Comparative evaluation of two commercially available antigen enzyme immunoassays (EIA) for the detection of Legionella pneumophila urinary antigen in frozen non-concentrated urine samples

We evaluated two commercial enzyme immunoassay kits, Binax EIA (for detection of soluble antigen of Legionella pneumophila serogroup 1) and Biotest EIA (for detection of antigens of Legionella pneumophila serogroups and other Legionella spp.) in order to introduce this test routinely for the diagnosis of Legionnaires' disease (LD) in our Laboratory. Frozen non-concentrated urine samples belonging to 45 patients with and without LD were tested. The sensitivity of Binax EIA and Biotest EIA was 47.4% and 42.1% respectively, the specificity was 95% by both tests. Biotest did not detect antigen from a patient with culture-proven infection of L. pneumophila serogroup 6. The detection of urinary antigen by both EIA tests is a useful tool for rapid diagnosis of LD, especially when samples are unavailable for culture; the sensitivity may be increased if the assay is performed on unfrozen and concentrated samples.     

Cross-reactivity and sensitivity of two Legionella urinary antigen kits, Biotest EIA and Binax NOW, to extracted antigens from various serogroups of L. pneumophila and other Legionella species

Legionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non-serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in-vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L pneumophila SG 1 antigens, but reacted to different sets of non-SG I L. pneumophila with different sensitivity. No cross-reactivity was observed to Legionella species other than L. pneumophila.     

Detection of hypersensitivity toLegionella pneumophila in guinea pigs by skin test

Cross-reacting antigens of three serogroups ofLegionella pneumophila differed serologically and immunologically from the serogroup-specific antigens. Intradermal injection of cross-reacting antigens into sensitized guinea pigs evoked skin hypersensitivity. Animals were sensitized either by injection of inactivatedL. pneumophila in adjuvant or by infection with live organisms. Skin reactions were measurable about 2–4 h after injection and continued to increase in intensity for the first 24 h, followed by a gradual decline over the next 48 h. Histological examination of skin reactions taken from test sites at 48 h revealed infiltration of mononuclear cells in and about the small subcutaneous blood vessels and throughout the dermis, compatible with a delayed-type reaction. The overall appearance and time course of the reaction resembled a combination of immediate and delayed types of hypersensitivity. Each cross-reacting antigen of the three serogroups evoked skin reactions in animals which had been sensitized to any of those serogroups, but was not reactive in nonsensitized animals. These observations indicate the possibility of detecting present or past infection ofL. pneumophila by skin tests.     

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria. To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit’s performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.     

Comparison and evaluation of the Binax EIA and Biotest EIA urinary antigen kits for detection of Legionella pneumophila antigen in urine samples

The Binax and the Biotest urinary antigen kits for detection of L. pneumophila antigen were compared by testing of selected 67 urine samples obtained from EWGLI as reference samples in External Quality Assessment Scheme. Thirty nine were positive with the Binax kit (100% of sensitivity), and 33 were positive with the Biotest (84.6% of sensitivity). The test specificities were 100% for the both kits. It was concluded that the Binax kit was more suitable for the routine diagnosis of Legionella infections than the Biotest kit.     

Antibodies isolated by using cloned surface antigens recognize antigenically related components of Legionella pneumophila and other Legionella species

We studied the antigenic cross-reactivity of surface proteins among various strains of Legionella pneumophila and other Legionella species by using a novel method of antibody purification. Anti-bacterial antibodies in hyperimmune sera were adsorbed to and eluted from the surface of recombinant E. coli cells that express individual L. pneumophila antigens on their surface. These affinity-purified antibodies were then used to probe protein immunoblots prepared from the test strains to detect cross-reactive domains. We found that antigenic proteins are generally conserved in all L. pneumophila serogroups. Although some of these antigenic domains are shared with members of other Legionella species, they are associated with proteins of different molecular mass. Our approach to the study of antigenic cross-reactivity has potential advantages over similar studies that use either monoclonal antibodies or monospecific antibodies prepared by immunization with purified antigens.     

Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model

Laboratory diagnostics of toxoplasmosis depends primarily on serological methods detecting specific antibodies. Since these methods do not always enable specific and sensitive recognition of the infection and phase of toxoplasmosis, the search for new diagnostic tools continues. Recombinant antigens promise a new alternative in diagnostics of Toxoplasma gondii infections. In this work the usefulness of six recombinant T. gondii antigens: GRA1, GRA6, GRA7, p35, SAG1, and SAG2 in the detection of primary murine toxoplasmosis was evaluated. Sera obtained from infected mice differing in their natural susceptibility to T. gondii infection, BALB/c (relatively resistant) and C57BL/6 (relatively susceptible), were tested using ELISA. During acute infection high response to GRA7, GRA6, and p35 antigens was noticed, whereas a strong reactivity with surface antigens SAG1 and SAG2 was characteristic for chronic toxoplasmosis. Our results show that the recombinant antigens are useful in distinguishing between acute and chronic toxoplasmosis regardless of the genetically determined susceptibility of the host.     

Antigenic variability of the outer membrane antigens of Legionella pneumophila serogroups 1 to 8

The outer membrane proteins of Legionella pneumophila serogroups 1 to 8 were prepared from broken cells by selective solubilization using sodium lauryl sarcosinate. The isolated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. Rabbit antisera against each of the eight serogroups of L. pneumophila were obtained by immunizing each animal with live bacteria. The transferred proteins were revealed using these antisera and peroxidase-labeled swine anti-rabbit immunoglobulins. Antigenic determinants common to all eight serogroups were found in at least three outer membrane antigens (19, 29, and 45 kilodaltons (kDa)). However, cross-absorption experiments revealed that these three antigens were immunologically related, but not identical among serogroups. The antigenic relationships observed with two of these three antigens correlated well with cross-reactions observed in immunofluorescence. When a monoclonal antibody directed against L. pneumophila serogroup 1 lipopolysaccharide was used to reveal a blot of serogroup 1 outer membrane antigens, the 29- and 45-kDa bands appeared. This demonstrates a strong association between lipopolysaccharide and outer membrane proteins.     

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

African swine fever virus (ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds. Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase (nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA (cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7% by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.     

The immunological properties of the main outer membrane protein of Legionella

The immunogenic properties of Legionella outer membrane main protein (OMMP) were studied by its effect on the proliferative activity of lymphocytes in guinea pigs. Preliminary immunization with OMMP activated only the specific and nonspecific proliferation of spleen cells. After infection with Legionella, secondary immune response developed in the spleen and lungs of previously immunized animals, in contrast to intact ones, and the nonspecific proliferative activity of lymphocytes in the spleen and lungs of previously immunized animals considerably increased. These results are indicative of the fact that Legionella OMMP, similarly to other Legionella antigens and immunomodulators, may be used for the formation of protective immunity.     

Commercial polyclonal and monoclonal histostaining PAP kits

Summary Universal, polyclonal and monoclonal immunoperoxidase staining kits from BioGenex, Dako and Ortho were employed for the localization of antigens such as gastrin, prostate specific antigen, IgA, IgG, AFP and CEA in histological sections from formaldehyde fixed and paraffin embedded human specimens. The kit components were controlled by immunohistological and serological assays and were also compared with self-prepared reagents. In connection with specific primary antibodies, universal/basic kits gave reliable localization of defined antigens. The optimal concentration of the primary antibodies had to be established by dilution experiments. In the case of polyclonal kits, typical antigen localization was obtained in selected tissue sections with all the respective kits. CEA kits also stained strongly NCA molecules present in organs such as colon, stomach and liver. BioGenex polyclonal kits gave almost stronger stainings than kits from Dako and Ortho. Irrespective of which kit from different commercial sources is used, development of peroxidase activity with AEC/H₂O₂ often had to be stopped far below the recommended incubation time of 40 min or overstaining with color change from reddish to muddy green occurred. The latter was attributed to insufficiently balanced kit reagents, an interpretation wich was supported by quantitative serological studies. Sensitivity of immunohistological reactivity was much enhanced by pretreatment of tissue sections with Pronase. Thus, stronger immunostainings and larger numbers of positive cells were detected than in conventionally rehydrated sections. Incubation of sections with self-prepared primary antibodies, linking antibodies and PAP complexes gave essentially the same antigen localization as with commercial kits, but antibodies isolated by our affinity chromatography led to a better staining contrast with absence of nonspecific background. The advantage of monoclonal over polyclonal kits was the background-free staining of sections. Other-wise, antigens were localized in the same cell types, although cellular reactivity was usually less intense than with polyclonal antibodies. This, however, could be overcome by Pronase treatment of the sections prior to incubation.     

