Indirect ELISA Method Research Articles

Pemberian suplemen Swissoats A3® meningkatkan kadar hormon estrogen pada tikus (Rattus norvegicus) betina menopause galur Wistar

Abstract: Aging is a physiological process that occurs in all living creatures. Swissoats A3® is an anti-aging supplement that contains phytoestrogens. This study was aimed to prove that Swiss oats A3® supplementation could increase estrogen levels in menopausal female Wistar rats (Rattus norvegicus). This was an experimental study with a completely randomized pretest-posttest control group design. Subjects were 20 menopausal female rats (Rattus norvegicus), Wistar strain, healthy, aged 15-16 months, weighing 200 g, divided into 2 groups: the control group (P0), treated with distilled water for 21 days and the treatment group (P1), treated with Swissoats A3® of 34.2 mg/rat/day for 21 days. Before and after 21 days of treatment, 1 ml venous blood was drawn through the medial canthus of orbital sinus using microcapillary tube and the estrogen levels were meassured by using indirect ELISA method. The results showed that before treatment, the mean estrogen level in P0 group was 14.96±2.0468 pg/ml and in P1 group was 14.21±2.3905 pg/ml (P > 0.05). Meanwhile, after 21-day treatment, the mean estrogen level in P0 group was 15.07±1.8851 pg/ml, whereas in P1 group was 28.33±3.2052 pg/ml (P < 0.01). The analysis showed that the Swissoats A3® could elevate the estrogen levels significantly (P < 0.01) in group P1 (2.3905±14.21 vs 28.33±3.205). Conclusion: Swissoats A3® supplementation could increase the estrogen levels in menopausal female Wistar rats (Rattus norvegicus).Keywords: Swiss oats A3®, estrogen, female Wistar rats, menopauseAbstrak: Penuaan (aging) merupakan suatu proses fisiologik yang dialami oleh seluruh mahluk hidup. Swissoats A3® merupakan suplemen anti-aging yang mengandung fitoestrogen. Penelitian ini bertujuan untuk membuktikan bahwa pemberian suplemen Swissoats A3® dapat meningkatkan kadar estrogen pada tikus (Rattus norvegicus) betina menopause galur Wistar. Jenis penelitian ialah eksperimental dengan randomized pretest-posttest control group design. Subjek penelitian ialah 20 ekor tikus putih betina (Rattus norvegicus) galur Wistar, sehat, menopause, umur 15-16 bulan, berat badan 200 gr. Tikus dibagi menjadi 2 kelompok yaitu kelompok kontrol (P0) yang diberikan aquadest secara sonde selama 21 hari dan kelompok perlakuan yang diberikan suplemen Swissoats A3® dosis 34,2mg/ekor/hari secara sonde selama 21 hari. Sebelum dan sesudah 21 hari perlakuan, darah vena diambil melalui medial canthus sinus orbitalis sekitar 1 ml dengan menggunakan tabung mikrokapiler dan dilakukan pemeriksaan kadar estrogen menggunakan metode indirect ELISA. Hasil penelitian menunjukkan bahwa sebelum perlakuan, rerata kadar estrogen kelompok P0 14,96±2,0468 pg/ml dan kelompok P1 14,21±2,3905 pg/ml (P > 0,05). Setelah perlakuan selama 21 hari, rerata kadar estrogen kelompok P0 15,07±1,8851 pg/ml dan kelompok P1 28,33±3,2052 pg/ml (P < 0,01). Analisis efek perlakuan menunjukkan terjadinya peningkatan kadar hormon estrogen yang sangat bermakna (P < 0,01) pada kelompok P1 (14,21±2,3905 vs 28,33±3,205). Simpulan: Swissoats A3® dapat meningkatkan kadar estrogen pada tikus (Rattus norvegicus) betina menopause galur wistar.Kata kunci: Swissoats A3®, estrogen, tikus wistar betina, menopause

Diagnosis of Cattle Brucellosis by PCR and Serological Methods: Comparison of Diagnostic Tests

