Enzyme-linked Immunosorbent Assay Test Kit Research Articles

New Route for Fast Detection of Antibodies against Zoonotic Pathogens in Sera of Slaughtered Pigs by Means of Flow-through Chemiluminescence Immunochips

The research on fast screening methods for antibodies against zoonotic pathogens in slaughter animals is important for food safety in farming and meat-processing industries. As a proof-of-concept study, antibodies against the emerging zoonotic pathogen hepatitis E virus (HEV) and enteropathogenic Yersinia spp. were analyzed in parallel using immobilized recombinant antigens (rAgs) of HEV genotypes 1 and 3 and Yersinia outer protein D (YopD) on a flow-through chemiluminescence immunochip. These rAgs are usually part of commercially available line immunoassays (LIAs) used for human diagnostics. In this study, sera from slaughtered pigs were tested on the microarray analysis platform MCR 3 to detect anti-HEV and anti-Yersinia IgG. The new method was characterized regarding signal reproducibility and specificity. The analytical performance was compared with in-house enzyme-linked immunosorbent assay (ELISA) and a LIA based on recomLine HEV (Mikrogen) or the ELISA test kit pigtype Yersinia Ab (Qiagen), respectively. The immunochip revealed the highest analytical sensitivity and was processed in 9 min automatically on the MCR 3. A comparative screening of swine serum samples from Bavarian slaughterhouses regarding anti-HEV and anti-Yersinia IgG seroprevalence was conducted. By using the LIA, 78% of the sera were tested positive for HEV antibodies. The immunochip and the ELISA identified anti-HEV IgG in 96% and 93% of the tested samples using the O2C-gt1 and O2C-gt3 rAg, respectively. The screening for anti-Yersinia IgG resulted in 86% positive findings using the immunochip and 57% and 48% for the ELISA methods, respectively, indicating a higher detection capability of the new method. Serum samples of slaughtered pigs could be analyzed faster and in an automated way on the microarray analysis platform MCR 3 which shows the great potential of the new immunochip assay format for multiplexed serum screening purposes.

Elaboration of Immunoenzymatic Test-Kit for Total Human ΙgΕ Assay and Investigation of Its Analytical Properties

Enzyme-linked immunosorbent assay (ELISA) test-kit has been developed based on complex immuno-chemical, including epitop mapping, characteristics of monoclonal antibodies (MAbs) to human IgЕ. “Sandwich” type ELISA based on usage of different epitop directionality MAbs. It has been founded correlation between affinity of different MAb pairs and its sorbtion-detection ability. Optimal configuration of MAbs in “sandwich” ELISA was follow-ing: 164H10 - 165C12 and 164H10 - 166B7. Cooperative usage of horseradish peroxidase conjugates of MAbs directed to different epitops (165C12-HRP + 166B7-HRP) increased analytical sensitivity of assay and constituted 4.5 IU/ml. Analytical characteristics of developed test-kit were following: dynamic range – from 4.5 to 1600 IU/ml, variation coefficient value during one procedure – 4.2±2.1%, and between procedures – 4.8±2.3%. Presences of human IgG, IgA, IgМ, albumin (1 μg/ml) were not effect on the assay specificity (test concentration of IgE – 50 IU/ml).

경기도 북부지역 젖소 사육농장의 bovine leukemia virus 감염 실태 조사

This study was carried out to investigate the prevalence of bovine leukemia virus (BLV) infection and to compare the results of enzyme-linked immunosorbent assay (ELISA) and nested polymerase chain reaction (nPCR) in dairy farms in northern Gyeonggi province from August through December 2011. A total of 625 dairy cattle from 14 dairy farms were tested for antibodies against BLV using commercially available ELISA test kit. The overall seroprevalence of BLV infection was 76.3%. The seroprevalence of diary cattle according to age was the highest at 61~72 months (88.0%, P<0.001). Two hundred fifty one dairy cattle from 7 diary farms were tested ELISA and nPCR. The kappa value of BLV between ELISA and nPCR was 0.765. The results indicate that BLV infection spread widely in dairy farms and the nPCR is rapid method for the early detection of BLV infection.

