|Year : 2018 | Volume
| Issue : 1 | Page : 7-13
Effective Aeromonas specific monoclonal antibody for immunodiagnosis
Yuvadee Mahakunkijcharoen1, Chakrit Hirunpetcharat2, Sunisa Malijunbua3, Watcharamat Muangkaew1, Suporn Paksanont1
1 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Thailand
2 Department of Microbiology, Faculty of Public Health, Mahidol University, Thailand
3 Department of Medical Technology, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University, Thailand
|Date of Submission||29-Sep-2016|
|Date of Decision||16-Nov-2017|
|Date of Acceptance||02-Dec-2017|
|Date of Web Publication||22-Dec-2017|
Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Ratchathewi, Bangkok 10400
Source of Support: None, Conflict of Interest: None
Objective: To identify the monoclonal antibody specific to Aeromonas spp., a Gram negative bacteria causing gastroenteritis and wound infection. Methods: The monoclone, namely 88F2-3F4, was produced from hybridoma technology. The specificity of antibody secreted from 88F2-3F4 was tested against other Gram negative bacteria frequently found in gastrointestinal tract. Then the antibody was used for searching Aeromonas antigens in artificial seeded rectal swab cultures by dot-blot enzyme linked immunosorbent assay. Results: 88F2-3F4 produced an antibody that recognized an antigen with a molecular mass of 8.5 kDa in all 123 isolates of the seven Aeromonas species tested, but recognized no epitope of any other Gram-negative bacterium typically found in the gastrointestinal tract. A dot-blot enzyme linked immunosorbent assay based on this antibody showed 86.49% sensitivity and 92.13% specificity. Conclusions: 88F2-3F4 monoclonal antibody could react with all Aeromonas isolates, but not other Gram negative bacteria, therefore it should be a useful tool for the detection of Aeromonas antigen in clinical and environmental samples.
Keywords: Aeromonas, Monoclonal antibody, Immunodiagnosis, Gastrointestinal tract
|How to cite this article:|
Mahakunkijcharoen Y, Hirunpetcharat C, Malijunbua S, Muangkaew W, Paksanont S. Effective Aeromonas specific monoclonal antibody for immunodiagnosis. Asian Pac J Trop Biomed 2018;8:7-13
|How to cite this URL:|
Mahakunkijcharoen Y, Hirunpetcharat C, Malijunbua S, Muangkaew W, Paksanont S. Effective Aeromonas specific monoclonal antibody for immunodiagnosis. Asian Pac J Trop Biomed [serial online] 2018 [cited 2019 Dec 6];8:7-13. Available from: http://www.apjtb.org/text.asp?2018/8/1/7/221128
Foundation project: This work was supported by Grant No.59/2549 from Mahidol University and Grant No. 04/2554 from the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
| 1. Introduction|| |
Aeromonas species are facultative anaerobic Gram-negative rod-shaped bacteria in the family Aeromonadaceae,. Their natural habitat is in the aquatic environment, including ground water, surface water, estuarine and marine water, treated waste water,,, and some certain strains of Aeromonas can also be found in chlorine-treated municipal drinking water supplies,. Apart from aquatic environment, the bacteria can be found in soil and foodstuffs, including seafood, raw meats and dairy products,,,. As wide spread of Aeromonas in the environment is surrounding humans and animals, the bacteria are recognized as the potential pathogenic agents causing food and/or water borne infections,,. The Aeromonas infectious diseases can be both gastroenteritis and non-gastroenteritis,,. The more serious illness is the non-gastroenteritis including skin and soft-tissue infections,, septicemia,,, peritonitis and various internal organ infections in humans and animals,,. Recently the frequency of Aeromonas infections increase and associate with high fatality, especially in immunocompromised patients,. Therefore the rapid diagnosis is urgently needed, since routine diagnosis for the disease is mainly the conventional culture methods, which are both time-consuming and labor-intensive.
