Search Results (11)

Background: The R21 malaria vaccine is a next-generation, WHO-prequalified vaccine that was introduced to reduce the burden of clinical malaria. In alignment with WHO recommendations, multi-dose vaccine presentations are preferred for large-scale immunization and inclusion in the Expanded Programme on Immunization (EPI). Accurate protein quantification is a critical quality control parameter for lot release, but it remains challenging when the antigen is present at low protein concentrations or formulated with complex matrices, including adjuvants, stabilizers, and preservatives. Methods: In this study, multiple protein estimation methods including Micro-BCA, BCA, and Bradford assays were evaluated to determine their suitability for quantifying the R21 antigen formulated with Matrix-M1 adjuvant and 2-PE preservative. The Bradford assay was selected as the most appropriate method, based on a comparative assessment of precision, accuracy, and linearity. Further optimization was undertaken to identify suitable buffer systems, and the method was validated in accordance with ICH Q2(R2) guidelines. Results: Validation results demonstrated that the assay is specific, accurate, precise, and repeatable, with a limit of quantitation (LOQ) of 2 µg/mL. The method demonstrated comparable performance to ELISA and was found to be sensitive enough to detect changes in antigen concentration resulting from unintended adsorption of R21 to vial surfaces. The assay offers a rapid, high-throughput, and cost-effective solution for protein quantitation in commercial manufacturing, lot release, and stability studies. The protein content of the drug product, quantified using the Bradford method, demonstrated robust in vivo immunogenicity in both release and stability studies. Conclusions: The robustness and reproducibility of the assay establish a new benchmark in quality control for virus-like particle (VLP)-based vaccines with complex formulations, thereby supporting the precision and reliability required for global malaria prevention efforts. Full article

Bisphenol A (BPA) is widely used as a hardener in plastics production and its release and circulation in ecosystems lead to negatively impacts the human endocrine system. Therefore, there is a need for simple and efficient BPA monitoring tools. This paper presents a combination of two approaches for this purpose: the use of magnetic particles (MPs) as antibody carriers and immunochromatographic test strips based on quantum dots (QDs) for recording labeled immune complexes. Initially, free antigen binds to the MP-specific antibody conjugate, concentrating the sample to a final volume of 200 µL. A competitive interaction then occurs in the analytical zone of the test strip with immersion in a QDs solution. The visual detection limit of BPA was 2.7 μg/mL, the instrumental detection limit was 0.03 μg/mL, and the working range of quantification was 0.3–100 μg/mL (reproducibility was 7.7%, R² = 0.985). Concentration using MP reduces the detection limit by 100-fold (0.3 ng/mL). The developed test was used for qualitative control of the presence and quantitative determination of BPA content in samples of drinking and natural water (the detection rate is in the range of 94–110%). Full article

Background/Objectives: An assay for protein content is essential but insufficient for quality control of acellular pertussis vaccines, which might consist of up to five components, each needing individual quantification. Generally, purified pertussis antigens such as pertussis toxin (PTx), filamentous haemagglutinin (FHA), and pertactin (PRN) should be detoxified or stabilized chemically before being formulated into vaccine bulk. The use of chemical agents like formaldehyde and glutaraldehyde can alter the immunological reactivity of these antigens, rendering direct assays by methods such as ELISA ineffective. Methods: In this study, a simple method based on single radial diffusion (SRD) using low concentrations of polyclonal antisera against PT toxoid (PTd), FHA, and PRN was developed. By adding a detergent, diffusible subunits are produced regardless of the original physical state of the antigens, making it suitable for quantifying these antigens after chemical treatment. Results: The assay has shown good specificity, accuracy, and precision. Furthermore, it can differentiate between preparations with the same protein concentration but different antigenic contents. A significant positive correlation between the antigen content and the in vivo immunogenicity has also been demonstrated. Conclusions: An assay for quality control and consistency monitoring of combined vaccines containing acellular pertussis antigen components has been established. Full article

