Immunofluorescence Assay for Detection of Human Metapneumovirus-Specific Antibodies by Use of Baculovirus-Expressed Fusion Protein

Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (κ = 0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.     

Correlation between Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay with Lytic Antigens for Detection of Antibodies to Human Herpesvirus 8

The purpose of this study was to evaluate, in Kaposi's sarcoma patients, the correlation between antibody titers to the lytic antigens of human herpesvirus 8, as assessed by immunofluorescence assay, and values obtained by an enzyme immunoassay. The methods showed a stringent correlation, r = 0.625 (P < 0.001).     

Immunofluorescence Microphotomerry for the Detection of Platelet Antibodies

The sensitivity of the immunofluorescence microphotometry method for the demonstration of antibodies against platelets was compared with that of the agglutination reaction, the indirect antiglobulin consumption test, and the complement fixation method. For this purpose titres of sera containing hetero- or iso-antibodies were measured by various methods, with of the same donor. For the immunofluorescence method an anti-rabbit immunoglobulin conjugate, and an antihuman immunoglobulin conjugate, anti-human IgG conjugate, and an anti complement conjugate were used. Fixation of platelets with acetone appeared to be unsuitable for the immunofluorescence complement method On the whole, the highest titres were obtained with immunofluorescence microphotometry.     

Immunofluorescence Microphotometry for the Detection of Platelet Antibodies

After a survey of the literature dealing with the demonstration of platelet autoantibodies by immunofluorescence techniques, the results are given of a study in which immunofluorescence microphotometry was used for this purpose. The serum of 58, the platelets of 34, and the megakaryocytes of. 2 patients -with thrombocytopenia were investigated. In 21 of 52 sera (40%) in which the presence of platelet autoantibodies could be expected, positive results were obtained that could not be due to isoantibodies, either because the patients had not been pregnant and had not received blood transfusions or because the reactivity of the serum with the patient's own platelets was demonstrated. The platelets of 28 patients with thrombocytopenia not due to a platelet defect or decreased thrombopoiesis were investigated. In the platelets of 15 (54%) of these, significant differences in fluorescence were'found with anti-immunoglobulin conjugate as well as with anti-IgG, -IgA, -IgM, or -complement reagents. It was concluded that in these patients in vivo sensitization of the platelets with autoantibody was demonstrated. In two patients an indication of the in vivo sensitization of the megakaryocytes was also obtained.     

New Lineal Immunoenzymatic Assay for Simultaneous Detection of p24 Antigen and HIV Antibodies

 The aim of this study was to evaluate a new lineal immunoenzymatic assay for the simultaneous detection of HIV-1/HIV-2 antibodies and p24 antigen. A total of 320 serum samples were obtained from individuals infected by HIV (HIV 1, n=183; HIV 2, n=2), individuals with risk factors for HIV infection (n=49), recipients of multiple transfusions (n=40), and blood donors (n=46). The Western blot for detection of HIV antibodies and an enzyme immunoassay for detection of p24 antigen, both established techniques, were used for direct comparison. In cases of recent infection, p24 antigen was generally detected at the same time by the lineal test and the established assay. The p24 antigen sensitivity was about 200 pg of HIV antigen per milliliter. The results seem to indicate that the new test could be used with sufficient reliability for screening biological samples (sensitivity, 99.5%; specificity, 94.8%). Use of the lineal immunoenzymatic assay may shorten the amount of time needed to diagnose acute infection with HIV to approximately 2 weeks.     

细胞ELISA检测血清抗内皮细胞抗体

用培养的人脐带静脉内皮细胞作为抗原，经ＥＬＩＳＡ检测了ＳＬＥ患者血清抗内皮细胞抗体，目的在于建立ＥＬＩＳＡ检测抗内皮细胞抗体的方法，并与细胞毒试验比了比较，结果表明，ＥＬＩＳＡ及细胞毒试验检测抗内皮细胞抗体，阳性率分别为７３％和５１％，差异显著显著；阳性血清用红细胞及淋巴细胞吸收后，抗内皮细胞抗体活性无明显降低，提示该法特异、灵敏、且重复性较好，可进行大批量标本筛选，为研究抗内皮细胞抗体特征及其致     

Detection of Cryptosporidium parvum in Horses: Thresholds of Acid-Fast Stain, Immunofluorescence Assay, and Flow Cytometry

Feces collected from three asymptomatic horses and seeded with Cryptosporidium parvum oocysts (10¹ to 10⁶/g of feces) were evaluated by acid-fast staining (AF), an immunofluorescent antibody (IFA) technique, and flow cytometry. The thresholds of detection were 5 × 10⁵ oocysts/g of feces for the IFA and AF techniques and 5 × 10⁴ oocysts/g for flow cytometry.     