Brucellosis is one of the most widespread zoonotic diseases globally. This chronic, contagious disease mainly transmitted from cattle, sheep, goats, pigs and camels through direct contact with blood, placenta, fetuses or uterine secretions or through consumption of contaminated raw animal products such as unpasteurized milk or cheese. Brucellosis is endemic in Iran. The aim of this study was to compare the molecular and serological tests for detection of brucellosis. In a cross-sectional study, the blood specimens were collected from 92 unvaccinated cattle during the time period September 2014 to July, 2015.. The serum samples were aliquoted and stored at −20°C until tested. The brucellosis diagnosis was established by indirect ELISA (iELISA), Rose Bengal, 2ME, Wright and polymerase chain reaction (PCR) method. In this investigation frequency of brucellosis by using Wright, 2ME, Rose Bengal, and iELISA methods were 42.3% (n= 39), 50% (n= 71), 78.2% (n=72), 75% (n=69), 86.2% (n=76), respectively. The specificity and sensitivity of iELISA was higher than the other tests. The 72 positive Rose Bengal samples in PCR were shown to be positive by both genes and 20 negative Rose Bengal samples were shown negative by both samples. Prevention of brucellosis mainly involves education, food quality and personal hygiene. Efforts should be made by responsible authorities. The results of this study showed specificity and sensitivity of indirect ELISA (iELISA) test is more than other methods. So, brucellosis diagnosis using of this test is recommended.

Prokaryotic expression of a codon-optimized capsid gene from duck circovirus and its application to an indirect ELISA

Duck circovirus (DuCV), as a causative agent of long-term immunosuppressive disease, has caused heavy damage to waterfowl breeding worldwide. In this study, the full-length Cap (capsid) gene of DuCV was expressed in Escherichia coli (E. coli) for the first time by optimizing the codons in its nuclear localization signal (NLS) regions. The recombinant protein was expressed as inclusion bodies, and the quantification of purified Cap protein could reach 0.29mgmL−1. Moreover, an indirect ELISA method (Cap-ELISA) was established based on the recombinant Cap protein. The results of the optimization for Cap-ELISA revealed that the optimum concentration of the coating antigen and serum dilution ratio were 19.5ng per well and 1:1280, respectively. The cut-off value of the Cap-ELISA for positive sample detection was 0.145, the sensitivity of that reached 1:25600, and it was specific for the detection of DuCV anti-sera. In comparative experiments using 56 clinically suspected DuCV-infection samples, the Cap-ELISA showed a 94.64% coincidence rate with the PCR test. These results indicated that codon optimization is a reasonable strategy to obtain an intact Cap protein, and this Cap-ELISA is suitable for extensive applications in DuCV serological diagnosis.

The correlation between the use of personal protective equipment and level wild-type p53 of dental technicians in Surabaya

Background: Exposure of metals among dental technicians that come from the working environment can lead to the formation reactive oxygen species (ROS). ROS can cause mutations in the p53 gene (p53). The mutation is transversion mutation GuanineThymine. p53 mutations can lead to low expression of the wild-type p53 protein (p53). Wild-type p53 involved in many biological processes such as regulation of genes involved in cell cycle, cell growth after DNA damage, and apoptosis. However, exposure to metals among dental technicians can be prevented through the use of personal protective equipment (PPE) during work. Purpose: The purpose of this study was to analyze the correlation between the use of personal protective equipment to wild-type p53 protein levels among dental technicians in Surabaya. Method: This study was observational analytic with cross sectional approach. 40 samples were taken by random sampling. Data were retrieved through interviews and observations. Wild-type p53 was analyzed from saliva with indirect ELISA method. Analysis of data used Kolmogorov Smirnov normality test and a Pearson correlation test. Value significance was p<0.05 (95% confidence level). Result: There was a significant association between the use of personal protective equipment with wild-type p53 levels with p=0.002 Conclusion: The use PPE properly is positively correlated with the wild-type p53 protein levels of dental technicians in Surabaya.

Performance of Leishmania braziliensis enolase protein for the serodiagnosis of canine and human visceral leishmaniosis

In the present study, Leishmania braziliensis enolase was cloned and the recombinant protein (rEnolase) was evaluated for the serodiagnosis of canine and human visceral leishmaniosis (VL). For the canine VL diagnosis, this study examined serum samples of Leishmania infantum-infected dogs, from non-infected animals living in endemic or non-endemic areas of leishmaniosis, as well as those from Leish-Tec®-vaccinated dogs and Trypanosoma cruzi or Ehrlichia canis experimentally infected animals. For the human VL diagnosis, this study analyzed serum samples from VL patients, from non-infected subjects living in endemic or non-endemic areas of leishmaniosis, as well as those from T. cruzi-infected patients. In the results, an indirect ELISA method using rEnolase showed diagnostic sensitivity and specificity values of 100% and 98.57%, respectively, for canine VL serodiagnosis, and of 100% and 97.87%, respectively, for human VL diagnosis. These results showed rEnolase with an improved diagnostic performance when compared to the recombinant A2 protein, the crude soluble Leishmania antigenic preparation, and the recombinant K39-based immunochromatographic test. In conclusion, preliminary results suggest that the detection of antibodies against rEnolase improves the serodiagnosis of human and canine visceral leishmaniosis.

