ELISA System Research Articles

Clinical, immune and genetic risk factors of malaria-associated acute kidney injury in Zambian children: A study protocol.

Acute kidney injury (AKI) affects nearly half of children with severe malaria and increases the risk of adverse outcomes such as death and poor cognitive function. The pathogenesis and predictors of malaria-associated acute kidney injury (MAKI) are not fully described. This study aims to determine the clinical, immune, and genetic correlates of risk to AKI in Zambian children admitted with malaria. In addition, we intend to assess a modified renal angina index (mRAI), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and soluble urokinase receptor (suPAR), when done on the first day of admission, for the ability to predict AKI two days later (day 3) in children admitted with malaria. This is an unmatched case-control study with a nested prospective observational study. A case-to-control ratio of 1:1 is used and 380 children with malaria and aged less than 16 years are being recruited from two hospitals in Zambia. Eligible children are recruited after obtaining written informed consent. Recruitment occurs during the malaria season and began on 6th March 2024 and will continue until July 2025. AKI is defined using the 2012 KIDGO AKI creatinine criteria, and cases are defined as children admitted with malaria who develop AKI within 72 hours of admission, while controls are children admitted with malaria but with no AKI. Serum creatinine is collected on Day 1 within 24 hours of admission, on Day 3 and then again on discharge or day 7, whichever comes sooner. Baseline biomarker concentrations will be determined using the Luminex multiplex Elisa system or high-sensitivity ELISA. SPSS version 29 will be used for data analysis. Descriptive statistics and inferential statistical tests will be run as appropriate. A p ≤ 0.05 will be considered as significant. The sensitivity, specificity, and estimates of the area under the curve (AUC) for the renal angina score will be determined.

The Role of Complement Activation in IgM M-Protein-Associated Neuropathies.

Polyneuropathy associated with an immunoglobulin M (IgM) monoclonal gammopathy is characterized by slowly progressive, predominantly distal sensorimotor deficits, sensory ataxia, and electrophysiologic features of demyelination. IgM antibodies against myelin-associated glycoprotein (MAG) are present in serum from most patients. Nerve damage most likely results from the concerted action of binding of anti-MAG antibodies to nerves, followed by complement activation. The interaction of anti-MAG antibodies with complement activation and their relation to clinical characteristics have not been studied in detail. We studied the correlation among anti-MAG antibody titers, complement activation, and IgM-associated polyneuropathy disease severity. We used serum samples from 101 patients with IgM-associated polyneuropathy to assess IgM anti-MAG titers by ELISA and antibody-mediated complement deposition using both an ELISA-based system and a cell-based system of primate peripheral nerve slides. We studied correlations of complement activation with anti-MAG ELISA titers and clinical characteristics. IgM anti-MAG titers varied from negative to strongly positive. Complement deposition in the ELISA-based system correlated significantly with anti-MAG antibody titer (Spearman rho 0.80; p < 0.0001) despite large variability between serum samples with comparable anti-MAG titers. This variability was even larger in the cell-based assay, which also showed complement deposition in IgM anti-MAG negative patients, indicating the presence of autoantibodies directed against epitopes other than MAG in a subset of patients with IgM-associated polyneuropathy. Clinical characteristics did not correlate with anti-MAG titers or complement activation. Anti-MAG antibody titers correlate with the level of complement activation in both ELISA and cell-based systems. However, clinical characteristics of IgM-associated polyneuropathy do not or only weakly correlate with titers or the level of complement deposition. The lack of clear correlations between complement activation and clinical characteristics does, at this stage, not support the use of complement inhibitors in the treatment of IgM-associated polyneuropathy.

Establishing a baseline titer for reporting immunoglobulin M seropositivity of diagnosis of scrub typhus in our region

