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Veterinary World Apr 2024Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires...
BACKGROUND AND AIM
Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis.
MATERIALS AND METHODS
rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting.
RESULTS
PCR was able to amplify the LipL32 gene from rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic . SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic serovar but not with or .
CONCLUSION
IgG anti-rLipL32 antibody has high specificity and sensitivity against pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis.
PubMed: 38798296
DOI: 10.14202/vetworld.2024.871-879 -
Archives of Pathology & Laboratory... May 2024Leptomeningeal disease (LMD) is a clinical sequela of central nervous system metastasis involving the cerebrospinal fluid (CSF), often seen in late-stage solid tumors....
CONTEXT.—
Leptomeningeal disease (LMD) is a clinical sequela of central nervous system metastasis involving the cerebrospinal fluid (CSF), often seen in late-stage solid tumors. It has a grave prognosis without urgent treatment. Standard of care methodologies to diagnose LMD include CSF cytology, magnetic resonance imaging, and clinical evaluation. These methods offer limited sensitivity and specificity for the evaluation of LMD. Here, we describe the analytic performance characteristics of a microfluidic-based tumor cell enrichment and detection assay optimized to detect epithelial cells in CSF using both contrived samples as well as CSF from patients having suspected or confirmed LMD from carcinomas.
OBJECTIVE.—
To demonstrate the feasibility of using a microfluidic, multi-antibody cell capture assay to identify and quantify tumor cells in CSF.
DESIGN.—
An artificial CSF solution was spiked with 34 different human carcinoma cell lines at different concentrations and assayed for the ability to detect tumor cells to assess analytic accuracy. Two cell lines were selected to assess linearity, intra-assay precision, interinstrument precision, and sample stability. Clinical verification was performed on 65 CSF specimens from patients. Parameters assessed included the number of tumor cells, coefficient of variation percentage, and percentage of tumor cell capture (TCC).
RESULTS.—
Among contrived samples, average tumor cell capture ranged from 50% to 82% (261 of 522; 436 of 531), and coefficients of variation ranged from 7% to 67%. The cell capture assay demonstrated a sensitivity of 92% and a specificity of 95% among clinical samples.
CONCLUSIONS.—
This assay demonstrated the ability to detect and enumerate epithelial cells in contrived and clinical specimens in an accurate and reproducible fashion. The use of cell capture assays in CSF may be useful as a sensitive test for the diagnosis and longitudinal monitoring of LMD from solid tumors.
PubMed: 38797516
DOI: 10.5858/arpa.2023-0295-OA -
Food and Chemical Toxicology : An... Jul 2024Infant formulas based on hydrolysed cow's milk proteins are used when breastfeeding is not feasible in cow's milk allergic infants. Camel milk has been shown to be...
Infant formulas based on hydrolysed cow's milk proteins are used when breastfeeding is not feasible in cow's milk allergic infants. Camel milk has been shown to be well-tolerated by the majority of children with cow's milk allergy (CMA) and may be a substitute in management of CMA. Here we aimed to evaluate the impact of processing on immunogenicity, sensitising, antibody-binding and cross-reactive capacity of cow's and camel milk. Cow's and camel milk were processed by means of enzyme hydrolysis or heat treatment. Brown Norway rats were immunised with PBS, non-processed, enzyme hydrolysed or heat-treated cow's or camel milk. In vivo tests were performed for evaluation of clinical signs. Blood and faecal samples were analysed for levels and specificity of antibody responses. Cow's and camel milk showed similar sensitising capacity. Processing decreased the sensitising capacity of cow's milk, yet only enzyme hydrolysis but not heat treatment decreased the sensitising capacity of camel milk. Processing affected the specificity of antibodies raised in the rats, though the effect differed between cow's and camel milk. The study showed a low cross-reactivity between cow's and camel milk, which was decreased with processing, suggesting that processing of camel milk may improve its usefulness in CMA management.
Topics: Animals; Camelus; Milk Hypersensitivity; Rats; Cross Reactions; Cattle; Milk; Milk Proteins; Female; Rats, Inbred BN; Food Handling; Male
PubMed: 38796088
DOI: 10.1016/j.fct.2024.114761 -
Pharmaceutics Apr 2024Monoclonal antibodies are commonly engineered with an introduction of Met428Leu and Asn434Ser, known as the LS mutation, in the fragment crystallizable region to improve...
Impact of LS Mutation on Pharmacokinetics of Preventive HIV Broadly Neutralizing Monoclonal Antibodies: A Cross-Protocol Analysis of 16 Clinical Trials in People without HIV.