Tau oligomers in cerebrospinal fluid in Alzheimer's disease.

ObjectiveWith an increasing incidence of Alzheimer's disease (AD) and neurodegenerative tauopathies, there is an urgent need to develop reliable biomarkers for the diagnosis and monitoring of the disease, such as the recently discovered toxic tau oligomers. Here, we aimed to demonstrate the presence of tau oligomers in the cerebrospinal fluid (CSF) of patients with cognitive deficits, and to determine whether tau oligomers could serve as a potential biomarker for AD.MethodsA multicentric collaborative study involving a double‐blinded analysis with a total of 98 subjects with moderate to severe AD (N = 41), mild AD (N = 31), and nondemented control subjects (N = 26), and two pilot studies of 33 total patients with AD (N = 19) and control (N = 14) subjects were performed. We carried out biochemical assays to measure oligomeric tau from CSF of these patients with various degrees of cognitive impairment as well as cognitively normal controls.ResultsUsing a highly reproducible indirect ELISA method, we found elevated levels of tau oligomers in AD patients compared to age‐matched controls. Western blot analysis confirmed the presence of oligomeric forms of tau in CSF. In addition, the ratio of oligomeric to total tau increased in the order: moderate to severe AD, mild AD, and controls.ConclusionThese assays are suitable for the analysis of human CSF samples. These results here suggest that CSF tau oligomer measurements could be optimized and added to the panel of CSF biomarkers for the accurate and early detection of AD.

Dioctophyme renale: prevalence and risk factors of parasitism in dogs of São Cristóvão district, Três Barras county, Santa Catarina State, Brazil.

Dioctophyme renale is a nematode that can be found parasitizing the kidney, peritoneal cavity and, rarely, other organs of canids and mustelids. This disease has high occurrence in the municipality of Três Barras, state of Santa Catarina, thus making this an interesting area to study the epidemiological aspects of infection by D. renale in dogs. Among 197 dogs, 14.2% showed the parasite eggs in urine and 16.4% showed IgG antibodies anti-D. renale in serum samples according to the indirect ELISA method; among seropositive dogs, 15 (37.5%) animals did not show any parasite eggs in their urine. Parasitism was more frequent in females, and there was no finding of interference from age on parasitism. Factors such as water potential and presence of paratenic hosts in the studied region were reported by the owners of dogs and may have contributed to the occurrence of parasitism.

Antisecretory Factor (AF) egg-yolk peptides reflects the intake of AF-activating feed in hens

Summary Objective The aim of the present investigation was to determine the level of the active form of the endogenous protein Antisecretory Factor (actAF), and also the active form of AF immunoreactive molecules in the affinity purified egg yolk. Design Determination of AF in affinity purified egg yolk by means of in vivo and in vitro methods. Setting Sahlgrenska University Hospital and Chalmers University of Technology, both units located in Gothenburg, Sweden. The farm housing the egg-laying hens is situated some 10 miles west to Stockholm. Samples Egg yolk collected weekly from hens subjected to AF-stimulating feed for up to 18 weeks. Methods and main outcome measures The methods used were: a/ The in vivo rat ligated jejunal loop assay. b/ Two variations of ELISA, i.e. 1.An indirect ELISA using a polyclonal antibody against AF-16 antibodies. 2.A competitive enzyme immunoassay for detection of the peptide AF-16 and also related immunoreactive molecules. c/ Matrix-Assisted Laser Desorption Ionization Mass Spectrometry, (MALDI-MS). The numeric variables registered represents: a/ The rat jejunal ligated loop assay demonstrates the influence of AF on the in vivo secretory response (mg/cm) to cholera toxin challenge. The ligated loop is some 12–15 cm long and placed on the mid part of jejunum, and the cholera toxin induced secretion is registered after a 5 h long challenge period. b/ The indirect ELISA method demonstrates the relative concentration of immunogenic AF peptides/AF-16 peptides and also related immunoreactive compounds by means of absorbance values, while the values of the competitive immunoassay represent the concentration of AF-16 peptides including similar immunoreactive peptides in ng/ml. c/ The MALDI-MS method provide information about the concentration of the AF-16 peptide down to nanogram per ml. levels after mass spectrometry analysis of the sample. Results All methods revealed similar results by demonstrating a continuous increase over time in the collected egg yolk samples. Thus, low AF activity was registered in egg yolk collected in the period of 1–10 weeks of AF-stimulated feeding, significantly higher AF values was registered in yolk collected between 10 and 15 weeks of feeding, while maximal AF concentration was determined after 15 weeks of feeding. Thus, in the period between 15 and 18 weeks of stimulated AF-feeding, no further increase of the endogenous AF activity could be registered despite continuous AF-stimulated feeding. Conclusion During the period of AF-stimulated feeding of the egg laying hens the registration of AF concentration in the affinity purified egg yolk samples must be continuously registered over time. The various methods used for determination of AF concentration in the affinity purified egg yolk might all serve as tools in order to achieve the optimal concentration of active AF. Together, these methods will provide information about the optimal AF concentration in the final product consisting of spray dried egg yolk (Salovum®) used for disease treatment.