滴金免疫渗滤法检测广州管圆线虫抗体的实验研究

目的建立一种检测广州管圆线虫抗体的简便、快速、敏感、特异的新方法。方法以广州管圆线虫成虫抗原为包被抗原，金标记SPA为显色剂，建立检测广州管圆线虫抗体的滴金免疫渗滤法（DIGFA）；并以ELISA平行检测比较。结果用DIGFA检测广州管圆线虫实验感染鼠血清50份，其阳性检出率为96．0％，50份正常鼠血清的阴性符合率达100％。DIGFA分别检测蛲虫阳性鼠血清13份。绦虫阳性鼠血清11份和粪类圆线虫阳性鼠血清18份，前二者均为阴性，后者交叉反应率为5．6％；DIGFA与ELISA平行检测30份广州管圆线虫阳性鼠血清，两法符合率达96，7％。结论DIGFA与ELISA有相似的敏感性和特异性。且具有简便、快速、不需特殊仪器设备等优点。是一种检测广州管圆线虫抗体的新方法。     

Studies on Antibodies to Histones by Immunofluorescence

When mouse kidney tissue sections were extracted with 0.1 N hydrochloric acid, sera with antibodies to certain nuclear antigens no longer stained tissue nuclei by immunofluorescence. This effect was due to removal of histones and nuclear acidic proteins Sm and nuclear ribonucleoprotein by the acid. DNA remained in the nuclei of the acid-extracted tissue sections. When solutions of calf thymus histones were reacted with acid-extracted tissues, histones combined with nuclear DNA to form complexes of DNA-histone. These complexes contained antigenic determinants which reacted with sera containing antibodies to deoxyribonucleoprotein to give nuclear staining demonstrated by immunofluorescence. The reaction was immunologically specific in that sera with antibodies to Sm and nuclear ribonucleoprotein were not reactive with reconstituted DNA-histone in nuclei. Other basic proteins such as protamine, poly-L-lysine, and poly-L-arginine could not substitute for histones. The method is introduced as a specific and reproducible assay for study of antibodies to histones.     

New Immunofluorescence Assays for Detection of Human Herpesvirus 8-Specific Antibodies

Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a “gold standard.” Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs for IFA (ORF73-IFA and K8.1-IFA). Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity. The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (κ > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV⁺) Kaposi's sarcoma (KS) patients, HIV⁺ KS⁻ patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.     

Novel Real-Time PCR Assay for Simultaneous Detection and Differentiation of Clostridium chauvoei and Clostridium septicum in Clostridial Myonecrosis

A real-time PCR assay based on the 16S rRNA gene sequence was designed for differentiation of blackleg-causing Clostridium chauvoei and Clostridium septicum, a phylogenetically closely related bacterium responsible for malignant edema. In order to exclude false-negative results, an internal amplification control was included in the assay. A set of three probes, one specific for C. chauvoei, one specific for C. septicum, and one specific for both species, permitted unequivocal detection of C. chauvoei in tests of 32 Clostridium sp. strains and 10 non-Clostridium strains. The assay proved to be sensitive, detecting one genome of C. chauvoei or C. septicum per PCR and 1.79 × 10³ C. chauvoei cells/g artificially contaminated muscle tissue. In tests of 11 clinical specimens, the real-time PCR assay yielded the same results as an established conventional PCR method.Clostridium chauvoei is a strictly anaerobic, Gram-positive, spore-forming rod and a common microbe found in soil as well as the guts of ruminants. It is the causal agent for blackleg, a peracute, nontraumatic, endogenous infection in cattle, presenting as gas gangrene. After blunt trauma, which reduces oxygen levels in muscle tissue, spores are able to germinate. In sheep, C. chauvoei is expressed mainly as an exogenous wound infection resulting from shearing, castration, or docking (3, 12, 18). Clostridium septicum belongs to the pathogens involved in the gas edema complex and is very similar to C. chauvoei.One case of human fulminant gas gangrene caused by C. chauvoei has been reported. The actual prevalence is suspected to be higher if genetic characterization is included in the diagnostic procedure. Routine clinical microbiology laboratory tests may falsely identify C. septicum instead of C. chauvoei (12). Differentiation of the two pathogens is also needed to obtain governmental financial support for blackleg in certain countries or districts (8).Conventional microbiological methods are hampered by the slow growth of C. chauvoei, which is often overgrown by other anaerobes or swarming C. septicum colonies, as both might be present in a sample (8, 15). Immunological detection methods are reported to be prone to cross-reactions between the two species (6). In addition, production of antisera for their detection is complex because it involves the use of laboratory animals.DNA-based PCR identification proved to be a good alternative, as it is more reliable, fast, and easy to perform. Conventional PCR systems target the 16S rRNA gene, the 16S-23S rRNA gene spacer region, or the flagellin gene (1, 6, 8, 13, 14, 15, 16, 18). On one hand, conventional PCR bears the risk of yielding false-positive results due to contamination during post-PCR handling of the amplicons for detection. On the other hand, published assays do not include internal amplification controls (IACs) to permit detection of false-negative results due to PCR-inhibitory components in the sample matrix.Real-time PCR employs fluorescently labeled probes for amplicon detection. Thus, no post-PCR processing of the samples is required. This reduces the risk of laboratory contamination and also saves time (4). Multichannel detection permits parallel analysis of more than one target region by use of differently labeled probes. Thus, an IAC may easily be integrated into the procedure.The aim of the present study was to develop a real-time PCR assay for discrimination of C. chauvoei and C. septicum. An IAC was included in the assay. Inclusivity and exclusivity, as well as the detection limit, were tested, and the optimized assay was applied to clinical specimens.     