Although modern molecular biological techniques have been used to diagnose Aeromonas infections,,,, they are costly and complex. Therefore, immunological techniques are considered to be more useful. These techniques require a specific antibody that recognizes a specific antigen expressed by the organism of interest. Monoclonal antibodies (MAbs) are commonly used for this purpose because they can be designed to recognize a specific epitope of an organism and can be produced with the same specificity in unlimited amounts. In this study, we produced a MAb, designated 88F2-3F4, from the splenocytes of a mouse immunized with Aeromonas hydrophila (A. hydrophila) ATCC 7965. This MAb specifically bound an 8.5 kDa molecule in all 123 Aeromonas isolates tested in this study. Therefore, it has potential utility as a diagnostic tool.
| 2. Materials and methods|| |
2.1. Bacterial strains
One hundred twenty-three Aeromonas isolates were used in this study: 27 A. hydrophila isolates, 26 Aeromonas sobria (A. sobria) isolates, 26 Aeromonas caviae (A. caviae) isolates, 26 Aeromonas trota (A. trota) isolates, 8 Aeromonas jandaei (A. jandaei) isolates, 7 Aeromonas veronii (A. veronii) isolates, and 3 Aeromonas media (A. media) isolates. The phenospecies of each isolate had previously been confirmed according to their biochemical properties at the Bamrasnaradura Infectious Disease Institute, Department of Disease Control, Ministry of Public Health, Thailand.
Other Gram-negative bacteria were used as controls: Plesiomonas shigelloides (ATCC 14029), Vibrio cholerae (V. cholera) O17SR, (9701), V. cholerae O139, V. cholerae non O1/non O139 (DMST2873), Salmonella More Details enteritidis, enterohemorrhagic Escherichia More Details coli (E. coli), Proteus vulgaris, Pseudomonas aeruginosa (ATCC 28763), Enterobacter cloacae, Klebsiella pneumoniae, and Shigella flexneri 1a.
The bacterial stock cultures were cryopreserved in 30% (v/v) glycerol (Bio Basic Inc., Markharn, Ontario, Canada) and 1% (w/v) peptone (Difco, Becton Dickinson, Sparks, USA), and stored at -80 °C until analysis.
2.2. Preparation of crude bacterial extracts
Bacteria from the frozen stocks were grown on tryptic soy agar plates at 37 °C for 18–24 h. One loopful of bacterial colonies was transferred into tryptic soy broth (TSB) containing 0.6% yeast extract and incubated in a shaking incubator (Scientific Series 25D, New Brunswick, NJ, USA) at 120 rpm for 4 h at 37 °C. The cells were harvested by centrifugation (Beckman Coulter, Miami, FL, USA) at 20 000 rpm for 20 min at 4 °C. The pellet was washed and centrifuged once with sterile phosphate-buffered saline (PBS; pH 7.4). The cells in the pellet were then lysed by sonication (Vibra-Cell™, Sonics&Materials, Danbury, CT, USA), and the bacterial debris were removed by centrifugation. The supernatant was collected and stored in aliquots at -20 °C until analysis. The dry weights of the crude bacterial extracts were determined.
2.3. Production of Aeromonas-specific monoclones
Inbred BALB/c female mice, aged 6–8 weeks, with a mean weight of 25 g, were purchased from the National Laboratory Animal Center, Mahidol University. The mice were subcutaneously inoculated three times at two-week intervals with 100 μL of 2 mg/ mL whole cell extract from A. hydrophila ATCC 7965 mixed with Montanide ISA 720 (SEPPIC SA, Paris, France) adjuvant. The titers of antibodies specific for A. hydrophila ATCC 7965 in the immunized mouse sera were determined with an indirect enzyme-linked immunosorbent assay (indirect ELISA). The study was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM 2008-009-01).
2.3.2. Hybridoma technology
Monoclones were produced from hybridoma technology modified from Kohler and Milstein. Splenocytes from immunized mice with serum antibody titers>204 000 were mixed with PS-X63-Ag8 myeloma cells (kindly provided by Professor Watchara Kasinrerk, Chiang Mai University, Thailand) in a ratio of 10: 1 in the presence of polyethyleneglycol (PEG, Sigma) for 90 s. The polyethyleneglycol was then removed by centrifugation. The cell mixture was suspended in Dulbecco’s modified Eagle’s medium containing hypoxanthine, aminoptherin, and thymidine (HAT medium) and 10% fetal bovine serum, and then distributed into the wells of a 96-well flat-bottom microculture plate (Corning Inc., Mexico) and incubated at 37 °C in a 5% CO2 atmosphere. The growing hybridomas were observed under an inverted microscope.
