Step-by-step full factorial design to optimize a quantitative sandwich ELISA.

In this work, a quantitative sandwich ELISA was optimized, through a full factorial design of experiments (DOE) in successive steps of a preliminary protocol obtained by the method of one factor at a time (OFAT). The specificity of the optimized ELISA, the lower limit of quantification, the quantification range and the analytical sensitivity of the antigen quantification curve were evaluated, in comparison with the curve obtained from the preliminary protocol. The full factorial DOE was linked to a simple statistical processing, which facilitates the interpretation of the results in those laboratories where there is no trained statistician. The step-by-step optimization of the ELISA and the successive incorporation into the protocol of the best combination of factors and levels, allowed obtaining a specific immunoassay, with an analytical sensitivity 20 times greater and with a lower limit of antigen quantification that decreased from 156.25 at 9.766 ng/mL. As far as we know, there are no reports of optimization of an ELISA following the step-by-step scheme used in this work. The optimized ELISA will be used for the quantification of the TT-P0 protein, the active principle of a vaccine candidate against sea lice.

Monoclonal antibody generated against Nile tilapia (Oreochromis niloticus) IgT heavy chain using a peptide-based strategy.

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

SARS-CoV-2 spike RBD-specific IgA and IgG antibodies in breast milk after vaccination with the protein subunit vaccine Abdala.

Background: COVID-19 vaccines that trigger a strong secretory antibody response in breast milk may achieve effective passive protection of vulnerable newborns and breastfed infants of immunized mothers. The aim of this work was to investigate the presence of SARS-CoV-2 spike RBD-specific IgA and IgG antibodies in breast milk, 5 and 9 weeks after vaccination with 3 doses of the protein subunit vaccine Abdala, compared to those found in breast milk from COVID-19-recovered women, collected at least 40 days after the infection. Methods: SARS-CoV-2 spike RBD-specific IgA and IgG antibodies were semi-quantified by indirect ELISA, using a homemade standard generated by pooling twenty breast milk samples with high absorbance values according to preliminary data. The validity of the standard curves was proved following the European Medicines Agency Guideline. Two breast milk samples from 2 unvaccinated women who had not been infected with COVID-19 were included as negative controls. Potentially neutralizing antibodies was assessed by a SARS-CoV-2 surrogate virus neutralization test. Results: High levels of anti-RBD IgA antibodies were detected in breast milk samples 9 weeks after vaccination and anti-RBD IgG antibodies rise from the fifth to the ninth week. In the post-COVID-19 time that was evaluated, the IgG-type response was notably higher compared to both post-vaccination periods. Neutralizing antibody titers were similar in breast milk from vaccinated and COVID-19 recovered women. Conclusions: This is the first report about the immune response in breast milk after the administration of a COVID-19 protein subunit vaccine, which could provide analogous protection to that conferred by SARS-CoV-2 infection. This implies a potential passive immunity that breastfed infants receive from their mothers vaccinated with Abdala.

Murine monoclonal antibodies against RBD of the SARS-CoV-2 spike protein as useful analytical tools for subunit vaccine development and clinical trials.

COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.

Assessment three constitutive promoters for GUS expression in rice (Oryza sativa L., var. J-104) / Evaluación de tres promotores constitutivos para la expresión GUS en arroz (Oryza sativa L., cv. J-104)

This work analyzed the constitutive expression of the ß-Glucuronidase (GUS) reporter gene fused to three promoters: the cauliflower mosaic virus (CaMV) 35S, the chimerical A9 promoter which contains rice Act1, and the Ubiquitine-1 promoter from maize. The activity of the promoters was qualitative and quantitatively obtained in different tissues and various growth stages of rice plants (cv J-104) transformed by biolistic. All the promoters were found to be active, with distinct patterns of relative activity in leaves, stem and roots from in vitro and ex vitro plants, and in plants of T1 progeny. The chimerical A9 promoter increased significantly levels of GUS expression in all the tissues and at all growth stages of the plants.

Se analizó la expresión constitutiva del gen reportero de la ß-Glucuronidasa (GUS) fusionado a tres promotores: el 35S del virus del mosaico de la coliflor (CaMV), el promotor quimérico A9 que contiene la actina-1 de arroz y el promotor ubiquitina-1 de maíz. La actividad de los promotores fue analizada cualitativa y cuantitativamente en diferentes tejidos y estadíos de crecimiento de plantas de arroz (variedad J-104) transformadas mediante biobalística. Se demostró la expresión constitutiva de GUS bajo los promotores estudiados, con distintos patrones de actividad relativa en hojas, tallos y raíces de plantas in vitro y ex vitro, y en plantas de la progenie T1. Bajo el promotor quimérico A9 se lograron los mayores niveles de expresión GUS en todos los tejidos y fases de crecimiento de las plantas.

Effective B-lactam antibiotics for Agrobacterium tumefaciens suppression in indica rice calli / Antibióticos beta-lactámicos efectivos para eliminar el Agrobacterium tumefaciens en callos de arroz índica

It was evaluated the antibacterial activity of seven β-lactam antibiotics against A. tumefaciens strain EHA105, living in indica rice calli. For detection of persistent Agrobacterium in callus tissues, homogenates of infected calli were spread over LB agar plates to count colony forming units per gram of calli. Agrobacterium growth was completely suppressed during plant regeneration at 250 mg/l of all of the antibiotics tested. Also it was appraised the effect of β-lactams on callus growth and plant regeneration. A similar tendency of increased calli fresh weight with 100 mg/l and 250 mg/l of all antibiotics was proven. But the use of 500 mg/l caused decreasing of callus growth. About 80% of calli formed shoots in a month when we used 100 mg/l or 250 mg/l of β-lactam during the regeneration stage. Two morphogenic responses were distinguished during regeneration stage: somatic embryogenesis and adventitious shoots organogenesis.

