RESUMEN
The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.
Asunto(s)
Dependovirus , Vectores Genéticos , Dependovirus/genética , Dependovirus/aislamiento & purificación , Vectores Genéticos/genética , Humanos , Cápside/química , Cápside/metabolismo , Virión/aislamiento & purificación , Virión/genética , Células HEK293 , Cromatografía de Afinidad/métodos , Ultracentrifugación/métodos , Proteínas de la Cápside/aislamiento & purificación , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismoRESUMEN
A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30-50 °C and pH range of 4.0-10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.
Asunto(s)
Cromatografía de Afinidad , Proteínas Recombinantes , Inhibidores de Tripsina , Tripsina , Tripsina/química , Tripsina/metabolismo , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Cromatografía de Afinidad/métodos , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/química , Concentración de Iones de Hidrógeno , Fagopyrum/química , Temperatura , Sefarosa/química , Estabilidad de EnzimasRESUMEN
The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases. KEY POINTS: ⢠Panel of mAbs for differentiating MTB versus NTM ⢠IC strips for diagnosing MTBC and NTM after the BACTEC MGIT ⢠Combined detection of MTP64 and Ag85B enhances diagnostic accuracy.
Asunto(s)
Anticuerpos Monoclonales , Antígenos Bacterianos , Proteínas Bacterianas , Mycobacterium tuberculosis , Micobacterias no Tuberculosas , Tuberculosis , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/genética , Anticuerpos Monoclonales/inmunología , Humanos , Micobacterias no Tuberculosas/aislamiento & purificación , Micobacterias no Tuberculosas/clasificación , Micobacterias no Tuberculosas/crecimiento & desarrollo , Antígenos Bacterianos/análisis , Antígenos Bacterianos/inmunología , Tuberculosis/diagnóstico , Tuberculosis/microbiología , Proteínas Bacterianas/genética , Cromatografía de Afinidad/métodos , Sensibilidad y Especificidad , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Aciltransferasas , Anticuerpos Antibacterianos/inmunologíaRESUMEN
Analysis of amatoxins is of great importance as these cyclic peptides contribute to a high number of fatalities each year. Development of analytical approaches needs to focus on rapid, sensitive, and reliable methods. By establishing an affinity column chromatography-based assay using the monoclonal amanitin antibody AMA9G3 and liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) for the trace detection of α-, ß-, and γ-amanitin in human urine samples to confirm ingestion, we report the first approach that extents the current status of amatoxin analysis. The presented procedure allows detection of amatoxins in human urine down to 1 ng/mL. The method was successfully validated qualitatively for α- and γ-amanitin according to international recommendations. A proof of concept was performed by analyzing 37 urine samples after suspected amatoxin consumption submitted for regular clinical toxicological analysis. Using this antibody-based enrichment strategy, acute amatoxin intoxications can be determined within 90 min and due to the high sensitivity and selectivity, a comparable approach using target specific antibodies may also be used for other toxicological relevant peptides.
Asunto(s)
Amanitinas , Cromatografía de Afinidad , Espectrometría de Masas en Tándem , Humanos , Amanitinas/orina , Cromatografía de Afinidad/métodos , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodosRESUMEN
Excellent pretreatments before instrumental analysis are critical for separation and determination of target compounds for discovery of new drugs from herb medicines. We developed a rapid and highly-selective method to separate the bioactive compounds from herbal extract using protein affinity-selection spin column, which was packed with the new sorbent materials from integrating the recombinant ß2-adrenoceptor (ß2-AR) directly out of cell lysates onto the surface of microspheres. Protein affinity-selection spin column was placed in a centrifugal tube, where after the non-specific binders were released to the filtrate under the operational centrifugation, the specific binders on the spin column were cleaned with a washing solvent for LC-MS analysis. The known agonists of ß2-AR were retained/released on protein affinity-selection spin column but not on control column, demonstrating the method with good recovery (79.4â¼95.7 %) and high repeatability (RSD < 3.5 %). The adsorption features of three ligands on the spin column were described best by Prism saturation binding model, and the high-affinity binding and the large binding capacity of the spin column make it feasible to trap the trace analytes effectively. It was applied in separating bioactive compounds from Alstoniae Scholaris extract, two of which were identified as picrinine and oleanolic acid in combination with LC-MS and verified as the potential agonists towards ß2-AR though molecular docking and cell experiments. Our study demonstrated that, the spin column with the immobilized protein sorbents in the centrifugal filter device represents a promising tool, enabling rapid and target-specific affinity separation of the bioactive compounds from herbal extract.
