Double Sandwich ELISA Research Articles

Detection of Syphilis Infection among Voluntary Blood Donors from 2010 to 2014 in Chinese Nanjing Area

To understand the epidemiological characteristics of syphilis infection in blood donors of Chinese Nanjing area, to strengthen control of blood quality and to provide the scientific evidence for working out the strategy of prevention and treatment of syphilis infection. Firstly the double antigen sandwich ELISA was carried out according to its instructions, test results showing positive will be examed by double orifice second-round. A total of 362425 blood samples were collected from 2010 to 2014, among them 1277 positive samples were detected. The average rate of syphilis infection was 0.35％. The positive rates of different old years were statistically different（X(2)=265.679,P<0.001）, and the change trend of 5 years was statistically different(X(2)trend=237.928,P<0.001）. Among all age groups in the detection of treponema pallidema(TP), the most amount of positive samples and the lowest positive ratio 0.23% were found in the group 18 to 29 years old. The highest positive ratio was shown in age 50 years old and above. There was statistical different in all age groups(X(2)=361.620,P<0.001）. The positive ratio of the detection of TP was 0.32% in male donors which was lower than that in female donors(0.42%). There was statistical different in all sex groups(X(2)=24.659,P<0.001）. Peasant group had the highest positive ratio(1.07%) in the detection of TP and the student group had the lowest positive ratio(0.19%). There was statistical different in all occupation groups(X(2)=300.198,P<0.001）. Junior and under group had the highest positive ratio(0.59%) in the detection of TP and the undergraduates group has the lowest positive ratio(0.19%). There was statistical different in all education groups(X(2)=241.734,P<0.001）. Through chi-square test and trend test analysis showed that there was statistical different in sex, age, educational level and occupation differences. The tendency of the groups' statistics in 5 years is equally important. It is necessary to publicize blood donation information consultation scientifically before blood donation, especially for high-risk population. To ensure blood supply safety, it is important to recruit low-risk blood donors and develop permanent voluntary blood donors for reducing blood waste.

A study of carcinoembryonic antigen concentrations in patients with coronary artery disease

Abstract Background & aims Carcinoembryonic antigen (CEA) is a tumour marker whose role is now being evaluated as a biomarker for early diagnosis of acute coronary syndromes. Its levels may rise even prior to rise of established biomarkers of myocardial necrosis. Methods Serum CEA concentrations were measured by double sandwich ELISA method in four groups of subjects with STEMI, NSTEMI/UA (24 patients of NSTEMI and 18 patients of unstable angina), stable angina and healthy controls with 42 males between 40 to 60 years of age in each group. Also qualitative Troponin T assay and CPK-MB concentrations measurement was done in groups with STEMI and NSTEMI/UA and correlations between CEA concentrations and Troponin T and CPK-MB were made. Results Mean serum CEA concentrations were 5.19 ± 0.39, 3.84 ± 0.34, 3.91 ± 1.40, 1.84 ± 0.35 and 2.12 ± 0.40 ng/ml respectively in groups with STEMI, NSTEMI/UA, UA alone, stable CAD and healthy controls respectively. STEMI, NSTEMI/UA and UA patients had significantly higher concentrations than stable CAD and healthy controls (p Conclusions CEA is a sensitive biomarker for early diagnosis of ACS. Its levels correlate with severity of ACS. It may play a crucial role in diagnosis of ACS in patients who present with atypical manifestations or when traditional biomarkers such as CPK-MB and Troponins are not raised.

Serological Evidence and Risk Factors Associated With Hepatitis E Virus Infection in Pigs and Human at an Intensive Piggery Complex, Lagos Nigeria

Hepatitis E virus is a leading cause of acute and chronic liver failure, and pigs are considered natural reservoir host of zoonotic genotypes 3 and 4. The zoonotic risk of HEV among occupationally exposed individuals is a public health concern especially in developing countries. This paper describes evidence and risks associated with HEV in an intensive pig farm estate in Lagos Nigeria. In a cross sectional study, blood samples were conveniently collected from populations of pigs and pig handlers along with a questionnaire survey. Sera obtained were tested for anti-HEV IgG and IgM antibodies by two step double antigen sandwich ELISA using Hep.EV ELISA kit according to manufacturer?s protocol. Two hundred and twenty one and 73 sera were obtained from pigs and handlers respectively. Two hundred and twelve (97%) swine, and 13 (17.8%) human were positive for anti-HEV IgG. Similarly, 3 (1.4%) and 1 (1.3%) of swine and human sera were positive for anti-HEV IgM. This study shows evidence of HEV in the study populations and emphasizes its zoonotic risk among pig handlers. Improvement in biosecurity practices including sanitation and proper animal waste disposal is strongly recommended as part of control measures against HEV.