2.3.3. Screening for Aeromonas-specific antibody-producing hybridomas
The supernatants from the growing hybridomas were tested for antibodies specific for antigens of A. hydrophila ATCC 7965 with an indirect ELISA (modified from Engvall and Perlmann). Briefly, 100 μL of crude bacterial extract (20 μg/mL) in coating buffer (carbonate/bicarbonate buffer, pH 9.6) was added to the individual wells of a 96-well microtiter plate (Greiner, Frickenhausen, Germany) and incubated in a moist chamber at 4 °C for 18–24 h. The plates were washed three times with washing buffer [PBS+0.05% Tween 20 (PBST)]. The unoccupied space in each well was filled with 200 μL/well blocking solution [3% skim milk (Difco, Becton Dickinson) in PBST], and the plates were incubated in a moist chamber at 37 °C for 1 h. After the blocking solution was removed, the wells were filled with 100 μL of two-fold serially diluted mouse immune serum, normal mouse serum (to determine Aeromonas-specific mouse antibody titer), or hybridoma/monoclone culture supernatant as a test sample, myeloma culture supernatant as the negative control, or diluent only as the blank control (to screen for clones producing Aeromonas-specific antibody). The plates were incubated at 37 °C for 1 h. The unbound antibodies were removed with four washes with PBST. Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG)+IgM+IgA (Invitrogen, Carlsbad, CA, USA), diluted 1: 5 000 in 1% skim milk/PBST (100 μL/well), was added and incubated at 37 °C for 1 h. After four washes (as described above), soluble substrates [1 mg/mL o-phenylenediamine dihydrochloride in citrate–phosphate buffer (pH 5.0) and 0.03% (v/v) H2O2] were added to the plates, and kept in the dark at room temperature for 30 min. The reaction was stopped with 2.5 N H2SO4 (50 μL/well). The reaction strength was determined by measuring the optical density at 492 nm (OD492) with a microplate reader (Tecan, Austria).
The antibody producing hybridomas were expanded and their culture supernatants were collected to test their antibody-binding activities with the other Aeromonas species and other Gram-negative bacteria listed above. The hybridomas that produced antibodies specific for either A. hydrophila only or for all the Aeromonas species, but did not bind other Gram-negative bacteria, were selected. Monoclones were obtained from the selected hybridomas with limiting dilution. The supernatants collected from the monoclone cultures were screened again for Aeromonas specificity. The positive Aeromonas-specific antibody-producing monoclones were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and also preserved in liquid nitrogen. The culture supernatants were collected for further characterization.
2.3.4. Characterization of Aeromonas-specific monoclonal antibody
The Aeromonas antigen specific to each MAb was characterized with a Western blotting analysis. First, antigens (10 μg/lane) from all the tested bacteria were fractionated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% (w/v) polyacrylamide or 5%–20% gradient gel (e-PAGEL, ATTO Corporation, Tokyo, Japan) and a constant current of 20 mA/gel. After electrophoresis, the fractionated proteins on the polyacrylamide gel were transferred to a prewetted polyvinylidene fluoride (PVDF) membrane with a 0.2 μm pore size (PALL Life Sciences) with an electroblotting unit [Hoefer Semiwet, Amersham Bioscience (SF) Corp., USA] at a constant voltage of 25 V for 4.5 h. The blotted PVDF membranes were then blocked with 3% skim milk/PBS for at least 1 h. The membranes were then allowed to react with the culture supernatants and/or myeloma culture supernatant (as the negative control) at room temperature. After 2 h, the reacted membranes were washed with PBST (about 3-4 changes in 3-5 min), and incubated with HRP-conjugated goat anti-mouse IgG+IgA+IgM (Invitrogen, Carlsbad, CA, USA), diluted 1: 5 000 in 1% skim milk/PBS, at room temperature for 1 h. After the membranes were washed with PBST, they were incubated with DAB substrate solution [1 mg/mL 3,3’-diaminobenzidine in 0.1 M Tris-HCl (pH 7.4) and 0.03% (v/v) H2O2] in the dark at room temperature for 5 min. The reaction was stopped by washing the membranes with distilled water.
The molecular masses of the reactive bands were determined by comparing their relative mobilities against a standard curve constructed from the molecular masses of standard protein markers run on the same gel. The cross-reactions between these MAbs and other related crude Gram-negative bacterial antigens were also tested.
