Structural analysis and biosynthesis gene cluster of an antigenic glycopeptidolipid from Mycobacterium intracellulare

Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1-->3)-4-O-methyl-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->2)-6-deoxy-alpha-L-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.     

Anti-cord factor (trehalose 6,6'dimycolate) IgG antibody in tuberculosis patients recognizes mycolic acid subclasses.

The detection of anti-cord factor (trehalose 6,6'-dimycolate) IgG antibody in active (smear-and/or culture-positive) and inactive (smear-and culture-negative) tuberculosis patients is a useful serodiagnostic tool that can be used for early clinical diagnosis of the disease. We estimated the titers of anticord factor IgG antibody in the sera of tuberculosis patients, and compared them with those of Mycobacterium avium-infected patients. Most of the serum samples obtained from the tuberculosis patients were highly reactive against M. tuberculosis (MTB) cord factor isolated from M. tuberculosis H37Rv, a human-type mycobacterial strain, whereas they were less reactive against M. avium (MAC) cord factor. Similarly, most of the serum samples of the MAC-infected patients were highly reactive against MAC cord factor and less reactive against MTB cord factor. These results suggest that anti-cord factor IgG antibody recognizes the mycolic acid subclasses as an epitope which comprises cord factor, since MTB and MAC cord factor differ in mycolic acid subclasses and molecular species composition. To clarify the exact antigenic epitope in cord factor and to find out a more sensitive and specific diagnostic test antigen, we examined the reactivity of patients' sera to glycolipids containing trehalose (cord factor and sulfolipid) obtained from various mycobacterial species. Furthermore, the reactivity of human antisera to various mycolic acid subclasses (alpha-, methoxy and keto mycolic acids) of MTB cord factor was compared. We found that anti-cord factor IgG antibody in the sera of human tuberculosis patients most strikingly recognized methoxy mycolic acid in the cord factor of M. tuberculosis, whereas it recognized alpha- and keto mycolic acids weakly. Pre-absorption studies of antibody with MTB cord factor or methoxy mycolic acid methyl ester showed that anti-cord factor antibody was absorbed partially, but consistently. This is the first report describing that the specific subclass of mycolic acid from mycobacteria is antigenic in the humoral immune system of human tuberculosis infection.     

Structural characterization of a specific glycopeptidolipid containing a novel N-acyl-deoxy sugar from mycobacterium intracellulare serotype 7 and genetic analysis of its glycosylation pathway

The nontuberculous Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is distributed ubiquitously in the environment and is an important cause of respiratory and lymphatic disease in humans and animals. These species produce polar glycopeptidolipids (GPLs), and of particular interest is their serotype-specific antigenicity. Structurally, GPLs contain an N-acylated tetrapeptide-amino alcohol core that is glycosylated at the C terminal with 3,4-di-O-methyl rhamnose and at the d-allo-threonine with a 6-deoxy-talose. This serotype nonspecific GPL is found in all MAC species. The serotype-specific GPLs are further glycosylated with a variable haptenic oligosaccharide at 6-deoxy-talose. At present, 31 distinct serotype-specific GPLs have been identified on the basis of oligosaccharide composition, and the complete structures of 14 serotype-specific GPLs have been defined. It is considered that the modification of the GPL structure plays an important role in bacterial physiology, pathogenesis, and host immune responses. In this study, we defined the complete structure of a novel serotype 7 GPL that has a unique terminal amido sugar. The main molecular mass is 1,874, and attached to the tetrapeptide-amino alcohol core is the serotype 7-specific oligosaccharide unit of 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-2-O-methyl-beta-hexose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->2)-alpha-l-6-deoxy-talose. Moreover, we isolated and characterized the serotype 7-specific gene cluster involved in glycosylation of the oligosaccharide. Nine open reading frames (ORFs) were observed in the cluster. Based on the sequence homology, the ORFs are thought to participate in the biosynthesis of the serotype 7 GPL.     

Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trehalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen

IgG antibodies against purified cord factor (trehalose-6,6'-dimycolate, TDM) in sera of 99 patients infected with mycobacteria (42 patients with tuberculosis excreting tubercle bacilli in the sputum, 11 patients with non-tuberculous mycobacteriosis excreting acid-fast bacilli in the sputum, and 46 patients without bacilli in the sputum but diagnosed as having pulmonary tuberculosis by chest X-ray films and physical examination), five patients with lung cancer, and 100 healthy controls which included subjects positive and negative for the tuberculin test were tested by the ELISA with TDM purified from Mycobacterium tuberculosis H37Rv as the antigen. Of the 99 cases of mycobacteriosis, 83 patients (83.8%) had positive results (48 samples from 53 patients, or 90.5%, with bacilli in the sputum, and 35 samples from 46 patients (76%) with tuberculosis diagnosed clinically). The sera of the five patients with lung cancer and the 100 controls all gave negative results. Thus, the sensitivity and specificity were 83.8% and 100%, respectively. ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis, because it does not involve the cultivation of bacteria.     

Structure and antigenicity of the specific oligosaccharide hapten from the glycopeptidolipid antigen of Mycobacterium avium serotype 4, the dominant Mycobacterium isolated from patients with acquired immune deficiency syndrome

A large number of patients with acquired immune deficiency syndrome develop disseminated infections due to member serotypes of the Mycobacterium avium complex. Seroagglutination on 181 such isolates followed by enzyme-linked immunosorbent assay and thin layer chromatography of the type-specific glycopeptidolipid (GPL) antigens demonstrated that the majority of serotypes were M. avium serotype 4. The specific GPL of serotype 4 was isolated in both the native, acetylated, and the deacetylated forms and its oligosaccharide hapten released as the oligosaccharide alditol by reductive beta-elimination. A comprehensive structural analytical approach developed for more complex carbohydrates was applied to the oligosaccharide alditol in order to reveal glycosyl and glycosyl-linkage composition, sequence arrangements, ring forms, and enantiomeric and anomeric configurations. The structure of the triglycosyl alditol was established as, 4-O-Me-L-Rhap-(alpha 1----4)-2-O-Me-L-Fucp-(alpha 1----3)-L-Rhap- (alpha 1----2)-6-deoxytalitol, in which the nonreducing-end disaccharide unit is unique to serotype 4. The native GPL antigen is diacetylated, presumably at other than the terminal disaccharide, since the antigenicity of both the acetylated and deacetylated antigens are comparable. The structure of the epitope of the type-specific antigen of serotype 4 will serve as the basis for synthetic antigen probes and the target for the monoclonal antibodies required to trace the origins in the environment of the infectious agent and study the epidemiology of human infections.     

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.     

Evaluation of the Korean isolate-1 tachyzoite antigen for serodiagnosis of toxoplasmosis

To evaluate the usefulness of the Korean Isolate-1 (KI-1) antigen for serodiagnosis of toxoplasmosis, antigen profiles of KI-1 tachyzoites were analyzed in comparison with RH tachyzoites by SDS-PAGE and immunoblotting. ELISA was performed on latex agglutination (LA)-positive and negative serum samples using KI-1 and RH antigens. Immunoblotting of the KI-1 antigen showed multiple antigen bands with molecular sizes of 22-105 kDa. Among them, 1 and 6 common bands were noted against a KI-1-infected and a RH-infected human serum, respectively, which represented differences in antigenic profiles between KI-1 and RH tachyzoites. However, all 9 LA-positive human sera were found positive by ELISA, and all 12 LA-negative sera were negative by ELISA; the correlation between the ELISA titers and LA titers was high (r = 0.749). Our results suggest that tachyzoites of KI-1 may be useful for serodiagnosis of human toxoplasmosis.     

Trehalose 6,6′-dimycolate on the surface of Mycobacterium tuberculosis modulates surface marker expression for antigen presentation and costimulation in murine macrophages

Trehalose 6,6′-dimycolate (TDM) is the most abundant lipid extracted from Mycobacterium tuberculosis (MTB). TDM promotes MTB survival by decreasing phagosomal acidification and phagolysosomal fusion in macrophages. Delipidation of MTB using petroleum ether removes TDM and decreases MTB survival within host cells. TDM reconstituted onto MTB restores its virulent wild-type characteristics. We investigated the role of TDM in regulating surface marker expression in MTB-infected macrophages. Macrophages were infected with wild-type, delipidated, and TDM-reconstituted MTB for 24 h and measured for changes in surface marker expression. TDM on MTB was found to specifically target MHCII, CD1d, CD40, CD80 and CD86. Both wild-type and TDM-reconstituted MTB suppressed or induced no change in expression of these surface markers, whereas delipidated MTB increased expression of the same markers. MTB-infected macrophages were also overlaid with MHCII-restricted T cell hybridomas which recognize Antigen 85B. Macrophages infected by wild-type and TDM-reconstituted MTB did not present antigen as well as delipidated MTB-infected macrophages. The evidence shown furthers supports the notion that TDM present on MTB promotes its survival and persistence in host macrophages.     