Seroprevalensi Toksoplasmosis di Kabupaten Gianyar, Bali

Seroprevalence of human toxoplasmosis in Gianyar Bali provincePurposeThe present study was designed to determine the seroprevalence and risk factors of human toxoplasmosis.MethodsThe research used a cross-sectional design and was conducted in Gianyar District, Bali Province. A total of 240 sera samples were collected and examined by indirect ELISA methods for IgG Anti-T. gondii. Demographic characteristics and risk factors were collected by interviews using a structured questionnaire. Data were analysed using the Poisson regression test with robust variance estimators.ResultsSeroprevalence of human toxoplasmosis in Gianyar District were 56.7%. Multivariate analysis showed that there was an association between meal of raw meat/Lawar (aPR: 1.40; 95%CI: 1.05-1.86); Male (aPR1:42; 95%CI: 1.12-1.81); existence of animals/wild cats (aPR: 1.48; 95%CI: 1.04-2.09); often washing their hand (aPR: 1.25; 95% CI: 1.01-1.56); elevation (aPR: 0.99; 95%CI: 0.99-0.99); and distance to the river from the house (aPR: 0.99; 95%CI: 0.99 to 0.99).ConclusionThere was found high seropositive toxoplasmosis in Gianyar District. Patients with seropositive toxoplasmosis should receive treatment and health information. There should be continued prevention efforts to increase health knowledge about toxoplasmosis and transmission.

Development and application of CK-MB specific monoclonal antibodies

The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mAb), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mAb pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche's CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mAbs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche's was 0.930 9. This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche's.

Investigation of diethylstilbestrol residue level in human urine samples by a specific monoclonal antibody.

Diethylstilbestrol (DES) is used as a kind of animal feed additive and affects people's health through the food chain. The purpose of this study is to detect the residue level of DES in 576 human urine samples directly. DES-BSA was used to immunize Balb/c mice. The monoclonal antibody was produced by hybridoma that was screened through cell fusion techniques. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. The IC50 of this method was 3.33ng/mL. The LOD and LOQ were 0.16 and 0.54ng/mL. Linear range of the standard curve was from LOD to 12.50ng/mL. There was no cross-reactivity with two kinds of estrogens and two structural analogs with DES. Five hundred seventy-six urine samples were analyzed by the indirect competitive ELISA method, and the detection rate was 98.78%. The mean concentration and geometric mean were 4.70 and 3.50ng/mL. The indirect competitive ELISA method based on monoclonal antibody was sensitive and reliable for the detection of DES in human urine samples. The results warned us to pay more attention to human health and food safety.

Development, identification and application of 33 monoclonal antibodies against cardiac troponin T

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs' epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche's was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche's.