Asunto(s)
Proteínas Inmovilizadas , Microesferas , Extractos Vegetales , Extractos Vegetales/química , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Cromatografía de Afinidad/métodos , Cromatografía Liquida/métodos , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Adsorción , Humanos , Espectrometría de Masas/métodos , Medicamentos Herbarios Chinos/química , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 1014 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy.
Asunto(s)
Cromatografía de Afinidad , Dependovirus , Péptidos , Serogrupo , Dependovirus/aislamiento & purificación , Dependovirus/genética , Dependovirus/química , Cromatografía de Afinidad/métodos , Péptidos/química , Péptidos/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Vectores Genéticos , Células HEK293RESUMEN
Canine Visceral Leishmaniasis (CVL) is the most fatal form of Leishmania infection in dogs and is caused by L. infantum in the Americas. This parasite follows a zoonotic life cycle, raising concerns within domestic households, where dogs act as the primary reservoir of the parasite. Accurately detecting infected dogs is vital for effective epidemiological control in both canine and human populations. However, existing diagnostic methods in Brazil have limitations, particularly in detecting asymptomatic and oligosymptomatic dogs, leading to ineffective disease control. To address this challenge, we evaluated a novel recombinant antigen from L. infantum, the rLiNTPDase2. Previous studies have confirmed its high performance via ELISA, leading us to assess its suitability for a Lateral Flow Immunochromatographic Assay (LFIA), which is ideal for point-of-care testing. Standardization of the assay involved testing two nitrocellulose membranes (HF135 and HF120, Millipore), three blocking protocols, and five sample dilutions (1:10, 1:20, 1:40, 1:80, and 1:160). Following the chosen conditions (HF120 membrane, 1-minute blocking protocol, and 1:80 sample dilution), we validated our assay with a sample size of 78 dogs, comprising 32 negatives and 46 positives, including symptomatic (n=23), oligosymptomatic (n=17), and asymptomatic (n=6) cases. The results revealed a sensitivity of 86.9â¯%, specificity of 62.5â¯%, and accuracy of 76.9â¯%, which is consistent with ELISA performance for the same samples. Compared to DPP-LVC, our assay demonstrated promising results in detecting asymptomatic and oligosymptomatic cases. This study underscores the suitability of the rLiNTPDase2 antigen for the LFIA format, suggesting its potential as a novel point-of-care diagnostic test for CVL.
Asunto(s)
Antígenos de Protozoos , Enfermedades de los Perros , Leishmaniasis Visceral , Sensibilidad y Especificidad , Animales , Perros , Leishmaniasis Visceral/veterinaria , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/parasitología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/análisis , Cromatografía de Afinidad/veterinaria , Cromatografía de Afinidad/métodos , Leishmania infantum/enzimología , Leishmania infantum/inmunología , Proteínas Recombinantes/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodosRESUMEN
BACKGROUND: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies. METHODS: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen. RESULTS: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation. CONCLUSIONS: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.
