Species-specific Monoclonal Antibodies Research Articles

A Rapid and Quantitative Detection System for Streptococcus mutans in Saliva Using Monoclonal Antibodies

A new semiquantitative enumeration system was developed to detect Streptococcus mutans in saliva. Using species-specific monoclonal antibodies, the system quickly detected salivary S. mutans in 30 min and classified the results into three levels. In this study, saliva samples collected from 28 young adults aged between 22 and 24 years were subjected to the monoclonal antibody-based detecting system and selective medium-based detecting methods. The results generated from the PCR-confirmed culture method indicated the mean salivary S. mutans counts at level 1, 2 and 3 were 5.7 × 10⁴, 1.3 × 10⁵ and 3.4 × 10⁶ CFU/ml, respectively. The differences between level 3 and 1 and level 3 and 2 were statistically significant (one-way ANOVA; p < 0.01). The results generated from the system were consistent with the data generated from two culture-based commercial products.

Enumeration and detection of aerosolized Aspergillus fumigatus and Penicillium chrysogenum conidia and hyphae using a novel double immunostaining technique

The identification of collected airborne unicellular fungal conidia and hyphae using nonviable techniques is subjective and an imprecise process. Similarly, to determine whether an individual is allergic to a particular genus requires a separate immunodiagnostic analysis. This study demonstrates the development of a novel double immunostaining halogen assay, which enables (1) the simultaneous identification of collected airborne fungal conidia and hyphae of Aspergillus fumigatus and Penicillium chrysogenum using monoclonal antibodies and (2) the demonstration of patient-specific allergy to the same particles using human serum IgE. The results demonstrate that when conidia were ungerminated the binding of antibodies was homogeneous and localized in close proximity around the entire conidia for both species. However, when conidia were germinated, the proportion expressing antigen increased ( P < 0.0001) for both species and the sites of binding of the two antibodies changed with double immunostaining restricted to the hyphal tips for A. fumigatus, in addition to the sites of germination for P. chrysogenum. The described immunoassay has the potential to identify fungal particles in personal environmental air samples, provided species-specific monoclonal antibodies are available, while simultaneously demonstrating allergic sensitization to the same particles by co-staining the samples with the patient's own serum. Such an immunoassay can use those fungi that the patient is actually exposed to and potentially avoids many problems associated with extract variability based on the performance of current diagnostic techniques for fungal allergy.

Evaluation of Nonculture Methods for the Detection of Prosthetic Hip Biofilms

Authors of previous studies have found that culture-based detection of prosthetic hip infections can be improved by adherence to strict anaerobic protocols and mild ultrasonication of retrieved prostheses to remove bacteria growing as an adherent biofilm. Furthermore, direct analyses of sonicate samples by immunofluorescence microscopy (with genus-specific and species-specific monoclonal antibodies or polyclonal antisera) produces significantly greater and more rapid detection rates for infection compared with culture alone. Despite its diagnostic value within a research setting, the practical advantages and limitations of immunofluorescence microscopy for a busy diagnostic laboratory needs consideration. In addition, the method must be evaluated against other nonculture-based techniques that may prove more appropriate. In this review, we describe how immunofluorescence microscopy presents an attractive and reliable method for routine detection of prosthetic hip biofilms compared, primarily, to polymerase chain reaction, although other nonculture methods of diagnosis based on fluorescent in situ hybridization and serologic analysis are considered. We describe how the immunofluorescence microscopy technique is robust, relatively simple and, in contrast to broad-range 16S recombinant-deoxyribonucleic-acid-based polymerase chain reaction, does not require a separate clean-room facility or strict adherence to aseptic techniques. More importantly we highlight how dislodged biofilm, which appears as large aggregates of bacteria, easily can be identified from skin contaminants that occur as single cells or small aggregates of only a few cells.

Partial characterization of a species-specific stachybotrys chartarum antigen

RATIONALE: The presence of Stachybotrys in water-damaged homes and businesses has received a great deal of attention because this mold produces several potent mycotoxins, but the association between Stachybotrys and building related disease is controversial. We have developed a species-specific spore-specific monoclonal antibody (MAb) 9B4 that could be used for monitoring environmental contamination. The purpose of this work was to characterize the antigen recognized by the MAb 9B4.

