Digital dermatitis in Swedish dairy herds assessed by ELISA targeting Treponema phagedenis in bulk tank milk.

BACKGROUND: Digital dermatitis (DD) is a contagious hoof infection affecting cattle worldwide. The disease causes lameness and a reduction in animal welfare, which ultimately leads to major decreases in milk production in dairy cattle. The disease is most likely of polymicrobial origin with Treponema phagedenis and other Treponema spp. playing a key role; however, the etiology is not fully understood. Diagnosis of the disease is based on visual assessment of the feet by trained hoof-trimmers and veterinarians, as a more reliable diagnostic method is lacking. The aim of this study was to evaluate the use of an enzyme-linked immunosorbent assay (ELISA) on bulk tank milk samples testing for the presence of T. phagedenis antibodies as a proxy to assess herd prevalence of DD in Swedish dairy cattle herds. RESULTS: Bulk tank milk samples were collected in 2013 from 612 dairy herds spread across Sweden. A nationwide DD apparent prevalence of 11.9% (8.1-14.4% CI95%) was found, with the highest proportion of test-positive herds in the South Swedish regions (31.3%; 19.9-42.4% CI95%). CONCLUSIONS: This study reveals an underestimation of DD prevalence based on test results compared to hoof trimming data, highlighting the critical need for a reliable and accurate diagnostic method. Such a method is essential for disease monitoring and the development of effective control strategies. The novelty of ELISA-based diagnostic methods for DD, coupled with the disease's polymicrobial origin, suggests an avenue for improvement. Developing an expanded ELISA, incorporating antigens from various bacterial species implicated in the disease, could enhance diagnostic accuracy. The significance of this study is underscored by the extensive analysis of a substantial sample size (612). Notably, this investigation stands as the largest assessment to date, evaluating the application of ELISA on bulk tank milk for DD diagnosis at the herd level.

1,25-Dihydroxyvitamin D3 Suppresses UV-Induced Poly(ADP-Ribose) Levels in Primary Human Keratinocytes, as Detected by a Novel Whole-Cell ELISA.

Photoprotective properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH)2D3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH)2D3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200-400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH)2D3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH)2D3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH)2D3 in skin to reduce UV-induced DNA damage.

A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

The effect of burosumab on intact and C-terminal FGF23 measurements.

OBJECTIVE: To investigate the effect of CRYSVITA® (burosumab-twza) on FGF23 measurements in an intact and a C-terminal immunoassay. METHODS: An intact serum FGF23 (MedFrontier) and a C-terminal plasma FGF23 assay (Immutopics) were used. Serum/plasma pools were spiked to span the burosumab therapeutic range (1.4-11.3 µg/ml) and FGF23 recovery was assessed. Patient serum and plasma samples obtained pre and post-burosumab treatment were evaluated on both assays and compared with corresponding phosphorus measurements RESULTS: Spiking burosumab (1.4-11.3 µg/ml) into sample pools resulted in a dose-dependent negative analytical interference on intact FGF23 measurements and no significant interference for C-terminal FGF23 measurements. However, more than a 500-fold median increase (post- vs. pre-burosumab administration) in in vivo FGF23 concentrations were observed by both assays. CONCLUSIONS: Therapeutic concentrations of burosumab result in a negative analytical interference of the intact, but not the C-terminal FGF23 immunoassay. Despite this in vitro analytical interference in the intact assay, relatively large elevations of both intact FGF23 and C-terminal FGF23 measurements were observed in vivo following burosumab administration. Following burosumab administration, FGF23 measurements must be interpreted within the clinical context of the patient and other relevant biomarker results. SUMMARY: This article describes a negative analytical interference by burosumab in an intact FGF23 immunoassay. The recovery of C-terminal FGF23 is not significantly affected by the presence of burosumab. In vivo, both assays demonstrate extreme FGF23 elevations in the presence of the drug. Furthermore, the measurement of FGF23 blocked by burosumab is not clinically useful regarding hypophosphataemia.