Characterization of Foot-And-Mouth Disease Viruses (FMDVs) from Ugandan Cattle Outbreaks during 2012-2013: Evidence for Circulation of Multiple Serotypes

To investigate the foot-and-mouth disease virus (FMDV) serotypes circulating in Uganda’s cattle population, both serological and virological analyses of samples from outbreaks that occurred during 2012–2013 were performed. Altogether, 79 sera and 60 oropharyngeal fluid (OP)/tissue/oral swab samples were collected from herds with reported FMD outbreaks in seven different Ugandan districts. Overall, 61/79 (77%) of the cattle sera were positive for antibodies against FMDV by PrioCHECK FMDV NS ELISA and solid phase blocking ELISA detected titres ≥ 80 for serotypes O, SAT 1, SAT 2 and SAT 3 in 41, 45, 30 and 45 of these 61 seropositive samples, respectively. Virus neutralisation tests detected the highest levels of neutralising antibodies (titres ≥ 45) against serotype O in the herds from Kween and Rakai districts, against SAT 1 in the herd from Nwoya district and against SAT 2 in the herds from Kiruhura, Isingiro and Ntungamo districts. The isolation of a SAT 2 FMDV from Isingiro was consistent with the detection of high levels of neutralising antibodies against SAT 2; sequencing (for the VP1 coding region) indicated that this virus belonged to lineage I within this serotype, like the currently used vaccine strain. From the Wakiso district 11 tissue/swab samples were collected; serotype A FMDV, genotype Africa (G-I), was isolated from the epithelial samples. This study shows that within a period of less than one year, FMD outbreaks in Uganda were caused by four different serotypes namely O, A, SAT 1 and SAT 2. Therefore, to enhance the control of FMD in Uganda, there is need for efficient and timely determination of outbreak virus strains/serotypes and vaccine matching. The value of incorporating serotype A antigen into the imported vaccines along with the current serotype O, SAT 1 and SAT 2 strains should be considered.     

Some serological properties ofActinobacillus pleuropneumoniae strains of serotypes 1 through 5

Rabbits were hyperimmunized with live, formalin-killed, and heat-treated antigen preparations of the reference strains of serotypes 1 through 5 ofActinobacillus pleuropneumoniae in order to study the antibody response to both soluble and particulate antigens. The antibody response was studied by means of precipitation, agglutination, coagglutination, indirect hemagglutination, and complement fixation tests.Serotyping ofA. pleuropneumoniae strains was done by ring precipitation (RP) and coagglutination (CoA) tests with unheated and heated cell-saline extract as antigens and rabbit hyperimmune sera produced against either live cultures or formalin-killed whole-cell suspensions. The results showed that live cultures provoked more cross-reactive antibodies in rabbits, thus making the antisera unsuitable for use in serotyping by the RP test when unheated wholecell saline extract was used as antigen. Rabbit hyperimmune serum produced against formalinkilled bacterial suspension gave serotype-specific reactions in the RP test. Boiled or autoclaved cell-saline extracts gave serotype-specific reactions in the RP test even when rabbit anti-livecell sera were used. Serotype-specific reactions were obtained in the CoA test in both rabbit anti-live or anti-formalin-killed cell sera with either unheated or heated bacterial cell suspensions as antigens.Live and formalin-killed whole-cell suspensions as well as their saline extracts provoked a high antibody response in rabbits. Heating the cell suspension at 100°C for 1 h caused a significant reduction in their immunogenic potency, whereas autoclaving (121°C) of the cell suspension for 1 h almost completely destroyed their serotype-specific immunogenic properties, since the antibody response was either absent or very poor and not type-specific. However, neither boiling nor autoclaving of the cell suspensions caused significant reduction in their ability to react with preformed antibodies. Phenol-water-extracted antigens gave the highest degree of serotype specificity in the complement fixation test.     