Tetracycline-regulated highly inducible expression of the human prion protein in murine 3T3 cells

To provide an in vitro system that allows inducible or conditional overexpression of human prion protein (PrP), we have established a tetracycline (Tc)-regulated system in murine 3T3 L1 fibroblast cells. A replacement-type gene targeting vector cassette was constructed to express the human fatal familial insomnia ( FFI) prion protein gene ( PRNP) under control of a Tc-responsive element. Following stable integration of the vector into 3T3 Tet-Off cells, we have isolated and characterised six 3T3 L1 pTet-Off FFI clones. These clones were analysed by PCR and their expression level was determined by Western blot using species specific monoclonal antibodies (anti-mouse and human 3B5, 4F2, 12F10, 11C6, 8G8, and 14D3; anti-mouse l3). Addition of the antibiotic Tc to the culture medium turned off expression of human PrP. This supression was repeatedly reversible. However, no significant transcriptional leakiness of repressed P minCMV promoter was observed. In the absence of Tc, expression of human PrP was induced 10- to 20-fold as estimated from densitometric analyses. PrP was analysed by Proteinase K (PK) digestions and found to be PK sensitive. Subcellular fractionation revealed that PrP was located mainly in the cytoplasmic membrane fraction. Furthermore, we partially purified PrP by PrP-specific copper-binding. After immobilised metal affinity chromatography, majority of PrP showed a molecular weight consistent with non-glycosylated PrP. These clones offer a new tool to facilitate the investigation of PrP interaction with potential cellular ligands and PrP ex vivo propagation.

Monoclonal Antibodies for Specific Detection of Encephalitozoon cuniculi

Seven species-specific monoclonal antibodies (MAbs) were produced against Encephalitozoon cuniculi and characterized. The MAbs were immunoglobulin G, and when used for indirect microimmunofluorescence microscopy and Western immunoblot assays, they detected E. cuniculi originating from clinical samples. They did not cross-react with other Encephalitozoon species (E. intestinalis and E. hellem) or with a collection of gram-negative bacteria, yeast, and other parasites. The MAbs reacted primarily with 121-, 56-, 45-, 43-, and 41-kDa protein epitopes of E. cuniculi. These epitopes were demonstrated to be E. cuniculi species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We developed MAbs to strains of E. cuniculi that can be used successfully to distinguish E. cuniculi from other microsporidial species in cultures established from clinical specimens. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification and diagnosis of infections due to microsporidia.

Anti-keratin Monoclonal Antibodies for Identifying Animal Hair Fibers

Type II keratins from cashmere are used as antigens to produce species-specific monoclonal antibodies for fiber identification purposes. Balb/c mice are immunized with the keratins purified by two-dimensional preparative gel electrophoresis. Hybridoma clones showing high immunological responses are subcloned by limiting dilution and then expanded to obtain antibodies in large amounts. When the antibodies are screened by two-dimensional immunoblotting for immunoreactivity with keratins isolated from cash mere and wool, several differences are detected, both quantitative and qualitative. Al though further work is needed to develop routine analytical protocols, the preliminary results reported in this paper appear to be extremely promising, and our approach could contribute to solving the problem of the objective identification of cashmere and other speciality animal fibers.

Origin of the bursal secretory dendritic cell.

The origin of vimentin-positive secretory dendritic cells of the bursa of Fabricius was studied by chick-quail chimera, parabiosis and immunohistochemistry using species-specific monoclonal antibodies. Quail bursal primordia of different ages were transferred to coelomic cavity of 3-day-old chicken embryos and further incubated for 18 days. In transplanted quail bursas the secretory dendritic cells of chicken and quail origin were detected by double staining of vimentin plus 74.3 and vimentin plus QCPN monoclonal antibodies, respectively. In bursal primordia of 5- and 6-day-old quail embryos both dendritic cells and B cells were of host, i.e. chicken origin. Mixed dendritic cell population of quail and chick origin emerged in chimeric birds of 6.5 days of age. In quail embryos transplanted at 7 and 8 days of age both dendritic cells and B cells were mixed i.e. of chicken and quail origin. Bursal secretory dendritic cells and medullary epithelial cells create "dendro-epithelial tissue" to receive pre-B cells. Colonization of dendro-epithelial tissue by pre-B cells initiates at day 7, thus the colonization of bursal anlage by blood-borne cells is a two-step process; entering of dendritic cells at day 6.5 is followed by that of B cells at day 7 and afterwards. It is discussed that bursal secretory dendritic cells and their product are key elements of bursal function therefore the mammalian bursa equivalent organ might be represented by a cell, which is analogous with the bursal secretory dendritic cell.

