Stereolithography-Based 3D Printed "Pillar Plates" that Minimizes Fluid Transfers During Enzyme Linked Immunosorbent Assays.

Hydatidosis, a chronic zoonotic disease, has a distribution worldwide and is caused by the larval stage of the Echinococcus helminth. The Dot-ELISA test can diagnose hydatidosis quickly and accurately. Additionally, unlike other hydatid disease tests now used, this quick and affordable enzyme immunoassay is very serum-conservative and antigen-conservative, needing just nanogram levels of parasite antigen. In the present cross-sectional study, crude and B antigens of hydatid cyst fluid were obtained to diagnose human hydatidosis using CIEP (Counter Immunoelectrophoresis), ELISA (Enzyme-linked Immuno Sorbent assay), and Dot- ELISA (Dot Enzyme linked Immuno Sorbent Assay) methods. Infected liver with a hydatid cyst was collected from Tehran's slaughterhouses to prepare cyst fluid in different stages. After extracting and purifying the Cyst fluid, it is centrifuged at 4ºc, then prepared to concentrate. The study also included sera from hydatidosis (n=60), samples of helminth parasites (n=55), fascioliasis (n=35), toxocariasis (n=20) and negative control (n=35) were tested by CIEP (Counter Immunoelectrophoresis), ELISA (Enzyme-linked Immune Sorbent assay), and Dot- ELISA (Dot Enzyme linked Immuno Sorbent Assay) methods. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) for Windows release 25.0 (SPSS Inc., Chicago, IL, USA). Crude antigen of hydatid cyst showed a specificity of 76.7%, a sensitivity of 93.3% using the ELISA method, and B antigen showed a specificity of 96.7% and sensitivity of 88.3% using the same method. The crude antigen of the hydatid cyst exhibited a specificity of 68.9% and a sensitivity of 86.7% using CIEP. The B antigen showed a specificity of 87.8% and sensitivity of 83.3% using the same method.The crude antigen of hydatid cyst having serum dilution at 1:800 exhibited a specificity of 83.3% and sensitivity of 100% using the Dot-ELISA method and B antigen having serum dilution at 1:800 serum showed a specificity of 100% and sensitivity of 98.3% using the same method. The results of this finding showed that B antigen has the maximum specificity to diagnose hydatid test using the Dot- ELISA method. Hydatid cysts present with varied symptomatology. History of exposure to infected animals may not be present. A high degree of clinical suspicion combined with meticulous history and clinical examination supported by laboratory investigations are required for its diagnosis. The Dot-ELISA system with native antigen B is a viable approach for the immunodiagnosis of human hydatidosis that is preferred to infection.

Development of silver nano-based indirect ELISA and Dot-ELISA methods for serological diagnosis of a bacterial fish pathogen Aeromonas veronii

Epstein-Barr Virus (EBV), play role in etiology of malignancies as Burkitt Lymphoma and Nasopharyngeal Carcinoma alongside very common situation like Infectious Mononucleosis. Also in patients groups, like transplant and oncologic patients whose immune system especially depressed detection of EBV reactivation is important. In this study, investigation of results defined by Immunofluorescent Antibody (IFA) and Enzyme-Linked ImmunoSorbent Assay (ELISA) methods aimed. Between 2017 July and 2020 July in our laboratory, With 7455 samples Anti-VCA IgM results detected by the ELISA method were 3.9% positive, 94.1% negative, and 3% borderline. With 5510 samples Anti-VCA IgG results detected by the ELISA method were 82,3% positive, 16.1% negative, and 1.6% borderline. With 449 samples, 32.9% of Anti-VCA IgM, 96.8% of Anti-VCA IgG,55% of Anti-EA IgG, and 93.5% of Anti-EBNA antibody results detected by the IFA method were positive. Positive Anti-VCA IgM results were 3% by ELISA and 25% by the IFA, positive Anti-VCA IgG results were 96.3% by ELISA and 98% by IFA. ELISA Anti-VCA IgG sensitivity was found to be 96.3% and ELISA Anti-VCA IgM sensitivity was found to be 12.9% in statistical analysis, considering IFA as the gold standard. For serological diagnosis of acute EBV infection or reactivation of latent infection EBV Anti-VCA IgM, Anti-VCA IgG, Anti-EBNA IgG, Anti-EA IgG, and Anti-VCA IgG avidity antibodies should be evaluated together.