Evaluation of Four Enzyme-Linked Immunosorbent Assays for the Serologic Survey of Avian Influenza in Wild Bird Species

Wild birds that reside in aquatic environments are the major reservoir of avian influenza viruses (AIVs). Since this reservoir of AIVs forms a constant threat for poultry, many countries have engaged in AIV surveillance. More and more commercial enzyme-linked immunosorbent assays (ELISA) are available for serologic surveillance, but these tests are often developed and validated for use in domestic poultry. However, for a correct interpretation of ELISA test results from wild bird sera, more information is needed. In the present study, four ELISA test kits (ID-Vet IDScreen, IDEXX FlockChek AI MultiS-Screen Ab Test Kit, Synbiotics FluDETECTBE, and BioChek AIMSp) were compared for the serologic analysis of 172 serum samples from mallard, mute swan, and Canada goose. Samples were selected based on ID-Vet IDScreen results to obtain an approximately equal number of positive and negative samples. In addition, 92 serum samples from experimentally infected specific-pathogen-free (SPF) chickens and Pekin ducks were included in the tests for validation purposes. Cohen's kappa statistics and Spearman correlation coefficients were calculated for each combination of two tests and for each bird species. Test agreement for mallard sera varied from poor to moderate, while test results for Canada goose and swan sera agreed from fair to almost perfect. The best agreement was obtained with sera from experimentally infected SPF chickens and Pekin ducks. This study shows that some care must be taken before using nucleoprotein ELISAs for the testing of sera from wild birds and that more reliable validation studies should be considered before their use in the serologic surveillance of wild birds.

Immunochemical detection of masked mycotoxins: A short review

Mycotoxin derivatives that escape conventional analytical detection of parent (free) forms because their structure has been changed are designated masked mycotoxins. Masking phenomena are due to a defensive response of the host plant or can occur during food processing. Failure to detect masked mycotoxins will lead to a significant underestimation of the mycotoxin content of a particular sample. To date, mainly chromatographic methods were developed for masked mycotoxin determination and quantification. However, for fast screening, it is important to develop on-site methods for detection of masked and parent (free) forms. Although immunochemical methods could provide a simple and economical alternative to chromatography, their use for masked forms is only at the start of development. The key-point for antibody-based methods for masked mycotoxin determination is cross-reactivity of the specific antibody towards masked mycotoxins. If the antibody does not show meaningful affinity for masked forms, they will be latent, and the total content of this mycotoxin will be underestimated. If the antibody shows affinity for masked forms, the sum of free and masked forms will be determined. Currently, neither antibodies nor immuno-based methods were specifically developed for masked mycotoxins, but some enzyme-linked immunosorbent assay test-kits and immunoaffinity columns for mycotoxins were evaluated for their detection. This paper describes possible applications of antibody-based techniques for masked mycotoxin detection on the basis of recent literature.

Detection of Mycoplasma gallisepticum in experimentally infected broiler chickens using Culture, SPA, ELISA, and PCR methods

Avian mycoplasmosis is caused by several pathogenic mycoplasmas of which Mycoplasma gallisepticum and Mycoplasma synoviae are the most important. This experiment was designed to study the diagnosis of M. gallisepticum infection in broiler chicken receiving M. gallisepticum. One hundred and twenty one-day-old chickens were raised in separate isolated rooms and equally allocated into two groups and renamed groups A and B were 121-day-old chickens. At 3 weeks old, group A was considered as the control group while group B was challenged with M. gallisepticum containing 1 × 106 CFU/bird via an eye drop. In this study, culture, serum plate agglutination (SPA) test, the commercially available enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) tests were compared for the detection of M. gallisepticum infection during the 1–7 weeks of age. Results revealed that M. gallisepticum was found in group B using culture and PCR during the 4–7-week-old period. The SPA results showed positive reactors in group B during the 5–7-week-old period. The positive reactors were detected by commercial ELISA test kit from 6 to 7 weeks of age. In this study, sensitivity of the PCR method was higher as compared to culture and serological tests. Its high sensitivity and fast turnaround time is making it the most frequently used test to monitor M. gallisepticum infection beside other tests.

