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.

Validation of Two-Tiered Enzyme-Linked Immunosorbent Assay for the Detection of Anti-HeberProt-P® Antibodies

Heberprot-P® is a therapeutic product approved in Cuba for the treatment of diabetic foot ulcer. In spite of its ample clinical use, its immunogenicity in patients has not been evaluated. Regulatory agencies have developed guidelines for the validation of immunoassays designed at the detection of anti-drug antibodies to biologicals. The aim on this work was to validate a method for the detection of binding antibodies in human serum to recombinant human epider-mal growth factor (rhEGF), the active ingredient of Heberprot-P®. A two-tiered enzyme-linked immunosorbent assay (ELISA) format was used. First tier was a screening assay, based on im-mobilized rhEGF and anti-human polyvalent alkaline-phosphatase conjugated as development reagent. Positive samples were then tested with a confirmatory assay which used a competitive soluble rhEGF in solution. As FDA and EMA guidelines recommend, minimum required dilution, quality controls, screening and confirmatory cut points, sensitivity, recovery, precision, speci-ficity, selectivity, robustness and stability of samples were determined. The assay was precise and its sensitivity was calculated to be 214.2 ng/mL. The majority of evaluated parameters fulfilled the figures recommended by current guidelines. The immunoassays described in this work could be reliable tools for the evaluation of immunogenicity of Heberprot-P® in well-de-signed clinical studies (AU)