Objective To investigate the clinical application value of enzyme-linked immunosorbent assay (ELISA) and time-resolved fluorescence analysis(TRFIA) and latex immune chromatography (GICA) in detecting hepatitis B serum markers.Methods One hundred and forty-five suspected patients of hepatitis B were detected serum markers of hepatitis B by ELISA,TRFIA and GICA method,and the results were compared and analyzed.Results When TRFIA method was as gold standard,the positive coincidence rate of ELISA and GICA method in HBsAg,HBeAb,HBcAb was 71％ (57/80),45％ (36/80),and in HBsAb,HBeAb,HBcAb was 33％( 1/3),0 (0/3),and there were significant differences between two methods (P＜0.05 ) ; the others were no significant differences (P ＞ 0.05 ).There was significant difference in the sensitivity of HBsAg between ELISA method and GICA method (P ＜ 0.05 ),but there was no significant difference in HBsAb,HBeAg,HBeAb,HBcAb(P ＞ 0.05 ).There was no significant difference in the specificity of HBsAg,HBsAb,HBsAg,HBeAb and HBcAb between ELISA method and GICA method (P ＞0.05).There was significant difference in HBsAg among three methods (P ＜0.05),but there was no significant difference between ELISA method and TRFIA method (P＞0.05),and there was significant difference between GICA method and TRFIA method (P＜0.05).There was no significant difference in HBsAb,HBeAg,HBeAb and HBcAb among three methods (P ＞ 0.05 ); there was significant difference in both HBsAb and HBeAb positive among three methods (P ＜ 0.05),and there was significant difference between ELISA method,GICA method and TRFIA method (P＜ 0.05).Conclusion TRFIA method has supreme measuring range,sensitivity and specificity supreme,but the price is higher;ELISA method is in the intermediate level of three methods and price is cheap,and it as well as TRFIA is suitable for the batch detection; GICA accuracy is low,but quick and simple,it is more suitable for the complement of the first two methods. Key words: Hepatitis B; Enzyme-linked immunosorbent assay; Time-resolvod fluorescence analysis; Latex immune chromatography

To determine whether the serum levels of anti-double strand DNA (anti-dsDNA) autoantibodies detected using a newly developed fluorescence immunoassay (FIA) in patients with systemic lupus erythematosus (SLE) correlated more with clinical parameters, such as SLE disease activity index (SLEDAI), complement and the occurrence of nephritis when compared with traditional enzyme-linked immunosorbent assay (ELISA), we prospectively collected 124 serum samples from 31 patients who had juvenile-onset SLE and were regularly monitored every 2 months at our outpatient clinic. At every visit, clinical manifestations and laboratory parameters were assessed and the SLEDAI was determined. Correlation analyses between the two different measurements of anti-dsDNA antibodies and SLEDAI, serum complement levels and the occurrence of nephritis were performed. The results showed that anti-dsDNA autoantibodies detected using both ELISA and FIA significantly correlated with SLEDAI, and significantly and inversely correlated with the serum levels of C3 and C4. FIA had significantly higher correlation with SLEDAI and C4 than did ELISA. The mean values of anti-dsDNA antibodies detected using FIA in patients with nephritis were significantly higher than in those without nephritis. In contrast, the values of anti-dsDNA antibodies detected using ELISA did not show significant differences between these two groups. We conclude that FIA had better correlation with SLEDAI, C4 and the occurrence of nephritis, and comparable correlations with C3 that were similar to the results found using ELISA. Thus, it is worthwhile developing the FIA method for clinical evaluation of disease activity in SLE patients.

