scholarly journals Sumateran wild boar (Sus scrofa vittatus) meat antibody production as immunodiagnostic reagent candidate

2019 ◽  
Vol 12 (4) ◽  
pp. 477-482
Author(s):  
Melani Wahyu Adiningsih ◽  
Retno Damajanti Soejoedono ◽  
Rahmat Setya Adji ◽  
Dwi Desmiyeni Putri ◽  
Trioso Purnawarman ◽  
...  

Aim: Meat authentication gives significance values in view of religious, food safety, public health, quality assurance, and legal concern. Most of the meat authentication is based on molecular assay; a simpler method to authenticate meat is needed to develop. An immunoassays technique may offer a solution for simpler test. The aim of our current study was to develop a polyclonal antibody of Sus scrofa vittatus (Sumateran wild boar) as an immunodiagnostic reagent candidate. Materials and Methods: Three male New Zealand white rabbits were used in this study for antibody production. Antigen used was meat extract of Sumateran wild boar, each rabbit was immunized with meat extract antigen (0.5 mg/ml) emulsified in Freund's complete adjuvant at a 1:1 (v/v) ratio as much as 1 ml at subcutaneous route. Booster was carried out 3 times with interval time of 14 days, using meat extract antigen emulsified in Freund's incomplete adjuvant at a 1:1 (v/v) ratio. Serum samples were taken every week, start from 1 week after the first immunization up to 1 week after the third booster. Antibody purification was performed using ammonium sulfate precipitation and Protein A. The presence of specific antibody was determined using agar gel precipitation test and enzyme-linked immunosorbent assay, while purified specific IgG was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. Results: Specific antibody was detected at 14 days after the first immunization and still detected until 2 weeks after the third booster. Highest absorbance of specific antibody was detected 1 week after the third booster. Conclusion: The present study demonstrated that specific antibody of Sumateran wild boar is favorable to be produced in rabbit and showed that antibody produced is applicable to detect Sumateran wild boar meat antigen in immunodiffusion assay, indicating that it is promising as a reagent candidate in immunodiagnostic assay/kit.

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 494
Author(s):  
Angela Petruccelli ◽  
Tiziana Zottola ◽  
Gianmarco Ferrara ◽  
Valentina Iovane ◽  
Cristina Di Russo ◽  
...  

Background: A retrospective sero-survey for evidence of West Nile virus (WNV) infection in European wild boar (Sus scorfa) was conducted in the Latium region, Italy, on stored serum samples of the period November 2011 to January 2012. Methods: Sera were collected from 168 European wild boars and screened for antibodies to WNV and other Flaviviruses by competitive enzyme linked immunosorbent assay (cELISA). All sera positive for Flavivirus antibodies by cELISA were further examined by virus neutralization test (VNT). To test the presence of Flavivirus RNA in samples, an RT-PCR was performed using a pan-Flavivirus primers pair. Results: Thirteen wild boars (7.73%) were seropositive for Flaviviruses. The hemolysis of serum samples limited the interpretation of the VNT for 7 samples, confirming the presence of specific antibody against WNV in a single European wild boar serum sample. The presence of ELISA positive/VNT negative samples suggests the occurrence of non-neutralizing antibodies against WNV or other antigen-related Flaviviruses. No samples resulted positive for Flavivirus by RT-PCR assay. Conclusion: Although a moderately high percentage of animals with specific antibody for WNV has been detected in wild boar in other surveillance studies in Europe, this has not been reported previously in Italy. Together, these data indicate that European wild boar are exposed to WNV and/or other related-Flavivirus in central Italy and confirm the usefulness of wild ungulates, as suitable Flavivirus sentinels.


2001 ◽  
Vol 8 (2) ◽  
pp. 454-459 ◽  
Author(s):  
Mitsushi Tsujimura ◽  
Chuzo Ishida ◽  
Yasuko Sagara ◽  
Takashi Miyazaki ◽  
Koichi Murakami ◽  
...  

ABSTRACT Although a serum thermolabile β-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide fromAerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of d-glucose,N-acetyl-d-glucosamine, d-mannose, and d-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56°C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.


2006 ◽  
Vol 13 (8) ◽  
pp. 913-922 ◽  
Author(s):  
Helle Friis Svenstrup ◽  
Jørgen Skov Jensen ◽  
Kris Gevaert ◽  
Svend Birkelund ◽  
Gunna Christiansen

ABSTRACT Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.