2.3.5. Identification of immunoglobulin isotype and subclass by indirect ELISA
The isotype and/or subclass of the MAb were identified with an ELISA using the Mouse MonoAb ID Kit (HRP) from Zymed Laboratories Inc. (South San Francisco, CA, USA). Briefly, the immunoglobulins in monoclone culture supernatants from the monoclone 88F2-3F4 culture were added into a 96-well flat-bottom microplates (Greiner, Frickenhausen, Germany) coated with the crude antigens of A. hydrophila ATCC 7965. After incubation for 2 h, the binding immunoglobulin was allow to react with rabbit IgG specific for mouse μ, γ 1, γ 2a, γ 2b, γ 3, α chain and κ or λ L chain. Goat anti-rabbit IgG conjugated with horseradish peroxidase was then added and the reaction was visualized by the enzymatic reaction between HRP and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid).
2.3.6. Determination of monoclonal antibody strength
The sensitivity of the MAb was determined with a dot-blot ELISA. Crude Aeromonas lysate was two-fold serially diluted and dotted onto pieces of PVDF membrane. After the membrane was air dried, it was reacted with the monoclone culture supernatants, as an indirect ELISA.
2.4. Efficacy of MAb determined by dot blot ELISA
One hundred twenty-six rectal swabs in Stuart’s transport medium were obtained from the Bamrasnaradura Infectious Diseases Institute, Thailand. The use of these samples as expedited samples was approved by the Institutional Review Board of the Faculty of Tropical Medicine. All rectal swabs were transferred into tubes containing 2 mL of TSB and incubated overnight at 35 °C. The TSB cultures were stored as aliquots at -75 °C until analysis. Of these 126 samples, none were positive for Aeromonas with conventional testing. Therefore, various concentrations of A. hydrophila ATCC 7965 were blindly seeded into 37 rectal swab cultures and 5 μL of each sample was dotted onto PVDF membrane. The Aeromonas antigens presented in the cultures were detected with the dot-blot ELISA. The procedure of the test was similar to indirect ELISA mentioned above.
The correlation between the results of dot-blot ELISA and the known Aeromonas seeded samples was statistical analyzed by the calculation of Kappa coefficience (κ),.
| 3. Results|| |
3.1. Monoclonal antibody production
Among the several hundred of hybridomas obtained from one fusion of splenocytes from a mouse immunized with A. hydrophila ATCC 7965 and P3-X63-Ag8 myeloma cells, hybridoma 88F2 was selected as its production of an antibody that reacted strongly to antigens extracted from all the Aeromonas strains tested. This hybridoma was subcloned until the monoclone designated 88F2-3F4 was obtained.
3.2. Determination of monoclonal antibody isotype
The immunoglobulin produced from monoclone 88F2-3F4 showed high reaction to rabbit anti-mouse γ 2a and κ L chain determined by ELISA, therefore its isotype should be IgG2a, κ L chain [Figure 1].
|Figure 1: Identification of immunoglobulin isotype of MAb 88F2-3F4 with an indirect ELISA.|
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3.3. Specificity of MAb 88F2-3F4
In an indirect ELISA, MAb 88F2-3F4 reacted strongly with 42 of 44 crude Aeromonas lysates prepared from seven Aeromonas species and reacted weakly with the remaining 2 isolates (OD492 of the reactions varied from 0.106 to 1.411) [Figure 2]A,[Figure 2]B,[Figure 2]C,[Figure 2]D,[Figure 2]E,[Figure 2]F,[Figure 2]G, indicating that the MAb recognizes a common antigen in all Aeromonas spp. In dot-blot ELISAs using the crude antigens produced from 123 isolates of seven Aeromonas species, the MAb reacted with all the Aeromonas isolates [Figure 3].
|Figure 2: Specificity of MAb 88F2-3F4.|
The reactivity of MAb 88F2-3F4 with 44 Aeromonas isolates was tested by ELISA. A) A. hydrophila, B) A. sobria, C) A. caviae, D) A. trota, E) A. jandaei, F) A. media, and G) A. veronii.
