Detection of antibodies to Sendai virus in mouse sera by enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) for diagnosis of Sendai virus infection in mice was evaluated. A large-scale survey of infected mice showed that ELISA is approximately 100 times more sensitive than the hemagglutination-inhibition or complement-fixation test. Although a few SPF mice showed false-positive reactions at a serum dilution of 1:40, further dilution to 1:80 eliminated the non-specific reaction. It was shown that ELISA is a highly satisfactory method for examination of Sendai virus infection in mice.     

Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana)

An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.     

Comparison of an enzyme-linked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to K-papovavirus in mice

The sensitivity of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of antibody to K virus was compared with the sensitivities of an immunofluorescence assay (IFA) and a hemagglutination inhibition assay (HIA). Specific pathogen-free BALB/c RIVM mice, 5 weeks old, were inoculated intraperitoneally with a mouse organ suspension containing 10(4.5) TCID50 of K virus per dose. Control animals were inoculated with a control mouse organ suspension. No clinical signs were observed during the 7 weeks they were followed for the development of serum antibody. The ELISA proved to be the most sensitive of the three assays and demonstrated K virus-specific antibodies as early as 3 days after infection.     

Application of dried whole blood collected on filter paper disks to ELISA for the detection of Sendai virus and mouse hepatitis virus antibodies in mice.

This study was undertaken to simplify the preparation procedures for test specimens by applying whole blood collected on filter paper disks. The results of ELISA obtained using specimens collected in this way for the detection of Sendai virus and mouse hepatitis virus antibodies in mice were comparable to those for ordinary ELISA using serum samples.     

仙台病毒黑龙江省地方株的分离与鉴定

目的自本省普通级实验动物中分离并鉴定出仙台病毒地方毒株,为建立仙台病毒血清抗体检测方法奠定基础。方法通过鸡胚尿囊腔传代自普通级小鼠肺脏分离病毒,经血凝实验、血凝阻断实验和结构基因序列测定对分离得到的病毒进行鉴定;大量繁殖病毒并通过蔗糖密度梯度离心纯化,免疫动物制备阳性血清,用标准试剂盒检测阳性血清效价。结果自150份小鼠肺脏分离到2株有血凝性的病毒,经形态学、血清学和结构基因序列测定鉴定为仙台病毒,命名为SV-HLJ。SV-HLJ与标准毒株Fushimi核蛋白基因(N)的核苷酸、氨基酸同源性分别为99·6%和99·0%。结论分离并鉴定出了仙台病毒黑龙江省地方毒株,为检测试剂盒的研制奠定了基础。     

Evaluation of mouse thymic virus antibody detection techniques

Three serologic test methods for detection of serum antibodies to mouse thymic virus (MTV) were compared, including enzyme-linked immunosorbent assay (ELISA), complement fixation (CF) test and indirect fluorescent antibody (IFA) test. Serum was collected at regular intervals from CD-1 mice inoculated intraperitoneally with approximately 200 infectious doses of MTV, and from uninoculated mice placed in cages with the inoculated animals. The inoculated mice became MTV antibody-positive by all three assay methods at 15 days post inoculation, while the cage-contacts were seropositive by all methods at 30 days after contact. Although the incidence of positive results was similar by all methods, titers measured by ELISA were substantially higher than those measured by CF and IFA tests. Because MTV can cause persistent infections that adversely affect the suitability of mice for research, it is recommended that testing for antibodies to this virus be performed routinely.     

An immunofluorescence test for detection of serum antibody to rodent coronaviruses

An indirect immunofluorescence test, using cultured cells infected with two strains of mouse hepatitis virus, was developed for detection of antibody to rodent coronaviruses. The immunofluorescence test detected serum antibody to mouse hepatitis virus and rat sialodacryoadenitis virus earlier than the neutralization test. In the case of mouse hepatitis virus, the results of the immunofluorescence test closely paralleled those obtained with a commercially available enzyme-linked immunosorbent assay.     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

Detection of Sendai virus antibody in mouse and guinea pig sera by an enzyme-linked immunosorbent assay with protein A

An enzyme-linked immunosorbent assay using horseradish peroxidase (HRPO)-labeled protein A (P-ELISA) was established for detection of Sendai virus (SV) antibody in mouse and guinea pig sera. Sensitivity and specificity of P-ELISA were compared with those of ordinary ELISA using HRPO-labeled immunoglobulin G (IgG-ELISA) and the hemagglutination inhibition (HI) test. P-ELISA was 100 to 1,000 times more sensitive than the HI test for detection of the antibody in SV-naturally infected mice. P-ELISA and IgG-ELISA showed similar sensitivities for detection of the antibody in naturally infected mouse and guinea pig sera. A high specificity was demonstrated in P-ELISA with a cut-off optical density value of 0.2 (492 nm), while a non-specific reaction was observed when IgG-ELISA was used to both mouse and guinea pig sera at a low dilution (1:10-20). The antibody in rat sera was not detected by P-ELISA although it was realized by IgG-ELISA.     

Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.     

Protection of mice from wild-type Sendai virus infection by a trypsin-resistant mutant, TR-2.

A trypsin-resistant mutant of Sendai virus, TR-2, which could be activated by chymotrypsin but not by trypsin or the protease present in mouse lung, was inoculated intranasally into mice after being activated in vitro. TR-2 hardly brought about clinical illness or lung lesions in mice; the protease present in the lung could not activate the progeny virus, and the infection terminated after one-step replication. Nevertheless, the immunoglobulin A antibody against wild-type Sendai virus was produced in the respiratory tracts as well as the serum immunoglobulin G antibody, and the mice were protected from the challenge of the wild-type Sendai virus. On the basis of these results, TR-2 may provide a new model of live vaccine for paramyxoviruses; its availability as a live vaccine is also discussed.     

Immunofluorescence for detection of viral antigen in mice infected with Sendai virus

Sendai virus infection transmitted by contact from cagemates was followed by virus titration and immunofluorescence. The virus grew in the respiratory tract and caused macroscopic lesions in all contact mice. The virus grew to a higher titer in the lung than in the trachea. Tracheal smears, however, were found to be the most suitable for the diagnosis of Sendai virus infection by immunofluorescence, since they contained a large number of cells with intense fluorescence. Diagnosis of Sendai virus infection was made by immunofluorescence within a few hours after autopsy made at early stages of infection.     

小鼠呼肠孤病毒Ⅲ型标准化血清制备及ELISA检测方法的建立

目的制备标准化小鼠呼肠孤病毒III型（reovirus 3,Reo-3）免疫血清,建立小鼠Reo-3抗体ELISA检测方法。方法使用动物来源的Reo-3病毒感染BALB/c小鼠,获得高效价免疫血清;以BHK-21细胞制备Reo-3病毒抗原,同时制备做正常对照抗原。滴定抗原和标准化小鼠Reo-3血清的最佳工作浓度,进行特异性、敏感性、重复性、稳定性等实验。结果制备18 mL抗Reo-3血清,经检测,IFA效价达1∶640,IEA效价达1∶160;Reo-3抗原和标准化小鼠Reo-3免疫血清最佳工作浓度分别为5μg/mL和1∶2400;该ELISA检测体系Reo-3特异抗原批内变异系数为3.8%,批间变异系数为4.0%;检测灵敏度〉1∶4400;与仙台病毒（SV）、小鼠肝炎病毒（MHV）、小鼠腺病毒（Mad）、小鼠脑脊髓炎病毒（TMEV）、小鼠多瘤病毒（POLY）均无交叉反应。经37℃破坏性试验,2 d稳定性试验相对偏差〈27%。结论本研究制备的Reo-3抗血清达到同批次大量高滴度的水平,可作为检测实验小鼠Reo-3病原的标准化质控血清。本研究建立的ELISA方法重复性、稳定性好,特异性、敏感性强。该体系可用于大、小鼠等实验动物Reo-3抗体的检测。     

小鼠脑脊髓炎病毒标准血清制备及间接ELISA检测方法的应用

目的制备小鼠脑脊髓炎病毒（TMEV）标准血清,建立ELISA检测方法,实现一种病毒多种检测方法的比对研究。方法用TMEV感染3周龄SPF级BALB／c小鼠,制备免疫用抗原。分别在0、7、14d以腹腔注射的方式免疫SPF级6-8周龄BALB／c小鼠,免疫血清用IFA、IEA法进行鉴定;TMEV感染BHK-21细胞,制备EHSA抗原,确定酶结合物、抗原和标准阳性血清的最佳工作浓度,建立TMEVEHSA检测方法,进行敏感性、特异性、重复性和稳定性实验。结果制备TMEV免疫血清,以IFA、IEA测定血清效价分别为1：640和1：320,与痘苗病毒、小鼠肺炎病毒、小鼠肝炎病毒、仙台病毒和呼肠孤病毒-Ⅲ型均无交叉反应;建立了ELISA检测方法,确立了各种试剂的最佳工作浓度,其中酶结合物最佳工作浓度为1：10000,抗原为2.5μg／mL,阳性参考血清为1：200;EHSA检测灵敏度为1：3200。板内特异抗原、正常抗原平均变异系数为4.67％和5.7％,板间为4.39％和7.61％。稳定性实验相对偏差均小于20％。结论本研究制备的TMEV免疫血清可作为血清学检测方法中的标准质控血清;建立的ELISA方法重复性、稳定性好,敏感性和特异性强,可用于小鼠脑脊髓炎病毒血清抗体检测。     

