Non-commercial ELISA Research Articles

Advanced Glycation End-Products in Blood Serum-Novel Ischemic Stroke Risk Factors? Implication for Diabetic Patients.

New predictors of ischemic incidents are constantly sought since they raise the awareness of patients and their doctors of stroke occurrence. The goal was to verify whether Advanced Glycation End Products (AGEs), in particular AGE10, could be one of them. The AGE10 measurement was conducted using a non-commercial ELISA assay in the blood serum of neurological patients without cerebrovascular event (n = 24), those with transient brain attack (TIA) (n = 17), and severe ischemic stroke (n = 35). Twice as many of the people with TIA or severe stroke presented high AGE10 serum concentrations compared to the patients with other neurological conditions (χ2 = 8.2, p = 0.004; χ2 = 8.0, p = 0.005, respectively). The risk of ischemic incident was significantly risen in people with higher levels of AGE10 (OR = 6.5, CI95%: 1.7-24.8; OR = 4.7, CI95%: 1.5-14.5 for TIA and stroke subjects, respectively). We observed a positive correlation (r = 0.40) between high AGE10 levels and diabetes. Moreover, all the diabetic patients that had a high AGE10 content experienced either a severe ischemic stroke or TIA. The patients with high levels of AGE10 exhibited higher grades of disability assessed by the NIHSS scale (r = 0.35). AGE10 can be considered a new biomarker of ischemic stroke risk. Patients with diabetes presenting high AGE10 levels are particularly prone to the occurrence of cerebrovascular incidents.

MO263: Multi-Antibody Composition in Lupus Nephritis: Isotype Prevalence and Clinical Oucome

Abstract BACKGROUND AND AIMS Lupus nephritis (LN) is the most serious organ manifestation of systemic lupus erythematosus (SLE), occurring in 40%–60% of subjects with SLE with worse outcomes. LN is mediated by antibody deposition in glomeruli, but the mechanisms leading to the immune deposits and renal lesions are not clarified. Previous findings suggested a role for circulating anti–double stranded DNA (dsDNA). However, anti-DNA deposition in LN accounts for not more than 10%–20% of eluted IgG. Therefore, renal targets of autoimmunity in LN are largely unknown. METHOD We studied 1052 patients with SLE, 459 with and 573 without LN, enrolled at different times from diagnosis (0–1 month, 2–12 m, 13–24 m, 25–48 m, 48–96 m and > 96m). These cohorts are part of a nation-wide study (The Zeus study, NCT02403115) to characterize the antibody multi-components in SLE and LN. A total of 92 LN patients recruited at T0, had a follow-up of 36 months. Laser capture microdissection were performed from fresh frozen renal samples of 20 patients with LN (classes III–V), and eluted Igs were analyzed by Western blot using podocyte extracts as fixed antigens. Recognized proteins were characterized by matrix-assisted laser desorption ionization (MALDI)–mass spectrometry (MS) and liquid chromatography (LC)-MS. Levels of anti-dsDNA, anti-ENO1, anti-AnnexinA1, anti-SOD, anti-C1q, anti-H2A histone were determined using non-commercial ELISA. RESULTS Glomerular IgGs recognized 11 proteins (Fig. 1A), which resulted positive also in serum of same subjects (Fig. 1B). Isotype analysis of glomerular eluates showed prevalence of IgG2 for anti-dsDNA, anti-Histone H2, anti-C1q, anti–α-enolase and anti-Annexin AI (Fig. 1C). Fig. 1D shows representative images of patients with LN class V: colocalization with IgG2 is positive for dsDNA, Histone H2, C1qα-enolase and annexin. Prevalence of IgG2 for the same proteins were confirmed also in serum samples of the same subjects (Fig. 1E). The presence in the serum of the IgG2 panel was highly discriminatory for SLE/LN versus healthy subjects (Fig. 2A). Heat map in Fig. 2B reports the association between the levels of circulating IgG2 antibodies and the clinical parameters for both LN and SLE together. While in Fig. 2C, the heat maps indicate the correlation between two parameters (an antibody and an organ pathology). Volcano plots indicate that anti-ENO1 and anti-Histone H2 IgG2 had the most significant changes in LN patients. In the prospective analysis, considering the 91 LN with 36 months of follow-up, decrements of each antibody at T12 related to proteinuria are summarized in Fig. 2E: major decrements were for anti-ENO1 and anti-Annexin A1 (−55% and − 70%, respectively) and 0% for anti-dsDNA. Serum levels of anti-SOD IgG2 at T0 resulted significantly higher in LN compared to SLE (Fig. 2F). The kinetics of reduction of anti-SOD IgG2 was in accordance with reduction of proteinuria (Fig. 2G). Of interest, anti-dsDNA IgG2 serum levels persistently maintained at high values during the same period (Fig. 2H). CONCLUSION Our study demonstrated higher sensitivity of anti-dsDNA IgG2 than classical IgGs antibodies for marginal SLE diagnosis. Positivity for the whole panel of circulating IgG2 antibodies is a specific signature of SLE/LN and distinguishes them from healthy people. Moreover, anti-ENO1 and anti-Histone H2 IgG2 serum levels represent the best discriminatory element for distinguishing between LN and SLE. From prospective analysis, we report that circulating anti-ENO1 IgG2 may represent a valuable markers of remittent LN. Similarly, circulating anti-SOD2 IgG2 are elevated in new onset LN. It might be hypothesized that increase is related to the activation of SOD2, as response to the oxidative stress induced by the active disease.