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|Figure 3: MAb 88F2-3F4 specificity.|
The reactivity of MAb 88F2-3F4 with 123 Aeromonas isolates was determined by Dot blot ELISA. A. hydrophila; A1-A9, B1-B9, C1-C9. A. caviae; D1-D9, E1-E9, F1-F8. A. veronir, G1-G7. A. media, G8-G9, M9. A. trota, H1-H9, I1-I9, J1-J8. A. sobria,, K1-K9, L1-L9, M1-M8. A. jandaei, N1-N8. TSB, F9, J9, N9.
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To examine the cross-reactivity of the MAb with other Gram-negative bacteria, it was tested against the crude lysates of 11 different Gram-negative bacteria: V. cholerae O1 (Vc O17SR), V. cholerae O139, V. cholerae non O1/non O139, Plesiomonas shigelloides, E. coli, Salmonella typhimurium, Shigella flexneri, Enterobacter cloacae, Klebsiella pneumoniae, and Proteus vulgaris. The MAb did not cross-react with these bacteria [Figure 4].
|Figure 4: Specificity of MAb 88F2-3F4.|
The reactivity of MAb 88F2-3F4 with A. hydrophila (ATCC 7965) and other 11 Gram-negative bacterial lysates was tested by ELISA.
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The antigen was recognized by the MAb using Western blotting. Seven crude Aeromonas lysates were fractionated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 5%–20% gradient polyacrylamide gel, then blotted onto PVDF membrane and reacted with the MAb. The MAb reacted with a band of relative molecular mass (Mr) ~8.5 kDa in the lysates of all seven Aeromonas species, but did not react with the lysates of the other 11 Gram-negative bacteria [Figure 5]. We concluded that the specific Aeromonas antigen recognized by MAb 88F2-3F4 has a relative molecular mass of about 8.5 kDa.
|Figure 5: MAb 88F2-3F4 specificity.|
MAb 88F2-3F4 specificity to Aeromonas antigen was determined by Western blot analysis using 8 Aeromonas crude lysates and 9 crude lysates prepared from other Gram negative bacteria. Lane M=Low molecular weight marker. Lane 1=A. hydrophila ATCC 7965. Lane 2=A. hydrophila 03036. Lane 3=A. sobria 03133. Lane 4=A. veronii 03086. Lane 5=A. caviae 03125. Lane 6=A. media 03132. Lane 7=A. trota 03165. Lane 8=A. jandaei 03168. Lane 9=V. cholerae O17SR. Lane 10=V. cholerae O139. Lane 11=Plesiomonas shigelloides. Lane 12=E. coli. Lane 13=Salmonella typhimurium. Lane 14=Shigella flexneri. Lane 15=Enterobacter cloacae. Lane 16=Klebsiella pneumoniae. Lane 17=Proteus vulgaris.
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3.4. Sensitivity of MAb 88F2-3F4
To determine the sensitivity of MAb 88F2-3F4 in detecting the Aeromonas antigen, a crude bacterial extract of A. hydrophila ATCC 7965 was two-fold serially diluted (from 250 to 0.49 μg/mL), dotted onto PVDF membrane, and reacted with the MAb. MAb 88F2-3F4 reacted with the antigen at a concentration of 3.9 μg/mL, indicating the modest sensitivity of this MAb [Figure 6].
|Figure 6: MAb 88F2-3F4 sensitivity.|
The sensitivity of MAB 88F2-3F4 was tested against two fold serial dilution of A. hydrophila lysate by dot-blot ELISA.
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3.5. Efficiency of MAb 88F2-3F4
The efficiency of MAb 88F2-3F4 in detecting the Aeromonas antigen was evaluated using artificially seeded rectal swab samples. Thirty seven of one hundred twenty-six Aeromonas negative rectal swab cultures were blindly seeded with various concentrations of crude antigens from A. hydrophila ATCC 7965. An aliquot (5 μL) of each seeded sample was dotted onto PVDF membrane (PALL Life Sciences) and reacted with MAb 88F2-3F4. Of the 37 Aeromonas antigen-seeded rectal swab samples, 32 samples (86.49%) were positive after detecting with MAb 88F2-3F4 and 5 samples were definitely negative. In the number of 89 unseeded samples, 82 samples were truly negative and 7 samples showed mildly positive.