Rapid Detection of Panton-Valentine Leukocidin in Staphylococcus aureus Cultures by Use of a Lateral Flow Assay Based on Monoclonal Antibodies

Panton-Valentine leukocidin (PVL) is a virulence factor of Staphylococcus aureus, which is associated with skin and soft-tissue infections and necrotizing pneumonia. To develop a rapid phenotypic assay, recombinant PVL F component was used to generate monoclonal antibodies by phage display. These antibodies were spotted on protein microarrays and screened using different lukF-PV preparations and detection antibodies. This led to the identification of the optimal antibody combination that was then used to establish a lateral flow assay. This test was used to detect PVL in S. aureus cultures. The detection limit of the assay with purified native and recombinant antigens was determined to be around 1 ng/ml. Overnight cultures from various solid and liquid media proved suitable for PVL detection. Six hundred strains and clinical isolates from patients from America, Europe, Australia, Africa, and the Middle East were tested. Isolates were genotyped in parallel by DNA microarray hybridization for confirmation of PVL status and assignment to clonal complexes. The sensitivity, specificity, and positive and negative predictive values of the assay in this trial were 99.7, 98.3, 98.4, and 99.7%, respectively. A total of 302 clinical isolates and reference strains were PVL positive and were assigned to 21 different clonal complexes. In summary, the lateral flow test allows rapid and economical detection of PVL in a routine bacteriology laboratory. As the test utilizes cultures from standard media and does not require sophisticated equipment, it can be easily integrated into a laboratory''s workflow and might contribute to timely therapy of PVL-associated infections.     

A Solid-Phase Assay for the Detection of Anti-Sperm Antibodies

PROBLEM: ELISA is an ideal assay method for a large-scale screening of anti-sperm antibodies among a large number of infertile males. However, conventional ELISA with whole spermatozoa needs time-consuming steps of centrifugation. METHOD: A solid-phase assay used for detecting anti-sperm antibodies was established. This assay is suitable not only for detecting circulating anti-sperm antibodies of IgG, IgM, and IgA subclass simultaneously but also for screening hybridomas secreting anti-sperm monoclonal antibodies (mAbs). The microtiter plates, on which solubilized sperm antigens are fixed, can be stored at ? 80°C for up to six months without losing reactivity with anti-sperm antibodies. RESULTS: Using this assay, 53 sera (13 were proven positive and 40 were proven negative for sperm agglutination antibody) were tested. Although the false-negative rate was 0%, the false-positive rate was 32%. One thousand one hundred sixty-five supernatants from hybridomas constructed with splenocytes of mice who were hyperimmunized with human sperm and nonsecreting myeloma cells were tested by this solid-phase assay and two anti-sperm mAb secreting clones were selected and established. CONCLUSIONS: It is recommended that for research work this assay could be used for the first screening of the hybridoma secreting anti-sperm mAb, and for clinical use this assay might be suitable for the first screening of sera of infertile patients. However, conventional bioassays should follow to confirm the biological meaning of the positivity.     