Near-infrared Spectroscopic Method to Identify &lt;I&gt;Cotesia flavipes&lt;/I&gt; and &lt;I&gt;Cotesia sesamiae&lt;/I&gt; (Hymenoptera: Braconidae)

Parasitoids of the Cotesia flavipes complex (C. flavipes and Cotesia sesamiae) are natural enemies of stemboring lepidopteran pests in sub-Saharan Africa. The two species are difficult to differentiate using morphological markers, and a quick, reliable test was sought for their correct identification. After numerous, unsuccessful attempts at developing species-specific monoclonal antibodies that could differentiate between the two species, we were successful in using near-infrared spectroscopy to distinguish the cocoons of the two species with an accuracy of better than 85%. Calibrations were established using partial least squares analysis, enabling identification of cocoons of known species, cocoons from an alternative host, as well as in blind tests. This technology would greatly expedite identification of field-caught insects used to determine ecological parameters and parasitization rates of an individual species.

Detection of circulating gp43 antigen in serum, cerebrospinal fluid, and bronchoalveolar lavage fluid of patients with paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is an important systemic fungal disease, particularly among individuals living and working in rural areas of endemicity in Latin America, who, without antifungal therapy, may develop fatal acute or chronic infection. For such patients, the detection of antibody responses by immunodiffusion is of limited value due to false-negative results. In contrast, the detection of Paracoccidioides brasiliensis gp43 circulating antigen may represent a more practical approach to the rapid diagnosis of the disease. Accordingly, an inhibition enzyme-linked immunosorbent assay (inh-ELISA) was developed for the detection of a 43-kDa P. brasiliensis-specific epitope incorporating a species-specific murine monoclonal antibody. With sera from patients with acute and chronic forms of the disease (n = 81), the overall sensitivity of the test was found to be 95.1%, while specificity was found to be 97.5% compared to that with normal human sera from blood donors (n = 93) and sera from patients with other chronic fungal infections (histoplasmosis [n = 33] and cryptococcosis [n = 20]). The inh-ELISA detected circulating antigen in 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM according to the patient's clinical presentation. Cerebrospinal fluid from 14 patients with neuroparacoccidioidomycosis and 13 samples of bronchoalveolar lavage fluid from patients with pulmonary unifocal PCM were also tested for gp43 detection, with the test showing 100% sensitivity and specificity. This novel, highly specific inh-ELISA represents a significant addition to the existing tests for the diagnosis of PCM.

Detection of R. felis and R. typhi in fleas using monoclonal antibodies.

Rickettsia typhi and R. felis are flea-transmitted human pathogenic rickettsial species. To investigate the distributional dynamics of these rickettsiae we designed a micro-immunofluorescence assay (MIF) using species-specific monoclonal antibodies (MAbs) applied to flea cryosections. Our assay was performed in less than 3 h and its applicability was demonstrated by the detection of R. typhi in 50 artificially infected human body lice but in none of 50 uninfected lice. With MIF, we identified 31 positive among 32 fleas proven with PCR to be naturally infected with R. felis; and 7 positive among 32 fleas proven with PCR to be naturally infected with R. typhi. No cross-detection was observed with both MAbs. Fresh R. felis-infected fleas were significantly more MIF-positive than long conserved R. typhi-infected fleas (31/32 vs. 7/32, P < 0.01). This discrepancy may be linked to degradation of antigens by long-term freezing. For R. typhi-infected fleas, our assay was significantly more efficient when applied to fleas in early stages of infection (less than 15 days) by comparison with fleas frozen more than 20 days after infection (7/15 vs. 0/17, P = 0.01). This difference may be related to an antigenic modification caused by selection pressure in the vector and host process. The sensitivity of the described method did not exceed 47% (7/15) for R. typhi but, in contrast, was 97% for R. felis. Thus, our method appears to be useful for surveillance in R. felis infections, but requires further studies for the detection of R. typhi.