Abstract Background: Clinical diagnosis of scrub typhus an agent of undifferentiated fever caused by Orientia tsutsugamushi often rests on serological (immunoglobulin [Ig] M enzyme-linked immunosorbent assay [ELISA]) test. This assay needs a baseline titer in the region to serve to be used as a reference value. Owing to the lack of studies in our area, the present study aims to bridge the gap of data of a baseline titer value for reporting IgM ELISA for O. tsutsugamushi. Materials and Methods: The study was conducted in a prospective manner in Bhubaneswar, Odisha, wherein a period of 1 year, 150 serum samples of patients who were hospitalized with a suspicion of scrub typhus and 200 serum samples from healthy blood donors were included. The IgM ELISA was performed by Scrub Typhus Detect™ IgM ELISA system, InBios International Inc. kit. IgG ELISA was performed on 100 healthy blood donor samples by Scrub Typhus Detect™ IgM ELISA system, InBios International Inc. kit. Statistical Analysis: The optical density (OD) obtained as the result of ELISA study was entered into MS Excel and cutoff was determined by SPSS software. Results: The cutoff value for IgM was 0.6 by receiver operating characteristic curve analysis with a sensitivity and specificity of 92.16% and 98.01%, respectively. Similarly, cutoff OD of 2.1 for IgG ELISA was determined in our area. Conclusion: Considering the need for a cost-effective serological method and unavailability of paired sera, these cutoffs can be used in our area and surrounding nearby areas of Eastern part of India as a reference value for reporting of scrub typhus.

Toxoplasma, Rubella and Cytomegalovirus Seroprevalence and Distribution by Age in Pregnant Women in Diyarbakir: Eight Years Experience

Objective: Toxoplasma, rubella and cytomegalovirus (CMV) infections can cause malformations in the fetus, usually in the first three months of pregnancy. In this study, we aimed to examine the distribution of Immunoglobulin M (IgM) type and immunoglobulin G (IgG) type antibodies against toxoplasma, rubella and CMV in pregnant women in XXX over an 8-year period according to age groups. Methods: Pregnant women aged 15-49 years who applied to our hospital between 2015-2022 were included in this study. IgM and IgG antibodies for toxoplasma, rubella and CMV were studied with the Triturus automatic ELISA (Grifols SA, Barcelona, Spain) system and the Dia. ProDiagnostic Bioprobes (Milan, Italy) brand kit. Relationships between age groups and antibody seropositivity were investigated with the chi-square method at the p

Myelin basic protein and TREM2 quantification in the CSF of patients with Multiple System Atrophy and other Parkinsonian conditions

BackgroundIt is well known that myelin disruption and neuroinflammation are early and distinct pathological hallmarks in multiple system atrophy (MSA) as well as in idiopathic Parkinson’s disease and in other atypical Parkinsonian syndromes. The objective of this study was to assess the value of non-neuronal biomarker candidates that reflect myelin disruption and neuroinflammation.MethodsMyelin basic protein (MBP) and the soluble form of TREM2 were quantified in a comprehensive movement disorder cohort from two different neurological centers, comprising a total of 171 CSF samples. Commercially available ELISA systems were employed for quantification.ResultsThe results of the MBP analysis revealed a significant increase in cerebrospinal fluid (CSF) MBP levels in all atypical Parkinsonian conditions compared to PD. This differentiation was more pronounced in the MSA-c subtype compared to MSA-p. Receiver operating characteristic (ROC) analysis revealed a significant discrimination between PD and MSA (p = 0.032, AUC = 0.70), PD and DLB (p = 0.006, AUC = 0.79) and PD and tauopathies (p = 0.006, AUC = 0.74). The results of the TREM2 analysis demonstrated no significant differences between the PD and atypical Parkinsonian groups if not adjusted for confounders. After adjusting for age, sex, and disease duration, the PD group exhibited significantly higher TREM2 levels compared to the DLB group (p = 0.002).ConclusionsIn conclusion, MBP, but not TREM2, is elevated in the CSF of not only MSA but in all atypical Parkinsonian conditions compared to idiopathic Parkinson’s disease. This highlights the value of the evaluation of myelin/oligodendrocyte-associated markers in neurodegenerative movement disorders.

C-reactive protein and association with disease severity in hospitalized adult patients with Lassa fever in Nigeria.

