Nanozyme-assisted molecularly imprinted polymer-based indirect competitive ELISA for the detection of marine biotoxin.

Saxitoxin (STX), which is produced by certain dinoflagellate species, is a type of paralytic shellfish poisoning toxin that poses a serious threat to human health and the environment. Therefore, developing a technology for the convenient and cost-effective detection of STX is imperative. In this study, we developed an affinity peptide-imprinted polymer-based indirect competitive ELISA (ic-ELISA) without using enzyme-toxin conjugates. AuNP/Co3O4@Mg/Al cLDH was synthesized by calcining AuNP/ZIF-67@Mg/Al LDH, which was obtained by combining AuNPs, ZIF-67, and flower-like Mg/Al LDH. This synthesized nanozyme exhibited high catalytic activity (Km = 0.24 mM for TMB and 132.5 mM for H2O2). The affinity peptide-imprinted polymer (MIP) was imprinted with an STX-specific template peptide (STX MIP) on a multi-well microplate and then reacted with an STX-specific signal peptide (STX SP). The interaction between the STX SP and MIP was detected using a streptavidin-coated nanozyme (SA-AuNP/Co3O4@Mg/Al cLDH). The developed MIP-based ic-ELISA exhibited excellent selectivity and sensitivity, with a limit of detection of 3.17 ng/mL (equivalent: 0.317 µg/g). Furthermore, the system was validated using a commercial ELISA kit and mussel tissue samples, and it demonstrated a high STX recovery with a low coefficient of variation. These results imply that the developed ic-ELISA can be used to detect STX in real samples.

Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.

A dual-mode fluorometric/colorimetric sensor for sulfadimethoxine detection based on Prussian blue nanoparticles and carbon dots.

A dual-mode (colorimetric/fluorescence) nanoenzyme-linked immunosorbent assay (NLISA) was developed based on Au-Cu nanocubes generating Prussian blue nanoparticles (PBNPs). It is expected that this method can be used to detect the residues of sulfonamides in the field, and solve the problem of long analysis time and high cost of the traditional method. Sulfadimethoxine (SDM) was selected as the proof-of-concept target analyte. The Au-Cu nanocubes were linked to the aptamer by amide interaction, and the Au-Cu nanocubes, SDM and antibody were immobilized on a 96-well plate using the sandwich method. The assay generates PBNPs by oxidising the Cu shells on the Au-Cu nanocubes in the presence of hydrochloric acid, Fe3+ and K3[Fe (CN)6]. In this process, the copper shell undergoes oxidation to Cu2+ and subsequently Cu2 + further quenches the fluorescence of the carbon point. PBNPs exhibit peroxidase-like activity, oxidising 3,3',5,5'-tetramethylbenzidine (TMB) to OX-TMB in the presence of H2O2, which alters the colorimetric signal. The dual-mode signals are directly proportional to the sulfadimethoxine concentration within the range 10- 3~10- 7 mg/mL. The limit of detection (LOD) of the assay is 0.023 ng/mL and 0.071 ng/mL for the fluorescent signal and the colorimetric signal, respectively. Moreover, the assay was successfully applied to determine sulfadimethoxine in silver carp, shrimp, and lamb samples with satisfactory results.

Novel monoclonal antibodies against house dust mite allergen Der p 21 and their application to analyze allergen extracts.

Background: Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Methods: Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. Results: MAbs raised against recombinant Der p 21 were characterized in detail and proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed and optimized. The allergen was detected and its concentration was determined in only three of six analyzed HDM allergen extracts from different manufacturers. Conclusion: HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy.

Evolution of methods to detect paraneoplastic antibodies.

An adaptive immune response in less than 1% of people who develop cancer produces antibodies against neuronal proteins. These antibodies can be associated with paraneoplastic syndromes, and their accurate detection should instigate a search for a specific cancer. Over the years, multiple systems, from indirect immunofluorescence to live cell-based assays, have been developed to identify these antibodies. As the specific antigens were identified, high throughput, multi-antigen substrates such as line blots and ELISAs were developed for clinical laboratories. However, the evolution of assays required to identify antibodies to membrane targets has shone a light on the importance of antigen conformation for antibody detection. This chapter discusses the early antibody assays used to detect antibodies to nuclear and cytosolic targets and how new approaches are required to detect antibodies to membrane targets. The chapter presents recent data that support international recommendations against the sole use of line blots for antibody detection and highlights a new antigen-specific approach that appears promising for the detection of submembrane targets.

Differential detection of H1N1 virus spiker proteins by two hexaphenylbutadiene isomers based on size-matching principle.