PCR and peptide based PCMV detection in pig - development and application of a combined testing procedure differentiating newly from latent infected pigs.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

Seroprevalence and associated factors of infectious bursal disease (IBD) in indigenous chicken in eastern province in Rwanda.

The status of Infectious bursal disease (IBD) in indigenous chickens and backyard poultry in Rwanda has not been previously elucidated. This cross-sectional study was to determine the seroprevalence of infectious bursal disease in indigenous chickens and to identify the associated factors. The study was been done in three districts in the Eastern province of Rwanda where blood from 364 indigenous chickens were collected. ID Screen® IBD indirect enzyme-linked immunosorbent assay (ELISA) test was used to detect IBD antibodies in these birds. 145 questionnaires were also administered to poultry farmers to obtain information on biosecurity measures and associated factors to IBD outbreaks. The study revealed 48.4% (176/364) prevalence of the chicken with IBDV antibodies with statistical significance (P < .05) among/between location and age groups. The questionnaire revealed that there were other important associated factors which included chicken scavenging for seed as a source of food (59.3% of farmers reported), absence of routine vaccination (53.8%), live chickens are purchased from the open market with no information about IBD outbreaks and vaccination (30.0%), open disposal of dead chickens suspected of IBD (58.9%). IBD virus antibodies are present in indigenous chicken in Eastern Rwanda hence further investigation to better understand the epidemiology of IBD virus in indigenous chickens is desired and more research is needed to identify the role of indigenous chickens in the spread of IBD virus in Rwanda.

Construction of a monoclonal antibody against glabridin (2G4) and development of an enzyme-linked immunosorbent assay.

INTRODUCTION: Glabridin is a unique isoflavonoid found only in Glycyrrhiza glabra L. The pharmacological effects of glabridin are well established, especially for beauty- and wellness-related uses, such as antioxidant, anti-inflammatory, ultraviolet (UV) protection, and skin-lightening effects. Therefore, glabridin is often found in commercial products such as creams, lotions, and dietary supplements. OBJECTIVE: This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) using a glabridin-specific antibody. METHOD: Immunogen conjugation of glabridin-bovine serum albumin was performed via the Mannich reaction, and the resulting conjugates were injected into BALB/c mice. Subsequently, hybridomas were produced. An ELISA method for glabridin determination was developed and validated. RESULT: A highly specific antibody against glabridin was produced using clone 2G4. The assay range for the determination of glabridin was 0.28-7.02 µg/ml, with a detection limit of 0.16 µg/ml. The validation parameters in terms of accuracy and precision met the acceptable criteria. Standard curves of glabridin in various matrices were compared to evaluate the matrix effect on human serum using ELISA. Standard curves of the human serum and water matrix were obtained in the same manner, and the measurement range was 0.41-10.57 µg/ml. CONCLUSION: The developed ELISA method was used to quantify glabridin in plant materials and products with high sensitivity and specificity, and has potential applications in quantifying compounds in plant-derived products and human serum samples.

Testing the link between isoaspartate and Alzheimer's disease etiology.

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts protein structures, making them prone to aggregation. Here we strengthened the link between isoAsp and Alzheimer's disease (AD) by novel approaches to isoAsp analysis in human serum albumin (HSA), the most abundant blood protein and a major carrier of amyloid beta (Aß) and phosphorylated tau (p-tau) in blood. We discovered a reduced amount of anti-isoAsp antibodies (P < 0.0001), an elevated isoAsp level in HSA (P < 0.001), more HSA aggregates (P < 0.0001), and increased levels of free Aß (P < 0.01) in AD blood compared to controls. We also found that deamidation significantly reduces HSA capacity to bind with Aß and p-tau (P < 0.05). These suggest the presence in AD of a bottleneck in clearance of Aß and p-tau, leading to their increased concentrations in the brain and facilitating their aggregations there.

