ELISA For Detection Research Articles

Regulation of Curcumin on Follicle Initiation Rate in Diminished Ovarian Reserve.

To study the mechanism by which curcumin regulates ovarian primordial follicle initiation in rats with triptolide-induced diminished ovarian reserve (DOR). An in vitro gelatin sponge culture was performed on 3-day-old rat ovaries. After the establishment of the DOR model with triptolide, curcumin was administered for 3 days. Histological analysis and follicle counts were performed using H&E staining. ELISA detection of ovarian hormones in the culture medium (E2, FSH and LH), western blotting and Q-PCR for protein and mRNA expression (LTCONS-00011173, TGF-β1, Smad1, AMH, PTEN and GDF-9). Ovarian primordial and growing follicles increased significantly after curcumin intervention (p < 0.05), FSH/LH and E2 levels were increased significantly (p < 0.05). Curcumin also significantly decreased the expression of LTCONS-00011173. Meanwhile, curcumin increased the expression of TGF-β, AMH, and GDF-9 (p < 0.05). In addition, curcumin increased Smad1 gene expression and protein phosphorylation in the ovary on the one hand (p < 0.05), but inhibited Smad1 and p-Smad1 protein expression on the other hand (p < 0.05). Moreover, curcumin decreased PTEN protein and mRNA expression (p < 0.05). Curcumin activates primordial follicles in DOR model rats through TGF-β1 and downstream AMH signaling pathways and may limit follicle exhaustion through LncRNA.

Uncovering the role of traditional Chinese medicine in immune-metabolic balance of gastritis from the perspective of Cold and Hot: Jin Hong Tablets as a case study

Chronic gastritis (CG) is a common inflammatory disease of chronic inflammatory lesion of gastric mucosa and in the diagnosis of gastritis in traditional Chinese medicine (TCM), CG can be classified into Cold ZHENG (syndrome in TCM) and Hot ZHENG. However, the molecular features of Cold/Hot ZHENG in CG and the mechanism of Cold/Hot herbs in formulae for CG remained unclear. In this study, we collected a transcriptomics data including 35 patients of Cold/Hot ZHENG CG and 3 scRNA-seq CG samples. And 25 formulae for CG and 89 herbs recorded in these formulae were also collected. We conduct a comprehensive analysis based on the combination of transcriptomics datasets and machine learning algorithms, to discover biomarkers for Cold/Hot ZHENG CG. Then the target profiles of the collected formulae and Cold/Hot herbs were predicted to uncover the features and biomarkers of them against Cold/Hot ZHENG CG. These biomarkers suggest that Hot ZHENG CG might be characterized by over-inflammation and exuberant metabolism, and Cold ZHENG CG showed a trend of suppression in immune regulation and energy metabolism. Biomarkers and specific pathways of Hot herbs tend to regulate immune responses and energy metabolism, while those of Cold herbs are more likely to participate in anti-inflammatory effects. Finally, the findings were verified based on public transcriptomics datasets, as well as transcriptomics and ELISA detection, taking Jin Hong tablets as a case study. Biomarkers like leptin and IL-6 together with proportions of immune cells showed significant changes after the intervention. These findings might reflect the mechanism and build a bridge between macro and micro views of Cold/Hot ZHENG as well as Cold/Hot herbs.Graphical abstract

Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.

Evaluation of Molecular Assays for Diagnosis of Amoebic Liver Abscess in India with Bayesian Latent Class Analysis.