Lassa fever (LF) poses a significant health burden in West Africa. The pathophysiology of LF and determinants of clinical spectrum of disease remain poorly understood. We performed a study to understand the correlation of blood inflammatory marker C-reactive protein (CRP), with LF disease severity. A cross-sectional controlled study of adult patients with LF presenting to the Institute of Lassa Fever Research and Control at Irrua Specialist Teaching Hospital, Nigeria between January and April 2023. LF was confirmed using real-time polymerase chain reaction. Disease severity was classified as: severe disease = blood urea level >100 mg/dl and moderate disease = <100 mg/dl. CRP was measured using Tecan Infinite F50 ELISA system. Disease severity was correlated with CRP levels. A total of 64 adult patients with LF and 60 healthy controls were enrolled. There was no difference in the mean age (37.6 vs 35.2 years, P = 0.10) and gender (male 53% vs 56%, P = 0.82). CRP levels were significantly elevated in patients with LF (mean: 36.23 mg/l, SD: 4.56, %coefficient of variation [CV]: 12.59) compared with controls (mean: 5.42 mg/l, SD: 0.53, %CV: 9.78) (P = 0.000). CRP levels varied with disease severity, being significantly higher in patients with severe disease (28.57 mg/l, SD = 2.34; %CV = 8.19) than in those with moderately severe disease (12.34 mg/l, SD = 0.98; %CV = 7.94); (P = 0.001). Inpatients with LF showed significant inflammation and elevated CRP levels, which correlated with disease severity. CRP could serve as a potential baseline marker in outbreak situations to trigger management decision-making to treat or not to treat considering limited ribavirin supplies. Given the inflammatory changes and correlation of elevated CRP levels with LF disease severity, larger controlled studies during outbreaks are required to assess use of CRP for more accurate triaging, including commencement of treatment.

Comparison of Conjugates Obtained Using DMSO and DMF as Solvents in the Production of Polyclonal Antibodies and ELISA Development: A Case Study on Bisphenol A.

When developing immunochemical test systems, it is necessary to obtain specific antibodies. Their quality depends, among other things, on the immunogen used. When preparing hapten-protein conjugates to obtain antibodies for low-molecular-weight compounds, the key factors are the structure of the hapten itself, the presence of a spacer, the size of the carrier protein and the degree of its modification by hapten molecules. This work shows that one additional factor-the conditions for obtaining the hapten-protein conjugate-is overlooked. In this work, we have synthesized conjugates of bisphenol A derivative 4,4-bis(hydroxyphenyl)valeric acid (BVA), the protein carrier soybean trypsin inhibitor (STI), and bovine serum albumin (BSA) in reaction media combining water with two organic solvents: dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Namely, BSADMF-BVA, STIDMF-BVA, BSADMSO-BVA and STIDMSO-BVA conjugates were obtained. Rabbit polyclonal antibodies against the BSADMF-BVA conjugate demonstrated basically different interactions in the developed ELISA systems using either STIDMF-BVA or STIDMSO-BVA conjugates. The use of the STIDMF-BVA conjugate demonstrated the absence of competition in combination with antisera obtained from BSADMF-BVA in an ELISA. A competitive interaction was observed only with the use of the STIDMSO-BVA conjugate. Under the selected conditions, the detection limit of bisphenol A was 8.3 ng/mL, and the working range of determined concentrations was 18.5-290.3 ng/mL. The obtained data demonstrate the possibility of achieving sensitive immunoassays by simply varying the reaction media for the hapten-protein conjugation, which could provide an additional tool in the development of immunoassays for other low-molecular-weight compounds.

Human microglia polarization following infection with the Lyme disease spirochete.

Infection with Borrelia burgdorferi can spread and cause central nervous system involvement, known as neuroborreliosis. Microglia phagocytose bacteria, mediate inflammation, and elicit an immune response toward the spirochete. Like other tissue macrophages, microglia can polarize into two different modulatory phenotypes, M1 and M2. We explored human microglial polarization changes upon infection with B. burgdorferi. HMC3 human microglia cell line was infected with B. burgdorferi for 24 h. Expression of polarization markers was evaluated via flow cytometry at 4 and 24 h. Secreted immunological mediators were evaluated using a multiplex ELISA system at 4, 18, and 24 h. An early decrease followed by a later increase in expression of M1 polarization marker iNOS was observed at 4 h, and 24 h, respectively. A decrease in M2 marker CX3CR1 occurred at 24 h. There were no changes in expression of M1 markers CD14, or in M2 markers CD163 and CD206. Multiplex ELISA evidenced an increase in secretion of activation markers MIP-1α, MIP- 1β, IP-10, chemotactic factor MCP-1, M1 polarization cytokine IL-8, and VEGF, at 4, 18, and 24 h. A decrease of iNOS at 4 h of infection suggests a diminished production of reactive nitrogen species that are a critical component of innate defense against infection. Increased iNOS and simultaneously decreased expression of CX3CR1 at 24 h, may suggest initiation of neuroprotective regulation of microglia recruitment to neuroinflammation. The dynamics of major inflammatory cytokines appear to be important in the microglial response to B. burgdorferi and should be further studied as these could become therapeutic targets in neuroborreliosis.