Evaluation of Protein Kinase Cβ and PPARγ Activity in Diabetic Rats Supplemented with Momordica charantia.

The present study was taken up to compare and evaluate the effect of Momordica charantia supplementation with pioglitazone on PKC-β and PPAR-γ activity in kidneys of diabetic rats. The hypoglycaemic and lipid lowering effect of Momordica charantia were screened in laboratory animal model and its potency was compared with a Thiazolidinedione (TZD) group antidiabetic drug like pioglitazone. Adult healthy albino rats of Wistar strain aged 3-4months, weighing between 170-250gm of either sex were divided into 4 groups; Group 1 (normal controls), Group 2 (diabetic controls), Group 3 (diabetic rats treated with pioglitazone) and Group 4 (diabetic rats treated with bitter melon juice). Type 1 Diabetes was induced in rats by intraperitoneal injection of streptozotocin at a dose of 55 mg/kg body weight, following which glucose levels were estimated by Accu chek- active glucometer on day 0, 7, 14, 21 and 28 days to assess the efficacy of Bitter Melon Juice (BMJ) and pioglitazone. After 28 days of treatment, the rats were sacrificed and blood collected from abdominal vena cava was used for estimation of triglycerides by Glycerol 3 phosphate oxidase phenol aminophenazone method and cholesterol by Cholestrol oxidase phenol aminophenazone method. PKC-β and PPAR-γ were estimated in the dissected kidneys by using double sandwich ELISA based kits on an automated plate reader. BMJ significantly reduced blood glucose levels in group 4 as compared to diabetic controls (p<0.001). Total cholesterol and triglycerides were significantly reduced in both group 3 and 4. In Group 4, there was reduction in PKC-β levels, when compared to Group 3(p=0.004). PPAR-γ levels were increased in both Group 3 and 4, when compared to Group 2. The results suggest that BMJ has hypoglycaemic and lipid lowering effect in diabetic animal models. BMJ increases PPAR-γ activity and decreases PKC-β activity in kidneys of diabetic rats, thereby preventing the complications of diabetes mellitus. Fresh BMJ mimics action of pioglitazone belonging to TZD group thus showing a potential for further research in identifying the active molecules responsible for glucose and lipid lowering action.

National serosurvey of encephalomyocarditis virus in healthy people and pigs in China.

Encephalomyocarditis virus (EMCV) is a zoonotic pathogen that has a wide spectrum of host range. The virus has been discovered on swine farms worldwide and can cause acute fatal myocarditis in piglets and reproductive disorders in sows. Although EMCV infection has been documented in farmed pigs in China, seroprevalence in humans has not been reported. In this study, we conducted nationwide serological surveys for EMCV in humans and farmed pigs in China in 2013, by the use of a double antigen sandwich ELISA method. A total of 3305 serum samples from healthy people were obtained from seven geographical regions in China, of which 1010 samples (30.56%) were positive for EMCV antibodies. The overall seroprevalence for EMCV in the age groups of 0-20, 21-40, 41-60 and >60 years were 13.5%, 30.25%, 36.83% and 38.71% respectively, showing a tendency of increasing with age (P = 0.000). A total of 3470 serum samples from farmed pigs were collected and tested for antibodies to EMCV. A high seroprevalence of 77% was recorded, and significant regional differences were observed. It was concluded that people and pigs in China were commonly infected by EMCV. In addition, in order to characterize changes of seroprevalence during natural EMCV infection in pigs, 240 serial serum samples were collected from 30 pigs (at 0, 15, 30, 60, 75, 90, 120, and 150 days of age) in a farrow-to-finish farm in China. The data showed that there were two EMCV antibody peaks: the first peak appeared at day 30, followed by a decrease in EMCV antibody titer, and the second occurred after day 75. Thus, the most susceptible period of pigs for EMCV infection was between day 30 and day 75 of age.