Amoebic liver abscess (ALA) is the most common extra-intestinal complication of Entamoeba histolytica, accounting for 50,000 deaths annually, and is endemic in South Asia. Diagnosis based on microscopic examination is insensitive, and serological assays are not discerning of current infections in endemic settings with high exposure. For a rapid and confirmatory laboratory diagnosis of ALA, the performance of a polymerase chain reaction (PCR), quantitative real time PCR (qPCR), digital droplet PCR (ddPCR), and a loop-mediated isothermal amplification (LAMP) assay that detects E. histolytica DNA in liver abscess pus, and a lectin antigen detection ELISA were evaluated against clinical diagnosis (based on predefined criteria) as the gold standard. Owing to the lack of a laboratory gold standard, a Bayesian latent class analysis approach was also used to determine sensitivity and specificity of these assays. In the latent class analysis, qPCR and ddPCR showed the highest sensitivity (98% and 98.1%) and specificity (both 96.6%), and although clinical diagnosis had a comparable sensitivity to qPCR and ddPCR (95.2%), poorer specificity (64.3%) was seen. Kappa agreement analysis showed that qPCR and ddPCR had a perfect agreement of 1 followed by an agreement of 0.76 (95% CI: 0.64-0.88) with PCR. Considering the performance characteristics and relative ease of setting up qPCR as well as the wide availability of qPCR equipment needed, this would be the most optimal assay for rapid, confirmatory, molecular diagnosis of ALA in the tertiary care laboratory setting in India, whereas further optimization of LAMP or antibody-based detection is required for use at smaller or secondary hospitals.

One Health Investigation into Mpox and Pets, United States.

Monkeypox virus (MPXV) is zoonotic and capable of infecting many mammal species. However, whether common companion animals are susceptible to MPXV infection is unclear. During July 2022-March 2023, we collected animal and environmental swab samples within homes of confirmed human mpox case-patients and tested for MPXV and human DNA by PCR. We also used ELISA for orthopoxvirus antibody detection. Overall, 12% (22/191) of animal and 25% (14/56) of environmental swab samples from 4 households, including samples from 4 dogs and 1 cat, were positive for MPXV DNA, but we did not detect viable MPXV or orthopoxvirus antibodies. Among MPXV PCR-positive swab samples, 82% from animals and 93% from environment amplified human DNA with a statistically significant correlation in observed cycle threshold values. Our findings demonstrate likely DNA contamination from the human mpox cases. Despite the high likelihood for exposure, we found no indications that companion animals were infected with MPXV.

Passive protection of chicken egg yolk immunoglobulin (IgY) against Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus)

IgY is an immunoglobulin primarily found in the serum and egg yolk of birds, amphibians, and reptiles. Recent years, IgY is considered to have a good application prospect in the immunodiagnostics and passive immunotherapy of aquatic diseases. In this study, we prepared a specific IgY against Streptococcus agalactiae in tilapia after immunizing the hens for 4 times. The result of ELISA detection showed that the IgY titers in water-soluble fraction (WSF) after 6 weeks of immunization reached 1:51200 and last for 4 weeks. Western blot (WB) analysis data showed that the specific IgY could recognize the target band, the specific IgY showed a concentration-dependent inhibitory effect on the growth of S. agalactiae, altered cell wall structure and aggluted of S. agalactiae. The quantitative reverse transcription PCR (qRT-PCR) analysis data suggested that the specific IgY downregulated the expression of pro-inflammatory factors (IL-8, TNF-α), upregulated the anti-inflammatory factors (IL-10, TGF-β). In addition, the histopathological results showed that the specific IgY significantly decreased the pathological manifestations, dramatically improved the survival rates of tilapia in injection, feeding, and immersion experiments. Collectively, our findings demonstrated that the broad potential of specific IgY for the prevention and treatment of S. agalactiae infection in tilapia.

Specific detection of crustacean allergens in food: Development of indirect competitive and sandwich ELISA targeting sarcoplasmic calcium binding protein

To overcome the problem of cross-reactivity between crustaceans, mollusks, insects, and mites in current crustacean allergen immunodetection, the significant stable crustacean allergen, sarcoplasmic calcium binding protein (SCP) was innovatively selected as the target of ELISA detection. An indirect competitive ELISA (icELISA) and a sandwich ELISA (sELISA) were constructed and evaluated with spiked model foods. The sELISA (LOD 0.001 mg/kg and LOQ 0.003 mg/kg) is more sensitive than icELISA (LOD 0.11 mg/kg and LOQ 0.56 mg/kg). But the icELISA presented a wider standard range (10–10000 ng/mL) and greater recoveries of SCP in matrices (84.04%–118.94%). Sufficient precision (repeatability and reproducibility <15%), excellent specificity distinguishing crustaceans from others, and adequate detectability in diverse commercial processed foods were observed from the SCP-based icELISA. These results demonstrated the feasibility and applicability of SCP-targeted icELISA and provided a new direction to improve the capacity for crustacean allergen detection.