Objective: This manuscript describes an innovative, non-destructive, high-throughput method for the quantification of aluminum hydroxide in aluminum-adjuvanted vaccines, eliminating the need of reagents and providing real-time results. The method is based on a spectrophotometric principle, and several model proteins were studied and tested with the aim to simulate the behavior of aluminum-adjuvanted antigens. Methods: As a proof of concept, the MenB vaccine was used, and the titration of aluminum hydroxide (AH) with ethylenediaminetetraacetic acid (EDTA) was used as an orthogonal reference, as it is one of the current release methods for the content determination of aluminum-hydroxide-adjuvanted vaccine drug products (DPs). The factors influencing the spectrophotometric analysis, such as different plate 96/well containers, variation in the sedimentation of the suspension due to component addition errors during formulation, and batch-to-batch variation were studied to assess the method’s robustness. Five concentration levels (ranging from 2.0 to 4.0 mg/mL AH) with two different batches of aluminum hydroxide were each measured with independent preparations performed by three different operators, for a total of four sessions/operator and 20 formulations/session. An in-depth statistical study was carried out with generated data to assess the precision (in terms of intermediate precision and repeatability), accuracy, linearity, and specificity of the method. Results: The novel spectrophotometric method and the official release one (potentiometric) yielded comparable results, demonstrating the potential of this new method as a release test for AH-adjuvanted products. A simple calibration curve enabled the measurement of samples in a 96-well plate in just a few minutes. Conclusions: We developed a novel method for Aluminum concentration determination in Aluminum-containing pharmaceutical products, like alum-adjuvanted vaccines. This method is fast, completely automatable, and as precise and accurate as already-in-place release methods. Full article

Gut bacteria release extracellular vesicles (BEVs) as an intercellular communication mechanism that primes the host innate immune system. BEVs from E. coli activate dendritic cells (DCs) and subsequent T-cell responses in a strain-specific manner. The specific immunomodulatory effects were, in part, mediated by differential regulation of miRNAs. This study aimed to deepen understanding of the mechanisms of BEVs to drive specific immune responses by analyzing their impact on DC-secreted cytokines and exosomes. DCs were challenged with BEVs from probiotic and commensal E. coli strains. The ability of DC-secreted factors to activate T-cell responses was assessed by cytokine quantification in indirect DCs/naïve CD4+ T-cells co-cultures on Transwell supports. DC-exosomes were characterized in terms of costimulatory molecules and miRNAs cargo. In the absence of direct cellular contacts, DC-secreted factors triggered secretion of effector cytokines by T-cells with the same trend as direct DC/T-cell co-cultures. The main differences between the strains influenced the production of Th1- and Treg-specific cytokines. Exosomes released by BEV-activated DCs were enriched in surface proteins involved in antigen presentation and T-cell activation, but differed in the content of immune-related miRNA, depending on the origin of the BEVs. These differences were consistent with the derived immune responses. Full article

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is controlled by vaccine policy in many countries. For vaccine potency, the content of intact virus particles (146S antigens) is critical, and the sucrose density gradient (SDG) fractionation is the gold standard for the quantification of 146S antigens. However, this method has several drawbacks. Although size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace the classic method, its application is generally confined to purified samples owing to the interfering signals. Therefore, we aimed to develop optimal pretreatment methods for SE-HPLC quantification in less purified samples. Crude virus infection supernatant (CVIS) and semi-purified samples with PEG precipitation (PEG-P) were used. Chloroform pretreatment was essential to remove a high level of non-specific signals in CVIS, whereas it caused loss of 146S antigens without the distinctive removal of non-specific signals in PEG-P. Benzonase pretreatment was required to improve the resolution of the target peak in the chromatogram for both CVIS and PEG-P. Through spiking tests with pure 146S antigens, it was verified that the combined pretreatment with chloroform and benzonase was optimal for the CVIS, while the sole pretreatment of benzonase was beneficial for PEG-P. Full article