Genetic polymorphisms of RAGE and risk of ulcerative colitis in a Chinese population

The receptor for advanced glycation end products (RAGE) and its proinflammatory ligands are critically implicated in the pathological progression of ulcerative colitis (UC). Functional polymorphisms in the regulatory elements and/or ligand-binding regions of the RAGE gene affect the expression and function of RAGE and thus may increase susceptibility to UC. In this study, a total of 266 unrelated UC patients and 247 control subjects were analyzed for 3 RAGE single nucleotide polymorphisms (SNPs) (−429 T/C, −374 T/A, and G82S) using an improved small-amplicon high resolution melting curve (HRM) analysis assay. Serum levels of soluble RAGE (sRAGE) were determined by a double sandwich ELISA system. The genotypes, alleles and haplotypes were analyzed and compared between UC patients and control subjects. Three pairs of genotyping primers for three RAGE polymorphism loci (−429 T/C, −374 T/A, and G82S) were developed based on HRM. Significant differences in the allele distribution of the G82S polymorphism was found among UC cases and controls from a Chinese population. Carriers of the RAGE G82S variant genotype were at higher risk of UC (OR=2.594, 95% CI: 1.778–3.784, P<0.001) than homozygous wild-type individuals. Further analyses revealed that the 82 (GS+SS) variant genotype was associated with patients who have extended UC (OR=1.924, 95% CI: 1.163–3.181, P=0.010), and a family history of inflammatory bowel disease (IBD) (OR=1.923, 95% CI: 1.049–3.521, P=0.032). The polymorphisms −374 T/A and −429 T/C did not demonstrate any association with UC, but an association was found between the −374(TA+AA) variant genotypes and the serum sRAGE level (P=0.002). Moreover, haplotypes T/A/A and T/A/G showed significantly different frequencies between UC patients and controls (OR=3.337, 95% CI: 1.892–6.091, P=0.026; OR=0.530, 95% CI: 0.351–0.801, P=0.002). The present study developed novel primers based on HRM to provide preliminary evidence in a Chinese population that the RAGE polymorphism is involved in genetic susceptibility to UC and that the 82(GS+SS) genotype of G82S is a risk factor for UC. Furthermore, RAGE polymorphisms may be related to the location of UC as well as a family history of IBD in a Chinese population.

Quantification of Peach Fruit Allergen Lipid Transfer Protein by a Double Monoclonal Antibody-based Sandwich ELISA

Fruit is one of the most commonly reported food allergy sources in China, and peach lipid transfer protein (Pru p 3) has been identified as the major allergen inducing systematic symptoms. Crude allergen extracts and single component allergens have been used in food allergy diagnosis and immunotherapy. Reliable and sensitive analytical methods to quantify Pru p 3 content in fruit will help to identify low-allergenic cultivars among the abundant peach genetic resources in China. In this study, we developed a sensitive sandwich ELISA method to measure Pru p 3 in peach fruit based on two monoclonal antibodies with high sensitivity and linearity. Significant variability was observed in peach cultivars, with a much higher Pru p 3 level in peach peel than in pulp. This method will be very useful to select peach varieties with low Pru p 3 content and as a diagnostic product to detect hidden allergens in processed food.