Development of a double-antibody sandwich ELISA for detection of SARS-CoV-2 variants based on nucleocapsid protein-specific antibodies.

The COVID-19 pandemic, driven by the SARS-CoV-2 virus, has posed a severe threat to global public health. Rapid, reliable, and easy-to-use detection methods for SARS-CoV-2 variants are critical for effective epidemic prevention and control. The N protein of SARS-CoV-2 serves as an ideal target for antigen detection. In this study, we achieved soluble expression of the recombinant SARS-CoV-2 N protein using an Escherichia coli expression system and generated specific monoclonal antibodies by immunizing BALB/c mice. We successfully developed 10 monoclonal antibodies against the N protein, designated 5B7, 5F2-C11, 5E2-E8, 6C3-D8, 7C8, 9F2-E9, 12H5-D11, 13G2-C10, 14E9-F6, and 15H3-E10. Using these antibodies, we established a sandwich ELISA with 6C3-D8 as the capture antibody and 5F2-C11 as the detection antibody. The assay demonstrated a sensitivity of 0.78 ng/mL and showed no cross-reactivity with MERS-CoV, HCoV-OC43, HCoV-NL63, and HCoV-229E. Furthermore, this method successfully detected both wild-type SARS-CoV-2 and its variants, including Alpha, Beta, Delta, and Omicron. These findings indicate that our sandwich ELISA exhibits excellent sensitivity, specificity, and broad-spectrum applicability, providing a robust tool for detecting SARS-CoV-2 variants.

Comparison of urine and serum IgG detection ELISA for tegumentary leishmaniasis diagnosis and prognosis

Laboratorial diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests, which are still hampered by blood́ invasive collection. In this context, in the present study, we develop a serum- and urine-based ELISA to TL diagnoses. A recombinant protein (rLiHyA), which was previously showed to be antigenic for the disease, as well as a B-cell epitope produced as synthetic peptide and a Leishmania antigenic extract (SLA), were used as antigens. A total of paired 205 urine and serum samples were used, which were comprised by samples from cutaneous (n = 30) and mucosal (n = 30) leishmaniasis patients, as well as from healthy individuals living in endemic region of disease (n = 45), of patients with Chagas disease (n = 30), leprosy (n = 35), malaria (n = 15) or HIV-infected (n = 20). Results showed that serum-based ELISA presented sensitivity of 24.0 %, 100 % and 41.0 %, when SLA, rLiHyA and synthetic peptide were used as antigens, and specificity of 98.4 %, 98.4 % and 98.4 %, respectively. The area under the curve (AUC) was calculated and results were 0.74, 1.0, and 0.71, respectively, when SLA, rLiHyA and synthetic peptide were used as antigens. Performing an urine-based ELISA, sensitivity was 28.0 %, 100 % and 75.0 %, respectively, when SLA, rLiHyA, and synthetic peptide were used, while specificity values were of 98.4 %, 98.4 % and 98.4 %, respectively. In addition, the AUC values were 0.82, 1.0, and 0.94, respectively. A significant drop in specific antibodies levels in both patientś serum and urine samples was found six months after treatment, suggesting a prognostic role of rLiHyA for TL. In conclusion, preliminary data suggest the potential of use patient urine to TL diagnoses.