汉滩病毒84Fli株DNA疫苗诱导小鼠免疫应答的初步研究

为了加强我国病毒性出血热的防治,本研究将汉滩病毒84Fli株核蛋白S和糖蛋白M编码片段分别克隆至pcDNA3.0载体,构建了pcDNA3/84S和pcDNA3/84M重组质粒,等量混合采用肌肉注射途径免疫C57BL/6小鼠,免疫3次,每次间隔2周,同时与双价出血热病毒灭活疫苗进行对比。ELISA及免疫荧光(IFA)分别检测小鼠血清中汉滩病毒核蛋白及糖蛋白特异性抗体,流式细胞仪和ELISPOT方法分析小鼠免疫后的细胞免疫水平。微量中和试验检测小鼠血清抗体的的中和活性。结果显示,DNA疫苗免疫组C57BL/6小鼠在初次免疫2周后即能检测到汉滩病毒核蛋白与糖蛋白的特异性抗体,与灭活疫苗组相比,重组质粒诱导的抗体滴度高,产生时间早,产生的抗体具有中和活性;同时可诱导产生特异性细胞免疫应答。研究表明,汉滩病毒pcDNA3/84S和pcDNA3/84M重组质粒能有效刺激小鼠产生特异性体液免疫和细胞免疫应答。     

Studies on the development of an ELISA kit for microbiological monitoring. 1. Evaluation of the reliability of the prototype kit by field tests

The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV)/sialodacryoadenitis virus (SDAV)/Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2 micrograms/ml and MHV Nu-67 strain, 5 micrograms/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10,094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.     

Serodiagnosis of microbiosis in mice with a quantitative assay of immunoglobulins by a microcomputer-introduced ELISA system. 1. Anti-Sendai virus antibodies in naturally infected mouse sera

An enzyme-linked immunosorbent assay system combined with microcomputer data analysis was established as a quantitative assay method of immunoglobulins. The assay system was applied to measure IgG and IgM levels of anti-microbe antibodies in animals, especially mouse and rat. And now the measurement of IgG and IgM levels (ng/ml) of anti-Sendai virus (HVJ) antibodies in naturally infected mice is available. The assay system could improve serodiagnosis in the specificity and sensitivity and in the rapid treatment of many serum samples. The operation of this system was performed by a microcomputer, FM 8 connected Titertek Multiskan MC. The limited sensitivity of this assay for IgG and IgM was 10 ng/ml and 30 ng/ml, respectively. Ninety-one of serum samples were positive for IgG and/or IgM (45 samples for IgG and IgM, 44 samples for IgG, 2 samples for IgM) to Sendai virus in the tested 279 mouse sera, and serum titers were ranged from 1: 10 to 1: 12,800 in the IgG, and from 1: 20 to 1: 160 in the IgM. In these titers, serum IgG and IgM amounts were estimated to be 0.1 to 154 micrograms/ml and 0.5 to 4.8 micrograms/ml, respectively. Relationships of serum titers and antibody amounts were almost consisted, being judged like that approximately 10 micrograms/ml is 1: 400, 30 micrograms/ml is 1: 1,600 in IgG, and 2.4 micrograms/ml is 1: 80, 4 micrograms/ml is 1: 160 in IgM.     

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.     

Immunoglobulin classes of anti-Sendai virus antibody detected by ELISA in infected nude mouse serum

Sendai virus-infected nude mouse sera obtained on the seventh day after infection or later, in which anti-Sendai virus antibodies were undetectable by hemagglutination-inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase-conjugated anti-mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti-Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti-Sendai virus antibody activity of fractions from Sephadex G-200 gel filtration was detected in the IgM fraction when anti-mouse mu chain-specific antiserum was used and in both IgG and IgM fractions when heavy and light chain-specific anti-mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A-Sepharose affinity chromatography of Sendai virus-infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti-Sendai virus anti-bodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.