Parasitology ◽  
2020 ◽  
Vol 147 (6) ◽  
pp. 715-720
Author(s):  
Dayane Costa de Souza ◽  
Lucas Silva de Faria ◽  
José Eduardo Neto de Sousa ◽  
Camila de Almeida Lopes ◽  
Vanessa da Silva Ribeiro ◽  
...  

AbstractDefinitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized with Ancylostoma ceylanicum saline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.


2006 ◽  
Vol 11 (5) ◽  
pp. 546-552 ◽  
Author(s):  
Jingyan Wei ◽  
Yang Liu ◽  
Songchuan Yang ◽  
Junjie Xu ◽  
Hangtian Kong ◽  
...  

A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.


Plant Disease ◽  
2005 ◽  
Vol 89 (5) ◽  
pp. 529-529 ◽  
Author(s):  
Y. K. Chen ◽  
C. C. Yang ◽  
H. T. Hsu

Allamanda (Allamanda cathartica L., family Apocynaceae) is native to Brazil and is a popular perennial shrub or vine ornamental in Taiwan. Plants showing severe mosaic, rugosity, and leaf distortion symptoms on leaves are common in commercial nurseries and private gardens. Examination of crude sap prepared from symptomatic leaves using an electron microscope revealed the presence of spherical virus particles with a diameter of approximately 28 nm. The virus was mechanically transmitted to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). The virus caused local lesions on inoculated leaves of Chenopodium quinoa and C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, Nicotiana benthamiana, N. glutinosa, N. rustica, and N. tabacum. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Tests of leaf sap extracted from naturally infected allamanda and inoculated indicator plants using enzyme-linked immunosorbent assay were positive to rabbit antiserum prepared to CMV. Viral coat protein on transblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis reacted with CMV subgroup I specific monoclonal antibodies (2). With primers specific to the 3′-half of RNA 3 (1), amplicons of an expected size (1,115 bp) were obtained in reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from infected allamanda and N. benthamiana. The amplified fragment (EMBL Accession No. AJ871492) was cloned and sequenced. It encompasses the 3′ part of the intergenic region of RNA 3 (158 nt), CP ORF (657 nt), and 3′ NTR (300 nt) showing 91.8–98.9% and 71.4–72.8% identities to those of CMV in subgroups I and II, respectively. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and the nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB, which is predominant on the island. To our knowledge, CMV is the only reported virus that infects allamanda and was first detected in Brazil (3), and this is the first report of CMV infection in allamanda plants occurring in Taiwan. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) H. T. Hsu et al. Phytopathology 90:615, 2000. (3) E. W. Kitajima. Acta. Hortic. 234:451, 1988.


2014 ◽  
Vol 143 (10) ◽  
pp. 2126-2130 ◽  
Author(s):  
H. M. VALE-GONÇALVES ◽  
J. A. CABRAL ◽  
M. C. FARIA ◽  
M. NUNES-PEREIRA ◽  
A. S. FARIA ◽  
...  

SUMMARYLeptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic spirochaetes of the genus Leptospira. The wild boar (Sus scrofa), an important hunting species in Europe, seems to play a significant role in the epidemiological cycle of leptospirosis. A total of 101 serum samples from wild boar hunted in Northern Portugal were analysed for leptospiral antibodies detection by microscopic agglutination test. Sera were collected during hunting seasons (2011–2013) and tested with 17 different pathogenic serovars of Leptospira. Antibodies against nine serovars were detected in 66 (65·4%) of these sera. Serovars Tarassovi and Altodouro exhibited the highest seroreactivity rates (23·8% and 16·8%, respectively), followed by Autumnalis (7·9%) and Bratislava (6·9%). Age and district of origin were found to be risk factors for the presence of leptospiral antibodies in contrast to gender. From a One Health perspective, this study revealed that wild boar should be considered as a potential source of leptospirosis dissemination for humans and animal species (domestic and wild) in shared environments, particularly in the Trás-os-Montes region.