Monoclonal antibodies are commonly engineered with an introduction of Met428Leu and Asn434Ser, known as the LS mutation, in the fragment crystallizable region to improve pharmacokinetic profiles. The LS mutation delays antibody clearance by enhancing binding affinity to the neonatal fragment crystallizable receptor found on endothelial cells. To characterize the LS mutation for monoclonal antibodies targeting HIV, we compared pharmacokinetic parameters between parental versus LS variants for five pairs of anti-HIV immunoglobin G1 monoclonal antibodies (VRC01/LS/VRC07-523LS, 3BNC117/LS, PGDM1400/LS PGT121/LS, 10-1074/LS), analyzing data from 16 clinical trials of 583 participants without HIV. We described serum concentrations of these monoclonal antibodies following intravenous or subcutaneous administration by an open two-compartment disposition, with first-order elimination from the central compartment using non-linear mixed effects pharmacokinetic models. We compared estimated pharmacokinetic parameters using the targeted maximum likelihood estimation method, accounting for participant differences. We observed lower clearance rate, central volume, and peripheral volume of distribution for all LS variants compared to parental monoclonal antibodies. LS monoclonal antibodies showed several improvements in pharmacokinetic parameters, including increases in the elimination half-life by 2.7- to 4.1-fold, the dose-normalized area-under-the-curve by 4.1- to 9.5-fold, and the predicted concentration at 4 weeks post-administration by 3.4- to 7.6-fold. Results suggest a favorable pharmacokinetic profile of LS variants regardless of HIV epitope specificity. Insights support lower dosages and/or less frequent dosing of LS variants to achieve similar levels of antibody exposure in future clinical applications.
PubMed: 38794258
DOI: 10.3390/pharmaceutics16050594 -
Vaccines May 2024The presence of the anti-SARS-CoV-2-RBD antibody (anti-RBD) prevents severe COVID-19. We aimed to determine the accuracy of a point-of-care anti-RBD testing implemented...
Accuracy of Anti-SARS-CoV-2 Antibody in Comparison with Surrogate Viral Neutralization Test in Persons Living with HIV, Systemic Lupus Erythematosus, and Chronic Kidney Disease.
The presence of the anti-SARS-CoV-2-RBD antibody (anti-RBD) prevents severe COVID-19. We aimed to determine the accuracy of a point-of-care anti-RBD testing implemented in persons living with HIV (PLWH), systemic lupus erythematosus (SLE), and chronic kidney disease (CKD). We enrolled 182 non-comorbid subjects and 335 comorbid subjects (PLWH, SLE, CKD) to test the anti-RBD assay compared to the surrogate viral neutralization test (sVNT) as the reference test. We performed linear correlation analysis between anti-RBD and sVNT, along with an ROC analysis to ascertain the anti-RBD cutoff at 30%, 60%, and 90% inhibition of sVNT, to calculate accuracy. The correlations between anti-RBD and sVNT among all groups were excellent, with R = 0.7903, R = 0.7843, and R = 0.8153 among the non-comorbid, SLE, and CKD groups, respectively, and with significantly higher correlation among the PLWH group (R = 0.8877; -value = 0.0072) compared to the non-comorbid group. The accuracy of the anti-RBD test among the PLWH and CKD groups was similar to that among the non-comorbid group but showed lower sensitivity in the SLE group ( = 0.000014). The specificity of the test remained high in all groups. In conclusion, the anti-RBD test had excellent correlation with the sVNT. The persistently high specificity in all groups suggests that this test can be reliably utilized to detect the presence of low neutralization capacity, prompting additional vaccination.
PubMed: 38793809
DOI: 10.3390/vaccines12050558 -
Vaccines Apr 2024Public antibody responses have been found against many infectious agents. Structural convergence of public antibodies is usually determined by immunoglobulin V genes.... (Review)
Review
Public antibody responses have been found against many infectious agents. Structural convergence of public antibodies is usually determined by immunoglobulin V genes. Recently, a human antibody public class against SARS-CoV-2 was reported, where the D gene (IGHD3-22) encodes a common YYDxxG motif in heavy-chain complementarity-determining region 3 (CDR H3), which determines specificity for the receptor-binding domain (RBD). In this review, we discuss the isolation, structural characterization, and genetic analyses of this class of antibodies, which have been isolated from various cohorts of COVID-19 convalescents and vaccinees. All eleven YYDxxG antibodies with available structures target the SARS-CoV-2 RBD in a similar binding mode, where the CDR H3 dominates the interaction with antigen. The antibodies target a conserved site on the RBD that does not overlap with the receptor-binding site, but their particular angle of approach results in direct steric hindrance to receptor binding, which enables both neutralization potency and breadth. We also review the properties of CDR H3-dominant antibodies that target other human viruses. Overall, unlike most public antibodies, which are identified by their V gene usage, this newly discovered public class of YYDxxG antibodies is dominated by a D-gene-encoded motif and uncovers further opportunities for germline-targeting vaccine design.