Seroepidemiological Study of Canine and Human Dirofilariasis in the Endemic Region of Northern Serbia.

Dirofilariasis is a vector-borne zoonotic disease caused mainly by Dirofilaria immitis and Dirofilaria repens that affect dogs and humans all over the world. Serbia is considered an endemic country to both forms of dirofilariasis, although most of the population is concentrated in the north of the country. The aims of this study were to show the prevalence of D. immitis and D. repens in dogs and the seroprevalence in humans compared to previous studies in Northern Serbia. In total, 346 dog sera samples and 265 human samples were analyzed. Dog blood samples were analyzed using the modified Knott's method to check whether there were Dirofilaria spp. microfilariae and serum samples were checked by a commercial D. immitis antigen test. Human serum samples were analyzed with a non-commercial ELISA for detection of specific anti-D. immitis, anti-D. repens, and anti-Wolbachia IgG antibodies, and confirmed by western blotting. The overall prevalence for Dirofilaria spp. in dogs was 29.19%. The overall prevalence for D. immitis was 26.30%. The percentages of D. immitis and D. repens microfilaremia in dogs were 25.72 and 1.45%, respectively, while D. immitis./D. repens microfilaremia co-infections were also 1.45%. The overall seroprevalence for Dirofilaria spp. in humans was 3.77%. The overall seroprevalence for D. immitis was 1.51, 1.13% for D. repens, and for D. immitis/D. repens co-infections was 1.13%. The results indicate that D. immitis and D. repens are present in dogs and humans in the province of Vojvodina, in the northern part of Serbia. It is most likely associated with the presence of many rivers, the climate, and presence of mosquitoes in the area, so there could be a real public health risk.

Current Situation of the Presence of Dirofilaria immitis in Dogs and Humans in Bucaramanga, Colombia.