Construction of glutathione peroxidase (GPx) DNA vaccine and its protective efficiency on the orange-spotted grouper (Epinephelus coioides) challenged with Vibrio harveyi

The main aims of this study were to construct glutathione peroxidase (GPx) DNA vaccine of Vibrio harveyi ZJ0603 and to investigate its immune protective efficiency as a vaccine candidate on the orange-spotted grouper (Epinephelus coioides) treated with V. harveyi. Base on the cloning of ZJ0603 GPx gene, a DNA vaccine, named as pcDNA-GPx, was constructed by inserting GPx gene into pcDNA3.1 (+) plasmid. Orange-spotted groupers were immunized with the pcDNA-GPx plasmid by injection intramuscularly. The relative percent of survival (RPS) of fish vaccinated with the DNA vaccine against pathogenic V. harveyi infection was 77.5%. The expression of DNA vaccine was analyzed in the tissues of orange-spotted grouper by PCR and RT-PCR. The results indicated that pcDNA-GPx distributed and expressed in the head kidney, liver, spleen, gill and injected muscle at 7 and 28 days after vaccination. Significant specific antibody responses were also detected in the vaccinated orange-spotted groupers by indirect ELISA method. In a conclusion, DNA vaccine pcDNA-GPx showed an effective immune protection to the orange-spotted grouper treated with V. harveyi. The GPx can be used as a candidate DNA vaccine for the control of vibriosis.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

BackgroundHeparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer.ObjectivesEvaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control).MethodsGlycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR).ResultsThe A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma.ConclusionThe glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

Determination of the serum antibody level of different age groups of broiler chicken against Infectious Bursal Disease (IBD)

A study on Infectious Bursal Disease (IBD) was conducted on broiler chicks (N=200+70) of strains: Cobb-500 and Hubbard classic at Laxmipur district of Bangladesh. The blood sample were collected at day old chicks (1st day), pre-vaccinated flocks (11th day) and post-vaccinated flocks (26th day), to measure the antibody titer by indirect ELISA method. Maternal derived antibody (MDA) mean titer of day old chicks (N=40) were found 9621.65 ± 780.78 and 4232.60 ± 301.66 for Cobb-500 and Hubbard classic respectively. MDA mean titer was measured at day eleven chicken (N=60) before vaccination and found 1963.00 ± 143 and 984.16 ± 126.4037 respectively. Of these two titers; Cobb-500 strain was found to be protective (>1000) where Hubbard classic was below protective (<1000) titer level. The two flocks of Cobb-500 and Hubbard classic strains of broiler chickens were vaccinated with intermediate (Bursine-2) and intermediate plus (IBD Blen) vaccine respectively at day twelve and serum was harvested on day twenty six. The mean serum titers were found 131.30 ± 36.04 and 7413.54 ± 569.39 in Cobb-500 and Hubbard classic respectively, where mean titer level of Cobb-500 was below the minimum protective level but Hubbard classic strain was developed 7413.54, protective level. These results were indicating that the maternal derived antibody titer persists above 1000 level hindering the development of antibody titer in vaccinated flocks. However, persistence of MDA titer above 1000 may causes of vaccination failure of IBD vaccine.International Journal of Natural Sciences (2014), 4(2) 22-27

Changes in body composition and Basic Metabolic Rate (BMR) following bariatric surgery

Changes in body composition and Basic Metabolic Rate (BMR) following bariatric surgery

Development of real‐time and quantitative QCM immunosensor for the rapid diagnosis of Aeromonas hydrophila infection

Since bacterial infection cause a significant economic loss in fish farms, it is necessary to develop rapid diagnostic tools. Interests on label-free biosensors have been raised for the rapid detection of aquatic pathogenic bacteria but have not been extensively studied yet. Here we report a quartz crystal microbalance (QCM) immunosensor system for the rapid and simple detection of Aeromonas hydrophila, a pathogen for fish and human, in comparison with a conventional indirect ELISA method. In QCM immunosensor system, an antibody against A. hydrophila was covalently cross-linked to the gold surface of sensor chip and bacterial attachment was monitored as real-time frequency shifts within 5 min. The frequency shifts were very positively related to the amounts of bacterial cells between 6.25 and 100 μg corresponding to 6 × 106 to 108 CFU with a high specificity. The QCM immunosensor was also able to detect bacterial cells in fish tissue extract in a dose-dependent manner. Indirect ELISA also showed the dose-dependent reaction and the amplified signal may allow a lower detection limit. However, QCM immunosensor system showed a more linear and reliable standard curve with R2 value of almost 1 (0.9999). Moreover, detection of the bacteria was much quicker and simpler.