Immunogenicity Analysis of PCV3 Recombinant Capsid Protein Virus-like Particles and Their Application in Antibodies Detection.

Porcine circovirus type 3 is a newly emerging pathogen of porcine circovirus associated disease (PCVAD). Currently, there is no commercially available vaccine, resulting in huge economic losses to the pig industry. Porcine circovirus type 3 capsid protein (Cap) can self-assemble into virus-like particles (VLPs). Therefore, the expression of the recombinant Cap protein is of great significance for the prevention, diagnosis and control of porcine circovirus type 3 associated diseases. In this study, the recombinant Cap protein was successfully expressed in Escherichia coli by deleting the nuclear localization sequence (NLS). The VLPs were observed by transmission electron microscopy. To evaluate the immunogenicity of the recombinant Cap protein, mice were immunized. As a result, the recombinant Cap protein can induce higher levels of humoral and cellular immune responses. A VLP-based ELISA method was developed for the detection of antibodies. The established ELISA method has good sensitivity, specificity, repeatability and clinical applicability. These results demonstrate the successful expression of the PCV3 recombinant Cap protein and the preparation of recombinant Cap protein VLPs, which can be used for the preparation of subunit vaccines. Meanwhile, the established I-ELISA method lays a foundation for the development of the commercial PCV3 serological antibody detection kit.

An Extremely Highly Sensitive ELISA in pg mL-1 Level Based on a Newly Produced Monoclonal Antibody for the Detection of Ochratoxin A in Food Samples.

In this study, an extremely highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a newly produced monoclonal antibody (mAb) for the detection of ochratoxin A (OTA) in food samples was developed. OTA-Bovine serum albumin (BSA) conjugate was prepared and used as the immunogen for the production of the mAb. Among four hybridoma clones (8B10, 5C2, 9B7, and 5E11), the antibody from 8B10 displayed the highest affinity recognition for OTA. Based on the mAb (8B10), the IC50 and LOD of the ELISA for OTA were 34.8 pg mL-1 and 1.5 pg mL-1, respectively, which was 1.53~147 times lower than those in published ELISAs, indicating the ultra-sensitivity of our assay. There was no cross-reactivity of the mAb with the other four mycotoxins (AFB1, ZEN, DON, and T-2). Due to the high similarity in molecular structures among OTA, ochratoxin B (OTB), and ochratoxin C (OTC), the CR values of the mAb with OTB and OTC were 96.67% and 22.02%, respectively. Taking this advantage, the ELISA may be able to evaluate total ochratoxin levels in food samples. The recoveries of the ELISA for OTA in spiked samples (corn, wheat, and feed) were 96.5-110.8%, 89.5-94.4%, and 91.8-113.3%; and the RSDs were 5.2-13.6%, 8.2-13.0%, and 7.7-13.7% (n = 3), respectively. The spiked food samples (corn) were measured by ELISA and HPLC-FLD simultaneously. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation of y = 0.918x - 0.034 (R2 = 0.985, n = 5) was obtained. These results demonstrated that the newly produced mAb-based ELISA was a feasible and ultra-sensitive analytical method for the detection of OTA in food samples.

Concentration of Urine Samples Improves Sensitivity in Detection of Strongyloides-Specific IgG Antibody in Urine for Diagnosis of Strongyloidiasis.