Performance evaluation of microfluidic microplate-based fluorescent ELISA for qualitative detection of SARS-CoV-2–specific IgG and IgM

We evaluated the diagnostic performance of newly developed microfluidic microplate-based fluorescent ELISA for anti-SARS-CoV-2 antibody detection: the Veri-Q opti COVID-19 IgG and IgM ELISAs (hereafter, “Opti IgG/M”; MiCo BioMed, Gyeonggi-do, Republic of Korea), in comparison with conventional ELISAs. A total of 270 serum samples were analyzed, among which 90 samples were serially obtained from 25 COVID-19 patients. Another 180 samples were collected from 180 SARS-CoV-2–negative individuals. As comparative assays, we used SCoV-2 Detect IgG/M ELISA (hereafter, “InBios IgG/M”; InBios, Seattle, WA, USA) and Veri-Q COVID-19 IgG/IgM ELISA (hereafter, “Veri-Q IgG/M”; MiCo BioMed). Compared with conventional ELISAs, the Opti IgG yielded 97.1–100.0% positive percent agreement, 95.2–98.0% negative percent agreement, 96.3–97.8% total percent agreement, and kappa values of 0.90–0.94. Between the Opti IgM and the InBios IgM, the values were 93.7%, 96.6%, 95.9%, and 0.89, respectively. For the Opti IgG, sensitivities for the samples collected from 0–7, 8–14, 15–21, and ≥ 22 days after symptom onset were 40.0, 58.3, 94.1, and 100.0%, respectively. The values for the Opti IgM were 30.0, 54.2, 88.2, and 80%, respectively. The diagnostic specificities of the Opti IgG and IgM were 99.4 and 97.2%, respectively. The microfluidic microplate-based fluorescent ELISAs showed comparable diagnostic performance to conventional ELISAs for detecting anti-SARS-CoV-2 antibodies. With the combination of high throughput, a simplified workflow, and the ability to analyze reduced volumes, this new technology has great potential for improving SARS-CoV-2 serologic testing.

The Frequency of Rotavirus Gastroenteritis in Children from West of Iran and Genotyping of Rotavirus Isolates: A Suggestion for Further Changes in Childhood Immunization Program.

Rotavirus is the most common cause of gastroenteritis among children. Currently, four oral live-attenuated vaccines are available to prevent rotavirus infection. The World Health Organization (WHO) has recommended including rotavirus vaccination in national immunization programs; however, it has not been introduced to the Iranian national immunization program. The study aimed to assess the frequency of rotavirus gastroenteritis in the west of Iran and investigate the necessity of rotavirus vaccination. Study Design: A case series study. In this case series study, 284 cases under six years of age who presented with acute gastroenteritis from March 2021 to 2022 to a referral hospital in the west of Iran were evaluated. Data on baseline characteristics, clinical manifestations, results of stool test, ELISA for rotavirus detection, and polymerase chain reaction (PCR) test for genotyping of rotavirus-positive samples were recorded. Results showed that the prevalence of rotavirus infection was 36.6%. The highest frequency was observed among children aged 6-12 months and during the autumn. According to the PCR results, G1P[8], G9P[8], G9P[4], and G1P [4] were the dominant genotypes, and 33.75% of samples were infected with multiple rotavirus genotypes. The study highlights the considerable prevalence of rotavirus infection among cases of acute gastroenteritis in children under six years of age who were referred to a referral hospital in the west of Iran and the high diversity of rotavirus genotypes in the targeted community. Consequently, physicians and health policymakers should prioritize strategies for the prevention and control of this infection, particularly by considering the rotavirus vaccine as a priority for the Iranian national immunization program.

The attenuation of gut microbiota-derived short-chain fatty acids elevates lipid transportation through suppression of the intestinal HDAC3-H3K27ac-PPAR-γ axis in gestational diabetes mellitus

Gut flora is considered to modulate lipid transport from the intestine into the bloodstream, and thus may potentially participate in the development of GDM. Although previous studies have shown that the intestinal microbiota influences lipid transport and metabolism in GDM, the precise mechanisms remain elusive. To address this, we used a high-fat diet (HFD)-induced GDM mouse model and conducted 16s rRNA sequencing and fecal metabolomics to assess gut microbial community shifts and associated metabolite changes. Western blot, ELISA, and chromatin immunoprecipitation (ChIP) were utilized to elucidate how gut microbiota affect intestinal lipid transport and the insulin sensitivity of hepatic, adipose, and skeletal muscle tissues. We found that HFD impaired the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) in pregnant mice. 16s rRNA sequencing demonstrated profound compositional changes, especially in the relative abundances of Firmicutes and Bacteroidetes. Metabolomics analysis presented a decline in the concentration of short-chain fatty acids (SCFAs) in the GDM group. Western blot analyses showed an upregulation of HDAC3 and a concurrent reduction in H3K27 acetylation in the intestine. ChIP-qPCR showed that PPAR-γ was inhibited, which in turn activated lipid-transporter CD36. ELISA and insulin signaling pathway detection in insulin-target organs showed high concentrations of circulating fatty acids and triglycerides and insulin resistance in insulin-target organs. Our results suggest that gut microbiota is closely associated with the development of GDM, partly because decreased gut flora-associated SCFAs activate CD36 by suppressing the HDAC3-H3K27ac-PPAR-γ axis to transport excessive fatty acids and triglycerides into blood circulation, thereby dysregulating the insulin sensitivity of insulin target organs.