2016 ◽  
Vol 1 (3) ◽  
pp. 612-621 ◽  
Author(s):  
Abdus Salam ◽  
Md Atiqul Haque ◽  
Md Mostafizer Rahman ◽  
Mir Rowshan Akter ◽  
Farzana Afroz

The present study was conducted on layer birds of different age groups to determine specific antibody titer level against avian reovirus (ARV) by indirect enzyme linked immunosorbent assay (iELISA) at Dinajpur district of Bangladesh. This study showed that ARV specific antibody positive cases were 84 out of 90 blood serum samples and the highest antibody titer was 26120 and lowest antibody titer was 288. The total 93.33% sera samples were showed positive result. The study showed that 100% sera sample were positive against ARV at 6 weeks of aged group and the highest, lowest and mean antibody titer were 13917, 4895 and 10269 respectively. On the other hand 88.88% sera sample were positive against ARV at 10 weeks of aged group and the highest, lowest and mean antibody titer were 9779, 288 and 5689.89 respectively. The sera sample collected from 14 weeks of aged group showed 88.88% positive and the highest, lowest and mean antibody titer were 11727, 871 and 5250 respectively. The sera sample collected from 18 weeks of aged group showed 88.88% positive against ARV and the highest, lowest and mean antibody titer were 24440, 1234 and 12648.89 respectively. The sera sample collected from 22 weeks of aged group were 100% positive against ARV and the highest, lowest and mean antibody titer were 26120, 1752 and 11373.89 respectively. The sera sample collected from 26 weeks of aged group showed 100% positive against ARV and the highest, lowest and mean antibody titer were 8566, 1630 and 4327.44 respectively. The sera sample collected from 30 weeks of aged group showed 100% positive against ARV and the highest, lowest and mean antibody titer were 13431, 1989 and 5890.56 respectively. The sera sample collected from 40 weeks of aged group showed 77.77% positive against ARV and the highest, lowest and mean antibody titer were 14618, 433 and 5103.22 respectively. The sera sample collected from 48 weeks of aged group showed 88.88% positive against ARV and the highest, lowest and mean antibody titer were 14553, 957 and 7436.5 respectively. In conclusion it is evident that avian reovirus-specific antibody was successfully detected through commercially available avian reovirus antibody test kit (ELISA kit) and the virus induced a significant antibody titer indicating the affecting virus was absolutely ARV.Asian J. Med. Biol. Res. December 2015, 1(3): 612-621


1993 ◽  
Vol 39 (4) ◽  
pp. 635-640 ◽  
Author(s):  
J Risteli ◽  
I Elomaa ◽  
S Niemi ◽  
A Novamo ◽  
L Risteli

Abstract We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.


1993 ◽  
Vol 74 (5) ◽  
pp. 2432-2442 ◽  
Author(s):  
G. M. Matuschak ◽  
C. A. Klein ◽  
T. L. Tredway ◽  
D. R. Schilly ◽  
A. J. Lechner

We analyzed differences in host regulation of tumor necrosis factor-alpha (TNF-alpha) production and pathophysiological responses in conscious rats after infection with two strains of pathogenic Candida albicans spp. (CA-1 and CA-2) compared with Escherichia coli serotype 055:B5 (EC). The hypothesis was tested that, in contrast to EC, hypotension, organ injury, and mortality after candidemia are not obligatorily dependent on TNF-alpha or TNF-alpha-induced cyclooxygenase pathway metabolites. Dose, viability, and strain-specific dependencies were established after intravenous 10(6) or 10(9) viable CA, as well as heat-killed (HK) or Formalin-inactivated (FI) CA blastospores, compared with live EC at the 24-h LD25 [10(9) colony-forming units (CFU)] and LD100 (10(10) CFU). Shock without endotoxemia developed 4–8 h after 10(9) live CA-1 or CA-2 (LD100 at 24 h) with disseminated yeast-mycelial transformation and increased microvascular permeability in multiple organs but not after HK or FI CA-1. Peak serum TNF-alpha after an LD100 of CA-1 or CA-2 was < 3% of LD25 EC values and was < 1% of peak values during lethal bacteremia. Similar pathogen-specific differences were found in liver- and lung-associated TNF. Production of functionally inactive TNF-alpha during candidemia was excluded by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blotting. Passive immunization against TNF-alpha 2 h before microbial challenge was not protective against CA but prevented otherwise lethal EC sepsis. Cyclooxygenase inhibition also failed to attenuate candidemic shock. We conclude that the magnitude and kinetics of TNF-alpha production and TNF-alpha-dependent immunophysiological responses are differentially regulated after lethal fungal vs. gram-negative bacterial infection. Thus TNF-alpha is not a pivotal mediator of the acute Candida septic shock syndrome with disseminated candidiasis.


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