Detection of IgG in urine is an efficient method comparable to that in serum for diagnosis of strongyloidiasis, but the effects of daily variation in urine dilution on diagnostic accuracy are not clearly known. This study evaluated the effects of urine concentration on the detection of parasite-specific IgG by urine enzyme-linked immunosorbent assay (ELISA), particularly in individuals with borderline results or false-negative diagnosis. Optimal concentration conditions were established by comparing Strongyloides-specific IgG antibody levels between unconcentrated and concentrated urine in participants with different infection intensities, namely, healthy control (HC), low-negative (LN), high-negative (HN), and low-positive (LP) groups. The optimal condition was selected and validated in a field trial study. The final urine concentration protocol required centrifugation at 4,000 × g at 4°C for 10 mins using the Amicon concentrator tube. This protocol was validated in groups of participants with various diagnoses according to urine ELISA and fecal examination (n = 148). The concentrated-urine ELISA increased the proportion of positive results in the LN group by 68.2% and by 100% in the HN group. Significantly elevated IgG antibody levels were seen in the LP group. In the group that was false negative by urine ELISA but positive by fecal examination (n = 28), concentrated-urine ELISA yielded 100% positive results. Overall, the frequency estimates of Strongyloides stercoralis were 23.6% by fecal culture, 27% by standard urine ELISA, and 90.5% by concentrated-urine ELISA. The concentration of urine samples prior to analysis by ELISA improved the sensitivity for diagnosis and is potentially useful in the diagnosis of strongyloidiasis in immunocompromised individuals or in low-prevalence areas.

Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

Towards harmonization of directly measured free 25-hydroxyvitamin D using an enzyme-linked immunosorbent assay.

The majority of circulating 25-hydroxyvitamin D (25(OH)D) is protein bound and perhaps less available than the free fraction of 25(OH)D; therefore, researchers have proposed that the measurement of free 25(OH)D in human serum may be a better indicator of vitamin D health status than total 25(OH)D. The availability of a new enzyme-linked immunosorbent assay (ELISA) for the determination of free 25(OH)D provides a method for direct measurement of the low levels of non-protein bound 25(OH)D. As an initial step towards harmonization of measurements of free 25(OH)D, the ELISA was used to measure free 25(OH)D in three existing Standard Reference Materials (SRMs): SRM 972a Vitamin D Metabolites in Frozen Human Serum, SRM 2973 Vitamin D Metabolites in Frozen Human Serum (High Level), and SRM 1949 Frozen Prenatal Human Serum. Target values for free 25(OH)D in the nine SRM serum pools, obtained by combining the results from two laboratories, ranged from 3.76 ± 0.36 to 10.0 ± 0.58 pg/mL. Of particular significance is the assignment of free 25(OH)D target values to SRM 1949, which consists of four serum pools from non-pregnant female donors of reproductive age and pregnant women in each of the three trimesters and which also has values assigned for vitamin D binding protein, which increases during pregnancy. The availability of target values for free 25(OH)D in these SRMs will allow researchers to validate new analytical methods and to compare their results with other researchers as an initial step towards harmonization of measurements among different studies and laboratories.

Application of a truncated ORF2 protein-based ELISA for diagnosis of hepatitis E in an endemic area.

Enterically transmitted waterborne hepatitis E (HE) caused due to hepatitis E virus (HEV) prevails as a significant public health problem endemic to India. Due to short-term viremia/fecal excretion and poor in vitro transmissibility of HEV, HE diagnosis depends on detection of specific IgM antibodies in serum. Present study evaluated performances of two in-house and six commercial IgM detection enzyme-linked immunosorbent assays (ELISAs) using sera collected from volunteers/acute hepatitis patients (n = 716). The in-house ELISAs were based on complete and truncated open reading frame 2 (ORF2) proteins containing neutralizing epitope/s region of genotype 1 HEV (ORF2p, 1-660 amino acid (a.a.) and T1NEp, 458-607 a.a., respectively). The commercial ELISAs included Wantai (China), MP Diagnostics (MPD) (Singapore), DIA.PRO Diagnostics (Italy), MBS (Italy), abia (Germany), and ImmunoVision (USA). T1NE ELISA showed 97.0% positive percent agreement (PPA), 99.4% negative percent agreement (NPA), and 98.6% concordance (κ = 0.97, P = 0.0000) with ORF2 ELISA. ORF2, T1NE, Wantai, and MPD ELISAs agreed on results for 88% of sera tested. Two percent sera showed reactivity in each combination of three and two of aforementioned four ELISAs. Remaining 8% sera were single ELISA reactive. PPA and NPA value ranges were 76.3-99.0% and 84.8-99.5%, respectively. Pairwise concordances between all the eight ELISAs ranged from 88.0 to 100% (κ: 0.74-1.00). Both the in-house ELISAs agreed better with Wantai over MPD ELISA. In conclusion, both ORF2 and T1NE ELISAs were equally efficient in diagnosing HEV infections. T1NEp proved to be an excellent tool in HE sero-diagnosis and is worth exploring in development of simple rapid tests. KEY POINTS: • In-house ELISA based on bacterially expressed neutralizing epitope/s region protein • In-house ELISA based on complete ORF2 protein expressed in insect cells • Comparison of two in-house and six commercial anti-HEV IgM antibody detection ELISAs.

