Use In Serodiagnosis Research Articles

Humoral and cellular immune responses against Type I cysteine proteinase of Leishmania infantum are higher in asymptomatic than symptomatic dogs selected from a naturally infected population

Canids are natural reservoirs of Leishmania infantum and have been promoted as experimental hosts to decipher the pathogenesis of human visceral leishmaniasis (VL). In this study, the presence of IgG antibodies as well as the presence of mononuclear leukocytes reactive to different cysteine proteinases (CPs) were examined in 13 L. infantum-infected dogs (six with symptoms, seven asymptomatic). Cysteine proteinases which belong to papain-like enzymes known as clan CA are the most studied CPs of parasite protozoa. These molecules are expressed by the intracellular stages of the parasite and could be immunogenic. We studied Type II CP (CPA) and Type I CP (CPB) with its long C-terminal extension (CTE) which could be highly immunogenic. We showed that the level of antibodies reactive to rCPA is low in both symptomatic and asymptomatic dogs. In contrast, when CPB and CTE were used as antigens, the level of total IgG (with IgG2 superior to IgG1) reached higher values in asymptomatic dogs than in dogs with VL. While the peripheral blood mononuclear cell (PBMC) reactivity was significant when cultured in the presence of freezed/thawed (F/T) lysate, it remained low in presence of CP although always higher for PBMC recovered from asymptomatic dogs. We showed the importance of CPB and CTE in particular as a target of immune response and their potential use for serodiagnosis in asymptomatic dogs.

Analysis of the shotgun expression library of the Mycobacterium tuberculosis genome for immunodominant polypeptides: potential use in serodiagnosis.

A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the lambdagt11 vector. DNA from a virulent M. tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides. beta-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis. The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%.

AFLMP1 encodes an antigenic cel wall protein in Aspergillus flavus.

We have previously reported the cloning and characterization of the MP1 gene in Penicillium marneffei and the AFMP1 gene in Aspergillus fumigatus and their use for serodiagnosis of penicilliosis and aspergilloma and invasive aspergillosis, respectively. In this study, we describe the cloning of the AFLMP1 gene, which encodes the homologous antigenic cell wall protein in Aspergillus flavus, the most common Aspergillus species associated with human disease in our locality and in other Asian countries and the second most common Aspergillus species associated with human disease in Western countries. AFLMP1 codes for a protein, Aflmp1p, of 273 amino acid residues, with a few sequence features that are present in Mp1p and Afmp1p, the homologous antigenic cell wall proteins in P. marneffei and A. fumigatus, respectively, as well as several other cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Aflmp1p antibody was generated with recombinant Aflmp1p protein purified from Escherichia coli to allow further characterization of Aflmp1p. Indirect immunofluorescence analysis indicated that Aflmp1p is present on the surface of the hyphae of A. flavus. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. flavus develop a specific antibody response against Aflmp1p. This suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with aspergilloma or invasive aspergillosis, and the protein may represent a good cell surface target for host humoral immunity.

Identification of a specific antigenic region of the P82 protein of Babesia equi and its potential use in serodiagnosis.

The efficacy of the Be82 gene product fused with glutathione S-transferase (GST/Be82) in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Babesia equi infection was reported previously (H. Hirata et al., J. Clin. Microbiol. 40:1470-1474, 2002). However, the ELISA with the GST/Be82 antigen cross-reacted with Babesia caballi-infected horse sera, despite the high rate of detection of B. equi. These results suggested that GST/Be82 has an antigen in common with B. caballi or antigenicity similar to that of B. caballi. In the present study, we constructed a series of five clones with deletions in the Be82 gene product, each of which was fused with GST, and used them in ELISAs in order to overcome the cross-reactivity seen with B. caballi. One of the deletion clones, a clone with a deletion of the Be82 gene from positions 236 to 381 (Be82/236-381), specifically and sensitively detected B. equi-infected horse sera without cross-reactivity with B. caballi-infected horse sera. Assays with clones from which other gene products were deleted showed decreased sensitivities or remained nonspecific for the detection of B. equi-infected horse sera. These results suggest that the Be82/236-381 gene product is a novel antigen for the diagnosis of B. equi infection in horses.

Candida albicans cell wall antigens for serological diagnosis of candidemia.