Production and characterization of species-specific monoclonal antibodies against Actinomyces naeslundii and Lactobacillus casei.

Dental caries is a disease induced by a few cariogenic bacterial species. Quantitative detection of these cariogenic bacteria could provide useful information for caries risk assessment. In this study, we developed highly species-specific monoclonal antibodies (MAbs) against the type strains of Actinomyces naeslundii ATCC 12104 and Lactobacillus casei ATCC 11578. Assayed with immunoprecipitation and immunofluorescent microscopy, these antibodies showed high sensitivities and specificities in detecting A. naeslundii and L. casei in saliva. Examining 100 saliva samples using these MAb-based techniques, we found that the number of A. naeslundii in saliva ranges from 0.5 x 10(4) to 4.8 x 10(5) and that the number of L. casei in saliva ranges from 1 x 10(4) to 1.2 x 10(6). With fluorescent imaging techniques and confocal laser scanning microscopy (CLSM), these antibodies were used to visualize A. naeslundii and L. casei within dental plaques derived from stimulated whole human saliva in vitro. The study demonstrated that these MAbs were also able to effectively detect A. naeslundii and L. casei in plaque.

Molecular and immunological evidence of oral Treponema in the human brain and their association with Alzheimer's disease

The purpose of this investigation was to use molecular and immunological techniques to determine whether oral Treponema infected the human brain. Pieces of frontal lobe cortex from 34 subjects were analyzed with species-specific PCR and monoclonal antibodies. PCR detected Treponema in 14/16 Alzheimer's disease (AD) and 4/18 non-AD donors (P < 0.001), and AD specimens had more Treponema species than controls (P < 0.001). PCR also detected Treponema in trigeminal ganglia from three AD and two control donors. Cortex from 15/16 AD subjects and 6/18 controls contained Treponema pectinovorum and/or Treponema socranskii species-specific antigens (P < 0.01). T. pectinovorum and/or T. socranskii antigens were also found in trigeminal ganglia and pons from four embalmed cadavers, and 2/4 cadavers also had Treponema in the hippocampus. These findings suggest that oral Treponema may infect the brain via branches of the trigeminal nerve.

Characterization of Agrobacterium vitis Strains Isolated from Turkish Grape Cultivars in the Central Anatolia Region.

Crown gall was detected in several vineyards in the Central Anatolia region of Turkey. Vineyards were planted to cultivars of grape that originated in Turkey and that were not grafted. The predominant species isolated from galls consisted of tumorigenic strains of Agrobacterium vitis. They were identified based on reactions to standard biochemical and physiological tests, by polymerase chain reaction amplification of specific Ti plasmid and chromosomal sequences, and by reaction to a species-specific monoclonal antibody. All strains utilized octopine, suggesting that they may carry similar types of Ti plasmids. Some of the strains exhibited a differential host range compared with others and were less virulent based on the numbers of galls that they induced on grape. When grapevines were treated with nontumorigenic A. vitis strain F2/5 prior to inoculation with the Turkish A. vitis strains, crown gall was effectively controlled. The genetic diversity of strains was evaluated by comparing DNA fingerprints that were generated by restriction enzyme digestion of the intergenic spacer region that lies between 16S and 23S rRNA genes. They segregated into two main groups, one that is similar to previously identified A. vitis strains carrying octopine type Ti plasmids and one that was more similar to strains carrying nopaline and vitopine Ti plasmids. The strains of A. vitis from Turkey may represent ancestral forms of the pathogen that will provide insight into the evolution of the bacterium.