Seroprevalence of Toxoplasma gondii in pregnant women

The present work aimed to evaluate the seroprevalence of Toxoplasma gondii and their associated risk factors among the pregnant women. This study was carried out from 2009 to 2010 in 232 pregnant women attending the private hospitals in and around Salem, Tamil Nadu. Anti-toxoplasma specific IgG and IgM were assessed using Enzyme-linked immunosorbent assay (ELISA) test kits. Of these 232 samples, anti-toxoplasma specificimmunoglobulin G (IgG) antibodies were detected in 23 (9.9%) cases while 9 (3.9%) had positive anti-toxoplasma specific immunoglobulin M (IgM) suggestive of acute infection during or just before pregnancy. A structured questionnaire interview for pregnant women was performed to investigate the risk factors associated with the T. gondii infection. The higher infection rate of 7.9% (for anti-toxoplasma IgG antibodies) was found with pregnant women living in rural area (15 subjects) than in urban area (8 subjects) and it was statistically significant (P<0.03). A few risk factors such as stillbirth (P<0.02), miscarriage (P<0.009), rearing cat as pet animal (P<0.005) and outdoor gardening (P<0.03) were statistically associated with the seroprevalence rate. Even with low seroprevalence rate of 9.9% latent infection and 3.9% acute infection there is a need for the pregnant women to be educated on the ways to minimize the exposure to T. gondii infection and to reduce the risk of congenital transmission. Key words: Toxoplasma gondii, seroprevalence, pregnancy, anti-toxoplasmaimmunoglobulin G (IgG), immunoglobulin M (IgM).

Comparison of three methods for detection of melamine in compost and soil

Recent product recalls and food safety incidents due to melamine (MM) adulteration or contamination have caused a worldwide food security concern. This has led to many methods being developed to detect MM in foods, but few methods haves been reported that can rapidly and reliably measure MM in environmental samples. To meet this need, a high performance liquid chromatography (HPLC) with UV detection method, an enzyme-linked immunosorbent assay (ELISA) test kit, and an enzyme-linked rapid colorimetric assay (RCA) test kit were evaluated for their ability to accurately measure MM concentrations in compost and soil samples. All three methods accurately detected MM concentrations if no MM degradation products, such as ammeline (AMN), ammelide (AMD) and cyanuric acid (CA), were present in an aqueous sample. In the presence of these MM degradation products, the HPLC yielded more accurate concentrations than the ELISA method and there was no significant (P>0.05) difference between the HPLC and RCA methods. However, if samples were purified by SPE or prepared with blocking buffer, the ELISA method accurately measured MM concentrations, even in the presence of the MM degradation products. The HPLC method generally outperformed the RCA method for measuring MM in soil extracts but gave similar results for compost extracts. The number of samples that can be analyzed by the ELISA and RCA methods in a 24-hour time period is much greater than by the HPLC method. Thus the RCA method would seem to be a good screening method for measuring MM in compost and soil samples and the results obtained could then be confirmed by the HPLC method. The HPLC method, however, also allows simultaneous measurement of MM and its degradation products of AMD, AMN and CA.

Prevalence and risk factors of Giardia duodenalis in dogs from Romania

The protozoan Giardia duodenalis is a mammalian-infecting parasite that produces diarrhoea and malabsorption in its hosts. A survey to investigate canine infections with G. duodenalis in Romania was undertaken between June 2008 and December 2009. The objectives of the study were to (i) estimate the prevalence of infection in different dog populations (kennels, shelters, shepherd, household) using microscopy and a commercially available enzyme-linked immunosorbent assay (ELISA) test kit; (ii) to establish the level of agreement and characteristics of the tests; and (iii) to identify risk factors for infection by multivariate logistic regression models. Faecal samples were collected from 614 dogs aged from 1 month to 16 years (mean±SD=2.88±2.86 years). Each sample was tested for the presence of cysts using a flotation method with saturated sodium chloride solution and 416 out of 614 stool samples were further examined for the presence of G. duodenalis specific antigens using Giardia Microwell ELISA (SafePath™ Laboratories).Giardia cysts were identified in 8.5% of total dogs (52/614) and statistical significantly more frequently in dogs living in communities. The cysts prevalence according with dog populations was as follows: 7.2%(9/125) in kennel dogs; 16.5%(27/164 in shelter dogs; 4.3%(2/46) in shepherd dogs; 4.8%(4/84) in household dogs from urban areas; and 5.1%(10/195) in household dogs from rural areas.The overall prevalence of Giardia infection by ELISA was 34.6% (144/416). The prevalence was significantly higher in kennel dogs (50%; 13/26), shelter dogs (47.7%; 74/155) and shepherd dogs (40.5%; 17/42) than in household dogs from urban areas (34.1%; 15/44) and household dogs from rural areas (16.8%; 25/149).It was noticed poor agreement between microscopy and ELISA (k=0.19). The microscopy performed best, with an Youden Index of 0.74, a Se of 73.68% and a Sp of 100%. ELISA had 100% Sp, but only 19.44% Se.Young dogs (up to 12 months age) and living in communities were identified as risk factors for infection by multivariate logistic regression analysis.71.2% (37/52) Giardia cysts positive dogs presented co-infections with other intestinal parasites: Toxocara canis (14/52; 26.9%), Isospora ohioensis (12/52; 23.1%), Ancylostoma caninum (9/52; 17.3%), Uncinaria stenocephala (7/52; 13.5%), Trichocephalus vulpis (6/52; 11.5%), Hammondia heydorni/Neospora caninum (5/52; 9.6%), Sarcocystis spp. (5/52; 9.6%), Isospora canis (4/52; 7.7%), Capillaria aerophila (3/52; 5.8%), Strongyloides stercoralis (2/52; 93.8%), Dipylidium caninum (1/52; 1.9%) and Toxascaris leonina (1/52; 1.9%).