人心脏型脂肪酸结合蛋白检测试剂盒的临床评价

目的评价人心脏型脂肪酸结合蛋白(H-FABP)检测试剂盒(胶体金法)在急性心肌梗死(AMI)诊断中的价值。方法采用平行、盲法、对照的对比试验设计,比较其试验产品和对比产品对诊断AMI的敏感性、特异性、准确性。结果共测定240份临床血液标本。试验产品和对比产品的阳性符合率为100%,阴性符合率为96.15%,总符合率为97.92%。对比产品和试验产品结果不一致的5例标本以临床诊断结果为标准进行验证后,试验产品与临床诊断结果的阳性符合率为100%,总符合率为100%。采用Kappa检验考核两种产品测定结果的一致性,Kappa指数为0.958。经McNamara's test分析,两产品之间无差异,χ2=3.20,P>0.05。结论试验产品显示出较好的诊断价值,可以作为AMI早期诊断标志物,试验产品与对比产品等效。     

Comparison of three ELISA kits for the differentiation of foot-and-mouth disease virus-infected from vaccinated animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals. Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits, Ceditest® FMDV-NS (Ceditest® kit), UBI® FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI® kit) and a FMDV 3ABC-I-ELISA kit developed at the Lanzhou Veterinary Research Institute. The test parameters (sensitivity and specificity) of the three kits were determined, and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits. The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest® kits was 98.05%, and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI® kits was 94.4%; the sensitivity of both Ceditest® and FMDV 3ABC-I-ELISA kit was 100%. However, the sensitivity of the UBI® kit was only 81.8%. With sera from naive or vaccinated non-infected animals, the specificity of all tests exceeded 90%.     

Antigenic relatedness of immunoglobulins toLegionella pneumophila serogroups 1–6 from humans and the nonhuman primateMacaca fascicularis

The purpose of this study was to determine whether cynomolgus monkey antisera toLegionella pneumophila serogroups 1–6 antigens could be used as positive controls in the indirect immunofluorescence assay (IFA) for legionellosis. Immunoelectrophoretic mobilities and Ouchterlony analyses with heavy chain-specific antisera and IFA titers with immunoglobulin class-specific conjugates were used to show antigenic relatedness of immunized monkey immunoglobulins to those produced as a result of infection in humans. Identical immunoelectrophoretic precipitation patterns were obtained for human and monkey sera with antihuman gamma, mu, and alpha heavy-chain-specific antisera. Ouchterlony analyses showed precipitin bands of partial identity between human and monkey IgG, IgM, and IgA classes. IFA titers in the monkey hyperimmune antisera were >16,000 with antihuman conjugate. These data suggest that hyperimmune cynomolgus monkey antisera are suitable alternatives to human sera for IFA-positive controls.     

A novel recombinant multiepitope protein as a hepatitis C diagnostic intermediate of high sensitivity and specificity

A novel recombinant multiepitope protein has been designed that consists of six linear, immunodominant, and phylogenetically conserved epitopes from hepatitis C virus. Five of these antigens (core, NS3, NS4I, NS4II, and NS5) are being used in many of the third-generation kits while sixth epitope (core3g) is an additional sequence from a newly identified Indian isolate. The genes for these epitopes have been joined together to code for a single multiepitope protein that has been evaluated for its diagnostic potential for the detection of anti-HCV antibodies in human plasma. Two separate synthetic genes have been designed, both encoding the same six epitopes in a single open reading frame along with spacers having additional amino acids to function as flexible (r-HCV-F-MEP) or rigid (r-HCV-R-MEP) linkers. High-level expression of hepatitis C multiepitope protein in Escherichia coli has been achieved. The protein has been purified using a single affinity step yielding >25 mg pure protein/liter culture and used as the coating antigen in anti-HCV EIA. The use of this multiepitope protein eliminates the requirement for multiple diagnostic intermediates for the development of anti-HCV diagnostic kit. The sensitivity and specificity of the HCV multiepitope protein was evaluated by Boston Biomedica Worldwide Performance Panels, HCV Seroconversion Panels and Viral Co-infection Panels, and was found to be comparable with commercially available anti-HCV EIA kits. This analysis indicated its unequivocal performance as capture antigen in anti-HCV EIA. The high epitope density, careful choice of epitopes and use of E. coli system for expression, coupled with simple purification protocol provides the potential for the development of an inexpensive diagnostic test with high degree of sensitivity and specificity.     

Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids ²²⁴HWPKPHTLW²³², was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.     

Study of the antibody response against Mycobacterium tuberculosis antigens in Warao Amerindian children in Venezuela

This study was aimed at investigating alternate methods for serodiagnosis of tuberculosis (TB), which are needed because bacteriologic diagnosis of childhood TB is difficult. A selection of 80 serum and saliva samples were tested from Warao indigenous children under 15 years of age; 34 high TB suspects (28 positive and 6 negative for the tuberculin skin test, TST) and 46 healthy contact children (32 positive and 14 negative for the TST). Several enzyme-linked immunosorbent assay (ELISA) serological tests were developed to test for Mycobacterium tuberculosis-specific antibodies, including serum IgA, IgG, IgE, and secretory IgA (sIgA) in saliva against 3 specific antigens (PPD, HSP60, 38 kDa). Of these, 2 antigens, PPD and 38 kDa, showed significantly higher reactivity. The sensitivity and specificity of these tests for diagnosis remained limited, between 26.5% and 38.2%, and 77.4% and 97%, respectively. Of all the samples studied and combinations realized between all isotypes and antigens combined with 3 isotypes (anti-PPD IgG, IgE, and anti-38kDa sIgA) managed to detect the largest number of patients, showing an improved sensitivity level of 64.7%, although specificity levels dropped to 81.8%. These results were compared with the Omega diagnostics commercial kit results. The commercial kits showed significantly lower reactivity (sensitivity of 20% and 13.33% to Myco G and Complex Plus, respectively) and a specificity of 100%. This study shows that in indigenous populations of Venezuela, where invasive procedures cannot be used to select samples but evaluation with a chest X-ray for radiological studies is available, the combination of 3 specific isotypes may be a useful tool to increase diagnostic accuracy with pulmonary TB in this population, when used together with clinical and epidemiological criteria.     

Commercial polyclonal and monoclonal histostaining PAP kits. Immunoperoxidase reagents and performance characteristics in comparison with self-prepared immunoreagents

Universal, polyclonal and monoclonal immunoperoxidase staining kits from BioGenex, Dako and Ortho were employed for the localization of antigens such as gastrin, prostate specific antigen, IgA, IgG, AFP and CEA in histological sections from formaldehyde fixed and paraffin embedded human specimens. The kit components were controlled by immunohistological and serological assays and were also compared with self-prepared reagents. In connection with specific primary antibodies, universal/basic kits gave reliable localization of defined antigens. The optimal concentration of the primary antibodies had to be established by dilution experiments. In the case of polyclonal kits, typical antigen localization was obtained in selected tissue sections with all the respective kits. CEA kits also stained strongly NCA molecules present in organs such as colon, stomach and liver. BioGenex polyclonal kits gave almost stronger stainings than kits from Dako and Ortho. Irrespective of which kit from different commercial sources is used, development of peroxidase activity with AEC/H2O2 often had to be stopped far below the recommended incubation time of 40 min or overstaining with color change from reddish to muddy green occurred. The latter was attributed to insufficiently balanced kit reagents, an interpretation which was supported by quantitative serological studies. Sensitivity of immunohistological reactivity was much enhanced by pretreatment of tissue sections with Pronase. Thus, stronger immunostainings and larger numbers of positive cells were detected than in conventionally rehydrated sections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the dnaJ gene for the detection and identification of all Legionella pneumophila serogroups and description of the primers used to detect 16S rDNA gene sequences of major members of the genus Legionella

We sequenced about 930 bp of the dnaJ gene from 15 Legionella pneumophila serogroups and some other members of the genus Legionella. As L. pneumophila 16S rDNA sequences could not discriminate between all subspecies and serogroups, we assessed the use of dnaJ gene sequences to differentiate between Legionella subspecies as well as between L. pneumophila serogroups. A phylogenetic analysis revealed that dnaJ gene sequences were more variable between the L. pneumophila serogroups than mip gene and 16S rDNA sequences. By studying 61 strains from 41 species of the genus Legionella, as well as other genera, we established a PCR method that could amplify 285 bp of dnaJ gene from all L. pneumophila serogroups. This primer set was more sensitive than mip gene primers and was able to detect 0.25 ng of purified DNA. We also describe the 16S rDNA primers that were used to detect most Legionella genus members.