Thyroid Cancer With Autocrine Sialyl-fibronectin Depletion Has a Poor Prognosis due to EMT Progression.

Elevated blood fibronectin (FN) levels have been observed in various cancers; however, their significance remains controversial. Herein, we measured the levels of sialyl-fibronectin (S-FN), a type of FN secreted by tumor cells, and investigated whether blood S-FN secretion is associated with recurrent metastasis and epithelial-mesenchymal transition (EMT). An ELISA system recognizing S-FN was constructed, and the amount of S-FN in blood samples from 63 patients with thyroid carcinoma was measured. The relationship between S-FN secretion and clinical prognosis was also examined. Vimentin immunostaining was performed to identify the mesenchymal status of the cells during EMT. After 12 years of observation, 17/63 patients had recurrent metastases, including nine cases of lymph node recurrence (LNR) and eight cases of remote metastasis (RM). LNR occurred in 7/39 (17.9%) of S-FN-negative cases, where 4/7 (57.1%) had two or more repeat recurrences. In S-FN-positive cases, LNR was observed in 2/24 cases (8.3%), and no repeat recurrence was observed. For RM, 6/39 (15.4%) patients were S-FN-negative, of which 5/6 (83.3%) had progressive disease even during treatment at metastasis. Of the S-FN-positive cases, RM was observed in 2/24 (8.3%) patients; progressive disease was observed in 1/2 (50.0%) patients. In 9/11 S-FN-negative recurrent metastasis cases (81.8%) and 2/4 S-FN-positive cases (50.0%), many vimentin-positive, FN-secreting cells were found in the interstitial tissue around the tumor. S-FN-negative thyroid cancer has a poor prognosis because of the progression of EMT associated with increased paracrine FN levels in the stroma.

Enhanced sensitivity in acute myocardial infarction diagnosis through optimized multiplex biomarker detection on a paper-based microfluidic platform with individual control capabilities

Enhanced sensitivity in acute myocardial infarction diagnosis through optimized multiplex biomarker detection on a paper-based microfluidic platform with individual control capabilities

Assessment of transthyretin instability in patients with wild-type transthyretin amyloid cardiomyopathy

The pathophysiology of variant transthyretin (TTR) amyloidosis (ATTRv) is associated with destabilizing mutations in the TTR tetramer. However, why TTR with a wild-type genetic sequence misfolds and aggregates in wild-type transthyretin amyloidosis (ATTRwt) is unknown. Here, we evaluate kinetic TTR stability with a newly developed ELISA system in combination with urea-induced protein denaturation. Compared with that in control patients, endogenous TTR in patients with wild-type transthyretin amyloid cardiomyopathy (ATTRwt-CM) exhibited thermodynamic instability, indicating that circulating TTR instability may be associated with the pathogenesis of ATTRwt as well as ATTRv. Our findings provide new insight into the underlying mechanisms of ATTRwt.

First Sero-Molecular Diagnosis of Toxoplasma gondii and Toxocara spp. Infections in the Police Dogs and Their Trainers in Iran.