Identification of antigenically distinct populations of Leishmania ( Viannia) guyanensis from Manaus, Brazil, using monoclonal antibodies

Eighty Leishmania isolates from patients who contracted cutaneous leishmaniasis in the Manaus region, Amazonas State, Brazil, were identified as Leishmania ( Viannia) guyanensis by the electrophoretic profiles of six enzymes. None reacted with the species-specific monoclonal antibody B19. Two L. ( V.) guyanensis subpopulations were detected with the monoclonals B2 and B12. The lack of B19 expression by the L. ( V.) guyanensis strains in the present study contrasts with that of the vast majority of the strains of the same parasite from eastern Amazonia and French Guyana that express the epitope.

The First Isolation ofChlamydia pneumoniaefrom a Korean Patient

Background : Chlamydia pneumoniae is one of common causes in upper and lower respiratory infections. Isolating C. pneumoniae from clinical specimens is very difficult due to the characteristics of the organism. Recently, we succeeded in isolating C. pneumoniae from a Korean patient, who suffered from acute pharyngitis. This is the first isolate from a clinical specimen in Korea. Methods : We attained a nasopharyngeal swab from a 22-year-old female patient, and inoculated it on a monolayer of the Hep-2 cell line. After 8 passages, we found the inclusion bodies of C. pneumoniae by an immunofluorescence(IF) test. The species-specific monoclonal antibody IF staining and species-specific PCR were done to confirm the species of the isolate, and electron microscopy was used to characterize the morphology. Results : The isolated was confirmed to be C. pneumoniae by species-specific IF and PCR, and the strain was named LKK-1. The shape of the elementary body was round and with a narrow periplasmic space, as shown by electron microscopy, which is similar to the Japanese strain, but not the Western strain. Conclusion : We succeeded in isolating C. pneumoniae from a 22-year-old patient with acute pharyngitis, which is the first isolate in Korea. In the future, this Korean strain will be useful to the study of C. pneumoniae.

Overexpression of the mouse Fas gene in human Hep3B hepatoma cells overcomes their resistance to Fas-mediated apoptosis

Background / Aims : Fas-induced apoptosis is one of the main forms of apoptosis occurring in hepatocytes. We have previously demonstrated that the human hepatoma cell line Hep3B is resistant to Fas-mediated apoptosis. In this study, we investigated whether the human Fas receptor itself, or the Fas transduction pathway was responsible for the resistant phenotype. Methods : Clones of Hep3B cells overexpressing the mouse Fas gene (Hep3B mfas ) were generated by transfection, and apoptosis was studied by (i) chromatin condensation and nuclear fragmentation, (ii) flow cytometry, (iii) DNA fragmentation and (iv) poly (ADP-ribose) polymerase cleavage. Results : Use of the species-specific and agonistic anti-mFas monoclonal antibody (JO2), showed that the mFas receptor was correctly routed to the plasma membrane of Hep3B mfas cells. Using the four above-mentioned criteria, we demonstrated that JO2 triggered mFas-mediated apoptosis of Hep3B mfas , but not of Hep3B pCi cells (transfected with an empty vector). Conclusions : Our data show (i) that the Fas signaling pathway can be completed when a functional mFas receptor is expressed in Hep3B cells, and thus, (ii) that the death-inducing signaling complex components and the effector caspases are functional in Hep3B cells. Moreover, they suggest that the Fas subunits are not pre-assembled at the cell membrane before receptor–ligand interaction.

First Report of Colletotrichum acutatum on Kalmia.