Antibody Response After a Single Dose of an AS03-Adjuvanted Split-Virion Influenza A (H1N1) Vaccine in Heart Transplant Recipients

Influenza A (H1N1) has emerged as a considerable threat for recipients of organ transplants. Vaccination against the novel influenza A (H1N1) virus has generally been advocated. There is limited experience with AS03-adjuvanted A/H1N1 pandemic influenza vaccines in immunosuppressed patients. We conducted an observational, nonrandomized single-center study to assess antibody response and vaccine-related adverse effects in 47 heart transplant recipients (44 men; age, 56±13 years). The AS03-adjuvanted, inactivated split-virion A/California/7/2009 H1N1v pandemic vaccine was administered. Antibody titers were measured using hemagglutination inhibition; immunoglobulin G (IgG) response was assessed using a new pandemic influenza A IgG enzyme-linked immunosorbent assay (ELISA) test kit and compared with hemagglutination-inhibition titers. Adverse effects of vaccination were assessed by a questionnaire. Postvaccination antibody titers of greater than or equal to 1:40 were found in only 15 patients, corresponding to a seroprotection rate of 32% (95% confidence interval, 19%-47%). Sensitivity, specificity, positive predictive value, and negative predictive value of ELISA testing were 80.0%, 68.8%, 54.5%, and 88.0%, respectively. Age, time posttransplantation, and immunosuppressive regimen did not impact antibody response. Vaccination was well tolerated. Single-dose administration of an AS03-adjuvanted vaccine against the novel influenza A (H1N1) virus did not elicit seroprotective antibody concentrations in a substantial proportion of heart transplant recipients; the new pandemic influenza A IgG ELISA test kit proved to be of limited clinical use.

Quantitative Detection of Human Tumor Necrosis Factor α by a Resonance Raman Enzyme-Linked Immunosorbent Assay

Tumor necrosis factor α is an inflammatory cytokine which has been linked with many infectious and inflammatory diseases. Detection and quantification of this key biomarker is commonly achieved by use of an enzyme-linked immunosorbent assay (ELISA). This fundamental technique uses the spectroscopic detection of a chromogen such as 3,3',5,5'-tetramethylbenzidine (TMB). Horseradish peroxidase (HRP), bound to the detection antibody, catalyzes the oxidation of TMB by hydrogen peroxide to generate colored products which may be measured spectrophotometrically. In this study we have used a conventional ELISA kit and shown that, by replacing the traditional colorimetric detection with resonance Raman spectroscopy, we can achieve 50 times lower detection limits and the potential for multiplexed analysis is increased. In this approach, the laser wavelength was tuned to be in resonance with an electronic transition of the oxidized TMB. The relative intensity of the enhanced Raman bands is proportional to the amount of TMB, thus providing a means of improved quantification. Furthermore, TMB is one of the most widely used chromogenic substrates for HRP-based detection and commercial ELISA test kits, indicating that this detection technique is applicable to a large number of target analytes.