Toxoplasma gondii (T. gondii) and Toxocara spp. are two types of parasites that can infect humans and various animals, including dogs. Police dogs and their trainers have a vital role in law enforcement, and their health and well-being are crucial for them to effectively carry out their duties. No study has yet been conducted on the prevalence of T. gondii and Toxocara spp. infections among police dogs and their trainers in Iran. The objective of this study was to determine the sero-molecular prevalence of T. gondii and Toxocara spp. infections in police dogs and their trainers in Tehran, the capital of Iran. In Tehran province, the anti-narcotics police have nearly 200 well-trained police dogs. Each dog is assigned a dedicated trainer and upon completing missions, is housed separately in a designated area. In the present study, a total of 150 samples were gathered. These included 50 blood samples from randomly selected police dogs, 50 fecal samples from the same dogs, and 50 blood samples from their trainers. The Modified Agglutination Test (MAT) was performed to detect T. gondii antibodies in dog blood samples and the ELISA system was utilized to identify anti-Toxoplasma and anti-Toxocara antibodies in the sera of the dog trainers. A specific segment of the SAG2 and ITS genes were amplified via nested-PCR in order to molecularly detect T. gondii in human blood samples and Toxocara spp. in dog fecal samples. Regarding serological findings, the prevalence of T. gondii in dog and human blood samples was 4% (2/50) and 10% (5/50), respectively. According to reports, the seroprevalence of Toxocara spp. in human blood samples was 6% (3/50). No statistically significant association was found between the prevalence of the examined parasites and variables (age, sex, and breed) in dogs, as well as the age variable in military personnel. Molecular findings showed that out of the 50 dog fecal samples and 50 human blood samples, there was no presence of Toxocara spp. and T. gondii, respectively. Understanding the prevalence of parasitic infections helps public health officials assess the risk to human and animal populations. This information can guide the development of prevention and control measures to reduce the spread of these infections. Overall, the prevalence of parasitic infections, particularly T. gondii and Toxocara spp., in police dogs and their trainers remains uncertain and necessitates further in-depth research.

Development of a novel sandwich immunoassay based on targeting recombinant Francisella outer membrane protein A for the diagnosis of tularemia.

Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.

The Association of Vitamin D Receptor Gene Polymorphism with Polycystic Ovary Syndrome in a Southeast Iranian Population

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women of reproductive age, characterized by hyperandrogenism, amenorrhea, and polycystic ovaries. Metabolic disorders, including insulin resistance (IR), are common comorbidities of PCOS, with vitamin D receptors playing a crucial role. Objectives: We investigated whether the rs2228570T &gt; C variant of the vitamin D receptor (VDR) gene is associated with the risk of PCOS. Methods: In the current study, 112 women with PCOS and 150 healthy women participated. Genomic DNA was extracted using the standard salting-out method. Genotyping of rs2228570T &gt; C was conducted via amplification-refractory mutation system polymerase chain reaction. Additionally, VD3 levels were measured using the ELISA system. Statistical analysis of genotyping data was performed using the SPSS V.22 package. Results: The statistical analysis showed that the T-allele of rs2228570C/T significantly decreased the risk of PCOS. Moreover, the T-allele in the codominant model significantly eliminated the risk of PCOS in our population. Our results also showed a statistically significant difference in VD3 levels between the CC and TT genotypes. Conclusions: Based on our result, rs228570C/T had a protective role for PCOS risk in our population.

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity.

The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

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.

IgE-Mediated Sensitization to Blo t 21 and Blo t 5 Is Associated With Asthma in the Tropics: A Case-Control Study.

Sensitization to Blomia tropicalis is associated with asthma in various tropical and subtropical countries; however, information about the specific molecular components associated with this disease is scarce. Using molecular diagnosis, we sought to identify B tropicalis allergens associated with asthma in Colombia. Specific IgE (sIgE) to 8 B tropicalis recombinant allergens (Blo t 2, 5, 7, 8, 10, 12, 13, and 21) was determined using an in-house ELISA system in asthma patients (n=272) and controls (n=298) recruited in a national prevalence study performed in several Colombian cities (Barranquilla, Bogotá, Medellín, Cali, and San Andrés). The study sample included children and adults (mean [SD] age, 28 [17] years). Cross-reactivity between Blo t 5 and Blo t 21 was evaluated using ELISA-inhibition. Specific IgE (sIgE) to 8 B tropicalis recombinant allergens (Blo t 2, 5, 7, 8, 10, 12, 13, and 21) was determined using an in-house ELISA system in asthma patients (n=272) and controls (n=298) recruited in a national prevalence study performed in several Colombian cities (Barranquilla, Bogotá, Medellín, Cali, and San Andrés). The study sample included children and adults (mean [SD] age, 28 [17] years). Cross-reactivity between Blo t 5 and Blo t 21 was evaluated using ELISA-inhibition. Although Blo t 5 and Blo t 21 are considered common sensitizers, this is the first report of their association with asthma. Both components should be included in molecular panels for diagnosis of allergy in the tropics.