Development of Viral-Vectored Vaccines and Virus Replicon Particle-Based Neutralisation Assay against Mayaro Virus.

Mayaro virus (MAYV) is an emerging alphavirus causing acute febrile illness associated with chronic polyarthralgia. Although MAYV is currently restricted to tropical regions in South America around the Amazon basin, it has the potential to spread globally by Aedes species mosquitoes. In addition, there are currently no specific therapeutics or licenced vaccines against MAYV infection. We have previously shown that an adenovirus based Mayaro vaccine (ChAdOx1 May) was able to provide full protection against MAYV challenge in vaccinated A129 mice and induced high neutralising antibody titres. In this study, we have constructed a replication deficient simian adenovirus (ChAdOx2) and a Modified Ankara Virus (MVA) based vaccine expressing the MAYV structural cassette (sMAYV) similar to ChAdOx1 May, and characterised recombinant MAYV E2 glycoprotein expressed in a mammalian system for immune monitoring. We demonstrate that ChAdOx2 May was able to induce high antibody titres similar to ChAdOx1 May, and MVA May was shown to be an effective boosting strategy following prime vaccination with ChAdOx1 or ChAdOx2 May. In order to measure MAYV neutralising ability, we have developed a virus replicon particle-based neutralisation assay which effectively detected neutralising antibodies against MAYV. In summary, our study indicates the potential for further clinical development of the viral vectored MAYV vaccines against MAYV infections.

[Preparation of monoclonal antibodies against Pinellia ternata lectin protein and establishment of double-antibody sandwich ELISA].

To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 µg·mL~(-1) working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL~(-1). The linear range was 75.000-4 800.000 pg·mL~(-1), and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g~(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g~(-1), 16.53 µg·g~(-1), and 122.63 ng·g~(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.

Seroprevalence of Chlamydia trachomatis Among Female Adults in the United States: The National Health and Nutrition Examination Surveys.

BACKGROUND: Chlamydia trachomatis is the most common nationally notifiable sexually transmitted infection in the United States; however, the seroprevalence of C. trachomatis infection is unknown. METHODS: This cross-sectional study was conducted among 1725 females aged 18 to 39 years who provided serum and urine samples in the 2013 through 2016 National Health and Nutrition Examination Surveys. Presence of anti-C. trachomatis Pgp3 immunoglobulin G (IgG) was determined using both an enzyme-linked immunosorbent assay (ELISA) and multiplex bead array (MBA). Weighted seroprevalence estimates were calculated. Correlates of seroprevalence were examined by multivariable Poisson regression. RESULTS: In 2013 through 2016, overall seroprevalence of C. trachomatis Pgp3 IgG was 30.0% (95% confidence interval [CI], 25.5-35.0) as measured by ELISA and 29.4% (95% CI, 25.8-33.0) as measured by the MBA assay. Overall agreement between tests was 87.1% (1503/1725). There was a high positive agreement by the MBA assay with current detection of chlamydia in urine (86% [36/42]), a past-year diagnosis of chlamydia (81.8% [27/33]), and a history of treatment for pelvic inflammatory disease (60.7% [37/61]). Seroprevalence of C. trachomatis Pgp3 IgG, as measured by MBA, was significantly higher among non-Hispanic Blacks (68.0%; adjusted prevalence ratio (aPR) = 2.7 [95% CI, 2.3-3.3]), Mexican Americans (30.9%; aPR = 1.5 [95% CI, 1.2-1.9]), and other Hispanics (35.0%; aPR = 1.9 [95% CI, 1.4-2.5]) compared with non-Hispanic Whites (21.4%). A higher lifetime number of sexual partners and a younger age at sexual debut was also associated with higher seroprevalence. CONCLUSION: Both the ELISA and MBA serologic assays revealed a high prevalence of antibodies to C. trachomatis Pgp3 in young adult females in the US household population. There were major racial/ethnic disparities in exposure to C. trachomatis, with increased vulnerability among non-Hispanic Black females.