Objective: To obtain purified protein antigen of guertu virus (GTV) nucleoprotein (NP) and establish a rapid and accurate enzyme-linked immunosorbent assay (ELISA) method for detection of GTV antibody. Methods: Codon optimized GTV NP encoding genes were synthesized, cloned into the pet32a (+) vector, and recombinant expression plasmids were constructed and transformed into BL21 (DE3). Recombinant protein (rNP) obtained from the optimized expression were purified over a Ni column and identified by SDS-PAGE and Western blot. The purified protein was used as the antigen to optimize the reaction conditions, and an indirect ELISA assay for GTV IgG antibody was developed and optimized, which was evaluated and initially applied. Results: The prokaryotic expression plasmid pet32a-NP was successfully constructed, the recombinant protein was highly expressed in E. coli in the form of inclusion bodies, the size was about 44 kD, and the results of Western blot indicated that the recombinant protein had good antigenicity with GTV positive serum. The optimized ELISA (GTV-rNP-iELISA) established in this study showed strong specificity, high sensitivity, and the coefficient of variation within and between batches is less than 10%, and has good repeatability; the detection results are consistent with the IFA detection results. Using the established ELISA method to detect 162 sheep sera from some regions of Xinjiang in 2017-2019, the total positive rate of antibodies was 39.8%. Conclusions: The GTV NP antibody detection ELISA method has good sensitivity, reproducibility, and specificity and has the potential to be a powerful tool for the diagnosis and serological investigation of GTV infection.

We measured anti-Chlamydia pneumoniae (C. pneumoniae) specific antibody titers by means of a newly-developed enzyme-linked immunosorbent assay (ELISA) method using an anti-C. pneumoniae specific antibody detection reagent. The clinical usefulness of this method was hereby evaluated. The IgG, IgA and IgM titers in 418 serum specimens obtained from patients with respiratory tract infections were measured by this new ELISA method, and the results were compared with the titers determined for the same specimens with the micro immunofluorescence (Micro-IF) method. The results showed good correlation coefficients for IgG, IgA and IgM. The two assay methods showed high agreement rates for positivity and for negativity. Specimens which did not yield the same results with the ELISA method and the Micro-IF method were subjected to analysis by the Western blot method, and the rates of agreement with the ELISA results were high. In addition, the child (0 approximately 15 yrs old; n = 122) and adult (16 approximately 90 yrs old; n = 133) cases were classified on the basis of being antigen-positive or antigen-negative at the initial examination, and their antibody-positive rates were determined. The adults showed no statistically significant differences in the antibody-positive rates for either IgG or IgA antibodies as a function of the pretreatment antigen status. However, the children showed statistically significant (p < 0.001) differences in the antibody-positive rates for both IgG and IgA antibodies as a function of the antigen status in the antigen-positive group compared with the rates in the antigen-negative group. Furthermore, the IgM-positive rates for the children were high in the antigen-positive group compared with the rates in the antigen-negative group, and the difference was statistically significant (p < 0.001). The IgM-positive rates in the adults were also significantly (p < 0.05) different between the antigen-positive group and the antigen-negative group. The Micro-IF method was applied to 34 specimens from antigen-positive patients, and 22 specimens were found to show an IgG titer of > or = 512 or an IgM titer of > or = 16. The diagnoses of these patients were acute respiratory disease in sixteen, pneumonia in four. Application of the ELISA-method to those 22 specimens showed all of them to exhibit IgG absorbance of > or = 0.6 and IgA absorbance of 0.2. The results described above indicate the clinical usefulness of our new ELISA method for the detection of antibodies specific for C. pneumoniae. The significance of this ELISA method for serological diagnosis of C. pneumoniae infections and the criteria for diagnosis of acute infections were also discussed.

Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevant Anisakis allergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-food-related allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-Anisakis IgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-Anisakis IgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P < 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and old Anisakis infections.

A new chemiluminescence immunoassay method (CLIA) for detecting IgA anti-transglutaminase (atTG IgA) in celiac disease (CD) has prompted inquiries into its diagnostic performance. We conducted a systematic review and meta-analysis comparing CLIA with traditional enzyme-linked immunosorbent assay (ELISA) and fluorescence enzyme immunoassay (FEIA). We searched PubMed, Medline, and Embase databases up to March 2024. The diagnostic references were intestinal biopsy and ESPGHAN guidelines. We calculated the sensitivity and specificity of atTG IgA assessed by CLIA and the odds ratio (OR) between the assays. Eleven articles were eligible for the systematic review and seven for the meta-analysis. Sensitivity and specificity of atTG IgA CLIA-assay were 0.98 (95% CI, 0.95-0.99) and 0.97 (95% CI, 0.94-0.99), respectively. The sensitivity of atTG IgA antibody detection did not significantly vary across the three assay modalities examined (CLIA vs. ELISA OR: 1.08 (95% CI, 0.56-2.11; p = 0.8); CLIA vs. FEIA OR: 6.97 (95% CI, 0.60-81.03; p = 0.1). The specificity of atTG IgA assessed by FEIA was higher than for CLIA (OR 0.17 (95% CI, 0.05-0.62); p < 0.007). According to the systematic review, normalization of atTG IgA levels in CD patients following a gluten-free diet was delayed when using CLIA compared to ELISA and FEIA methods. Conflicting findings were reported on the antibody threshold to use in order to avoid biopsy confirmation.

Objective We detected the enterovirus in the CSF of patients with sporadic aseptic meningitis and viral encephalitis in to investigate the enterovirus infection in this area, and two methods were used in this study. Methods CSF samples of 36 patients with sporadic aseptic meningitis and viral encephalitis were collected during 2009-2013. All the samples were frozen and stored at -80℃ immediately before RNA extraction. Total viral RNA extraction was taken within 3 month after the sample was stored by using an RNA extraction kit according to the method described by the instruction of the kit.Then RNA was reversed into cDNA immediately after the extraction of RNA. cDNA was stored at -80℃. Finaly, we used a rapid and sensitive method for the detection of all members of the enterovirus genus by using real-time PCR. We also detected the IgM of 7enterovirus including Echo virus and Coxsackie virus 1-6 serotype by Enzyme-Linked Immunosorbent Assay(ELISA) method. Results Of all the 36 CSF samples, 9(25%) were positive for enterovirus by PCR method. For patients with aseptic meningitis, viral encephalitis and meningoencephalitis, the proportion were 42.86%, 12.50% and 11.11%. Only 2 samples were positive for Coxsackie virus by ELISA method, and both of them had aseptic meningitis. Conclusion Enterovirus infection is a common cause of sporadic CNS virus infection among patients with viral encephalitis, aseptic meningitis and aseptic meningoencephalitis in Beijing, especially for the patients with aseptic meningitis.Real-time PCR was more sensitive than ELISA, while the better way is to use both of them to detect the serotype of the enterovirus. Key words: Enterovirus; Meningitis; Encephalitis; Infection