As one of the high pathogenic influenza viruses, H1N1 virus easily induces to serious diseases, even leading to death. To date, all detection methods for H1N1 virus had shortcomings, including high equipment cost, time consumption, and etc. Therefore, a novel detection method should be established to achieve more convenient, rapid, and low-cost detection. In this work, an isomer of HPBmN-I with aggregation-induced emission characteristic was firstly synthesized on the basis of our previous reported HPBpN-I. The results showed that HPBmN-I only selectively binds to N1 in the presence of H1, while HPBpN-I can exhibit total fluorescence response to H1 and N1 in H1/N1 mixture. The limited of detection (LOD) of HPBmN-I to N1 was estimated to be 20.82 ng/mL in normal saline (NS) according to the IUPAC-based approach. The simulation calculations based on molecular docking revealed that four HPBmN-I molecules combine well with the hydrophobic cavity of N1 and achieve the fluorescence enhancement due to size matching with each other. The combination of HPBpN-I and HPBmN-I as probes was successfully used to quantitatively detect H1 and N1 in real H1N1 virus. Compared to enzyme-linked immunosorbent assay (ELISA) method, the established method not only showed the same detection accuracy but also had the advantages of real-time, ease of preparation, and low-cost, demonstrating potential market prospects.

Establishment and evaluation of detection methods for process-specific residual host cell protein and residual host cell DNA in biological preparation.

To establish accurate detection methods of process-specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific-process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process-specific enzyme-linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non-process-specific commercial ELISA kit and the process-specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process-specific HCPs, could not be detected by the non-process-specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process-specific ELISA has high sensitivity in detecting process-specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future.

Evaluating cross-reactivity of new psychoactive substances (NPS) in human whole blood by enzyme-linked immunosorbent assay (ELISA).

Due to the increase in the use of novel psychoactive substances (NPS) and their overall prevalence, it is important to have effective and reliable screening technologies to detect NPS in biological matrices. Enzyme-linked immunosorbent assays (ELISA) are among the most popular screening methods. To evaluate the effectiveness of ELISA for NPS detection, five subclasses of NPS (novel synthetic opioids, fentanyl analogs, stimulants, benzodiazepines and hallucinogens) were evaluated in whole blood for their cross-reactivity on commercially available ELISA kits. A variety of novel synthetic opioids were tested at concentrations of 1-80 ng/mL and 50-2000 ng/mL and demonstrated no cross-reactivity to a morphine ELISA plate at either concentration range. Fentanyl analogs were tested at concentrations ranging from 0.01 to 1 ng/mL and had cross-reactivities ranging from 8% to 178% on the fentanyl ELISA kit used. Both para-chloro fentanyl (178%) and acryl fentanyl (164%) showed cross-reactivities well above that of fentanyl. Novel stimulants were tested at concentrations of 0.5-40 ng/mL and 20-2,000 ng/mL. 4-Fluoroamphetamine was the only novel stimulant with cross-reactivity (3,354%) to the amphetamine ELISA plate. Novel benzodiazepines were tested at concentrations of 1-40 ng/mL on a benzodiazepine plate. Cross-reactivities ranged from 36.1% to 263%, with desalkylflurazepam having the highest cross-reactivity. Finally, novel hallucinogens were tested at concentrations of 0.5-10 ng/mL on a phencyclidine (PCP) ELISA plate, which produced no cross-reactivity and then with 10-1,000 ng/mL, which gave results from 56.6% to 151%. Both hydroxy-PCP (151%) and chloro-PCP (137%) showed cross-reactivities above that of PCP. This research has demonstrated the utility of using ELISA-based screening for novel benzodiazepines, hallucinogens and for fentanyl analogs; however, there is limited application and risk of false-negative results for the other drug classes due to low or non-existent cross-reactivities.

Quantification of Hepatitis E Virus ORF2 Protein by a Novel Sandwich ELISA.

Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1-4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin-streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2-3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.

Preparation of limonin monoclonal antibody and establishment of a sensitive icELISA for analyzing limonin in citrus and herbal samples.

Limonin is an intensely bitter and highly oxidized tetracyclic triterpenoid secondary metabolite, which is abundant in the Rutaceae and Meliaceae, especially in Citrus. In order to detect limonin content in complex substrates such as citrus and traditional Chinese medicine, monoclonal antibodies specifically recognizing limonin were prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The median inhibition concentration (IC50) was 5.40 ng/mL and the linear range was 1.25-23.84 ng/mL. The average recoveries from citrus peel and pulp samples were 95.9%-118.8% and 77.5%-113.1%, respectively. Moreover, the contents of limonin in 6 citrus samples and 4 herbal samples were analyzed by icELISA and UPLC-MS, and the results of the two methods were consistent. This validation is sufficient to demonstrate that the developed immunoassay is applicable for the detection of limonin in citrus and herbal samples and has the advantage of high efficiency, sensitivity, and convenience.