Quantitation of milk proteins in thermally treated milk samples and commercial food products by ELISA test kits

This study evaluated four ELISA kits for quantitation of milk proteins in thermally treated milk samples and food products. How reference materials may be used for comparison of kit performance was examined. Protein contents determined by Veratox Total Milk generally reflected those determined by the 660nm total protein assay. BioKits BLG Kit was less affected by thermal treatment but resulted in overestimation of protein contents in samples that were boiled, autoclaved or dry-heated at ≤149°C, while ELISA Systems Casein (ES Casein) and Beta-Lactoglobulin (ES BLG) assays underestimated protein levels in these samples. The four kits gave similar results for ice cream. Veratox registered higher concentrations in all products tested but its sensitivity was greatly lowered in retorted products. ES Casein underperformed Veratox for baked and retorted products. BioKits BLG maintained a better sensitivity towards fried, baked and retorted products while ES BLG exhibited reduced sensitivity for these products.

Lysosomal degradation of PD-L1 is associated with immune-related adverse events during anti-PD-L1 immunotherapy in NSCLC patients.

Background: Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events (irAEs). Liquid biomarkers to predict irAE occurrence are urgently needed. We previously developed an ELISA system to specifically detect soluble PD-L1 (sPD-L1) with PD-1-binding capacity (bsPD-L1). Here, we investigated the relationship between sPD-L1 and bsPD-L1 in gastric cancer (GC) and non-small cell lung cancer (NSCLC) treated with PD-1/PD-L1 blockade and their association with irAEs. Methods: We examined sPD-L1, bsPD-L1, matrix metalloproteinases (MMPs), and proinflammatory cytokine levels by ELISA in plasma samples from 117 GC patients prior to surgery and 72 NSCLC patients prior to and at 2 months after ICI treatment (anti-PD-1, n = 48; anti-PD-L1, n = 24). In mice treated with anti-PD-1/PD-L1 antibodies (Abs), sPD-L1 levels and localization of Abs were examined by ELISA and immunohistochemistry, respectively. Results:sPD-L1 was detected with higher frequency in GC patients than in NSCLC patients, whereas bsPD-L1 was detected with similar frequencies in GC and NSCLC patients. sPD-L1 levels were correlated with IL-1α, IL-1β, TNF-α, and IL-6 levels, while bsPD-L1 levels were correlated with MMP13, MMP3, and IFN-γ levels. In NSCLC patients, anti-PD-L1, but not anti-PD-1, treatment increased sPD-L1, which was associated with irAE development, but not with clinical outcomes. In mice, trafficking of anti-PD-L1 Abs to lysosomes in F4/80+ macrophages resulted in sPD-L1 production, which was suppressed by treatment with lysosomal degradation inhibitor chloroquine and macrophage depletion. Conclusion: Anti-PD-L1-mediated lysosomal degradation induces sPD-L1 production, which can serve as an indicator to predict irAE development during anti-PD-L1 treatment.

Study of Correlation between Serum Vitamin B12 Level and Aberrant DNA Methylation in Infertile Males.

Altered DNA methylation pattern in sperms has been associated with infertility in males demonstrating defective spermatogenesis or low semen quality. Vitamin B-12, by affecting 1-carbon metabolism pathways, might alter the DNA methylation pattern. We aimed to study the correlation of serum vitamin B12 levels with aberrant DNA methylation in infertile male patients. A cross-sectional study was conducted on 17 oligozoospermic infertile males (WHO criteria, 2010) and 10 healthy fertile males. Serum vitamin B12 levels were estimated using the chemiluminescence method. Global methylation was determined using the ELISA system (Imprint Methylated DNA Quantification Kit, Sigma-Aldrich). The levels of global DNA methylation were calculated and compared relative to the methylated (100%) control DNA provided with the kit. Mean serum vitamin B12 concentration in the control group was higher than that of the case group. This difference in serum vitamin B12 concentration in both groups was found statistically significant. Although the results of this study show that oligozoospermic men have relatively lower global DNA methylation as compared to normozoospermic control, the values could not reach a statistically significant level. A small positive correlation was found between serum vitamin B12 levels and percent methylation defect (r = 0.14) but was statistically insignificant. Our study concludes that oligozoospermic infertile males have a significant deficiency of vitamin B12 as compared to normozoospermic fertile males. This study did not find any significant difference in global DNA methylation between the two groups. The present study does not suggest any correlation between serum vitamin B12 level and percent DNA methylation.