Asunto(s)
Virus de la Fiebre Hemorrágica de Crimea-Congo , Proteínas Recombinantes , Humanos , Virus de la Fiebre Hemorrágica de Crimea-Congo/inmunología , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Células HEK293 , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificación , Fiebre Hemorrágica de Crimea/diagnóstico , Fiebre Hemorrágica de Crimea/virología , Ensayo de Inmunoadsorción Enzimática , Animales , Cromatografía de Afinidad/métodos , Cromatografía en Gel , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangreRESUMEN
OBJECTIVE: Cryptococcosis predominantly presents as a meningoencephalitis in Thailand. Early and expeditious diagnosis is essential for reducing both mortality and morbidity associated with cryptococcal meningitis. We aim to define and establish the diagnostic performances between the benchmark commercially available diagnostic kit (CrAg® LFA) and the large-scale prototype of an inexpensive in-house immunochromatographic test (ICT) based on monoclonal antibody (MAb) 18B7. METHODS: We have developed the large-scale prototype for the rapid detection of cryptococcal polysaccharide antigens by utilizing a single antibody sandwich ICT format employing MAb 18B7, which is highly specific to Cryptococcus neoformans glucuronoxylomannan (GXM) antigens. An in-house MAb18B7 ICT was manufactured in accordance with industry standards under the control of the International Organization for Standardization (ISO) 13485. RESULTS: The diagnostic sensitivity, specificity, and accuracy for the in-house MAb 18B7 ICT were 99.10%, 97.61%, and 97.83%, respectively. The agreement kappa (κ) coefficient was 0.968 based on the retrospective evaluation of 580 specimens from patients living in northern Thailand with clinically suspected cryptococcosis. CONCLUSION: The data suggest that this in-house MAb 18B7 ICT will be highly beneficial for addressing the issue of cryptococcal infection in Thailand. Moreover, it is anticipated that this inexpensive ICT can play a pivotal role in various global strategies aimed at eradicating cryptococcal meningitis among individuals living with HIV by 2030.
Asunto(s)
Anticuerpos Monoclonales , Antígenos Fúngicos , Cromatografía de Afinidad , Criptococosis , Cryptococcus neoformans , Sensibilidad y Especificidad , Humanos , Tailandia , Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad/métodos , Criptococosis/diagnóstico , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/aislamiento & purificación , Antígenos Fúngicos/análisis , Antígenos Fúngicos/inmunología , Estudios Retrospectivos , Anticuerpos Antifúngicos/sangre , Polisacáridos/análisis , Polisacáridos/inmunología , Masculino , Femenino , Adulto , Pruebas Diagnósticas de Rutina/métodos , Persona de Mediana Edad , Anciano , Adulto JovenRESUMEN
Porphyromonas gingivalis is the most pathogenic periodontal bacterium in the world. Recently, P. gingivalis has been considered responsible for dysbiosis during the development of periodontitis. This study aimed to evaluate a novel immunochromatographic device using monoclonal antibodies against P. gingivalis in subgingival plaques. A total of 72 patients with chronic periodontitis and 53 periodontally healthy volunteers underwent clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis and compared using real-time polymerase chain reaction (PCR). In the periodontitis group, a significant positive correlation was observed between the test device scores and the real-time PCR results. The specificity, positive predictive value, negative predictive value, and accuracy of the test device for P. gingivalis, as determined by real-time PCR, were 98%, 94%, 89%, and 90%, respectively. There were significant differences in bacterial counts by real-time PCR among the groups with different ranges of device scores. Additionally, there was a significant positive correlation between the device scores for P. gingivalis and periodontal parameters. These results suggest that this novel immunochromatographic device can be effectively used for rapid detection and semi-quantification of P. gingivalis in subgingival plaques.
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Cromatografía de Afinidad , Porphyromonas gingivalis , Humanos , Porphyromonas gingivalis/aislamiento & purificación , Porphyromonas gingivalis/inmunología , Masculino , Femenino , Persona de Mediana Edad , Adulto , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades Periodontales/microbiología , Enfermedades Periodontales/diagnóstico , Placa Dental/microbiología , Periodontitis Crónica/microbiología , Periodontitis Crónica/diagnóstico , Sensibilidad y EspecificidadRESUMEN
Bio-separation is a crucial process in biotechnology and biochemical engineering for separating biological macromolecules, and the field has long relied on bead-based and expanded bed chromatography. Printed monolith adsorption (PMA) is a new alternative to which uses a 3D-printed monolithic structure containing self-supporting, ordered flow channels. PMA allows for direct purification of biological molecules from crude cell lysates and cell cultures, and like the other technologies, can functionalized to specifically target a molecule and enable affinity chromatography. Here we have combined PMA technology with an immobilized metal affinity ligand (iminodiacetic acid) to provide selectivity of binding to polyhistidine-tagged proteins during PMA chromatography. Two different PMA structures were created and tested for both static and dynamic protein-binding capacity. At comparative linear flow rates, the dynamic binding capacity of both columns was ≈3 mg/mL, while static capacity was shown to differentiate based on column voidage. We show that a polyhistidine-tagged protein can be directly purified from crude lysate with comparable results to the available commercial providers of IMAC, and with a substantially reduced purification time.