The cardiopulmonary dirofilariosis caused by Dirofilaria immitis, is a vector-borne infection, which can be transmitted to humans. The main hosts are both domestic and wild canids. This species mainly occurs in tropical and subtropical climates, and temperature and humidity are the main factors that favor the presence and proliferation of culicid mosquitoes as vectors of the disease. There are few reports of cardiopulmonary dirofilariosis in dogs and humans in Colombia, a region with favorable climatic conditions which favors the presence of mosquitoes that act as vectors of the disease. Therefore, this study aimed to examine its current prevalence in dogs and the risk of human exposure to the disease in Bucaramanga, one of the most populated areas in Colombia located at the center of the country. Furthermore, its demographic and environmental characteristics could be useful as a study model for other similar locations and neighboring countries. Serum samples from 351 dogs and 506 humans from the Bucaramanga Metropolitan area were analyzed. All dog samples were analyzed by Knott's technique and tested with a commercial immunochromatographic to detect the presence of circulating antigens of D. immitis. Human samples were analyzed using a non-commercial ELISA test kit to detect IgG against the somatic antigens of adult D. immitis and Wolbachia. Positive results were further confirmed using western blot analysis. Thirty-eight dogs tested positive with a overall prevalence of 10.82%. Of these dogs, 18 showed D. immitis microfilariae, being 5.12% of the total population. The overall seroprevalence in humans was 6.71%; seroprevalence was significantly higher in individuals aged 16–34 years-old and in women than in men. To our knowledge, this study describes seropositivity to D. immitis for the first time in a Colombian human population located in the same area as that of dogs infected with D. immitis, which represents a potential threat to public health. In humans, age and gender can be considered risk factors for exposure to D. immitis.

Evaluation of a standardised Vi poly-l-lysine ELISA for serology of Vi capsular polysaccharide antibodies

Typhoid vaccines based on protein-conjugated capsular Vi polysaccharide (TCVs) prevent typhoid in infants and young children. Analysis of the serum anti-Vi IgG response following immunisation against typhoid confirms the immunogenicity of TCVs and forms an important part of the pathway to licensing. Comparative studies could expedite the licencing process, and the availability of a standardised ELISA method alongside the 1st International Standard (IS) 16/138 for anti-typhoid capsular Vi polysaccharide IgG (human) will facilitate this process. To this end, a non-commercial ELISA based on a coat of Vi and poly-l-lysine (Vi-PLL ELISA) was evaluated by 10 laboratories. Eight serum samples, including IS 16/138, were tested in the standardised Vi-PLL ELISA (n = 10), a commercial Vi ELISA (n = 3) and a biotinylated Vi ELISA (n = 1). Valid estimates of potencies relative to IS 16/138 were obtained for all samples in the Vi-PLL ELISA and the commercial ELISA, with good repeatability and reproducibility evident from the study results and concordant estimates obtained by the two ELISA methods. The study demonstrates that the Vi-PLL ELISA can be used in clinical trial studies to determine the immunogenicity of TCVs.

Seroepidemiological survey of human exposure to Dirofilaria spp. in Romania and Moldova

The present study aimed to evaluate the extent of Dirofilaria immitis and D. repens exposure in humans from eastern and southern areas of Romania and central Moldova by serological methods. The serological screening was performed on a total of 450 serum samples (187 from Romania and 263 from Moldova). The sera were collected using a convenience sampling with the help of physicians from the hospitals of the study areas. All samples were analysed by a non-commercial ELISA test for the detection of IgG antibodies against adult somatic antigens of D. immitis and D. repens. The results showed a total of 49 (10.9%; 95% CI = 8.3–14.1%) individuals from Romania and Moldova with a positive response to IgG antibodies against both adult somatic antigens of D. immitis and D. repens. Specifically, 48 (10.7%; 95% CI = 8.0–14.0%) patients were positive for IgG-antibodies against adult somatic antigens of D. immitis, one (0.2%; 95% CI = 0.4–1.2%) against D. repens antigens, and four (0.9%; 95% CI = 0.4–3.3%). were positive for antigens of both parasites.At country level, out of 187 samples from Romania, 13 (6.9%; 95% CI = 4.1–11.5%) were positive for anti-D. immitis IgG with high exposure in the southern part of the country (Bucharest). Of the 263 people from Moldova, 36 (13.7%; 95% CI = 10.0–18.4%) were positive for D. immitis antigens from which three (1.1%, 95% CI = 0.4–3.3%) were positive for the antibodies against antigens of both parasites. Only one sample was found positive for anti-D. repens IgG.Positive IgG-ELISA results were confirmed by Western blot analysis. In addition, for further confirmation, a complementary ELISA was performed for anti-WSP IgG antibodies against Wolbachia endosymbionts. Our findings showed a noticeable exposure of humans from Romania and Moldova to Dirofilaria parasites. Serology can be useful for indicating exposure to Dirofilaria spp. in a healthy population in order to obtain useful data on the epidemiological scenario of human dirofilariosis in Eastern Europe.