Serological Characterization of Haemophilus Pleuropneumoniae (Actinobacillus Pleuropneumoniae) Strains and Proposal of a New Serotype: Serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain. In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses. In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

An enzyme-linked immunosorbent assay to detect serum IgG to Pasteurella multocida in naturally and experimentally infected rabbits

An enzyme-linked immunosorbent assay (ELISA) was evaluated for efficacy in detecting serum IgG against Pasteurella multocida in both naturally and experimentally infected rabbits. Blood samples and nasal cultures were taken concurrently from 58 rabbits from four conventional rabbitries. Nine rabbits from a pasteurella-free colony served as negative controls. Fifty-six rabbits were ELISA positive. Of these, 46 were P. multocida culture positive, 10 were culture negative. Two rabbits were ELISA negative, culture negative. There were no ELISA negative, culture positive animals. Serotyping by the gel diffusion precipitin test demonstrated that of the 44 typed P. multocida isolates, 57% were serotype 4, 27% were serotype 12 and 16% were serotype 3. In rabbits experimentally infected intranasally with P. multocida, serum IgG against P. multocida began to rise 21 to 33 days after infection and remained elevated until the animals were euthanized 90 days post infection. Two enzyme-linked immunosorbent assays were compared which used potassium thiocyanate extracts of different serotypes of P. multocida as antigen. The results obtained were similar, suggesting the presence of antigens common to both serotypes.     

Identification of a novel tetrapeptide structure of the Mycobacterium avium glycopeptidolipid that functions as a specific target for the host antibody response

Mycobacterium avium complex (MAC) is a group of non-tuberculous mycobacteria that cause tuberculosis-like diseases in humans. Unlike Mycobacterium tuberculosis, MAC expresses high levels of glycopeptidolipids (GPLs) containing a well-defined tetrapeptide-amino alcohol core, composed of D-phenylalanine, D-allo-threonine, D-alanine, and L-alaninol, that is modified with a fatty acid and sugar residues. Surprisingly, however, a careful scrutiny of the mass spectrum of MAC GPLs revealed the presence of ions that could not readily accountable for the known GPL structure. The magnitude of the ions was increased prominently when GPLs were isolated from the valine-supplemented culture, and the ions representing the authentic GPL species were diminished, suggesting the possibility that the basic structure of the peptide backbone might be altered in response to the exogenously added valine. Indeed, further mass spectrometry (MS)/MS and gas chromatography-MS analysis indicated a substitution of D-valine for the N-terminal D-phenylalanine of the tetrapeptide core, and the presence of D-valine and the absence of D-phenylalanine was confirmed by high-performance liquid chromatography, using the derivatized amino acid residues that were released from the tetrapeptide. Finally, specific antibodies to the purified valine-containing GPL species were detected in the serum of a MAC-infected guinea pig. Therefore, these results identify a new molecular species of MAC GPLs with immunogenic potential.     

Evaluation of three serological tests for the detection of human plague in northeast Brazil.

The passive haemagglutination (PHA) test, enzyme-linked immunosorbent assay (ELISA) and the dot enzyme-immunosorbent assay (DOT-ELISA) were used to detect the levels of IgG antibodies against the Fraction 1 (F1) antigen of Yersinia pestis in sera of plague-infected patients from Northeast Brazil. Twenty three selected PHA-positive sera of subjects with bacteriological confirmation of plague were also positive in the DOT-ELISA but only 19 were detected by the conventional ELISA technique. Another group of 186 serum samples from subjects diagnosed as plague-infected by clinical and epidemiological parameters, but PHA-negative, were screened with DOT-ELISA and 11 gave positive results. The specificity of the assays on the serological detection of plague was confirmed in inhibition tests using purified F1 antigen. These results suggest that DOT-ELISA can be an useful, simple and more sensitive alternative for the serodiagnosis of plague in Northeast Brazil.     