Diagnosis of Invasive Aspergillosis: Use of the Galactomannan Assay

Invasive aspergillosis is the cause of severe morbidity and mortality in immunocompromised patients. Given the challenges of fungal cultures, non-culture surrogates are crucial to the timely diagnosis of invasive aspergillosis (IA) to initiate expedited treatment. The Platelia™ Aspergillus EIA (Bio-Rad, Hercules, California) is a double-sandwich ELISA that detects the galactomannan (GM) of the fungal cell wall and was cleared by the FDA for use in serum and bronchoalveolar fluid (BAL) in 2003 and 2011, respectively. The population in which GM has been studied the most and has shown the greatest accuracy is that of hematologic malignancy. The optimal optical density index (ODI) cutoff to define test positivity in the serum is still a matter of debate because this value influences test performance. Using a lower ODI threshold (≥0.5 vs. ≥1 vs. ≥1.5) optimizes sensitivity at the expense of specificity and vice versa using a higher ODI threshold. One must be alert to the potential for false-positive results, particularly if a ODI cutoff of 0.5 is used in the serum. False positives can occur due to medications, i.e., piperacillin/tazobactam, though there is increasing evidence that newer formulations are less cross-reactive with GM, and false-positive results occur in the presence of other molds that cross-react with GM. As the use of mold-active antifungal prophylaxis increases, one must be aware that GM may not perform as well due to lower pre-test probability of IA and lower test sensitivity. Emerging evidence indicates that use of GM in combination with other tests, e.g., Aspergillus PCR or lateral flow device (LFD), may enhance diagnostic accuracy beyond GM alone; however, further validation of these diagnostics in combination are required before routine implementation can be recommended. BAL GM performs better than serum as it is significantly more sensitive, though the optimal ODI cutoff is also debated (≥0.5 vs. ≥1).False-positive results can be due to use of medications, as with serum GM. False negatives can occur with the use of certain agents which decrease the viscosity of the BAL fluid, and use of such agents need to be considered by the clinician when evaluating a test result. Again, BAL GM, in conjunction with other tests, e.g., PCR and LFD, are promising, but further studies are needed. GM in other fluids, i.e., CSF, urine, and tissue, may be useful, but the studies are very limited. In summary, when employed in the right clinical context and interpreted appropriately, serum and BAL GM can facilitate the diagnosis and early treatment of IA. While there are significant limitations and the landscape is evolving, the test has an important role in clinical practice today.

A Double-Sandwich ELISA for Identification of Monoclonal Antibodies Suitable for Sandwich Immunoassays.

The sandwich immunoassay (sELISA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated into the test define most of the performance parameters of any subsequent immunoassay regardless of the assay format: traditional ELISA, lateral-flow immunoassay, various bead-based assays, antibody-based biosensors, or the reporting label. Here we describe an approach for identifying monoclonal antibodies (mAbs) suitable for use as capture antibodies and detector antibodies in a sELISA targeting bacterial protein toxins. The approach was designed for early identification of monoclonal antibodies (mAbs), in the initial hybridoma screen.

Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1.

BackgroundInfluenza H5N1 is one subtype of the influenza A virus which can infect human bodies and lead to death. Timely diagnosis before its breakout is vital to the human health. The current clinical biochemical diagnosis for influenza virus are still flawed, and the diagnostic kits of H5N1 are mainly based on traditional monoclonal antibodies that hardly meet the requirements for clinical applications. Nanobody is a promising tool for diagnostics and treatment due to its smallest size, high specificity and stability. In this study, a novel Nanobody-based bioassay was developed for rapid, low-cost and sensitive detection of the influenza H5N1 virus.MethodsNanobodies specific to H5N1 virus were selected from a VHH library by phage display technology. In this system, the biotinylated Nanobody was directionally captured by streptavidin coated on ELISA plate, which can specifically capture the H5N1 virus. Another Nanobody conjugated with HRP was used as a detector. A novel directional enzyme-linked immunosorbent assay for H5N1 using specific Nanobodies was established and compared to the conventional undirected ELISA assay.ResultsWe have successfully constructed a high quality phage display Nanobody library and isolated two Nanobodies against H5N1 with high affinity and specificity. These two Nanobodies were further used to prepare the biosensor detection system. This streptavidin-biotin-based directional double Nanobodies sandwich ELISA for H5N1 detection showed superiority over the commonly undirectional ELISA protocol. The linear range of detection for standards in this immunoassay was approximately 50–1000 ng/mL and the detection limit was 14.1 ng/mL. The average recoveries of H5N1 virus from human serum samples were in the range from 94.58% to 114.51%, with a coefficient of variation less than 6.5%.ConclusionCollectively, these results demonstrated that the proposed detection system is an alternative diagnostic tool that enables a rapid, inexpensive, sensitive and specific detection of the influenza virus.

Plasma T-cadherin negatively associates with coronary lesion severity and acute coronary syndrome.