These results indicate that MAb 88F2-3F4 has 86.49% sensitivity, 92.13% specificity, 92.68% accuracy, 82.05% positive predictive value and 94.25% negative predictive value. The dot blot ELISA using MAb 88F2-3F4 could detect Aeromonas antigens in the artificial seeded rectal swab cultures with high correlation to the known number of the Aeromonas seeded samples as κ value was 0.774.
| 4. Discussion|| |
Aeromonas spp. survive at a wide range of temperatures, pHs, salt concentrations, and chlorine concentrations,,. They occur throughout the environment, especially in water and soil, and are one of the indicators of fecal and waste contamination. Aeromonas spp. produces hemolysin and enterotoxin, as V. cholera and Salmonella spp. Therefore, the bacteria are recognized as pathogens that cause diseases in humans and animals. Their prevalence in humans has not been extensively studied, but according to our observations based on the collection of rectal swabs from a laboratory service, it is about 3%. Aeromonas can infect wounds and induce serious clinical problems, including septicemia and the infection of various internal organs. In some cases of infection in immunocompromised hosts, it has more serious consequences. Therefore, sensitive diagnostic tests for this infection may have utility in the surveillance of this re-emerging disease and consequently its prevention.
Although the technology of MAb production has been established for more than 35 years, the broad application of MAbs is still increasing. The production of MAbs directed against Aeromonas spp. has been achieved by many investigators, including Delamare et al.. They used splenocytes from a mouse immunized with A. hydrophila ATCC 7966 and obtained MAb 5F3 with specificity for a 110 kDa antigen on the whole cells of A. hydrophila. The specificity of MAB 5F3 was determined with an ELISA. The researchers reported that the MAb reacted strongly only with A. hydrophila but produced a basal reaction to A. sobria, A. trota, Aeromonas salmonicida, Aeromonas enteropelogenes, Aeromonas ichtiosmia, and another five Gram-negative bacteria: Salmonella typhimurium, E. coli, Enterobacter cloacae, Pseudomonas aeruginosa, and Pseudomonas putida. The sensitivity of MAb 5F3 for detection of A. hydrophila was 105 cell/mL.
In 2007, Longyant and colleagues also produced Aeromonas-specific MAbs, which were classified into three groups according to their recognition of different Aeromonas antigens. MAbs in Group 1 reacted with 10–200 kDa Aeromonas antigens. The MAbs in Group 2 reacted with 10–200 kDa antigens from two of the three A. hydrophila strains tested in the study. The MAbs in Group 3 reacted with all the Aeromonas strains tested in the study, i.e., three A. hydrophila, two A. sobria, and one A. caviae. However, its reaction with A. caviae was weak and only six Aeromonas isolates were tested in the study. The sensitivities of these three MAbs, determined with dot-blot ELISAs, were about (106–107) cfu/mL.
In the present study, we produced the Aeromonas-specific MAb 88F2-3F4. This antibody reacted with all 123 isolates of seven Aeromonas species tested, and did not cross-react with other Gram-negative bacteria typically found in the gastrointestinal tract. Therefore, MAb 88F2-3F4 is an Aeromonas-specific MAb and can be used as an immunodiagnostic tool for the detection of Aeromonas infection or contamination in clinical or environmental samples.
On a dot-blot ELISA, MAb 88F2-3F4 detected concentrations of crude Aeromonas antigen as low as 3.9 μg/mL. The efficacy of the MAb may be rather low because the specific antigen with which it reacts is very small (8.5 kDa).
When this MAb was used to detect the Aeromonas antigen in artificial seeded rectal swab cultures, the sensitivity, specificity, and accuracy of MAb 88F2-3F4 were 86.49%, 92.13%, and 92.68%, respectively. Therefore, this MAb may be useful for the rapid detection of Aeromonas in clinical samples from both humans and animals and for screening bacterial contamination of foodstuffs, waste, and the environment.
Conflict of interest statement
We declare that we have no conflict of interest.
We gratefully acknowledge Mr. Boonchuay Eampokalap and the staff of the Microbiology Section, Bamrasnaradura Infectious Disease Institute, Department of Disease Control, Ministry of Public Health, for kindly providing Aeromonas from clinical isolates and rectal swabs that were available after routine diagnoses. We also express our sincere thanks to Professor Watchara Kasinrerk of Chiang Mai University for his generous gift of the PS-X63-Ag8 myeloma cells.
This work was supported by Grant No.59/2549 from Mahidol University and Grant No. 04/2554 from the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]