoV treated with either heat or Triton X-100.
Assuntos
Coronavirus/imunologia , Técnicas Imunoenzimáticas/métodos , Nucleocapsídeo/análise , Nucleocapsídeo/imunologia , Síndrome Respiratória Aguda Grave/virologia , Animais , Anticorpos Monoclonais/imunologia , Chlorocebus aethiops , Cromatografia , DNA Recombinante , Escherichia coli , Técnicas Imunoenzimáticas/instrumentação , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Células VeroRESUMO
3-Deoxyglucosone (3-DG) is a metabolite of glucose that is thought to lead to the production of advanced glycation end products in diabetes. The previous assay for 3-DG in serum was based on a multi-step protocol, including derivatization, extraction, HPLC separation, and detection. In the current studies, we established a monoclonal antibody that recognizes the 3-DG-derivative, which is generated by the reaction of 3-DG and a 2,3-diamino-benzene derivative. Attachment of a biotin moiety to the 2,3-diamino-benzene ring via a linker allowed development of a highly sensitive chemiluminescent enzyme immunoassay for 3-DG equivalents. Unlike the previous assay, this method does not require extraction of 3-DG derivatives from serum. Treatment of 3-DG in serum with the DAB-link-biotin produced a quinoxaline derivative, which was specifically recognized by the monoclonal antibody. Using this assay, we found that serum 3-DG was higher in streptozotocin-induced diabetic rats than in normal control rats (25+/-5.6 vs. 9.8+/-1.1 microg/L). This simple assay may allow the monitoring of conditions leading to the accumulation of advanced glycation end products and evaluation of the risk of complications in diabetic patients.