Serological tests for diagnosis of disseminated fungal infections in the immunocompromised host are used with varying results. In the present study, the relative ability of antibodies to specifically recognize Candida albicans cell wall components was evaluated in order to find antigenic markers for serological diagnosis of candidemia. Native C. albicans cell wall fragments (CW), periodate- (CWIO4) and proteinase-K- (CWP) treated CW, a mildly extracted phosphopeptidomannan (PPM), and beta(1-3)(1-6)-glucan were used as antigens in ELISA with sera from rabbits immunized with C. albicans (n = 10), patients with culture proven candidemia (n = 8) and healthy individuals (n = 8). The antibody response in rabbits consisted predominantly of anti-PPM antibodies, a finding that was substantiated by inhibition-ELISA. Consistently, periodate treatment (CW104) destroyed a major proportion of the antigenic epitopes. Low rabbit antibody levels were found against glucan, the major Candida cell wall component. These results supported the conclusion that glucan is localized mainly in the inner part of the C. albicans cell wall. In contrast to rabbits' serum IgG antibody response against PPM, which was at least tenfold higher than that raised against CW, patients with candidemia had similar IgG antibody levels against both antigens. These levels were significantly higher than those seen in healthy controls (CW, P = 0.0005 and PPM, P < 0.0001). Although the human anti-glucan and anti-CWIO4 IgG antibody levels were low overall, they were nonetheless significantly increased in the patient group (P = 0.0159 for antiglucan and P = 0.0491 for anti-CWIO4). In addition, a correlation was noticed between levels of these antibodies. No significant differences were found between patients and controls for IgM antibodies when CW, CWIO4, PPM and Glu were used as antigens. In conclusion, IgG antibodies to PPM and native cell wall fragments (CW) were highly discriminatory for recognition of candidemia and these antigens are thus promising candidates for use in serodiagnosis.

Expression of Cysteine Proteinase of Clonorchis sinensis and Its Use in Serodiagnosis of Clonorchiasis

Expression of Cysteine Proteinase of Clonorchis sinensis and Its Use in Serodiagnosis of Clonorchiasis

Cloning and expression of a Helicobacter bilis immunoreactive protein.

In an effort to identify immunoreactive Helicobacter bilis antigens with potential for serodiagnosis, sera from mice experimentally infected with H. bilis were used to screen an H. bilis genomic DNA expression library. Among 17 immunoreactive clones, several contained sequences that encoded a predicted 167-kDa protein (P167). Five overlapping P167 peptides (P167A to P167E) of approximately 40 kDa each were generated and tested. Immune sera reacted with fragments P167C and P167D at dilutions of 1:1,600 and 1:6,400, respectively, and reacted with an H. bilis membrane extract at a dilution of 1:800 in an enzyme-linked immunosorbent assay. Sera from mice experimentally infected with H. hepaticus did not react with P167C and P167D. Sera from mice naturally infected with H. bilis but not sera from mice naturally infected with H. hepaticus reacted with P167C and P167D. Hyperimmune sera against P167C peptide reacted with recombinant P167C and with a 120-kDa band in H. bilis lysates but did not react with a protein of the same size on immunoblots prepared from H. hepaticus, H. muridarum, or unrelated Borrelia burgdorferi and Campylobacter jejuni whole-cell lysates. Nevertheless, the P167A, P167B, P167C, and P167D primers, but not the P167E primers, amplified DNA from H. hepaticus, and all five primer sets amplified DNA from H. muridarum. These results suggest that P167 is an immunodominant, H. bilis-specific antigen that may have potential for use in serodiagnosis.

Evaluation of Chlamydia pneumoniae 43- and 53-kilodalton recombinant proteins for serodiagnosis by Western Blot.

Chlamydia pneumoniae is a common cause of respiratory infection. It has also been shown to be associated with coronary heart disease. Two proteins that have been reported to be recognized frequently during human infection are proteins having molecular masses of 43 and 53 kDa. In order to develop a useful alternative serological test to the microimmunofluorescence (micro-IF) assay, recombinant 43-kDa and 53-kDa chlamydia-specific proteins were evaluated in dot blot and/or for comparison to the standard micro-IF test. Primers for amplification were derived from genome sequence information for two C. pneumoniae genes (CPn0809 and CPn0980) encoding 53-kDa proteins and four C. pneumoniae genes (CPn0562, CPn0927, CPn0928, and Cpn0929) encoding 43-kDa proteins of unknown function, which were Chlamydia specific and not found in Chlamydia trachomatis. The 53-kDa protein product of CPn0809 or the N-terminal 18-kDa portion had better specificity than any of the 43-kDa recombinants but was much less sensitive than micro-IF. In contrast, the 53-kDa protein encoded by CPn0980 was recognized by 11 of 12 (92%) acute-phase sera, 35 of 46 (76%) chronic sera, 0 of 12 micro-IF-negative sera (C. pneumoniae and C. trachomatis negative), and 1 of 12 (8%) C. pneumoniae negative, C. trachomatis positive sera. Thus, it appears that the 53-kDa protein encoded by CPn0980 has potential use for serodiagnosis of C. pneumoniae infection.