Rapid and sensitive detection of SARS-CoV-2 IgM through luciferase luminescence on an automatic platform.

SARS-CoV-2 has brought a global health crisis worldwide. IgM is an early marker in sera after the infections, and the detection of IgM is crucial to assist diagnosis and evaluate the vaccination clinically. Herein, we developed an automated platform to identify IgM against SARS-CoV-2 in sera. Streptavidin-magnetic beads were utilized to bind to a biotinylated anti-IgM antibody, which was employed to capture IgM in sera. RBD fused luciferase hGluc was employed to label the trapped IgM against RBD and the signal of luminescence of hGluc with the substrate of coelenterazine corresponded to the amount of SARS-CoV-2 IgM conjugated to the magnetic beads. An appropriate cut-off value of the designed method was defined by a set of negative samples and positive samples with 100 % sensitivity and 100 % specificity. Through serial dilution of a positive sample, it was found that the method has a better sensitivity than ELISA. The application to determine IgM against SARS-CoV-2 demonstrated a good performance of the method. The developed system can complete the analysis of SARS-CoV-2 IgM within 25 min. Through the substitution of RBD antigen with antigens of other pathogens in this platform, the automated detection of IgM against the corresponding pathogens can be realized.

Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody.

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies. The developed method exhibited a linear detection range of approximately 6-200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%-102%. These results indicate robust reproducibility of the method. To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits. In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.

Nucleic Acid and Nanomaterial Synergistic Amplification Enables Dual Targets of Ultrasensitive Fluorescence Quantification to Improve the Efficacy of Clinical Tuberculosis Diagnosis.

Interferon-γ (IFN-γ) release assays (IGRAs) are constrained by the limited diagnostic performance of a single indicator and the excessive Mycobacterium tuberculosis (Mtb) antigen stimulation time. This study presents a simultaneous, homogeneous, rapid, and ultrasensitive fluorescence quantification strategy for IFN-γ and IFN-γ-induced protein 10 (IP-10). This method relies on the high-affinity binding of aptamers to IFN-γ and IP-10, the enzyme-free catalytic hairpin assembly reaction, and the heightened sensitivity of CdTe quantum dots to Ag+ and hairpin structure C-Ag+-C and carbon dots to Hg2+ and hairpin structure T-Hg2+-T. Under optimized conditions, the selectivity of IFN-γ and IP-10 was excellent, with a linear range spanning from 1 to 100 ag/mL and low limits of detection of 0.3 and 0.5 ag/mL, respectively. Clinical practicality was confirmed through testing of 57 clinical samples. The dual-indicator combination detection showed 92.8% specificity and 93.1% sensitivity, with an area under the curve of 0.899, representing an improvement over the single-indicator approach. The Mtb antigen stimulation time was reduced to 8 h for 6/7 clinical samples. These findings underscore the potential of our approach to enhance the efficiency and performance of a tuberculosis (TB) clinical diagnosis.

Preparation and Immunochemical Characterization of a Water-Soluble Gluten Peptide Fraction for Improving the Diagnosis of Celiac Disease.

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood.

Generation of novel anti-apoE monoclonal antibodies that selectively recognize apoE isoforms.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid ß in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110-117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

On-site rapid detection of perfluorooctanoic acid by visual immunochromatographic strip biosensor in domestic water and real human samples.

The International Agency for Research on Cancer (IARC) classifies PFOA as a Class 1 carcinogen. Here, a new naked-eye PFOA immunochromographic strip was developed to recognize PFOA in domestic water and real human samples within 10 min based on a novel custom designed anti-PFOA monoclonal antibody (mAb) 2A3, which was firstly an immune rapid detection method for PFOA has been proposed. Using computer simulation techniques such as quantum computing to assist in designing the structural formula of PFOA semi antigen, which hapten was firstly proposed. The half maximal inhibitory concentration of PFOA monoclonal antibody (mAb) 2A3 was 2.4 µg/mL. Using mAb 2A3, we developed an immunochromatographic strip (ICS) for detecting PFOA in real samples. The developed method generated results in 10 min, with visual detection limits of 20, 20, and 200 µg/mL and limit of detection of 50, 200, and 500 µg/mL for water, blood and urine samples, respectively. The established ICS and indirect competitive enzyme-linked immunosorbent assay were used to analyze the actual samples, and the results were confirmed by LC-MS/MS. Our study findings showed that the ICS and ic-ELISA can quickly detect PFOA in actual samples.

The serodiagnositic value of Chlamydia trachomatis antigens in antibody detection using luciferase immunosorbent assay.

