Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N' protein and (N)Delta(121) protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N' protein-based ELISA showed a highly nonspecific reaction. The (N)Delta(121) protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N' protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV (N)Delta(121) protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the (N)Delta(121) protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.     

人冠状病毒229E、OC43与SARS-CoV血清学相关性的研究

目的 检测正常人和SARS患者血清中3种人冠状病毒（229E、OCA3和SARS-CoV）特异性抗体．分析3种冠状病毒血清学相关性。方法 采用免疫印迹、免疫荧光和ELISA方法检测100例健康献血员、34例SARS患者恢复期以及11例SARS患者双份血清中229E、OCA3和SARS-CoV3种冠状病毒核衣壳（N）蛋白抗体。结果 用免疫荧光方法检测100例健康献血员血清中229E、OCA3和SARS-CoV IgG阳性率分别为98％、100％和1％，34例SARS患者恢复期血清中3种冠状病毒IgG的阳性率均为100％；免疫印迹检测100例健康献血员血清中229E、OCA3和SARS-CoVN蛋白IgG阳性率分别9r7％、99％和2％，34例SARS患者恢复期血清中229E、OCA3和SARS-CoVN蛋白IgG阳性率分别97％、100％和100％；11例SARS患者的急性期和恢复期双份血清中，免疫荧光检测有5例出现229E IgG滴度4倍或以上升高，10例出现OC43 IgG滴度4倍或以上升高，ELISA检测2例出现229EN蛋白IgG滴度4倍以上升高，没有一例出现OCA3N蛋白抗体滴度升高。结论 正常人群中普遍存在229E和OCA3两种人冠状病毒抗体，SARS-CoV感染者存在对人冠状病毒229E和OCA3血清学交叉反应，提示核衣壳蛋白不是引起血清学交叉反应的主要抗原，结果对研究SARS溯源有重要意义。     

False-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid enzyme-linked immunosorbent assay due to HCoV-OC43 and HCoV-229E rectified by Western blotting with recombinant SARS-CoV spike polypeptide

Using paired serum samples obtained from patients with illness associated with increases in anti-human coronavirus OC43 (HCoV-OC43) or anti-HCoV-229E antibodies, we examined the possibility of false-positive results detected in a recombinant severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein immunoglobulin G enzyme-linked immunosorbent assay (ELISA). Three of the 21 and 1 of the 7 convalescent-phase serum samples from persons with increases in antibodies against HCoV-OC43 and HCoV-229E, respectively, tested positive by the recombinant SARS-CoV nucleocapsid protein-based ELISA. None of these samples were found to contain a specific antibody in the recombinant SARS-CoV spike polypeptide-based Western blot assay.     

Evaluation of Inapparent Nosocomial Severe Acute Respiratory Syndrome Coronavirus Infection in Vietnam by Use of Highly Specific Recombinant Truncated Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N′ protein and NΔ₁₂₁ protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N′ protein-based ELISA showed a highly nonspecific reaction. The NΔ₁₂₁ protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N′ protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV NΔ₁₂₁ protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the NΔ₁₂₁ protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.     

Comparison of immunoglobulin G responses to the spike and nucleocapsid proteins of severe acute respiratory syndrome (SARS) coronavirus in patients with SARS

Both the nucleocapsid (N) and the spike (S) proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) are able to induce strong humoral responses in humans following an infection. To compare the immunoglobulin G (IgG) responses to the S and N proteins of SARS-CoV in SARS patients during the manifestation/convalescent period with those during the postinfection period, serum samples were collected from hospitalized SARS patients within 6 weeks after the onset of illness (set 1; 57 sequential samples from 19 patients) or 2 to 3 months after their recovery (set 2; 33 postinfection samples from 33 subjects). Serum samples from 100 healthy blood donors (set 3), collected in 2002, were also included. The specific IgG response to whole virus, the fragment from positions 450 to 650 of the S protein (S450-650), and the full-length N protein of SARS-CoV were measured by enzyme-linked immunosorbent assays (ELISAs). Western blot assays were carried out to confirm the ELISA results. Fifty-one of the serum samples in set 1 (89%) bound to the N protein, a proportion similar to that which recognized whole virus (79%) and the S-protein fragment (77%). All 33 serum samples from set 2 were strongly positive for N-protein-specific IgG, while 27 (82%) were positive for anti-S450-650 IgG. Two of the serum samples from set 3 were strongly positive for anti-N-protein IgG but not anti-S450-650 IgG. Similar levels of IgG responses to the S and N proteins were observed in SARS patients during the manifestation and convalescent stages. In the postinfection period, however, a number of patients had much lower serum IgG levels against S450-650 than against the N protein.     

Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome

A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.     

Cloning, sequencing, expression, and purification of SARS-associated coronavirus nucleocapsid protein for serodiagnosis of SARS

A novel coronavirus has been associated with a worldwide outbreak of atypical pneumonia referred to as Severe Acute Respiratory Syndrome (SARS-CoV). SARS-CoV nucleocapsid (N) protein has been cloned sequenced and expressed in Escherichia coli strain. Purified N protein was used to measure the SARS-CoV specific IgG antibodies from 16 SARS-CoV infected patients' sera and from 131 control subjects using ELISA assay. Specific antibody responses to the purified recombinant N protein after 10, 20, and 30 days of disease onset were observed in 13 of 16 (81.3%), 16 of 16 (100%) and 16 of 16 (100%) SARS patients sera, respectively. Comparison of detection results with a commercially available diagnostic kit coated with a mixture of SARS-CoV viral proteins showed 9 of 16 (56.3%), 13 of 16 (81.3%), and 15 of 16 (93.7%) positive responses, respectively. None of 131 control sera gave positive reaction in either assay. This data suggests that the N protein of SARS-CoV is immunodominant and this ELISA based test assay for detecting the SARS-CoV N antigen may hold a significant value for SARS diagnosis.     

Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.     

Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): identification of neutralizing and antibodies reactive to S, N, M and E viral proteins

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.     

Monoclonal Antibody-Based Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Sensitivity of the Nucleocapsid Protein in Acute-Phase Sera of Severe Acute Respiratory Syndrome Patients

Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum samples from healthy donors and non-SARS febrile patients. The results of the N protein detection analysis were directly related to the serological analysis data. From 27 SARS patients who tested positive with the neutralization test, 100% of the 24 sera collected from 1 to 10 days after the onset of symptoms were positive for the N protein. N protein was not detected beyond day 11 in this group. The positive rates of N protein for sera collected at 1 to 5, 6 to 10, 11 to 15, and 16 to 20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients were 92.9, 69.8, 36.4, and 21.1%, respectively. For 294 sera from 248 serological test-negative patients, the rates were 25.6, 16.7, 9.3, and 0%, respectively. The N protein was not detected in 66 patients with cases of what was initially suspected to be SARS but serologically proven to be negative for SARS and in 197 serum samples from healthy donors and non-SARS febrile patients. The specificity of the assay was 100%. Furthermore, of 16 sera collected from four patients during the SARS recurrence in Guangzhou, 5 sera collected from 7 to 9 days after the onset of symptoms were positive for the N protein. N protein detection exhibited a high positive rate, 96 to 100%, between day 3 and day 5 after the onset of symptoms for 27 neutralization test-positive SARS patients and 298 serologically confirmed patients. The N protein detection rate continually decreased beginning with day 10, and N protein was not detected beyond day 19 after the onset of symptoms. In conclusion, an antigen capture ELISA reveals a high N protein detection rate in acute-phase sera of patients with SARS, which makes it useful for early diagnosis of SARS.     

Monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay reveals high sensitivity of the nucleocapsid protein in acute-phase sera of severe acute respiratory syndrome patients

Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum samples from healthy donors and non-SARS febrile patients. The results of the N protein detection analysis were directly related to the serological analysis data. From 27 SARS patients who tested positive with the neutralization test, 100% of the 24 sera collected from 1 to 10 days after the onset of symptoms were positive for the N protein. N protein was not detected beyond day 11 in this group. The positive rates of N protein for sera collected at 1 to 5, 6 to 10, 11 to 15, and 16 to 20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients were 92.9, 69.8, 36.4, and 21.1%, respectively. For 294 sera from 248 serological test-negative patients, the rates were 25.6, 16.7, 9.3, and 0%, respectively. The N protein was not detected in 66 patients with cases of what was initially suspected to be SARS but serologically proven to be negative for SARS and in 197 serum samples from healthy donors and non-SARS febrile patients. The specificity of the assay was 100%. Furthermore, of 16 sera collected from four patients during the SARS recurrence in Guangzhou, 5 sera collected from 7 to 9 days after the onset of symptoms were positive for the N protein. N protein detection exhibited a high positive rate, 96 to 100%, between day 3 and day 5 after the onset of symptoms for 27 neutralization test-positive SARS patients and 298 serologically confirmed patients. The N protein detection rate continually decreased beginning with day 10, and N protein was not detected beyond day 19 after the onset of symptoms. In conclusion, an antigen capture ELISA reveals a high N protein detection rate in acute-phase sera of patients with SARS, which makes it useful for early diagnosis of SARS.     