THE SEROPREVALENCE OF HEPATITIS E VIRUS INFECTION IN WILD BOARS IN SERBIA

Hepatitis E (HEV) belongs to one of fi ve so far described types of viral hepatitis caused by human agents (hepatitis A, B, C, D and E). The disease is characterized by clinical and epidemiological signs of acute hepatitis and is transmitted primarily by fecal-oral route via contaminated food and water. The infection is mainly detected in the developing countries of the Middle East, Asia and Africa, especially in countries with poor sanitary conditions of life. HEV infection in pigs was fi rst recorded in 1990. Numerous studies that followed proved that HEV can skip species barrier and can be transmitted from pigs to humans. HEV has been demonstrated as a new zoonotic agent. Hepatitis E virus has infected people in Japan who consumed insuffi ciently cooked meat of deer, pig liver and meat of wild boar. In humans four genotypes have been determined: I, II, III and IV, while so far tested swine isolates belong to genotypes III and IV. It is also important to note that the swine HEV isolates from one geographical region are genetically closer to human isolates from the same area than to the other isolates from pigs in the world. Th e aim of this paper is to show whether and how much HEV infection is present in the population of wild boars in Serbia, which represent a reservoir of this disease caused by a signifi cant zoonotic agent. Preliminary serological tests included the examination of 92 blood serum samples of wild boars. In 32 animals, or 34.78%, the presence of specifi c antibodies against HEV genotype was detected. Th e blood samples were collected during 2009, 2010 and 2011 from 15 hunting sites in Serbia. Laboratory testing was performed by non-commercial ELISA (in-house ELISA), where the used antigen was recombinant capsid protein-HEV genotype 3 ΔORF 2, which was obtained by laboratory cloning procedure. The test results showed that the hepatitis E virus is present in wild boars in Vojvodina, that are a potential source of this infection, as well as for many other infections of diferrent etiology.

Do Commercial Serologic Tests for Trypanosoma cruzi Infection Detect Mexican Strains in Women and Newborns?

We sought to determine the serological test that could be used for Trypanosoma cruzi seroprevalence studies in Mexico, where lineage I predominates. In a previous study among pregnant women and their newborns in the states of Yucatan and Guanajuato, we reported a 0.8-0.9% of prevalence for T. cruzi -specific antibodies by Stat-Pak and Wiener ELISA. We have expanded this study here by performing an additional non-commercial ELISA and confirming the seropositives with Western blot, using whole antigens of a local parasite strain. We found a seroprevalence of 0.6% (3/500) in Merida and 0.4% in Guanajuato (2/488). The 5 seropositive umbilical cord samples reacted to both non-commercial ELISA and Western blot tests, and only 1 of the maternal samples was not reactive to non-commercial ELISA. A follow-up of the newborns at 10 mo was performed in Yucatan to determine the presence of T. cruzi antibodies in children as evidence of congenital infection. None of the children was seropositive. One newborn from an infected mother died at 2 wk of age of cardiac arrest, but T. cruzi infection was not confirmed. The T. cruzi seroprevalence data obtained with both commercial tests (Stat-Pak and ELISA Wiener) are similar to those from non-commercial tests using a local Mexican strain of T. cruzi.