Objective To investigate the expression of Caspase-9 and heat-shock protein-90 (HSP 90) in rats after focal cerebral ischemia-reperfusion injury. Methods The male SD rats (200-250 g) were divided into three groups by the random number table: normal group, sham group and cerebral ischemia-reperfusion (CIR) group. Each group was sorted into four subgroups including group 6 h, group 24 h, group 48 h and group 72 h according to the reperfusion time. Suture-occluded method was adopted to prepare focal cerebral ischemia-reperfusion(CIR) injury in rat model. Enzyme-linked immunosorbent assay (ELISA) method was used to detect the variations of Caspase-9 and HSP-90 expression in rats. Results The changes in Caspase-9 and HSP 90 expression in the brain cells were observed by ELISA method. The expression of Caspase-9 and HSP-90 was weakly expressed in sham group, and was at peak in CIR group within 24 h-48 h, then began to decline at 72 h after the reperfusion time. The differences in the expression of caspase-9 and HSP-70 between sham group and normal group were not statistically significant. Conclusions Apoptotic cells gradually increase along with reperfusion time and reach the peak at 48 h after cerebral ischemia-reperfusion. In ischemia half dark stripe, the expression of Caspase-9 and HSP 90 is increased in neuronal cells after cerebral ischemia-reperfusion, and the positive cells number is at peak at 48 h after cerebral ischemia-reperfusion. Apoptosis of neuronal cells after cerebral ischemia and reperfusion is a dynamic evolutionary process. The expression of Caspase-9 and HSP 90 in nerve cells plays an important role in regulating cell apoptosis. Key words: Reperfusion injury; Caspase-9; Heat-shock proteins

A collaborative study was performed by 30 laboratories in 3 sets of trials to validate a modified colorimetric monoclonal enzyme-linked immunosorbent assay (ELISA) method for Salmonella detection. The modifications to the current methodology included incubation of enrichments and post-enrichments at an elevated temperature, addition of novobiocin to the M-broth post-enrichment, and elimination of the centrifugation and agitation steps. Five artificially contaminated foods (nonfat dry milk, milk chocolate, dried egg, ground black pepper, and soy flour) and 1 naturally contaminated food (raw ground turkey) were analyzed. The artificially contaminated foods were inoculated with individual Salmonella serotypes at a high (10-50 cells/25 g) and low (1-5 cells/25 g) contamination level. Results from the modified ELISA method were compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method. In 2 of the food products, milk chocolate and pepper, a number of laboratories isolated Salmonella from un-inoculated control samples, thus invalidating their data. As a result, there were too few laboratories remaining with valid data, and these foods were repeated. In the completed study, there were 11 false negative results obtained by the modified ELISA method, while there were 28 false negatives produced by the BAM/AOAC procedure. There were 11 ELISA positive assays which could not be confirmed by culture methods. Statistically, there were no differences between the modified, colorimetric, monoclonal ELISA and the reference culture method in all foods except raw turkey, where the ELISA method was more productive. The colorimetric monoclonal enzyme immunoassay (Salmonella-Tek) method for detecting Salmonella in all foods has been adopted first action by AOAC INTERNATIONAL.

OBJECTIVE: The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin (aCL) antibodies is the most important test for the diagnosis of antiphospholipid syndrome (APS). However, the test is also positive in some infectious diseases and other non-related syndromes. It has been suggested that the detection of antibodies to a mixture of phospholipids or to beta2-glycoprotein I (beta2-GP I) has higher specificity for APS than the standard aCL ELISA. The aim of the present work is to compare the diagnostic specificity of three different antiphospholipid (aPL) assays in patients with infectious diseases. METHODS: Antiphospholipid antibodies were searched by three ELISA techniques, namely standard aCL, APhL® ELISA kit and anti-beta2-GP I, in sera of patients with infectious diseases, including syphilis (69), leptospirosis (33) and visceral leishmaniasis (30). RESULTS: The frequency of positivity of IgG aPL in patients with syphilis, leptospirosis and Kala-azar was 13/69 (19%), 9/33 (27%) and 2/30 (6%), respectively, using standard ELISA, versus only 1/69 (1.4%), 0/33 (0%) and 0/30 (0%) positivity by the APhL® ELISA kit. The positivity of the isotype IgM aPL was 10/69 (14%), 4/33 (12%) and 1/30 (3%), respectively, by the standard ELISA, and 1/69 (1.4%), 0/33 (0%) and 0/30 (0%) by the APhL® ELISA kit. The presence of significant levels of IgG anti-beta2GPI was observed in 14/69 cases of syphilis (20%), 6/33 cases of leptospirosis (18%) and 16/30 cases of Kala-azar (53%). The APhL® ELISA kit had superior performance showing the highest specificity: 97% (95% CI: 92%-99%) for IgG compared to 81% (95% CI: 74%-87%) for standard ELISA and 72% (95% CI: 64%-79%) for anti-beta2 GPI assay. CONCLUSIONS: The APhL® ELISA kit proved to be significantly more specific than the aCL standard ELISA and the anti-beta2GPI ELISA, and it should be used to help in the diagnosis and confirmation of APS.