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7.

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49)...

Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis.

Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora lambda gt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecular masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined "gold standard" panel of bovine sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate-based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.

Serodiagnosis of tuberculosis and leprosy by enzyme immunoassay.

OBJECTIVE: To evaluate the use of serodiagnosis for tuberculosis and leprosy using mycobacterial antigen 38 kDa, with kits from Omega laboratories, to detect IgG by enzyme immunoassay (EIA). METHODS: The study population consisted of 58 patients with evidence of tuberculous infection (culture of Mycobacterium tuberculosis complex or microscopic evidence), of whom 23 had pulmonary and 35 had extrapulmonary disease. There were six subjects who had recently been treated for tuberculosis, 11 patients on treatment for leprosy and 137 patients suspected of having tuberculosis on clinical or radiologic grounds (without laboratory evidence). A control group comprised 35 healthy individuals or patients suffering from diseases other than tuberculosis. RESULTS: The tests showed that there was a significant difference in antibody levels between the patients with active pulmonary disease, extrapulmonary tuberculosis and leprosy in comparison with the control group (p<0.001). The sensitivities of the two tests together for proven pulmonary tuberculosis were 100% and 95.7% at 1.0--1.5 and >1.6 EIA cut-off points respectively, while the specificities were 88.5% and 100% at the same cut-off points. The sensitivities for extrapulmonary tuberculosis were 71.4% and only 51.4% at 1.0--1.5 and >1.6 EIA cut-off points. The test was positive in 30 (21.9%) of the 137 suspected patients, while 43 (31.4%) had an equivocal result and the remaining 64 (47.7%) suspects were definitely negative. There was again a significant difference in positivity rates between suspects and the control group. CONCLUSIONS: Omega IgG test is useful in the serodiagnosis of active pulmonary tuberculosis and leprosy, but less sensitive in extrapulmonary disease, particularly in children. Equivocal results may only add to the evidence of tuberculosis in early or minimal disease.

Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium

Cowdria ruminatium, the causative agent of heartwater disease, expresses an immunodominant and conserved 32-kilodalton protein (MAP1; formerly called Cr32), which is currently in use for serodiagnosis of the disease. The gene encoding this protein, designated map1, was detected, cloned, and characterized. The gene is conserved between four different stocks of C. ruminantium originating from Senegal, Sudan, South Africa, and Zimbabwe. Homology searches revealed MAP1 to be homologous to the Anaplasma marginale surface protein MSP4, a potential protective antigen. The MAP1 protein, expressed in Escherichia coli fused with glutathione S-transferase, is specifically recognized by sera from animals infected with seven different stocks of C. ruminantium.

Antigenic analysis of Leishmania isolates from Tachira state, Venezuela

Studies of cutaneous leishmaniasis in 3 endemic foci in Tachira state, western Venezuela have revealed sympatric populations of parasites causing both cutaneous and mucocutaneous disease. Immunological techniques and measurement of protease/acid phosphatase activities have been used to detect species-specific parasite antigens from 3 isolates from Tachira. Identified antigens of particular interest had molecular masses of 100, 82, 66, 50 and 27 kDa, but there was a high degree of heterogeneity between the antigens of the Tachira isolates and other Venezuelan strains of Leishmania braziliensis and L. mexicana. This heterogeneity has implications concerning the selection of antigens for use in serodiagnosis of leishmaniasis.