PubMed: 38793718
DOI: 10.3390/vaccines12050467 -
Viruses May 2024The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is...
The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.
Topics: Hendra Virus; Animals; Nipah Virus; Antibodies, Monoclonal; Enzyme-Linked Immunosorbent Assay; Ephrin-B2; Henipavirus Infections; Antibodies, Viral; Swine; Humans; Sensitivity and Specificity; Receptors, Virus; Antigens, Viral
PubMed: 38793674
DOI: 10.3390/v16050794 -
Viruses May 2024The West Nile Virus (WNV), a member of the family , is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the... (Comparative Study)
Comparative Study
The West Nile Virus (WNV), a member of the family , is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.
Topics: West Nile virus; Antibodies, Viral; Humans; West Nile Fever; Sensitivity and Specificity; Enzyme-Linked Immunosorbent Assay; Serologic Tests; Immunoglobulin G; Fluorescent Antibody Technique, Indirect; Cross Reactions; Animals
PubMed: 38793670
DOI: 10.3390/v16050788 -
Molecules (Basel, Switzerland) May 2024Aflatoxins (AFs) including AFB, AFB, AFG and AFG are widely found in agriculture products, and AFB is considered one of the most toxic and harmful mycotoxins. Herein, a...
Aflatoxins (AFs) including AFB, AFB, AFG and AFG are widely found in agriculture products, and AFB is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB in agricultural and aquiculture products was developed. The AFB derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB was produced by hybridoma technology, and the mAb-based ELISA for AFB was established. IC and limit of detection (LOD) of the ELISA for AFB were 90 pg mL and 18 pg mL, respectively. The cross-reactivities (CRs) of the assay with AFB, AFG, and AFG 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 AFB were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB in spiked samples were 78.3-116.6% and 1.49-13.21% ( = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB, AFB, AFG, AFG) 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 (AFB, AFB, AFG, AFG) in wheat flour samples.
Topics: Enzyme-Linked Immunosorbent Assay; Animals; Antibodies, Monoclonal; Aflatoxin B1; Mice; Food Contamination; Limit of Detection; Zea mays; Flour; Agriculture; Serum Albumin, Bovine
PubMed: 38792140
DOI: 10.3390/molecules29102280 -
Experimental and Molecular Pathology Jun 2024Little information is available concerning protein expression of the free fatty acid receptor 2 (FFAR2), especially in tumours. Therefore, the aim of the present study...
OBJECTIVE
Little information is available concerning protein expression of the free fatty acid receptor 2 (FFAR2), especially in tumours. Therefore, the aim of the present study was to comprehensively characterise the expression profile of FFAR2 in a large series of human normal and neoplastic tissues using immunohistochemistry thus providing a basis for further in-depth investigations into its potential diagnostic or therapeutic importance.
METHODS
We developed a novel rabbit polyclonal anti-FFAR2 antibody, 0524, directed against the C-terminal region of human FFAR2. Antibody specificity was confirmed via Western blot analyses and immunocytochemistry using the FFAR2-expressing cell line BON-1 and FFAR2-specific small interfering RNA as well as native and FFAR2-transfected HEK-293 cells. The antibody was then used for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic human tissues.
RESULTS
In normal tissues, FFAR2 was mainly present in distinct cell populations of the cerebral cortex, follicular cells and C cells of the thyroid, cardiomyocytes of the heart, bronchial epithelia and glands, hepatocytes and bile duct epithelia of the liver, gall bladder epithelium, exocrine and β-cells of the endocrine pancreas, glomerular mesangial cells and podocytes as well as collecting ducts of the kidney, intestinal mucosa (particularly enteroendocrine cells), prostate epithelium, seminiferous tubules of the testicles, and placental syncytiotrophoblasts. In neoplastic tissues, FFAR2 was particularly prevalent in papillary thyroid carcinomas, parathyroid adenomas, and gastric, colon, pancreatic, hepatocellular, cholangiocellular, urinary bladder, breast, cervical, and ovarian carcinomas.
CONCLUSIONS
We generated and characterised a novel rabbit polyclonal anti-human FFAR2 antibody that is well-suited for visualising FFAR2 expression in human routine pathology tissues. This antibody is also suitable for Western blot and immunocytochemistry experiments. To our knowledge, this antibody enabled the first broad FFAR2 protein expression profile in various normal and neoplastic human tissues.
Topics: Humans; Neoplasms; HEK293 Cells; Animals; Rabbits; Immunohistochemistry; Receptors, G-Protein-Coupled; Female; Male
PubMed: 38788249
DOI: 10.1016/j.yexmp.2024.104902