Do serum GDNF levels correlate with severity of Alzheimer's disease?

INTRODUCTION: A growing body of evidence that glial cell line-derived neurotrophic factor (GDNF) levels are probably involved in pathogenesis and disease course of Alzheimer's disease (AD) suggested that its blood levels could potentially be used as a biomarker of AD. The aim of this study was to compare serum GDNF levels in patients with AD and age-matched controls. METHODS: Serum concentrations of GDNF were compared in 25 AD patients and 25 healthy volunteers using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Severity of the disease in AD patients was assessed using Functional Assessment Staging (FAST). Cognitive assessment of the patients was done using the Mini-Mental State Examination (MMSE). RESULTS: Mean GDNF levels were found to be 2.45 ± 0.93 ng/ml in AD patients and 4.61 ± 3.39 ng/ml in age-matched controls. There was a statistically significant difference in GDNF serum levels in patients with AD compared to age-matched controls (p = 0.001). Moreover, GDNF serum levels were significantly correlated with disease severity (p < 0.001) and cognitive impairment (p < 0.001). CONCLUSION: This study showed that serum levels of GDNF are significantly decreased in AD patients in comparison with age-matched controls, thus suggesting a potential role of GDNF as a disease biomarker. However, a comprehensive study of changes in serum levels of multiple neurotrophic factors reflective of different neurobiological pathways in large-scale population studies is recommended.

Viral Detection: Past, Present, and Future.

Viruses are essentially composed of a nucleic acid (segmented or not, DNA, or RNA) and a protein coat. Despite their simplicity, these small pathogens are responsible for significant economic and humanitarian losses that have had dramatic consequences in the course of human history. Since their discovery, scientists have developed different strategies to efficiently detect viruses, using all possible viral features. Viruses shape, proteins, and nucleic acid are used in viral detection. In this review, the development of these techniques, especially for plant and mammalian viruses, their strengths and weaknesses as well as the latest cutting-edge technologies that may be playing important roles in the years to come are described.

Serological responses to koi herpesvirus (KHV) in a non-cyprinid reservoir host.

Koi herpesvirus (KHV) is a highly contagious virus that causes KHV disease (KHVD) inducing high mortality in carp and koi (Cyprinus carpio L.). In the late stage, latency occurs with very low, often non-detectable virus concentrations, which represents a challenge for virus detection. After validation according to OIE recommendations, an antibody ELISA was established to recognize antibodies of C. carpio against KHV infection. In this study, the ELISA was modified to detect anti-KHV antibodies from a non-cyprinid fish. Experimentally infected rainbow trout (Oncorhynchus mykiss) were able to transmit KHV to naïve carp at two different temperatures, demonstrating their potential as a reservoir host. At 20°C, KHVD was induced in carp but not at 15°C. Unexpectedly, rainbow trout developed humoral response against KHV at both temperatures. In contrast to carp, at 15°C trout produced neutralizing antibodies but not at 20°C. While antibodies obtained from infected carp sera reacted in a similar way against all KHV, antibodies from rainbow trout sera reacted differently to the same isolates by ELISA. The data show that even when non-cyprinid fish species are infected with KHV, they can produce antibodies that differ from those observed in carp.