Effect of Heat Treatment on the Quantitative Detection of Egg Protein Residues by Commercial Enzyme-Linked Immunosorbent Assay Test Kits

This study examined the changes in the solubility of egg proteins as affected by different heat treatments and compared the performances of three commercial test kits for the quantitation of protein residues in heat-treated samples. National Institute of Standards and Technology (NIST) whole egg standard reference material #8415 and Henningsen spray-dried whole egg powder were subjected to heating in the presence of water at 60 and 100 degrees C, autoclaving for 5 or 10 min, or dry heating at 60-400 degrees C for 10 min. The amount of protein in the heated samples was assayed using the bicinchoninic acid total protein assay as well as egg-specific commercial enzyme-linked immunosorbent assay (ELISA) kits. Elevated heat resulted in a lower level of proteins extracted. Neogen's Veratox kit, which is reactive to multiple proteins in egg, greatly underestimated the amount of residual proteins in the boiled or autoclaved samples. Tepnel BioSystems' Biokits assay, which employs antibodies specific to a heat-stable marker protein (ovomucoid), registered a higher level of protein in these samples. Both test kits substantially underestimated the amount of residual proteins in samples dry-heated at temperatures >176 degrees C. The Morinaga test, using an improved extraction buffer, registered the highest level of protein in the heat-treated NIST samples but not the Henningsen samples. The underestimation by the commercial test kits was attributed to changes in the immunoreactivity of residual proteins after heat treatments and not the differences in the amount of protein extracted. These results suggest that thermal processing may affect the quantitative analysis of allergens and needs to be taken into account in the validation of commercial ELISA test kits.

Survey and risk assessment of the mycotoxins deoxynivalenol, zearalenone, fumonisins, ochratoxin A, and aflatoxins in commercial dry dog food

The aim of the present study was to investigate the occurrence of mycotoxins in commercial dog food, as a basis to estimate the risk of adverse effects. Seventy-six dry dog food samples from 27 producers were purchased from retail shops, supermarkets, and specialized pet food shops in Vienna, Austria. The frequency and levels of deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUM), ochratoxin A (OTA). and aflatoxins (AF) in dry dog food were determined. Mycotoxin analysis were performed by commercial enzyme-linked immunosorbent assay (ELISA) test kits. Confirmatory analyses were done for DON, ZEA, and FUM by high performance liquid chromatography (HPLC) after extract clean-up with immunoaffinity columns. The correlations between ELISA and HPLC results for DON and ZEA were acceptable and indicated that ELISA could be a simple, low cost, and sensitive screening tool for mycotoxins detection, contributing to quality and safety of pet food. DON was the mycotoxin most frequently found (83% positives; median 308µg/kg, maximum 1,390µg/kg). ZEA (47% positives, median 51µg/kg and maximum 298µg/kg) and FUM (42% positives, median 122µg/kg and maximum 568µg/kg) were also frequently detected in dog food. OTA was less frequently found (5%, median 3.6µg/kg, maximum 4.7µg/kg. AF were not detected (<0.5µg/kg) in any sample. The results show that dry dog food marketed in Vienna are frequently contaminated with mycotoxins (DON > ZEA > FUM > OTA) in low concentrations, but do not contain AF. The high frequency of Fusarium toxins DON, ZEA, and FUM indicates the need for intensive control measures to prevent mycotoxins in dog foods. The mycotoxin levels found in dry dog food are considered as safe in aspects of acute mycotoxicoses. However, repeated and long-time exposure of dogs to low levels of mycotoxins may pose a health risk.

Detection of hazelnuts and almonds using commercial ELISA test kits

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.

Surveying infections among pregnant women in the Niger Delta, Nigeria

Background:There is paucity of epidemiological data on infectious diseases among antenatal mothers in Bayelsa State of the Niger Delta, Nigeria.Aims:The aim of this study was to determine the seroprevalence of the serological markers Human immunodeficiency virus-antibody (HIV-Ab), Hepatitis B surface antigen(HBsAg), Hepatitis C virus antibody(HCV-A)and antibodies to T. pallidum among pregnant women in Yenagoa, Bayelsa State, South–South Nigeria.Settings and Design:This is a cross-sectional study which was carried out in Yenagoa city, the heart of the Niger Delta, Nigeria.Materials and Methods:Human immunodeficiency virus (HIV) antibodies were detected by using “Determine” HIV-1/2 test strip (Abbott Laboratories, Japan); hepatitis B surface antigen (HBsAg), antibodies to hepatitis C virus (anti-HCV) and antibodies to T. pallidum were carried out using ACON rapid test strips (ACON Laboratories, USA). All positive samples for HIV, HBV and HCV were confirmed using the Clinotech diagnostic enzyme-linked immunosorbent assay (ELISA) test kits (Clinotech Laboratories, USA), while all reactive samples to Treponema pallidum antibodies were confirmed by the Treponema pallidum hemagglutination (TPHA) test (Lorne Laboratories Ltd., UK). All test procedures were carried out according to the manufacturers’ instructions.Statistical Analysis Used:The data generated were coded, entered, validated and analyzed using Statistical Package for Social Science (SPSS), version 12.0, and Epi info. The seroprevalence of syphilis, HBsAg, HCV and HIV was expressed for the entire study group by age, sex and other demographic features using Pearson chi-square analysis. Values below 0.05 were considered statistically significant.Results:A total of 1,000 apparently healthy pregnant women aged between 15 and 44 years with a mean of 27.34±5.43 years were screened. In terms of percentage, 89.4% of the subjects were married, and 10.6% were without formal husbands. The overall seroprevalence of HIV, HBsAg, HCV and syphilis was found to be 4.1%, 5.3%, 0.5% and 5.0%, respectively.Conclusions:High prevalence of some infectious diseases was observed in the present study, which may pose serious health risk to women of reproductive age in this region. It is important to point out that there is need to improve antenatal care of pregnant women by mandatory screening for these infectious diseases.