This study evaluated associations between plasma T-cadherin levels and severity of atherosclerotic disease. Three hundred and ninety patients undergoing coronary angiography were divided into three groups based on clinical and angiographic presentation: a group (n=40) with normal coronary arteries, a group (n=250) with chronic coronary artery disease and a group (n=100) with acute coronary syndrome. Plasma T-cadherin levels were measured by double sandwich ELISA. Intravascular ultrasound data of the left-anterior descending artery were acquired in a subgroup of 284 patients. T-cadherin levels were lower in patients with acute coronary syndrome than in normal patients (p=0.007) and patients with chronic coronary artery disease (p=0.002). Levels were lower in males (p=0.002), in patients with hypertension (p=0.002) and inpatients with diabetes (p=0.008), and negatively correlated with systolic blood pressure (p=0.014), body mass index (p=0.001) and total number of risk factors (p=0.001). T-cadherin negatively associated with angiographic severity of disease (p=0.001) and with quantitative intravascular ultrasound measures of lesion severity (p<0.001 for plaque, necrotic core and dense calcium volumes). Significant associations between T-cadherin and intravascular ultrasound measurements persisted even if the regression model was adjusted for the presence of acute coronary syndrome. Multivariate analysis identified a strong (p=0.002) negative association of T-cadherin with acute coronary syndrome, and lower T-cadherin levels significantly (p=0.002) associated with a higher risk of acute coronary syndrome independently of age, gender and cardiovascular risk factors. A reduction in plasma T-cadherin levels is associated with increasing severity of coronary artery disease and a higher risk for acute coronary syndrome.

Pharmacokinetics of the Sri Lankan hump-nosed pit viper (Hypnale hypnale) venom following intravenous and intramuscular injections of the venom into rabbits

The knowledge of venom pharmacokinetics is essential to improve the understanding of envenomation pathophysiology. Using a double-sandwich ELISA, this study investigated the pharmacokinetics of the venom of hump-nosed pit viper (Hypnale hypnale) following intravenous and intramuscular injections into rabbits. The pharmacokinetics of the venom injected intravenously fitted a three-compartment model. There is a rapid (t1/2π = 0.4 h) and a slow (t1/2α = 0.8 h) distribution phase, followed by a long elimination phase (t1/2β = 19.3 h) with a systemic clearance of 6.8 mL h−1 kg−1, consistent with the prolonged abnormal hemostasis reported in H. hypnale envenomation. On intramuscular route, multiple peak concentrations observed in the beginning implied a more complex venom absorption and/or distribution pattern. The terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were nevertheless not significantly different (p > 0.05) from that of the venom injected intravenously. The intramuscular bioavailability was exceptionally low (Fi.m. = 4%), accountable for the highly varied median lethal doses between intravenous and intramuscular envenomations in animals. The findings indicate that the intramuscular route of administration does not significantly alter the pharmacokinetics of H. hypnale venom although it significantly reduces the systemic bioavailability of the venom.

Analysis of combined CSF biomarkers in AD diagnosis.