Different levels of humoral immunoreactivity to different wheat cultivars gliadin are present in patients with celiac disease and in patients with multiple myeloma

BackgroundImmunity to food antigens (gliadin, cow's milk proteins) is in the centre of the attention of modern medicine focused on the prevention of diseases, prevention which is based on the use of appropriate restriction diet. Detection of the enhanced levels of the immune reactions to antigen(s) present in food is from this point of view of great importance because there are reports that some of health disturbances, like celiac disease (CD) and some premalignant conditions, like monoclonal gammopathy of undetermined significance (MGUS), were vanished after the appropriate restriction diets.It is well known that gliadin is toxic to small bowel mucosa of relatively small population of genetically predisposed individuals, who under this toxic action develop celiac disease (CD). As the quantity of immunogenic gliadin could vary between different wheat species, the first aim of this work was to determine the percentage of immunogenic gliadin in ten bread wheat cultivars and in three commercially grown durum wheat cultivars. The second part of the study was initiated by results of previous publication, reporting that sera of some of multiple myeloma (MM) patients showed the presence of elevated levels of anti-gliadin IgA, without the enhanced levels of anti-gliadin IgG antibodies, determined with commercial ELISA test. It was designed to assess is it possible to reveal is there any hidden, especially anti-gliadin IgG immunoreactivity, in serum of mentioned group of patients. For this purpose we tested MM patients sera, as well as celiac disease (CD) patients sera for the immunoreaction with the native gliadin isolated from wheat species used for bread and pasta making in corresponding geographic region.ResultsGliadin was isolated from wheat flour by two step 60% ehanolic extraction. Its content was determined by commercial R5 Mendez Elisa using PWG gliadin as the standard. Results obtained showed that immunogenic gliadin content varies between 50.4 and 65.4 mg/g in bread wheat cultivars and between 20 and 25.6 mg/g in durum wheat cultivars.Anti-gliadin IgA and IgG immunoreactivity of patients' sera in (IU/ml) was firstly determined by commercial diagnostic Binding Site ELISA test, and then additionally by non-commercial ELISA tests, using standardized ethanol wheat extracts -gliadin as the antigen.In both patients groups IgA immunoreactivity to gliadin from different cultivars was almost homogenous and in correlation with results from commercial test (except for one patient with IgA(λ) myeloma, they were more then five times higher). But, results for IgG immunoreactivity were more frequently inhomogeneous, and especially for few MM patients, they were more then five times higher and did not correlate with results obtained using Binding Site test.ConclusionResults obtained showed different content of immunogenic gliadin epitopes in various species of wheat.They also point for new effort to elucidate is there a need to develop new standard antigen, the representative mixture of gliadin isolated from local wheat species used for bread production in corresponding geographic region for ELISA diagnostic tests.

Tissue transglutaminase ELISA positivity in autoimmune disease independent of gluten-sensitive disease

BackgroundOur aim was to understand why some sera from patients with a broad spectrum of autoimmune diseases or non-autoimmune diseases involving enhanced apoptosis, cell lysis and/or putative secondary autoimmune processes show reactions in the tissue transglutaminase (TGc) ELISA used for diagnosis of gluten-sensitive disease. MethodsSera were compared from groups of patients with autoimmune diseases, diseases involving organ specific enhanced cell death, celiac disease or dermatitis herpetiformis, diseases of non-autoimmune origin, and a group without known disease. IgA antibodies against TGc were detected using human antigen (produced recombinantly in bacterial or human cells) in different systems (non-commercial ELISA with buffers of differing NaCl concentrations, and anti-TGc sandwich ELISA). Anti-gliadin and anti-endomysium antibodies were also determined. ResultsMany sera from patients with autoimmune disorders gave a positive signal in the human TGc ELISAs. The signal appeared related to minor impurities in the recombinant human TGc used and to raised serum IgA antibody levels rather than to the occurrence of TGc specific antibodies in these patients. ConclusionsNo association of anti-TGc Abs and autoimmune conditions independent of gluten-sensitive disease could be shown. Care should be taken to exclude copurification of chaperones, like heat shock protein 70, where preparing antigens for TGc ELISAs.