The external quality assessment scheme: Five years experience as a participating laboratory

Background and Aim:Quality assurance in blood banking includes active participation in the external quality program. Such a program offers valuable benefits to patient care, their safety, and an overall quality of laboratory practices. In the year 2002, we participated in the External Quality Assessment Scheme (EQAS) under the World Health Organization (WHO), Bureau of Laboratory Quality Standards, Thailand.Materials and Methods:In the current study we evaluated our EQAS test result of the past five years, from 2003 to 2007. Test results of all blood samples such as ABO grouping, D typing, antibody screening, antibody identification, and transfusion transmitted infection (TTI) testing were analyzed and documented.Results:Discordant results in one or more instances were observed with antibody identification, weak D testing, and tests for anti-HIV1/2 and HBsAg. Twice we failed to detect the ‘anti-Mia’ antibody in the issued sample and that could be attributed to the absence of the corresponding antigen in the used cell panel. HBsAg was missed due to its critically low titer in the serum and the comparatively low sensitivity of our Enzyme-Linked Immunosorbent Assay (ELISA) test kit.Conclusion:All these failures in the last five years have helped us to significantly improve our transfusion service in terms of performance evaluation, patient care and safety issues, and the overall quality of laboratory practices. We therefore recommend all laboratories and hospitals to participate in the EQAS program, which will definitely help them to improve from what they learn.

Prevalence of Potentially Pathogenic Enteric Organisms in Clinically Healthy Kittens in the UK

Faecal samples were collected from 57 clinically healthy kittens presented for initial vaccination, in the UK. Routine bacteriological examination identified Salmonella species in one and Campylobacter species in five samples. Polymerase chain reaction (PCR) detected the presence of Campylobacter species in a further four samples. Routine parasitological examination revealed Toxocara species ova in nine (including four kittens stated to have been administered an anthelmintic) and Isospora species in four samples. No Giardia or Cryptosporidium species were detected by routine methods. A Giardia species enzyme-linked immunosorbent assay (ELISA) test kit designed for use in cats was positive in three kittens. A similar test kit designed for use in humans was negative in all samples and produced negative results even when known positive samples were tested. Potentially pathogenic enteric organisms were detected in 19 kittens by routine methods and 26 (prevalence 45%) by all methods. The high prevalence in asymptomatic kittens highlights the possibility that the detection of these organisms in kittens with gastrointestinal disease may be an incidental finding.

Assessment of Five Interleukins in Human Synovial Fluid as Possible Markers for Aseptic Loosening of Hip Arthroplasty

One of the most important factors that seems to be involved in total hip replacement is periprosthetic osteolysis. As it is well documented that several interleukins (ILs) are triggered in periprosthetic osteolysis, this article investigates the role of five ILs in primary and replacement total hip arthroplasty, understanding if one of them can also predict hip implant loosening, secondary surgery, and prosthesis breakage. The levels of IL-1alpha, 1beta, 6, 8, and 10 in synovial fluid were examined, using a high sensitivity enzyme-linked immunosorbent assay (ELISA) test kit (Pierce Biotechnology, Inc., Rockford, IL, USA) to determine whether these cytokines could be used as markers of enhanced periprosthetic osteolysis, leading to aseptic loosening of total/partial hip arthroplasty or revision surgery. Synovial fluid was harvested from 23 patients undergoing primary total hip arthroplasty and 35 patients undergoing total/partial hip revision due to aseptic loosening. In the revision group, four cases had suffered a prosthesis fracture and five were second revisions. ILs 6 and 8 were significantly higher in the revisions (305 and 817 pg/mL) compared with the primary arthroplasties (151 and 151 pg/mL), including cases with prosthesis fracture and those requiring a second revision. IL-10 levels were lower (not significantly) in second revision samples compared with those of revision samples. IL-1beta levels were significantly higher in prosthesis fracture samples compared with those of all the other revision samples. No statistically significant differences in IL levels were found between osteoarthritis samples and those of other diseases. These results are a step forward to elucidating the complex network of events that are involved in loosening of hip implants.