Introduction: Among the different antigens used in the detection of anti-Chlamydia trachomatis antibodies, significant differences in sensitivity and specificity have been observed. Further evaluation of C. trachomatis antigens in antibody detection is urgently needed for the development and application of C. trachomatis serologic assays. Methods: Chlamydia trachomatis antigens Pgp3, TmeA, InaC, and HSP60 were selected and used in luciferase immunosorbent assay (LISA). The detection results obtained from well-defined C. trachomatis positive and negative samples were compared with the commercial C. trachomatis ELISA (Mikrogen) for performance evaluation. Results: Pgp3, TmeA, InaC, and HSP60-based LISA showed sensitivity of 92.8, 88.8, 90.4, and 94.4%, and specificity of 99.2, 99.2, 99.2, and 92%, respectively. ROC analysis indicated that Pgp3-based LISA showed similar performance to Mikrogen ELISA (AUC 0.986 vs. 0.993, p = 0.207). Furthermore, four C. trachomatis antigens achieved strong diagnostic efficiency, i.e., positive likelihood ratios [+LR] ≥ 10 in C. trachomatis-infected women and negative likelihood ratios [-LR] ≤ 0.1 in C. trachomatis negative low exposure risk children, but only Pgp3 and TmeA showed strong diagnostic value in general adults. In addition, Pgp3, TmeA, and InaC, but not HSP60, achieved high performance, i.e., both positive predictive value (PPV) and negative predictive value (NPV) ≥ 90.9%, and showed no significant cross-reactivity with anti-Chlamydiapneumoniae. Conclusion: Three C. trachomatis species-specific antigens Pgp3, TmeA, and InaC show superior performance in the detection of anti-C. trachomatis antibody, indicating the potential to be used in developing C. trachomatis serologic tests.

[Etiological diagnostic methods and research progress of forest encephalitis].

Forest encephalitis is a natural focal disease transmitted through the bite of hard ticks, and its pathogen is the tick-borne encephalitis virus from the Flaviviridae family. The mortality rate of forest encephalitis is relatively high, making laboratory testing significant in diagnosing this disease. This article elaborates on the etiological diagnostic methods and recent research progress in forest encephalitis. Laboratory tests for forest encephalitis mainly include routine examinations, serological tests, virus isolation, and molecular biological testing. The detection of serum-specific IgM antibodies against the forest encephalitis virus is of great importance for early diagnosis, and specific IgG antibodies serve as a "gold standard" for differentiation from other diseases. Techniques such as enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay for detecting specific IgM antibodies in serum and/or cerebrospinal fluid, the serum hemagglutination inhibition test or serum complement fixation test, and the double serum hemagglutination inhibition test or complement fixation test all contribute to the early diagnosis. The development of molecular testing methods is rapid, and techniques such as metabolomics, digital PCR, and matrix metalloproteinases are also applied in the early diagnosis of forest encephalitis.

Potency, immunogenicity, and efficacy of rabies vaccine: In vitro and in vivo approach.

BACKGROUND: Rabies, a potentially lethal virus, affects more than 150 countries. Although the rabies vaccine and immunoglobulin have been available since 1908, Bangladesh is new to vaccine manufacturing. We checked the quality of the local manufacturing rabies vaccine for substandard. METHODS: The potency and immunogenicity of 20 vaccines were analyzed by three in vivo and in vitro methods from March 2020 to May 2023. Single radial immunodiffusion, fluorescent antibody virus neutralization, and national institutes of health tests were carried out to evaluate the vaccine's efficacy to provide sufficient protection against the rabies virus. RESULTS: The potency of the rabies vaccine was determined by the in vitro SRID method by measuring glycoprotein content. An average of 16 articles from each batch was calculated. The minimum and maximum average mean values of the 20 batches were 5.058 and 5.346, respectively. The variance was calculated at 0.00566. We found a coefficient of variation (CV) between 9.36% and 14.80%. The 100% sample was satisfactory, as these samples had a potency of over 2.5 IU/mL. To observe immunogenicity, we applied the FAVN method for determining antibody titers. An average of 16 articles from every batch were counted to quantify antibody titers. The mean quantity of antibody titers ranged from 2.389 to 3.3875. The CV was slightly lower because of the dispersion of the data. At last, we performed an in vivo method, the NIH test method, to determine potency based on mortality rate. We found a mean value of 4.777 IU/SHD with a standard deviation of 1.13 IU/SHD. All 20 batches were found 100% satisfactory in the NIH test. CONCLUSION: The study implies that the rabies human vaccines manufactured in Bangladesh are potent enough to provide sufficient immunogenicity. Our research is warranted testimony for healthcare providers who work to extirpate rabies.

Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus.

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.