Colletotrichum acutatum J. H. Simmonds was isolated from diseased leaves of ornamental Kalmia latifolia L. (mountain laurel) cvs. Carousel and Peppermint on plants imported from the United States to Edinburgh, Scotland, in December 1999. Symptoms included sunken, desiccated, darkened necrotic areas, primarily at the leaf tip. Necrotic areas advanced toward the leaf base and were bordered by purple/red pigmentation. Isolations were made from salmon colored conidiomata that developed on abaxial leaf surfaces following incubation in a humidity box at 25°C for 7 to 10 days. White aerial mycelia, becoming gray to grayish beige, and producing salmon to orange colored conidial masses, formed on potato dextrose agar after 10 to 14 days. Conidia were hyaline, aseptate, fusiform to slightly irregular, and measured 13.4 to 13.8 × 4.3 to 4.9 μm. Both morphological and conidial characteristics were consistent with the description of C. acutatum (2). The identity of isolates was further verified by positive plate-trapped antigen ELISA of conidial preparations using a species-specific monoclonal antibody (1). Pathogenicity was assessed by inoculating the adaxial surface of healthy leaves of both cultivars of the imported plants with colonized agar disks and a range of spore suspensions (30, 300, and 3,000 spores delivered in 30 μl volumes) from test fungal isolates and a confirmed laboratory strain (three replicates per treatment). To ensure inoculum uptake, two 5 mm2 areas of cuticle on either side of the mid-rib of each leaf were lightly scratched with a sterile hyperdermic needle prior to inoculation. Inoculated leaves were incubated in a humidity box at 25°C for up to 3 weeks. Symptom development was progressive but relatively slow on both cultivars. The relatively slow development on artificially infected leaf material may be partly attributable to residual fungicide treatment as prescribed by the Scottish Plant Health Service at the time of planting out. Symptoms produced on fruits (apple, banana, and strawberry), inoculated with both test and laboratory strains of the fungus, were identical. Symptoms did not occur on control leaves or fruits inoculated with sterile distilled water or uninoculated agar disks. Koch's postulates were confirmed by consistently reisolating isolates with morphological and immunological characteristics identical to the fungal isolate used to initially inoculate test material. Over the same period, additional symptoms, identical to those originally described at the time of interception, continued to develop on leaf tips of both Kalmia cultivars. Additional isolations from this material were characterized as C. acutatum. Identification of representative isolates was confirmed by CABI Bioscience, Egham, UK, where a reference culture (accession number IMI 384569) has been deposited. As advanced symptoms were observed immediately on arrival of this consignment in the UK, original infection is thought to have occurred prior to importation. This is the first report of C. acutatum infecting K. latifolia.

Specificities and sensitivities of four monoclonal antibodies for typing of Borrelia burgdorferi sensu lato isolates.

Borrelia burgdorferi, the agent of Lyme borreliosis, is genetically more heterogeneous than previously thought. In Europe five genospecies have been described from the original B. burgdorferi sensu lato (sl): B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. lusitaniae, and B. valaisiana. In the United States, B. burgdorferi ss as well as B. bissettii in California and B. andersonii on the East Coast were differentiated. In Asia, B. japonica has been identified along, with B. garinii, B. afzelii, and B. valaisiana. In order to evaluate sensitivity and specificity of four species-specific monoclonal antibodies, we analyzed 210 B. burgdorferi sl isolates belonging to eight genospecies by immunoblot and confirmed genospecies by restriction fragment length polymorphism (RFLP) of rrf (5S)-rrl (23S) intergenic spacer amplicon. Monoclonal antibody H3TS had 100% sensitivity for 55 B. burgdorferi ss isolates but showed reactivity with all four isolates belonging to B. bissetii. Monoclonal antibody I 17.3 showed 100% specificity and sensitivity for 45 B. afzelii isolates. Monoclonal antibody D6 was 100% specific for B. garinii but missed 1 of 64 isolates (98.5% sensitivity). Monoclonal antibody A116k was 100% specific for B. valaisiana but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with results of reactivity and confirmed efficacy of the phenotypic typing of these antibodies. Some isolates showed atypical RFLP. Therefore, both phenotypic and genotypic analyses are needed to characterize new Borrelia isolates.

Production of a species specific monoclonal antibody against a 56-kDa Mycobacterium marinum antigen which is potentially useful in culture identification.

A monoclonal antibody (MoAb) designated 1H11 has been produced against a 56-kDa antigen from Mycobacterium marinum. This MoAb has been shown to be species specific by both enzyme-linked immunosorbent assay and Western blot. The relationship of this 56 kDa to other known mycobacterial antigens is currently under investigation. MoAb 1H11 recognised bacilli taken from cultures by both immunofluorescence and immunoperoxidase staining. This MoAb 1H11 may provide a simple method for rapid identification of M. marinum from cultures of clinical isolates.