SARS-CoV核衣壳蛋白抗原性鉴定和基因免疫研究

目的　了解SARS冠状病毒 (SARS CoV)核衣壳蛋白 (N蛋白 )的抗原性及其基因疫苗的免疫原性。方法　用大肠杆菌表达SARS CoV的N蛋白 ,用SARS患者恢复期血清对其进行抗原性鉴定。再构建N蛋白基因疫苗 ,肌肉注射接种小鼠 ,检测小鼠血清中的抗N蛋白IgG抗体、脾细胞增殖和迟发型超敏反应。结果　大肠杆菌重组表达的SARS CoVN蛋白具有强抗原性 ,其基因疫苗能在小鼠有效诱导N蛋白特异性抗体和CD4 、CD8 T淋巴细胞免疫应答。结论　SARS CoV的N蛋白可作为重要的SARS血清学诊断抗原 ,并对于SARS的特异性预防和治疗有潜在的应用价值。     

Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.     

SARS冠状病毒N蛋白单克隆抗体的制备及鉴定

目的 制备SARS冠状病毒(SARS-CoV)N蛋白特异性单克隆抗体(McAb)，为SARS的快速诊断及致病机制的研究提供实验材料。方法 用纯化的重组SARS-CoVN蛋白免疫BALB/c小鼠，经细胞融合和亚克隆后获得分泌针对N蛋白的杂交瘤细胞株，用Western blot和间接免疫荧光法检测这些细胞株分泌的单克隆抗体特异性，并将N蛋白分3段表达初步定位单克隆抗体识别表位所在区域。结果 通过细胞融合和3轮克隆化，筛选出分泌抗N蛋白的6个杂交瘤细胞株。Western blot及免疫荧光显示，获得的McAb可与SARS-CoVN蛋白及SARS-CoV发生特异性反应，有4个细胞株分泌的抗体的识别位点位于N蛋白N端，2个位于C端。结论 获得了SARS-CoV特异性单克隆抗体并进行了初步定位，可用于SARS的早期诊断及致病机制研究。     

SARS冠状病毒N蛋白基因的表达及其在SARS诊断中的应用

目的 获得重组SARS冠状病毒（SARS-CoV）N蛋白抗原，建立特异性诊断SAILS病毒感染的免疫学方法。方法 大肠埃希菌中表达SARS病毒N蛋白基因，用金属螯合层析纯化N蛋白，建立检测SARS抗体的EUSA方法。结果 大肠埃希菌中表达了SARS病毒全长N蛋白抗原，经包涵体洗涤和金属螯和纯化后得到纯度较高的重组蛋白。用重组抗原EUSA检测30名SARS患者抗体全部为阳性，30名正常人血清为阴性，30名发热非SARS患者血清为阴性。结论 SARS表达核蛋白可以在大肠埃希菌中得到高效表达，纯化的重组N蛋白具有良好抗原性，可用于检测SARS抗体。     

Persistent shedding of viable SARS-CoV in urine and stool of SARS patients during the convalescent phase

In order to further the present knowledge of the emerging severe acute respiratory syndrome-associated coronavirus (SARS-CoV), 486 different specimens from 54 patients with a clinical diagnosis of SARS were investigated for the presence of viral RNA, and 314 plasma specimens of 73 patients were examined for IgM and IgG antibodies specific against SARS-CoV using an indirect ELISA. Viral RNA was detectable in 28 of the 54 patients tested. Cumulative data showed that 67 of the 73 SARS patients demonstrated seroconversion by week 5 of illness. In contrast, only 1 of 278 healthy subjects enrolled in the study was found to be positive for the IgG antibody. Coexistence of viral RNA in plasma and specific antibodies was simultaneously observed over three consecutive weeks in two critical cases. In three convalescent patients in particular, cultivable SARS-CoV was detected in stool or urine specimens for longer than 4 weeks (29–36 days). These findings suggest that SARS-CoV may remain viable in the excretions of convalescent patients.     