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Cromatografía de Afinidad , Histidina , Histidina/química , Cromatografía de Afinidad/métodos , Adsorción , Iminoácidos/química , Proteínas/aislamiento & purificación , Proteínas/química , Impresión Tridimensional , Unión ProteicaRESUMEN
This study investigates the impact of 2-methyl imidazolium dihydrogen phosphate (2-MIDHP) on monoclonal antibody (mAb) aggregation during the Protein A purification stage, at a low pH (pH 3.0), and the viral inactivation phase. Size-exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) were used to assess the mAb aggregation. Additionally, the influence of 2-MIDHP on mAb recovery, host cell protein (HCP) clearance, and Protein A leaching was investigated. Thermal stability of mAb, eluted in buffers containing 5 % to 25 % 2-MIDHP was analysed, using differential scanning calorimetry (DSC). Structural insights were obtained via circular dichroism (CD) and fluorescence spectroscopy. Our findings indicated that 2-MIDHP exerted a concentration-dependent protective effect against mAb aggregation, at the pH of 3.0. As the concentration of 2-MIDHP was increased from 0 % to 25 %, the aggregation was significantly reduced from 3.8 ± 0.01 % to 0.56 ± 0.002 %, as analysed by SE-HPLC. Addition of 2-MIDHP did not significantly impact the mAb recovery, HCP clearance, or Protein A leaching. DSC data supported these results, with higher 2-MIDHP concentrations leading to increased melting temperatures of mAb. CD and fluorescence spectroscopy revealed no significant changes in the secondary structure or aromatic residue environment in 2-MIDHP-treated samples, despite the observed reduction in aggregation. The results suggested that 2-MIDHP mitigated mAb aggregation during Protein A purification, possibly by stabilizing the protein structure under acidic stress conditions. These findings offer valuable insights for improving the robustness of mAb purification processes, enhancing product quality and yield.
Asunto(s)
Anticuerpos Monoclonales , Imidazoles , Estabilidad Proteica , Proteína Estafilocócica A , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Imidazoles/química , Proteína Estafilocócica A/química , Animales , Cromatografía Líquida de Alta Presión/métodos , Concentración de Iones de Hidrógeno , Células CHO , Cricetulus , Dicroismo Circular , Cromatografía en Gel/métodos , Cromatografía de Afinidad/métodos , Rastreo Diferencial de Calorimetría , Espectrometría de Fluorescencia , Agregado de ProteínasRESUMEN
Background: Detecting monoclonal protein (M-protein), a hallmark of plasma cell disorders, traditionally relies on methods such as protein electrophoresis, immune-electrophoresis, and immunofixation electrophoresis (IFE). Mass spectrometry (MS)-based methods, such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-quadrupole time-of-flight (ESI-qTOF) MS, have emerged as sensitive methods. We explored the M-protein-detection efficacies of different MS techniques. Methods: To isolate immunoglobulin and light chain proteins, six types of beads (IgG, IgA, IgM, kappa, lambda, and mixed kappa and lambda) were used to prepare samples along with CaptureSelect nanobody affinity beads (NBs). After purification, both MALDI-TOF MS and liquid chromatography coupled with Synapt G2 ESI-qTOF high-resolution MS analysis were performed. We purified 25 normal and 25 abnormal IFE samples using NBs and MALDI-TOF MS (NB-MALDI-TOF). Results: Abnormal samples showed monoclonal peaks, whereas normal samples showed polyclonal peaks. The IgG and mixed kappa and lambda beads showed monoclonal peaks following the use of daratumumab (an IgG/kappa type of monoclonal antibody) with both MALDI-TOF and ESI-qTOF MS analysis. The limits of detection for MALDI-TOF MS and ESI-qTOF MS were established as 0.1 g/dL and 0.025 g/dL, respectively. NB-MALDI-TOF and IFE exhibited comparable sensitivity and specificity (92% and 92%, respectively). Conclusions: NBs for M-protein detection, particularly with mixed kappa-lambda beads, identified monoclonal peaks with both MALDI-TOF and ESI-qTOF analyses. Qualitative analysis using MALDI-TOF yielded results comparable with that of IFE. NB-MALDI-TOF might be used as an alternative method to replace conventional tests (such as IFE) to detect M-protein with high sensitivity.