Background α-solanine (SO) and α-chaconine (CHA) are natural toxins of potato and often cause food poisoning worldwide. We have previously developed a polyclonal antibody (anti-Sold antibody) that bind to SO and CHA and used it in an enzyme-linked immunosorbent assay (ELISA). Our previously constructed ELISA (conventional ELISA) has a weak point of low sensitivity in detecting SO and CHA, we have tried to improve it in this study. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Anti-Sold antibody was then purified from rabbit serum using affinity column chromatography. In our ELISA method, named “B-S direct ELISA”, biotin-labeled anti-Sold antibody is bound to SO and CHA in the sample, followed by the binding of peroxidase-labeled streptavidin. To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results The absorbance of the samples containing SO and CHA prepared in 10 mM phosphate buffer saline (PBS) was about 5-fold higher than that of the conventional ELISA. Furthermore, the absorbance of the samples containing SO and CHA prepared in human serum and urine was enhanced about 2.5- and 1.6-fold, respectively, compared to that of the conventional ELISA. The limit of detection in B-S direct ELISA was 0.32 ng/mL in 10 mM PBS, 2.08 ng/mL in serum, and 4.88 ng/mL in urine, respectively. Using this ELISA, it was also possible to measure the content of SO and CHA in extracts of potato tubers, peels, and sprouts. Both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.4- and 2.2-fold higher, respectively, in each part than those in the extracts from fresh potatoes. Furthermore, the potato sprout extract contained approximately 50-fold more SO and CHA than the tuber and 9-fold more than the peel. On the other hand, B-S direct ELISA cross-reacted with solanidine and solasodine, which have similar chemical structures to SO and CHA, and also with cholesterol and vitamin D3 in serum. Conclusions Based on our results, B-S direct ELISA presented greater performance in detecting SO and CHA than our conventional ELISA. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although this ELISA has a detection performance applicable to human biological samples, cross-reactivity should be fully considered when judging the results.

Evaluating Fusarium head blight (FHB) involves inoculating barley (Hordeum vulgare L.) with Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.) Petch] followed by visual observation of disease and analysis for deoxynivalenol (DON). Disease symptoms and DON are not always correlated because both are affected by environmental variables. The objective of this study was to develop an enzyme‐linked immunosorbent assay (ELISA) for quantification of FHB in barley. Antibodies to F. graminearum were tested for reaction with other Fusarium spp. Antibodies from cell line IF8 reacted with Fusarium spp. tested, but not other Ascomycota. The ELISA method was developed using seed lots with no, low, and high levels of DON. Quantity of seed, volume of extraction buffer, and agitation time were tested and Fusarium quantified with ELISA. Five genotypes each for high, medium, and low ELISA values were selected from a field experiment using a doubled‐haploid mapping population in 2003. The lines were grown in 2004 and scored for FHB, DON, and ELISA. ELISA had lower error than FHB or DON. Lines selected for low, medium, and high ELISA in 2003 had low, medium, and high ELISA values in 2004. ELISA and DON were correlated in both field experiments (r = 0.51). ELISA and DON were correlated (r = 0.71) in samples selected from grain elevators in 1993 through 2003 indicating naturally occurring Fusarium spp. outside the B clade had no effect on the performance of the ELISA analysis.