Pharmacokinetic and Pharmacodynamic Profile of New Biosimilar Filgrastim XM02 Equivalent to Marketed Filgrastim Neupogen®

Filgrastim XM02 is a biosimilar non-glycosylated recombinant methionyl form of human granulocyte colony-stimulating factor (r-MetHuG-CSF) expressed in Escherichia coli for subcutaneous and intravenous administration in the treatment of different forms of neutropenia and stem cell mobilization. This study was conducted to compare the pharmacokinetic and pharmacodynamic characteristics of the new biosimilar filgrastim XM02 with the marketed filgrastim (Neupogen). Two filgrastim doses (5 and 10 microg/kg) of the new biosimilar filgrastim XM02 and the marketed filgrastim were administered either as intravenous infusion or subcutaneous injection in four single-dose, crossover, randomized substudies, conducted in 36 subjects each. Serum concentrations of filgrastim were determined using an enzyme-linked immunosorbent assay test kit on samples taken at intervals up to 48 hours after administration. The CD34+ stem cell count up to 15 days after administration was determined by flow cytometry using a validated CD34+ cell enumeration kit, and the absolute neutrophil count (ANC) up to 96 hours after dosing was determined by the Beckman Coulter AcT differential automated hematology analyzer. The primary pharmacokinetic endpoint was the AUC (48 h) (area under the serum concentration-time curve) of filgrastim serum concentration determined by the linear trapezoidal rule. Equivalence (biosimilarity) between the two filgrastim products was assessed by 90% confidence limits obtained from analyses of variance of log-transformed pharmacokinetic and pharmacodynamic endpoints, applying 80-125% equivalence intervals. The mean serum concentration profiles of filgrastim, ANC and CD34+ cells over time were similar for the two filgrastims. The 90% confidence intervals for all test/reference ratios for pharmacokinetic and pharmacodynamic endpoints lay within the accepted bioequivalence range of 80-125%. Both filgrastims showed similar safety profiles and were well tolerated. Equivalence of the two filgrastims was clearly demonstrated for all four dose/route of administration groups. Equivalence could be demonstrated for the serum concentration profile, for the ANC profile and, even more importantly, for the CD34+ cell count, which is a marker for the ability of the granulocyte colony-stimulating factor to mobilize stem cells.

Deoxynivalenol and its conjugates in beer: A critical assessment of data obtained by enzyme-linked immunosorbent assay and liquid chromatography coupled to tandem mass spectrometry

Enzyme-linked immunosorbent assays (ELISAs) are often employed for the control of deoxynivalenol (DON) in barley and other intermediates involved in beer production chain. Because of the occurrence of high levels of DON-3-glucoside (DON-3-Glc) in malt and beer that have been reported for the first time in our earlier study, research focused on the accuracy of DON determination by immunoassays in cereal-based matrices has been initiated. DON-3-Glc was strongly cross-reacting in all examined commercial ELISA test kits (Ridascreen ® DON (R-Biopharm), Veratox 5/5 DON ® (Neogen Corporation), Deoxynivalenol EIA (Euro-Diagnostica), and AgraQuant ® DON Assay 0.25/5.0 Test Kit (Romer Labs). The highest overestimation in beer analysis, up to 1000%, when taking the DON content determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) as a reference method, was obtained by AgraQuant assay. Besides of DON-3-Glc and 3- and 15-acetyldeoxynivalenol (ADONs), also other, not known yet, matrix components contributed to false positive results. Similar phenomenon, although in a lesser extent due to lower content of these substances, was observed for using ELISA in the analysis of wheat. The relationship between a way of sample handling and DON overestimation was demonstrated; higher ELISA response was measured in an aqueous extract compared to that prepared by acetonitrile–water (84:16, v/v). Most of cross-reacting co-extracts were removed by MycoSep™# 226 cartridge, what leads us to the hypothesis on the presence of currently unknown cross-reactive species.