The new proposed diagnostic criteria for early diagnosis of Alzheimer's Disease (AD) underline the value of cerebrospinal fluid (CSF) biomarkers. The first aim of the study was to determine the diagnostic accuracy of CSF biomarker Abeta1-42, T-tau, and P-tau in differentiating AD patients in our cohort by means of "pure" biomarkers and in form of a combined analysis of these biomarkers. The second aim of the study was to determine the diagnostic accuracy of these markers for predicting incipient AD in patients with mild cognitive impairment (MCI). We studied 102 CSF samples: 33 AD [mean age at baseline 71.2 (54-86)], 16 MCI [mean age at baseline 71.3 (57-78)], 24 non AD dementia, including 7 vascular dementia, 4 frontotemporal degeneration, 5 dementia with Lewy Body, and 8 with other dementia [mean age at baseline 72.7 (51-87)] and 32 non-demented neurological patients [mean age at baseline 71.3 (45-87) referred to as control (CO) later in the text]. A double sandwich ELISA (Innotest beta amyloid Abeta1-42, hTau and P-tau181 by Innogenetics, Gent, Belgium) was performed to quantify the concentration of the above biomarkers. The three biomarkers were then combined in the IATI index [(measured Ab1-42)/(240 + 1.18 *measured tau)], and in the ratios Abeta1-42/T-tau, Abeta1-42/P-tau, T-tau/Abeta1-42 and P-tau/Abeta1-42. Abeta1-42, T-tau and P-tau181 concentration showed statistically significant differences between AD and CO (327.2 pg/mL +/- 150.2 pg/mL and 659.4 pg/mL +/- 254.2 pg/mL; 508.2 pg/mL +/- 360.2 pg/mL and 305.3 pg/mL +/- 228.9 pg/mL; 82.2 pg/mL +/- 26.1 pg/mL and 45.3 pg/mL +/- 26.4 pg/mL, respectively, p < 0.05), while the difference between AD and MCI was statistically different only for Abeta1-42 (327.2 pg/mL +/- 150.2 pg/mL and 600.8 +/- 271.9 pg/mL, respectively, p < 0.05). The IATI index was 0.5 +/- 0.3 in AD, 0.9 +/- 0.6 in MCI, 1.37 +/- 0.9 in non AD dementia and 1.26 +/- 0.8 in non-demented neurological patients. With a cut-off fixed at 1 the sensitivity and specificity of the IATI index in discriminating AD from CO was 84% and 52%, respectively. This study confirms the great significance of CSF biomarker measurements in AD diagnosis in clinical routine. It is understood that a clinical diagnostic work-up is necessary in the process. Moreover, a biochemical profile of CSF biomarkers requires further investigations.

Abstract 297: Cyclic GMP Reduces Vascular Smooth Muscle Migration through Inhibition of TGF-ß1/Smad Signaling

Abnormal vascular smooth muscle (VSM) growth remains an elemental foundation of vasculoproliferative disorders including atherosclerosis and restenosis. Many second messenger, cytokine, and growth factor signals mediate control of VSM growth, and among these is transforming growth factor (TGF)-β1, a pluripotent cytokine with wide-ranging yet often opposite effects in VSM. Cyclic nucleotide signaling also exerts powerful growth control of VSM, and our previous work has helped establish a biological link between cyclic GMP and TGF-β1 in injured carotid arteries. The current study characterized the influence of cyclic GMP on TGF-β1 and its receptor-activated Smad3 in rat primary VSM cells. The heme-dependent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY41) significantly increased cyclic GMP and site-specific phosphorylation of vasodilator-activated serum phosphoprotein (VASP) in manner indicative of active protein kinase G (PKG) and PKA signaling. Recombinant TGF-β1 (10 ng/ml) significantly stimulated phospho-Smad3 (Ser 423/425 ) and decreased inhibitory Smad7 in VSM cell homogenates, and using flow cytometry significantly increased cells in G 2 /M and expression of cyclins D and E and Cdk2 and Cdk4 while decreasing expression of inhibitory p21 and p27 after 24 hours compared to vehicle controls. TGF-β1 also significantly increased cell numbers compared to controls after 48 hours, thus confirming growth promoting capacities of TGF-β1 in VSM. In cell lysates double-sandwich ELISA revealed that BAY41 significantly reduces total and active TGF-β1, and Western analyses showed it significantly decreases total and phospho-Smad3 Ser423/425 expression and reduces MMP-2 and MMP-9 expression and activity (via column zymography) in both cell lysates and conditioned media after 1 and 48 hours. BAY41 also significantly reduced serum- and PDGF-stimulated cell migration between 6 and 18 hours using an in vitro scrape injury and a transwell assay. In comparison, inclusive effects of BAY41 were replicated by its prototype YC-1 and by the heme-independent sGC activator BAY 60-2770. These data clearly support growth protective capacities of cGMP in VSM and propose it operates through attenuation of TGF-β1/Smad3 signaling.