Longitudinal profile of immunoglobulin G (IgG), IgM, and IgA antibodies against the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in patients with pneumonia due to the SARS coronavirus

By using a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and serum specimens serially collected (from day 0 to day 240 after symptom onset) from patients with pneumonia due to SARS-CoV, we analyzed the longitudinal profiles of immunoglobulin G (IgG), IgM, and IgA antibodies against the SARS-CoV nucleocapsid protein in patients with pneumonia due to SARS-CoV. For IgG, the median optical density at 450 nm (OD450) turned positive at day 17 and a biphasic response was observed. At day 240, all patients were still positive for anti-nucleocapsid protein IgG antibody. For IgM, the median OD450 turned positive at day 20.5, peaked at about day 80, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgM antibody. For IgA, the median OD450 turned positive at day 17, peaked at about day 50, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgA antibody. The time of seroconversion detected by the recombinant SARS-CoV nucleocapsid protein-based ELISA and that detected by indirect immunofluorescence assay were similar. The median times of seroconversion for IgG, IgM, and IgA detected by the indirect immunofluorescence assay were 17 days (17 days by ELISA), 16.5 days (20.5 days by ELISA), and 17.5 days (17 days by ELISA), respectively, after disease onset. One, four, and one of the six patients who died did not produce any IgG, IgM, and IgA antibodies against the nucleocapsid protein of SARS-CoV, respectively, although these antibodies were detected in all six patients by the indirect immunofluorescence assay. Further studies should be performed to see whether SARS-CoV nucleocapsid protein antibody positivity has any prognostic significance.     

Characterization of monoclonal antibody against SARS coronavirus nucleocapsid antigen and development of an antigen capture ELISA

This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417-422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5 pg of purified N protein and 50 TCID(50) of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.     

Longitudinal Profile of Immunoglobulin G (IgG), IgM, and IgA Antibodies against the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in Patients with Pneumonia Due to the SARS Coronavirus

By using a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and serum specimens serially collected (from day 0 to day 240 after symptom onset) from patients with pneumonia due to SARS-CoV, we analyzed the longitudinal profiles of immunoglobulin G (IgG), IgM, and IgA antibodies against the SARS-CoV nucleocapsid protein in patients with pneumonia due to SARS-CoV. For IgG, the median optical density at 450 nm (OD₄₅₀) turned positive at day 17 and a biphasic response was observed. At day 240, all patients were still positive for anti-nucleocapsid protein IgG antibody. For IgM, the median OD₄₅₀ turned positive at day 20.5, peaked at about day 80, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgM antibody. For IgA, the median OD₄₅₀ turned positive at day 17, peaked at about day 50, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgA antibody. The time of seroconversion detected by the recombinant SARS-CoV nucleocapsid protein-based ELISA and that detected by indirect immunofluorescence assay were similar. The median times of seroconversion for IgG, IgM, and IgA detected by the indirect immunofluorescence assay were 17 days (17 days by ELISA), 16.5 days (20.5 days by ELISA), and 17.5 days (17 days by ELISA), respectively, after disease onset. One, four, and one of the six patients who died did not produce any IgG, IgM, and IgA antibodies against the nucleocapsid protein of SARS-CoV, respectively, although these antibodies were detected in all six patients by the indirect immunofluorescence assay. Further studies should be performed to see whether SARS-CoV nucleocapsid protein antibody positivity has any prognostic significance.     

False positive antibody results against human T-cell lymphotropic virus in patients with severe acute respiratory syndrome

Taiwan suffered from the outbreak of severe acute respiratory syndrome (SARS) in 2003. Our laboratory performed a series of virology and serology tests for SARS patients admitted to our hospital. Cross-reactivity was found when testing for antibody against human T-cell lymphotropic virus (HTLV) in one patient with SARS. Therefore, antibodies against HTLV were examined in paired-sera from 26 SARS patients. ELISA and a neutralization test were used to measure anti-SARS antibodies. Seroconversion for antibody against SARS-CoV was observed in all patients. Surprisingly, with the use of ELISA for HTLV, sera for 13 patients were positive for HTLV (50%), and seroconversion for HTLV was also observed in 10 patients (38.5%). Western blot for HTLV on those 26 paired-sera from 13 HTLV-positive patients displayed 5 positive results for HTLV-I, 7 positive results for HTLV-II, 1 positive result for both HTLV-I and II, 9 negative results for either HTLV-I or HTLV-II, and 4 "indeterminate" results. The findings that antibody to HTLV can be detected in blood samples collected from SARS patients provide important information for safe handling of blood products. Without such knowledge, blood products can be discarded mistakenly even though they contain anti-SARS-CoV antibodies that may be potentially valuable for SARS therapy.