Comparaison de Deux Antigénes de Nature polysaccharidique pour le Diagnostic Sérologique par ELISA de la Pleuropneumonie Porcine (Sérotype 5)

Two antigens of polysaccharidic nature were evaluated for their specificity in serodiagnosis of porcine pleuropneumonia (serotype 5). Recently, long chains lipopolysaccharides (LC-LPS) was shown to increase the specificity of the ELISA test for A. pleuropneumoniae serotype 5 compared to a saline extract of boiled-formalinized whole cells. Nevertheless, some cross reactions were noticed with LC-LPS. We have thus cleaved the LPS molecule at the ketosidic bond between the lipid A and the core. The lipid A was separated from the polysaccharide (PS) by centrifugation. With an indirect sandwich ELISA, it was possible to demonstrate that the PS yielded positive responses with homologous sera (serotype 5) and negative responses with heterologous sera (serotype 3 and undeterminated serotype). Overall, the use of the PS did not notably increase the specificity of the ELISA test. Since production of PS from LC-LPS and its separation from the lipid A is not simple, we do not recommend its use in serodiagnosis.

Molecular diagnosis of parasites

New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene cloning and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.

Comparison of adult somatic and excretory-secretory antigens in enzyme-linked immunosorbent assay for serodiagnosis of human infection with Paragonimus heterotremus

Adult somatic antigen extract of Paragonimus heterotremus was compared with excretory-secretory (ES) antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of human paragonimiasis. The absorbence values in ELISA using the adult somatic antigen were not significantly different from the values obtained using ES antigens ( P>0·05). The sensitivity of the ELISA using either antigen was 100%, but the specificity was 96% with somatic and 98% with ES antigens due to a cross reaction with fascioliasis sera. It appears that both somatic and ES antigens are effective antigens for use in serodiagnosis of human paragonimiasis heterotremus.

Overproduction and purification of Treponema pallidum recombinant-DNA-derived proteins TmpA and TmpB and their potential use in serodiagnosis of syphilis.

We report the construction of expression plasmids carrying two Treponema pallidum genes encoding for the 42-kilodalton membrane protein TmpA (treponemal membrane protein A) and the 34-kilodalton membrane protein TmpB. Using the leftward promoter of bacteriophage lambda, which is controlled by a thermosensitive repressor, we obtained a high level of heat-inducible synthesis of TmpA and TmpB in Escherichia coli K-12. Both proteins were purified to near homogeneity, and the presence of antibodies to TmpA and TmpB in human sera was determined by an enzyme-linked immunosorbent assay. Whereas in all 44 serum samples from untreated patients in the secondary and early latent stages of syphilis, high levels of anti-TmpA antibodies were detected, only 34 serum samples contained anti-TmpB antibodies. As has been previously observed for TmpA, a correlation was found between the presence of anti-TmpB antibodies and anti-cardiolipin antibodies, suggesting that the level of antibodies to TmpB drops soon after successful antibiotic treatment. We concluded that, in contrast to TmpA, TmpB is not suitable for serodiagnostic purposes as a single antigen, because a significant fraction of sera from syphilitic patients was nonreactive with TmpB.

Application of indirect hemagglutination test and indirect fluorescent antibody test for IgM antibody for diagnosis of melioidosis in Thailand.

In hyperendemic areas such as Thailand, rapid diagnosis of melioidosis depends upon both bacteriological culture and serological methods. However, interpretation of indirect hemagglutination (IHA) for melioidosis which is the only test available, is seriously hampered by increased IHA titers present in one-third to one-half of the population. In order to get the best results from the available tests, IHA and indirect fluorescent antibody for IgM (IFA-IgM) were evaluated in controls and patients in Thailand. IHA titers of greater than or equal to 1:40 were considered remote or recent exposure to P. pseudomallei. IHA titers of this level were found in 47.1% of 227 blood donors and 29.5% of 210 sera submitted for other tests, while IFA-IgM was positive in only one donor who had an IHA titer of 1:1,280. IHA was positive in eight out of nine patients with melioidosis with IHA titers of less than 1:20 to 1:2,560. IFA-IgM was positive in six out of seven melioidosis patients whose sera were available for this test including a serum with IHA titer of less than 1:20. Six patients were predisposed by diabetes mellitus. Among sera serologically tested for melioidosis, 33 had IHA titers of 1:80-1:1,280, 10 of which were positive for IFA-IgM. This study demonstrates high background IHA titers among IHA titers among Thai people which greatly limits its use for serodiagnosis of melioidosis. In sharp contrast, serodiagnosis by IFA-IgM was more successful. Positive IFA-IgM among healthy Thais did exist indicating that serologic tests for melioidosis at best are only supplementary to bacteriological culture and clinical awareness.

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl-β-d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl-β-d-glucopyranoside in 65% yield, and with absolute stereospecificity for the β anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl-β-d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl-α-d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the β-linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3′,6′-di-O-methyl-β-d-glucopyranosyl)-α-l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.