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Cadenas kappa de Inmunoglobulina , Cadenas lambda de Inmunoglobulina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Humanos , Espectrometría de Masa por Ionización de Electrospray , Proteínas de Mieloma/análisis , Inmunoglobulina G , Cromatografía de Afinidad/métodos , Cromatografía Liquida , MicroesferasRESUMEN
Immunoglobulin G (IgG) purification is a critical process for evaluating its role in autoimmune diseases, which are defined by the occurrence of autoantibodies. Affinity chromatography with protein G is widely considered to be the optimal technique for laboratory-scale purification. However, this technique has some limitations, including the exposure of IgG to low pH, which can compromise the quality of the purified IgG. Here, we show that alternative methods for IgG purification are possible while maintaining the quality of IgG. Different techniques for IgG purification from serum were evaluated and compared with protein G-based approaches: Melon Gel, caprylic acid-ammonium sulfate (CAAS) precipitation, anion-exchange chromatography with diethylamino ethyl (DEAE) following ammonium sulfate (AS) precipitation, and AS precipitation alone. The results demonstrated that the purification yield of these techniques surpassed that of protein G. However, differences in the purity of IgG were observed using GeLC-MS/MS. The avidity of purified IgG against selected targets (SARS-CoV-2 and topoisomerase-I) was similar between purified IgG obtained using all techniques and unpurified sera. Our work provides valuable insights for future studies of IgG function by recommending alternative purification methods that offer advantages in terms of yield, time efficiency, cost-effectiveness, and milder pH conditions than protein G.
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Sulfato de Amonio , Cromatografía de Afinidad , Inmunoglobulina G , Humanos , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Cromatografía de Afinidad/métodos , Sulfato de Amonio/química , Cromatografía por Intercambio Iónico/métodos , Espectrometría de Masas en Tándem/métodos , SARS-CoV-2/inmunología , Caprilatos/química , Precipitación Química , COVID-19/sangre , COVID-19/inmunología , COVID-19/virología , Afinidad de AnticuerposRESUMEN
The development of a rapid, sensitive, and accurate screening method for staphylococcal enterotoxin B (SEB) in food is urgently needed because trace amounts of SEB can pose a serious threat to human health. Here, we developed a ultrasensitive triple-modal immunochromatographic assay (ICA) for SEB detection. The AuNFs@Ir nanoflowers exhibited enhanced colorimetric, photothermal, and catalytic performance by modulating the sharp branching structure of the gold nanoflowers and depositing high-density Ir atoms. Subsequently, the combination of AuNFs@Ir and ICA promoted colorimetric, catalytic amplified colorimetric, and photothermal-assisted quantitative detection. The results showed detection limits of 0.175, 0.0188, and 0.043 ng mL-1 in the colorimetric/photothermal/catalytic mode, which increased the sensitivity by 16.5-fold, 153.7-fold, and 67.2-fold, respectively, compared with the AuNPs-ICA. Furthermore, the proposed strategy was verified in milk, milk powder, pork, and beef successfully. This strategy improves significantly the sensitivity, accuracy, flexibility and offers an effective insight for foodborne bacterial toxin monitoring.