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids. Full article

Colorectal cancer (CRC) is the third leading cause of cancer death and the fourth most common cancer in the world. Colonoscopy is the most sensitive test used for detection of CRC; however, their procedure is invasive and expensive for population mass screening. Currently, the fecal occult blood test has been widely used as a screening tool for CRC but displays low specificity. The lack of rapid and simple methods for mass screening makes the early diagnosis and therapy monitoring difficult. Extracellular vesicles (EVs) have emerged as a novel source of biomarkers due to their contents in proteins and miRNAs. Their detection would not require invasive techniques and could be considered as a liquid biopsy. Specifically, it has been demonstrated that the amount of CD147 expressed in circulating EVs is significant higher for CRC cell lines than for normal colon fibroblast cell lines. Moreover, CD147-containing EVs have been used as a biomarker to monitor response to therapy in patients with CRC. Therefore, this antigen could be used as a non-invasive biomarker for the detection and monitoring of CRC in combination with a Point-of-Care platform as, for example, Lateral Flow Immunoassays (LFIAs). Here, we propose the development of a quantitative lateral flow immunoassay test based on the use of magnetic nanoparticles as labels coupled to inductive sensor for the non-invasive detection of CRC by CD147-positive EVs. The results obtained for quantification of CD147 antigen embedded in EVs isolated from plasma sample have demonstrated that this device could be used as a Point-of-Care tool for CRC screening or therapy monitoring thanks to its rapid response and easy operation. Full article

Foot-and-mouth disease (FMD) is considered one of the highly contagious viral infections affecting livestock. In Korea, an FMD vaccination policy has been implemented nationwide since 2010 for the prevention and control of FMD. Since the vaccines are imported from various countries, standardized quality control measures are critical. In this study, we aimed to validate a high-performance liquid chromatography (HPLC) device in the Animal and Plant Quarantine Agency lab and identify an appropriate FMD vaccine pretreatment method for HPLC—a simple, reliable, and practical method to measure antigen content. Based on the analyses of specificity, linearity, accuracy, repeatability, intermediate precision, limits of detection, and limits of quantification using FMD standard samples, we validated the method using a standard material. Overall, we confirmed that the HPLC technique is effective for the quantitative assessment of the FMD virus 146S antigen in Korea. Using commercial FMD vaccines, we evaluated three separation methods and identified the method using n-pentanol and trichloroethylene as optimal for HPLC analysis. Our HPLC method was effective for the analytical detection of the antigen content in FMD vaccine, and it may be useful as a reference method for national lot-release testing. Full article

Clostridium tetani is the etiological agent of tetanus, a life-threatening bacterial infection. The most efficient protection strategy against tetanus is a vaccination with the C. tetani neurotoxin, which is inactivated by formaldehyde-crosslinking. Since we assumed that besides the tetanus toxin, other proteins of C. tetani may also be present in toxoid preparations, we analyzed commercially available vaccines from different countries in respect to their protein content using mass spectrometry. In total 991 proteins could be identified in all five analyzed vaccines, 206 proteins were common in all analyzed vaccines and 54 proteins from the 206 proteins were potential antigens. The additionally present proteins may contribute at least partially to protection against C. tetani infection by supporting the function of the vaccine against the devastating effects of the tetanus toxin indirectly. Two different label-free protein quantification methods were applied for an estimation of protein contents. Similar results were obtained with a Total Protein Approach (TPA)-based method and Protein Discoverer 2.2 software package based on the minora algorithm. Depending on the tetanus toxoid vaccine and the quantification method used, tetanus neurotoxin contributes between 14 and 76 % to the total C. tetani protein content and varying numbers of other C. tetani proteins were detected. Full article

Aflatoxins are considered unavoidable natural mycotoxins encountered in foods, animal feeds, and feed grains. In this study, we demonstrate the application of our recently developed real-time immunoquantitative PCR (RT iq-PCR) assay for sensitive detection and quantification of aflatoxins in poultry feed, two types of dairy feed (1 and 2), horse feed, whole kernel corn feed grains, and retail yellow ground corn meal. Upon testing methanol/water (60:40) extractions of the above samples using competitive direct enzyme linked immunosorbent assay, the aflatoxin content was found to be <20 μg/kg. The RT iq-PCR assay exhibited high antigen hook effect in samples containing aflatoxin levels higher than the quantification limits (0.1–10 μg/kg), addressed by comparing the quantification results of undiluted and diluted extracts. In testing the reliability of the immuno-PCR assay, samples were spiked with 200 μg/kg of aflatoxin B₁, but the recovery of spiked aflatoxin was found to be poor. Considering the significance of determining trace levels of aflatoxins and their serious implications for animal and human health, the RT iq-PCR method described in this study can be useful for quantifying low natural aflatoxin levels in complex matrices of food or animal feed samples without the requirement of extra sample cleanup. Full article