IMMUNOGENICITY ASSESSMENT FOR DETERMINATION OF THE MOST POTENTIAL ISOLATES OF KOIHERPES VIRUS

Common carp and koi (Cyprinus carpio) are the main consumption fish commodity and ornamental fish in Indonesia. Diseases due to koiherpes virus (KHV) infection had caused a huge loss in both common carp and koi culture industries world-wide. This study was generated to select the most potential candidate from out of three koiherpes virus isolates based on their serological performances for vaccine development to control Koiherpes virus disease in koi. Virus collection, isolation, and propagation in cell culture have been conducted from common and koi samples originated from Banjarmasin, South Kalimantan; Takalar, South Sulawesi; Cirata Lake-Cianjur, West Java; Depok, West Java; Ciseeng, Bogor, West Java; and Purwokerto, Central Java. Determination of the causative agent of the disease was done by PCR (Polymerase Chain Reaction) assay and inoculation of filtrate homogenates from infected fish into cell culture. There are three koiherpes virus isolates (BJMN-1, BJMN-2, and SKBM) which are being successfully isolated, cultured and propagated in Koi Fin (KF-1) cell line. Calculation of tissue culture infection dose from each isolates were 104.55 TCID50/mL, 104.72 TCID50/mL, and 103.28 TCID50/mL. Immunoassay of the three isolates was conducted by Indirect-ELISA method. Polyclonal antibody was made by injecting each isolate to the experiment animals (rat/Rattus norwegicus strain Sprague Dawley). It was shown that all of three isolates have high level of immunogenicity, as seen from the absorbance values at 490 nm wavelength. BJMN-1 isolates have a higher potential for it’s ability to cross-react with the other isolates and provided the highest absorbance values. In future, all isolates can be used as KHV vaccine candidates to prevent KHV infection.

Characterization of Sarcoptes scabiei cofilin gene and assessment of recombinant cofilin protein as an antigen in indirect-ELISA for diagnosis

BackgroundScabies impairs the health of humans and animals and causes heavy economic losses. Traditional diagnostic methods for scabies are inefficient and ineffective, and so far there is no commercial immunodiagnostic or molecular based test for scabies.MethodsHere, we used recombinant Sarcoptes scabiei cofilin protein as an antigen to establish indirect ELISA. S. scabiei cofilin is highly homologous to Dermatophagoides farinae Der f 31 allergen (90 % identity). The S. scabiei cofilin gene was cloned and expressed in Escherichia coli to obtain recombinant protein. Western blotting and fluorescence immunohistochemistry were carried out, and we established an indirect ELISA method and detected 33 serum samples from scabies infected rabbits and 30 serum samples from naïve rabbits.ResultsWestern blotting demonstrated that S. scabiei cofilin possessed good immunogenicity and fluorescence immunohistochemistry showed the S. scabiei cofilin is widespread in the splanchnic area of mites. In ELISA, a cut-off value of 0.188 was determined to judge experimental positive and negative serum values. Specificity and sensitivity of the ELISA were 87.9 and 83.33 %, respectively.ConclusionsRecombinant S. scabiei cofilin showed potential value as a diagnostic antigen. The ELISA method established could be used in clinical diagnosis and provide experimental information in minimal or asymptomatic infection.

Development of an indirect ELISA method based on the VP3 protein of duck hepatitis A virus type 1 (DHAV-1) for dual detection of DHAV-1 and DHAV-3 antibodies

An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the recombinant VP3 protein of duck hepatitis A virus type 1 (DHAV-1) was developed and evaluated in this study. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:160 (0.94μg), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% gelatin. The cutoff value was determined to be 0.332, and the analytical sensitivity was 1:1280 (OD450–630=0.37). The results of the specificity evaluation showed that no cross-reactivity existed between DHAV-1 antiserum and other common duck-sensitive pathogens, except for duck hepatitis A virus type 3 (DHAV-3), suggesting that this could be a common approach for the simultaneous detection of DHAV-1 and DHAV-3 antibodies. The coefficients of variation (CVs) for all of the tested samples were lower than 10%. The concordance between the I-ELISA based on the VP3 subunit of DHAV-1 and that based on the whole DHAV-1 particle was 96%. These results indicate that the VP3-based I-ELISA method has high sensitivity, specificity, and repeatability and is as effective as the DHAV-1-based I-ELISA method for sero-surveillance. Thus, it may be a convenient and novel method for DHAV antibody detection and epidemiological surveillance of DHAV prevalence.