Toxicokinetics of Naja sputatrix (Javan spitting cobra) venom following intramuscular and intravenous administrations of the venom into rabbits

Existing protocols for antivenom treatment of snake envenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of venoms. The toxicokinetics of Naja sputatrix (Javan spitting cobra) venom was investigated following intravenous and intramuscular injections of the venom into rabbits using double-sandwich ELISA. The toxicokinetics of the venom injected intravenously fitted a two-compartment model. When the venom was injected intramuscularly, the serum concentration–time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were not significantly different (p > 0.05) from that of the venom injected intravenously. The systemic bioavailability of the venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some cobra venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn't significantly alter the toxicokinetics of N. sputatrix venom although it significantly reduces the systemic bioavailability of the venom.

Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs

There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.

240. Development of a Double Sandwich Flourescent ELISA to Detect Rattlesnake Venom In Biological Samples from Horses with a Clinical Diagnosis of Rattlesnake Bite

240. Development of a Double Sandwich Flourescent ELISA to Detect Rattlesnake Venom In Biological Samples from Horses with a Clinical Diagnosis of Rattlesnake Bite

Immunological properties of Hypnale hypnale (hump-nosed pit viper) venom: Antibody production with diagnostic and therapeutic potentials

Envenomation by hump-nosed pit viper (Hypnale hypnale, Hh) in Sri Lanka has caused significant morbidity and mortality, attributed to 35% of total venomous snakebites. In Southwestern India (Kerala), H. hypnale was increasingly identified as a dangerous and common source of envenomation, second to the Russell's viper but ahead of the cobra bites. Unfortunately, there is still no specific antivenom to date. This study aims to investigate the immunological properties of the venom and to assess the feasibility of specific Hh antivenom production as well as the development of a diagnostic assay. Hh venom elicited satisfactory titers of anti-Hh IgG in rabbits after 3rd immunization. The anti-Hh IgG, isolated with caprylic acid precipitation method, was effective in neutralizing the venom lethality (potency=48 LD50 per ml IgG) as well as its procoagulant, hemorrhagic and necrotic effects, indicating the possibility to produce the specific antivenom using the common immunization regime. Cross-reactivity studies using indirect ELISA showed that anti-Hh IgG cross-reacted extensively with several Asiatic crotalid venoms, particularly that of Calloselasma rhodostoma (73.6%), presumably due to the presence of venom antigens common to both snakes. Levels of immunological cross-reactivity were vastly reduced with double-sandwich ELISA. Further work demonstrated that the assay was able to distinguish and quantify venoms of H. hypnale, Daboia russelii and Echis carinatus sinhaleyus (three common local viperid) used to spike human sera at various concentrations. The assay hence may be a useful investigating tool for diagnosing biting species and studying the time course profile of venom concentrations in blood.

Differential diagnosis of pandemic (H1N1) 2009 infection by detection of haemagglutinin with an enzyme-linked immunoassay

A sensitive and convenient immunoassay that can directly differentiate pandemic (H1N1) 2009 (pH1N1) virus from seasonal influenza virus can play an important role in the clinic. In the presented study, a double-sandwich ELISA (pH1N1 ELISA), based on two monoclonal antibodies against haemagglutinin (HA) of the pH1N1 virus, was developed. After laboratory determination of the sensitivity and specificity characteristics, the performance of this assay was evaluated in a cohort of 904 patients with influenza-like illness. All seven strains of pH1N1 virus tested were positive by pH1N1 ELISA, with an average lower detection limit of 103.0 ± 0.4 tissue culture infective dose (TCID)50/mL (or 0.009 ± 0.005 HA titre). Cross-reaction of the assay with seasonal influenza virus and other common respiratory pathogens was rare. In pH1N1-infected patients, the sensitivity of the pH1N1 ELISA was 92.3% (84/91, 95% CI 84.8–96.9%), which is significantly higher than that of the BD Directigen EZ Flu A + B test (70.3%, p <0.01). The specificity of pH1N1 ELISA in seasonal influenza A patients was 100.0% (171/171, 95% CI 97.9–100.0%), similar to that in non-influenza A patients (640/642, 99.7%, 95% CI 98.9–100.0%). The positive predictive value for pH1N1 ELISA was 97.7% and the negative predictive value was 99.1% in this study population with a pH1N1 prevalence of 10.1%. In conclusion, detection of HA of pH1N1 virus by immunoassay appears to be a convenient and reliable method for the differential diagnosis of pH1N1 from other respiratory pathogens, including seasonal influenza virus.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.