Recombinant 3AB3 nonstructural protein-based indirect ELISA for detection of foot-and-mouth disease virus infection-elicited antibodies in goat.

Foot-and-mouth disease (FMD) is one of the most important animal diseases of economic significance globally. It is a highly infectious and contagious disease of cloven-hoofed animals including sheep and goat. For sero-diagnosis of FMD, recombinant antigen-based assays are considered as alternatives to conventional approaches such as the liquid phase blocking ELISA (LPBE). The early interventions towards control measures cannot be implemented unless the disease gets promptly diagnosed. It is relatively difficult to clinically diagnose FMD in goat due to the usual milder form or unapparent nature of symptoms. Under such situations where clinical samples are not available, demonstration of infection-specific FMD virus (FMDV) antibodies in serum sample may help identifying the animals exposed to the virus in retrospect. Antibody to 3AB nonstructural protein (NSP) has been considered to be the most reliable indicator for FMD diagnosis. The current study extended the earlier designed recombinant 3AB3 protein-based indirect ELISA originally validated on bovine serum samples to testing serum samples of goat. The performance of the indirect ELISA was validated using internationally accepted PrioCHECK® FMDV NS kit. The overall diagnostic sensitivity (DSn) of the indirect ELISA was estimated to be 95.52% (619/648), while the diagnostic specificity (DSp) on naïve and vaccinated animals varied at 98.06% (557/568) and 94.15% (435/462), respectively. In India, where FMD is prevalent and the goat population is so high, this 'in-house' optimized assay can be considered to be an adjunct in sero-epidemiological investigation of FMD in goat.

Livestock keeping, mosquitoes and community viewpoints: a mixed methods assessment of relationships between livestock management, malaria vector biting risk and community perspectives in rural Tanzania

BackgroundLivestock keeping is one of the potential factors related to malaria transmission. To date, the impact of livestock keeping on malaria transmission remains inconclusive, as some studies suggest a zooprophylactic effect while others indicate a zoopotentiation effect. This study assessed the impact of livestock management on malaria transmission risks in rural Tanzania. Additionally, the study explored the knowledge and perceptions of residents about the relationships between livestock keeping and malaria transmission risks in a selected village.MethodsIn a longitudinal entomological study in Minepa village, South Eastern Tanzania, 40 households were randomly selected (20 with livestock, 20 without). Weekly mosquito collection was performed from January to April 2023. Indoor and outdoor collections used CDC-Light traps, Prokopack aspirators, human-baited double-net traps, and resting buckets. A subsample of mosquitoes was analysed using PCR and ELISA for mosquito species identification and blood meal detection. Livestock's impact on mosquito density was assessed using negative binomial GLMMs. Additionally, in-depth interviews explored community knowledge and perceptions of the relationship between livestock keeping and malaria transmission risks.ResultsA total of 48,677 female Anopheles mosquitoes were collected. Out of these, 89% were Anopheles gambiae sensu lato (s.l.) while other species were Anopheles funestus s.l., Anopheles pharoensis, Anopheles coustani, and Anopheles squamosus. The findings revealed a statistically significant increase in the overall number of An. gambiae s.l. outdoors (RR = 1.181, 95%CI 1.050–1.862, p = 0.043). Also, there was an increase of the mean number of An. funestus s.l. mosquitoes collected in households with livestock indoors (RR = 2.866, 95%CI: 1.471–5.582, p = 0.002) and outdoors (RR = 1.579,95%CI 1.080–2.865, p = 0.023). The human blood index of Anopheles arabiensis mosquitoes from houses with livestock was less than those without livestock (OR = 0.149, 95%CI 0.110–0.178, p < 0.001). The majority of participants in the in-depth interviews reported a perceived high density of mosquitoes in houses with livestock compared to houses without livestock.ConclusionDespite the potential for zooprophylaxis, this study indicates a higher malaria transmission risk in livestock-keeping communities. It is crucial to prioritize and implement targeted interventions to control vector populations within these communities. Furthermore, it is important to enhance community education and awareness regarding covariates such as livestock that influence malaria transmission.

Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens.

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

Development and Bayesian validation of a competitive inhibition ELISA for detection of antibodies against Brucella abortus in cattle

Bovine brucellosis is a zoonotic disease caused by Brucella abortus, responsible for abortions in cows. It is endemic in low- and middle-income countries, where the brucellosis control and eradication programs are based on compulsory vaccination, detection of infected cattle through serologic assays, and culling of infected animals at slaughterhouses. The development of high sensitivity and specificity, and low-cost serologic assays guarantee their implementation in countries where the disease is endemic. The aim of the present study was to develop and validate a competitive inhibition enzyme-linked immune assay (ciELISA) to detect anti-B. abortus antibodies in sera from cattle. The developed ciELISA was validated using 2833 serum samples from dairy and beef cattle. From these, 1515 sera were from uninfected cows that belonged to free of brucellosis herds and 1318 were from infected cows that belonged positive to brucellosis herds. Sera were analyzed with the developed ciELISA, the buffer plate antigen (BPA) test, and the complement fixation test (CFT). The brucellosis status of the herds was officially established according to the country legislation and consistent for at least 5 years and was defined for each cow using the CFT as gold standard. The cutoff for the ciELISA was calculated using a ROC curve and its sensitivity and specificity were analyzed using the Bayesian Latent Class Model (BLCM) approach. The agreement among tests was calculated using the kappa (κ) value. In addition, 15 calves were vaccinated with 3 × 1010 viable cells of B. abortus Strain 19 vaccine, and the dynamics of antibodies were measured by CFT, buffered plate antigen (BPA) test, and the developed ciELISA. The obtained cutoff for ciELISA was ≥ 47 percentage of inhibition (% I), at the BLCM approach the sensitivity was 99.01 % (95 % CI: 97.55–100) and the specificity 98.74 % (95 % CI: 97.68–99.8). The κ between the ciELISA and BPA was κ = 0.88 and between the ciELISA and CFT κ = 0.95. Antibodies against B. abortus were detected in all the vaccinated calves 7 days after vaccination (AV) by the three assays, at day 135 AV all the calves were negative to CFT (15/15), 93.3 % (14/15) to ciELISA and 73.3 % (11/15) to BPA, and at day 190 AV all the calves were negative to the three assays. The developed ciELISA showed a very good performance, could detect the majority of vaccinated animals as negative after 135 days and could be used for the detection of anti-B. abortus antibodies in serum samples for the brucellosis control and eradication program.

Enzyme immunoassay system for serological diagnosis of hemorrhagic fever with renal syndrome based on inactivated purified Puumala virus (Hantaviridae: Orthohantavirus)

Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS. Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent. Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA. For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays. The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.

Bioluminescent aptamer-based microassay for detection of melanoma inhibitory activity protein (MIA).

Melanoma inhibitory activity protein (MIA) does obviously offer the potential to reveal clinical manifestations of melanoma. Despite a pressing need for effective diagnosis of this highly fatal disease, there are no clinically approved MIA detection ELISA kits available. A recommended MIA threshold has not yet been defined, mostly by reason of variability in immunoglobulins' affinity and stability, the difference in sample preparation and assay conditions. Here we present a pair of high-affinity DNA aptamers developed as an alternative recognition and binding element for MIA detection. Their stability and reproducible synthesis are expected to ensure this analysis under standard conditions. The devised aptamer-based solid-phase microassay of model standard and control human sera involves luciferase NLuc as a highly sensitive reporter. Bioluminescence dependence on MIA concentration ranges in a linear manner from 2.5 to 250 ng mL-1, providing a MIA detection limit of 1.67 ± 0.57 ng mL-1.