Se evaluó la actividad antibacteriana de siete antibióticos β-lactámicos sobre la cepa EHA105 de Agrobacterium tumefaciens inoculada en callos de arroz. Para detectar el Agrobacterium persistente en los callos, se cultivó en medio LB sólido un extracto de callos infectados, y se contó el número de unidades formadoras de colonias por gramo de callo. La bacteria se eliminó totalmente en la fase de regeneración utilizando 250 mg/l de cualquiera de los antibióticos probados. Se evaluó además el efecto de los β-lactámicos sobre el crecimiento de los callos y la regeneración de plantas. El incremento del peso de los callos fue similar cuando se utilizó 100 y 250 mg/l de antibiótico, pero disminuyó significativamente cuando la concentración se elevó a 500 mg/l. La regeneración de plantas ocurrió a partir del 80% de los callos cultivados con 100 y 250 mg/l de antibiótico. En la regeneración se distinguieron dos respuestas morfogénicas: embriogénesis somática y organogénesis adventicia.

Quantitative transient GUS expression in J-104 rice calli through manipulation of in vitro culture conditions

This paper purposes suitable conditions for callus induction and co-cultivation with Agrobacterium tumefaciens of J-104 rice cultivar. It was evaluated the effect of different concentrations of 2.4-D and agar, and the inclusion of L-proline and L-glutamine in callus culture medium. The use of 2.5 mg/L 2.4-D and 0.8% agar allowed the highest percentage of embryogenic calli. Callus formation was improved considerably with 500 mg/L of L-proline and L-glutamine in the culture medium. Different factors were studied throughout co-cultivation of calli with A. tumefaciens: inoculation time, co-cultivation temperature, concentration of acetosyringone and co-cultivation period. Transient GUS expression was quantified by fluorometry in all co-cultivated calli. The best results were obtained with the following conditions: 10 min as inoculation time, 100µM acetosyringone in co-cultivation medium, temperature of 20ºC, and 3 days as co-cultivation period.

Se describen las condiciones óptimas para la callogénesis y cocultivo de callos con Agrobacterium tume-faciens de la variedad de arroz J-104. Se determinó el efecto de diferentes concentraciones de 2.4-D, agar y de L-prolina y L-glutamina en el medio de cultivo de callos. El uso de 2,5 mg/L de 2.4-D y 0,8% de agar permitió lograr el porcentaje más alto de callos embriogénicos. La formación de callos fue mejorada considerablemente con la adición de 500 mg/L de L-prolina e igual concentración de L-glutamina en el medio de cultivo. Se estudiaron diferentes factores en el cocultivo de los callos con A. tumefaciens: tiempo de inoculación, concentración de acetosiringona, temperatura y tiempo de cocultivo. Para comparar el efecto de cada factor sobre la expresión GUS se cuantificó la actividad transitoria mediante fluorimetría. Los valores más altos de actividad fluorimétrica fueron obtenidos con las siguientes condiciones: 10 min de inoculación, 100µM de acetosiringona en el medio de cocultivo y 3 días de cocultivo a 20 ºC.

Evaluación morfológica de brotes regenerados de callos de arroz (variedad IACuba-28) resistentes a higromicina / Morphological evaluation of shoots regenerated from hygromycin-resistant rice callus (cv IACuba-28)

Se estableció un sistema de evaluación de brotes de arroz regenerados de callos resistentes a higromicina, que permitió relacionar las características morfológicas de dichos brotes con su resistencia al antibiótico. Callos embriogénicos de arroz se transformaron con Agrobacterium tumefaciens (EHA105/ pCAMBIA1300),con el gen de la higromicina-fosfotransferasa como marcador de selección. Después de dos semanas en medio de cultivo con higromicina, los callos resistentes fueron transferidos a regeneración. Durante 30 días se realizaron extracciones sucesivas de brotes emitidos de los callos, en seis intervalos de cinco días cada uno. Estos brotes se clasificaron según su morfología en: clase I – brote vigoroso con una estructura bipolar típica, de ápice y raíz con longitudes proporcionales; clase II – brote con raíz pequeña respecto al ápice, o sin raíz; clase III – brote con alteraciones fenotípicas, albinismo, hojas muy anchas o enrolladas. Los brotes clasificados se transfirieron a medio MS con higromicina para evaluar su viabilidad. Los declase I, que predominaron en las primeras extracciones, presentaron la mayor viabilidad durante el enraizamiento y crecimiento del follaje. En las últimas dos extracciones ocurrió una drástica reducción de los brotes clase I, y aumentaron los de clases II y III, simultáneamente disminuyó la viabilidad de estos últimos en MS con higromicina. Este resultado puede aplicarse para mejorar la eficiencia de obtención de plantas transgénicas de arroz en estas condiciones, debido a que precisa el momento óptimo para lograr brotes con características morfológicas normales y que sean resistentes a higromicina.