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Cromatografía de Afinidad , Colorimetría , Enterotoxinas , Contaminación de Alimentos , Oro , Leche , Enterotoxinas/análisis , Oro/química , Animales , Leche/química , Contaminación de Alimentos/análisis , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/instrumentación , Bovinos , Límite de Detección , Nanopartículas del Metal/química , Porcinos , CatálisisRESUMEN
The desire to reach ever-diminishing lower limits of quantification (LLOQ) to probe changes in low abundance protein targets has led to enormous progress in sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrumentation. To maximize signal and reduce noise, many approaches have been employed, including specific immunoaffinity (IA) enrichment and reducing the LC flow to the nanoflow (nLC) level; however, additional sensitivity gains may still be required. Recently, a technique termed "echo summing" has been described for small-molecular-weight analytes on a triple quadrupole (QqQ) MS where multiple iterations of the same, single selected reaction monitoring (SRM) transition are collected, summed, and integrated, yielding significant analyte dependent signal-to-noise (S/N) improvements. Herein, the direct applicability of echo summing to protein quantification by sequential IA combined with nLC-MS/MS (IA-nLC-MS/MS) is described for a beta nerve growth factor (NGF) and a soluble asialoglycoprotein receptor (sASGPR) assay from human serum. Five iterations of echo summing outperformed traditional collection in relative average accuracy (-1.5 ± 7.7 vs -41.7 ± 10.7% bias) and precision (7.8 vs 18.4% coefficient of variation (CV)) of the low-end quality control (QC) sample (N = 4) for NGF and improved functional sensitivity of serially diluted serum QC samples (N = 5 each population) approximately 2-fold (1.96 and 2.00-fold) for two peptides of sASGPR. Echo summing also extended the minimum quantifiable QC level for sASGPR 4-fold lower. Similar gains are believed to be achievable for most protein IA-nLC-MS/MS assays.
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Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Cromatografía de Afinidad/métodos , Factor de Crecimiento Nervioso/análisis , Cromatografía Liquida/métodos , Límite de DetecciónRESUMEN
ASFV is the only known double-stranded insect-borne DNA virus, which can rapidly infect domestic pigs and wild boars with ticks as transmission medium. Since it was first discovered in 1921, it quickly spread to all parts of the world and brought huge economic losses to the pig industry all over the world. At present, there is still no safe and effective vaccine for ASFV. Here, we developed a quantum-dot labeled antibody test strip for the detection of antibodies against ASFV pp62. The pp62 protein was labeled with quantum dots, and the antibody test strip was developed uses it in a detection mode of labeled antigen-SPA interceptor-monoclonal antibody quality control. The test strip showed high sensitivity, the positive detection limit of the strip was 1: 106 by continuous multiple dilution using the positive standard serum of ASFV antibody as reference. The test strip showed good specificity, and there was no cross reaction with other swine diseases virus (PCV2, PRRSV, CSFV, PPV). Using the detection results of commercialized kit for African swine fever virus as reference, 80 ASFV antibody negative serum and 4 different ASFV antibody positive serum were detected using the ASFV pp62 quantum-dot labeled antibody test strip. The results were consistent with the commercial kit. This study provides a new detection method for the prevention and control of African swine fever.
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Virus de la Fiebre Porcina Africana , Anticuerpos Antivirales , Puntos Cuánticos , Puntos Cuánticos/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Animales , Virus de la Fiebre Porcina Africana/inmunología , Porcinos , Cromatografía de Afinidad/métodos , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/inmunología , Tiras Reactivas , Anticuerpos Monoclonales/inmunologíaRESUMEN
A colloidal gold immunochromatographic assay (CGIA) based on single-chain variable fragments (scFvs) has been successfully developed for the detection of monensin (MON). Colloidal gold probes were conjugated to anti-MON scFvs through electrostatic interaction, with the conjugated objects serving as the visual signals. The detection lines were formed by capturing the antibody with MON-OVA. This assay offers a rapid detection time of 15 min, a wide linear range from 2.19 to 10.76 ng mL-1, and boasts high accuracy, precision, and an absence of cross-reactivity. By homology modeling and molecular docking, we predicted the interaction patterns between the scFv and monensin, and the amino acid residues involved in the recognition of MON by the antibody were analyzed. These key amino acid sites are presumed integral to ligand recognition per current interaction models. This hypothesis was confirmed by computer-aided alanine scanning mutation, MM/P(G)BSA molecular dynamics simulation, and in vitro binding experiments. In this study, we successfully developed the scFvs-based CGIA system for rapid and easy quantification of monensin, providing a simple, efficient routine detection of chicken muscle samples.