Prevalence of Human Papillomavirus Infection and its Association with the Risk of Cervical Cancer among Hiv-Positive Women in Plateau State, North-Central Nigeria

Study’s Novelty/Excerpt This study investigates the prevalence and risk factors of HPV among HIV-infected women in Plateau State, Nigeria, highlighting a significant correlation between low CD4+ counts, high viral loads, and increased HPV infection rates. By utilizing comprehensive diagnostic methods including ELISA for HPV detection and cytology for cervical abnormalities, the research offers robust data linking immunosuppression and HPV-related cervical pathology in a high-risk population. The findings emphasize the urgent need for targeted interventions to improve sexual health behaviors and further research on how low immunity accelerates cervical cancer progression in both HIV-positive women and the broader population. Full Abstract Human papillomavirus (HPV) is one of the most common sexually transmitted infections (STI) associated with cervical, uterine, and anogenital cancers. Persistent infection with HPV is associated with abnormal cervical cells, which can develop into cervical cancer if left untreated. Human papillomaviruses are the first viruses to have been acknowledged to prompt carcinogenesis, and they are linked with cancers of the uterine cervix, anogenital tumours, and head and neck malignancies. A hospital-based study of HIV-infected women across the three senatorial zones of Plateau State, Nigeria, was conducted between November 2018 to November 2020. Ethical approval for the study was first obtained from the ethical committee of Plateau State Specialist Hospital Jos, and informed consent to participate in the research was also obtained from each participant. HIV status confirmation was first done through standard rapid test procedures, followed by cytology testing via the Pap smear procedure to detect any precancerous or malignant changes in the cervix. Subsequent detection of HPV utilized the ELISA procedure, while CD4+ cell count and viral load estimations were done using flow cytometry and nucleic acid amplification techniques, respectively. Questionnaires were administered to obtain information on cervical cancer risk factors and clinical presentations. The overall prevalence of HPV was 28% among HIV-infected women. More HPV infection (31.9%) occurred in women with low CD4+ count (0-200 cells/mm3), and also highest (50.0%) among women with the highest HIV viral load (&gt;100 copies/mL). The possible risk factors identified in this study include multiple sexual partnering, low condom usage, and coinfection with other STIs, among others. In conclusion, this study identified a high HPV prevalence, low CD4+ counts, and coinfection with other STIs among high-risk populations (HIV-infected women). We, therefore, recommend improved sexual behaviours and further research on the impact of low immunity on the rate of progression of cervical abnormality to cervical cancer, not just in HIV-positive women but in the general population.

No detection of tick-borne encephalitis virus RNA in blood, urine or saliva of hospitalised immunocompetent tick-borne encephalitis patients.

Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by vaccination or cross-reactivity to previous flavivirus infections may result in false positive TBEV serology. Detection of TBEV RNA may be an alternative diagnostic approach to detect viral presence and circumvent the diagnostic difficulties present when using serology. Viral RNA in blood is commonly detectable only in the first viremic phase usually lasting up to two weeks, and not in the second neurologic phase, when the patients contact the health care system and undergo diagnostic work-up. TBEV RNA has previously been detected in urine in a few retrospective TBE cases in the neurologic phase, and furthermore RNA of other flaviviruses has been detected in patient saliva. In this study, blood, saliva and urine were collected from 31 hospitalised immunocompetent patients with pleocytosis and symptoms of aseptic meningitis and/or encephalitis, suspected to have TBE. We wanted to pursue if molecular testing of TBEV RNA in these patient materials may be useful in the diagnostics. Eleven of the 31 study patients were diagnosed with TBE based on ELISA detection of TBEV specific IgG and IgM antibodies. None of the study patients had TBEV RNA detectable in any of the collected patient material.