A Solid-Phase Radioimmunoassay for Anti-Histone Antibodies in Human Sera: Comparison with an Immunofluorescence Assay

A solid-phase assay for anti-histone antibodies is described, in which antibodies bound to calf thymus total histones, specific histone classes, or chromatin were detected by radioactive anti-human IgG, IgA or IgM. The reactivity of human sera from various rheumatic diseases was analysed by this assay and compared with results obtained from an indirect immunofluorescence assay (FANA) previously described. Sera that were positive in the FANA assay were usually positive in the solid-phase assay when total histones were the solid-phase antigen. However, many sera had IgM antibodies to total histones in the solid-phase assay but gave no detectable reaction in the FANA assay. Results from the solid-phase assay, using individual histones and chromatin, showed that the anti-histone antibodies in these sera were predominantly reactive with histones H3-H4 and did not bind well to histones complexed to DNA in the form of chromatin.     

Diagnosis of Granulocytic Ehrlichiosis in Humans by Immunofluorescence Assay

Serodiagnostic tests are widely available for tick-borne diseases. We evaluated a cell-free antigen of the human granulocytic ehrlichiosis agent. Immunofluorescence assay (IFA) with this antigen is as efficient as with the MRL kit and allows a one-step IFA with other cell-free antigens that is useful when testing sera from patients bitten by ticks.     

Detection by Immunofluorescence Assay of Bartonella henselae in Lymph Nodes from Patients with Cat Scratch Disease

Laboratory diagnosis of Bartonella henselae infections can be accomplished by serology or PCR assay on biopsy samples. The purpose of our work was to assess immunofluorescence detection (IFD) in lymph node smears using a specific monoclonal antibody directed against B. henselae and a commercial serology assay (IFA) compared with PCR detection. Among 200 lymph nodes examined from immunocompetent patients, 54 were positive for B. henselae by PCR, of which 43 were also positive by IFD. Among the 146 PCR-negative lymph nodes, 11 were positive by IFD. Based on PCR results, the specificity of this new technique was 92.5%, the sensitivity was 79.6%, and the positive predictive value was 79.6%. At a cutoff titer of 64, the sensitivity of the IFA was 86.8% and the specificity was 74.1%. Diagnosis of cat scratch disease (CSD) may be improved, with a specificity of 100%, when the two tests (IFD and IFA) were negative; the sensitivity was 97.4% if one of the two tests was positive. Since PCR-based detection with biopsy samples is available only in reference laboratories, we suggest using IFD coupled with the commercial serology test for the diagnosis of CSD.     

单克隆抗体间接荧光法分型检测单纯疱疹病毒的实验研究

本文选择３株抗单纯疱疹病毒型共同性和型特异性单克隆抗体，建立了分型检测单纯疱疹病毒的单克隆抗体间接荧光法（ＭｃＡｂ－１ＦＡ）．特异性试验和重复性试验证实，ＭｃＡｂ－ＩＦＡ特异性强、重复性好．用此法检测了不同部位来源的５１份临床分离株，分型检测结果与ＥＬＩＳＡ法检测结果一致．提示ＭｃＡｂ－ＩＦＡ有可能成为实验室分型检测单纯疱疹病毒可靠的方法．     

Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection and Differentiation of Antibodies against European and North American Porcine Reproductive and Respiratory Syndrome Virus

Two types of porcine reproductive and respiratory syndrome virus (PRRSV) have been reported, the European type (EU PRRSV) and the North American type (US PRRSV). We developed a dual enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection and differentiation of serum antibodies directed against either of the two PRRSV types. This tandem PRRS ELISA is based on affinity-purified recombinant nucleocapsid protein expressed in Escherichia coli. Sensitivity and specificity were assessed by using the IDEXX HerdChek PRRS ELISA and the indirect immunofluorescence assay as reference tests. A total of 1,571 sera originating from the United States, Europe, and two PRRS-free countries, i.e., Switzerland and New Zealand, were used for validation of the tandem PRRS ELISA. The new test performed at least as well as the reference tests in regard to sensitivity (0.94 for the US PRRS ELISA and 0.93 for the EU PRRS ELISA) and specificity (0.96 for the US PRRS ELISA and 0.99 for the EU PRRS ELISA). Positive sera were correctly differentiated in 582 of 591 cases, indicating a high differentiation capability of this dual ELISA. The robustness and repeatability of the test were assessed and found to be appropriate for diagnostic applications. Taken together, the data indicate that the tandem PRRS ELISA described here is the first differentiation ELISA for PRRSV serology based on recombinant antigen. It is convenient with respect to antigen production, and it is reliable, economical, and highly sensitive and specific. Thus, it is considered to be a powerful tool for routine diagnostics, epidemiological surveys, and outbreak investigations.