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Pollos , Simulación del Acoplamiento Molecular , Monensina , Músculos , Anticuerpos de Cadena Única , Animales , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Músculos/química , Músculos/metabolismo , Simulación de Dinámica Molecular , Oro Coloide/química , Cromatografía de Afinidad/métodosRESUMEN
Large scale manufacture of complex biotherapeutic formats such as multi-specific antibodies can require development of custom biomanufacturing platforms, particularly for purification processes. Substantial advances in affinity chromatography media have allowed monoclonal antibody-like processes for these formats, and simplified process development to enable fast speed to the clinic. Thorough assessment of chromatography media performance and stability is critical to ensure robust operation and consistent product quality over repeated cycles throughout its lifetime. However, evaluation of repeated cycling and extended storage for chromatography media is resource consuming, which typically delays rigorous study to later development stages and often is acquired through increased operational experience. These areas can present quality risks if not properly understood. In this work, a class of affinity chromatography media employing camelid antibody-fragment ligands were evaluated for extended storage in benzyl alcohol solution as an alternative to ethanol storage. Initial laboratory studies revealed resin discoloration after 12 months of exposure to ambient light at room temperature. Resin photo-stress studies confirmed light exposure as the root cause, with benzyl alcohol storage conditions producing a substantially greater degree of discoloration compared to ethanol. Extreme photo-stress over the course of 7 days was also found to negatively impact resin dynamic binding capacities, with more severe declines observed with benzyl alcohol storage conditions. Binding capacity loss of 54 % was observed for photo-stressed CaptureSelect Kappa XP compared to control conditions. Addition of antioxidants reduced or eliminated resin discoloration during photo-stress, indicating that benzyl alcohol storage accelerates photo-oxidation of the affinity chromatography ligands. The addition of l-methionine to benzyl alcohol solution prevented resin discoloration and maintained a dynamic binding capacity of 41 g/L for CaptureSelect Kappa XP even after extreme photo-stress. Of practical importance, a study of LambdaFabSelect resin used and stored in benzyl alcohol solution, under recommended conditions (2-8 °C storage, protected from light) in an internal GMP facility over a period of three years, showed no impact to resin color, performance, or product quality.
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Anticuerpos Monoclonales , Alcohol Bencilo , Cromatografía de Afinidad , Oxidación-Reducción , Cromatografía de Afinidad/métodos , Alcohol Bencilo/química , Anticuerpos Monoclonales/química , LuzRESUMEN
Magnetic nanoparticles are widely employed as signal labeling reporters in immunochromatographic test strips (ICTS) for detecting foodborne pathogens due to their outstanding anti-interference and magnetic enrichment performance. However, the insufficient colorimetric signal brightness of magnetic nanoparticles results in poor sensitivity, hindering their ability to meet the growing demand for advanced ICTS. Herein, we synthesized Fe3O4@CuS core-shell structure nanoparticles using a facile in-situ growth method. These Fe3O4@CuS nanoparticles exhibit a superior photothermal conversion efficiency of 42.12 % and a magnetization strength of 35 emu/g. We developed a dual-readout format ICTS based on Fe3O4@CuS, incorporating both colorimetric and photothermal formats to enhance sensitivity for Salmonella typhimurium detection. The limit of detection for Fe3O4@CuS-ICTS in the colorimetric and photothermal format was 5 × 104 CFU/mL and 7.7 × 10³ CFU/mL, respectively. Additionally, the average recoveries ranged from 91.25 % to 103.39 %, with variations from 2.2 % to 11.1 %, demonstrating good accuracy and precision. Therefore, this work suggests that Fe3O4@CuS nanoparticles, with their superior magnetic, optical, and photothermal properties, can serve as promising signal labeling reporters to improve the detection performance of ICTS and hold potential for constructing more accurate and sensitive point-of-care testing platforms.