Comparison of cell surface antigen extracts from two serotypes of Pasteurella haemolytica

Cells of Pasteurella haemolytica serotypes A1 and A6 were extracted with sodium salicylate and the chemical and antigenic composition of both extracts determined. The extracts were concentrated by ultrafiltration and the serotype antigen, measured by the indirect haemagglutination test, was estimated to have a molecular weight between 100 000 and 300 000. The chemical composition of sodium salicylate extracts (SSEs) from both serotypes was similar, having protein, carbohydrate, fatty acid and phosphorus present in the ratio 10:1:0.5:0.1. SDS-PAGE of both SSEs gave similar profiles with at least 48 bands present. These results suggest that sodium salicylate removes the outer membrane of P. haemolytica. Crossed immunoelectrophoresis indicated that a major serotype-specific antigen was present in SSEs of both strains. This antigen was extracted from the SSE with hot phenol/water and analysed by gas chromatography. The sugar composition of A1 and A6 phenol/water extract (PWE) was qualitatively identical although some differences in proportions were observed. A1 and A6 PWE antigens protected mice against homologous serotype challenge and A6 PWE protected against heterologous (A1) challenge.     

A novel serotype-specific gene cassette (gltA-gltB) is required for expression of teichoic acid-associated surface antigens in Listeria monocytogenes of serotype 4b

Listeria monocytogenes serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes, gltA and gltB, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either gltA or gltB. The gltA and gltB mutants were characterized by loss and severe reduction, respectively, of glucose in the teichoic acid, whereas galactose, the other serotype-specific sugar substituent in the teichoic acid, was not affected. Within L. monocytogenes, only strains of serotypes 4b, 4d, and 4e harbored the gltA-gltB cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp gltA-gltB cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of L. monocytogenes, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the teichoic acids in the cell wall.     

Antibody mediated suppression of secondary IgM response in nude mice against vesicular stomatitis virus

Nude mice immunized with either of the two serotypes of vesicular stomatitis virus (VSV), VSV Indiana (VSV-Ind) or VSV-New Jersey (VSV-NJ), showed an early T cell independent immunoglobulin (Ig) M antibody response comparable with normal euthymic mice. Unlike euthymic mice, however, nude mice reinjected with the homologous serotype were unable to mount a second measurable serum neutralizing (SN) antibody response; a second injection with the heterologous serotype induced a normal primary type of SN antibody response. The serotype-specific refractoriness to a second challenge recovered at about 10 wk after primary infection. When antibody responses were assayed by enzyme-linked immunoabsorbent assay (ELISA), suppressive effects by previous immunization could be observed even after challenge with the heterologous serotypes; this finding probably reflects the known existence of common nonneutralizing determinants shared between the two serotypes. A weak 2-mercaptoethanol (2-ME)-resistant anti-VSV IgG SN antibody response was noticed during the primary response in nude mice and was also found in ELISA; after second infections, this 2-ME-resistant response did not develop. Serum transfer studies in nude and +/+ mice confirmed that the serotype-specific transitory refractoriness of a second response in nude mice was mediated through the anti-VSV-specific IgM antibodies.     

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

Validity of an enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies for serotyping human rotavirus in stool specimens

We recently developed a method for serotyping human rotavirus (HRV) by an enzyme-linked immunosorbent assay with HRV serotype-specific neutralizing monoclonal antibodies (ELISA serotyping). In the present study this method was compared with the fluorescent focus neutralization test with serotype-specific rabbit antisera (NT serotyping) in the sensitivity and specificity of the test. Direct serotyping of HRVs which were contained in stool specimens indicated that while only 37% of the samples were successfully serotyped in NT, 78% of the samples could be serotyped in ELISA. Regarding the samples whose serotype could be determined in the two tests, the assigned serotypes were identical in both tests. The results obtained indicated the utility of ELISA serotyping in clinical and epidemiologic studies of HRV infection.     

梅毒螺旋体重组抗原酶联免疫吸附试验的建立及应用

建立梅毒螺旋体重组抗原酶联免疫吸附试验（ELISA）,用于梅素血清学诊断和调查。其法,用表达的重组抗原IPN17和TmpA,建立检测血清特异抗体的间接ELISA,并与其它检测方法比较,分别检测梅毒参比血清、病人及献血员血清。其结果,敏感性、特异性均为100％。新建ELISA与TPHA的总符率为95．7％,明显高于RPR与TPHA的总符合率（89．1％）。献血员人群抗体阳性率为0．3％－0．69％,健康人群中抗体阳性率较低。