Does tetanus vaccination contribute to reduced severity of the COVID-19 infection?

We present the hypothesis to the scientific community actively designing clinical trials and recommending public health guidelines to control the pandemic that - "Tetanus vaccination may be contributing to reduced severity of the COVID-19 infection" - and urge further research to validate or invalidate the effectiveness of the tetanus toxoid vaccine against COVID-19. This hypothesis was revealed by an explainable artificial intelligence system unleashed on open public biomedical datasets. As a foundation for scientific rigor, we describe the data and the artificial intelligence system, document the provenance and methodology used to derive the hypothesis and also gather potentially relevant data/evidence from recent studies. We conclude that while correlations may not be reason for causation, correlations from multiple sources is more than a serendipitous coincidence that is worthy of further and deeper investigation.

Tetanus vaccine-induced human neutralizing antibodies provide full protection against neurotoxin challenge in mice.

Clostridium tetani causes life-threatening disease by producing tetanus neurotoxin (TeNT), one of the most toxic protein substances. Toxicosis can be prevented and cured by administration of anti-TeNT neutralizing antibodies. Here, we identified a series of monoclonal antibodies (mAbs) derived from memory B cells of a healthy adult immunized with the C-terminal domain of TeNT (TeNT-Hc). Thirteen mAbs bound to both tetanus toxoid (TT) and TeNT-Hc, while two mAbs recognized only TT. VH3-23 was the most frequently used germline gene in these TT-binding mAbs, and the pairwise identity values of the VH gene sequences ranged from 27% to 69%. Three of these mAbs-T3, T7, and T9-6-completely protected mice from challenge with 2× LD50 of TeNT, and two (T2 and T18) significantly prolonged the survival time. The five neutralizing mAbs recognized distinct epitopes on TT, with binding affinities ranging from 0.123 to 11.9 nM. Our study provides promising therapeutic candidates for tetanus.

SARS - CoV-2: Reasons of epidemiology of severe ill disease cases and therapeutic approach using trivalent vaccine (tetanus, diphtheria and Bordetella pertussis).

The novel coronavirus Covid-19 follows transmission route and clinical presentation of all community-acquired coronaviruses. Instead, the rate of transmission is significative higher, with a faster spread of the virus responsible of the worldwide outbreak and a significative higher mortality rate due to the development of a severe lung injury. Most noteworthy is the distribution of death rate among age groups. Children and younger people are almost protected from severe clinical presentation. Possible explanation of this phenomenon could be the ability of past vaccinations (especially tetanic, diphtheria toxoids and inactivated bacteria as pertussis) to stimulate immune system and to generate a scattered immunity against non-self antigens in transit, as coronaviruses and other community-circulating viruses and make immune system readier to develop specific immunity against Covid-19. The first support to this hypothesis is the distribution of mortality rate during historical pandemics ("Spanish flu" 1918, "Asian flu" 1956 and "the Hong Kong flu" 1968) among age groups before and after the introduction of vaccines. The immunological support to the hypothesis derives from recent studies about immunotherapy for malignancies, which propose the use of oncolytic vaccines combined with toxoids in order to exploit CD4 + memory T cell recall in supporting the ongoing anti-tumour response. According to this hypothesis vaccine formulations (tetanus, diphtheria, Bordetella pertussis) could be re-administrate after the first contact with Covid-19, better before the development of respiratory severe illness and of course before full-blown ARDS (Acute Respiratory Distress Syndrome). The CD4 + memory exploiting could help immune system to recall immunity of already know antigens against coronaviruses, avoiding or limiting "lung crash" until virus specific immunity develops and making it faster and prolonged. Finally, this administration could be helpful not only in already infected patients, but also before infection. In fact, people could have an immune system more ready when the contact with the Covid-19 will occur.

History, extensive characterization and challenge of anti-tetanus serum from World War I: exciting remnants and deceived hopes : Centenarian IgGs lost their neutralization capacity.

During World War I (WWI), infectious diseases including tetanus were among the most important causes of death. Even though its efficacy was somewhat controversial before the war, tetanus antiserum played a key role in reducing the mortality of this disease. A vial of tetanus antiserum dating back from WWI, left behind on the French battlefield by the US Army, was borrowed from a private collection and opened. The serum contained within was characterized by orthogonal biochemical techniques to determine if any neutralizing IgGs could remain after 100 years of storage. In vitro analysis by Size Exclusion Chromatography and Serum Protein Electrophoresis suggested the presence of residual IgG. In spite of our hopes, these IgGs were not able to protect mice against tetanus toxin challenge in a neutralizing assay. Even though our results indicate the presence of remaining IgGs inside the serum, they were functionally disabled. These results show that obscurity alone is insufficient to protect IgGs from degradation over very long periods of time at room temperature. HIGHLIGHTS: Tetanus antiserum found its place in the therapeutic arsenal during World War I A century-old vial of tetanus antiserum was opened for biochemical and in vivo characterization Biochemical assays revealed the presence of proteins having all the characteristics of IgGs The serum was unable to protect mice against toxinic challenge.

Tetanus Immunoglobulin G Assessment in Adults Trauma Patients.

BACKGROUND: Clostridium tetani is an anaerobic, gram-positive bacillus that causes tetanus infection. It usually enters the body through injury with contaminated objects. Tetanus differs from other diseases that can be prevented by vaccination in that it is not contagious and does not spread from person to person. The aim of this study is to evaluate the levels of Tetanus IgG in trauma patients admitted to the emergency department (ED). METHODS: The study was planned as cross-sectional, prospective, and single-center. The study was conducted from January to July 2018 in the Kahramanmaras Sütçü Imam University Hospital. Totally, 178 patients aged ≥18 years were included. For measurement of the level of Tetanus IgG, Clostridium tetani toxin 5S IgG kit (NovaLisa, NOVATEC) was used to quantitatively detect IgG type antibodies by micro-ELISA method in accordance with the manufacturer's recommendation. RESULTS: In total, 143 cases were male and 35 were female. The mean age of the cases was 40 ± 16 years. Tetanus IgG levels were found to be 0.29 ± 0.6 IU/mL in cases from rural areas and 2.14 ± 1.64 IU/mL in cases from urban areas (P < 0.001). There was a negative correlation between age and Tetanus IgG level (r: (-) 0.479; P < 0.001). The protective level of Tetanus IgG was observed to be even lower, especially in patients aged ≥40 years (n = 43, 78.9%). CONCLUSION: Measurements of Tetanus IgG levels should be performed as far as possible in the ED. In this way, unnecessary vaccination can be avoided.

A fluorescent nanosphere-based immunochromatography test strip for ultrasensitive and point-of-care detection of tetanus antibody in human serum.

Tetanus still possesses a high infection risk and leads a number of human deaths in poor nations. Point-of-care and ultrasensitive detection of tetanus antibody levels in serum is the key to decrease the risk of tetanus infection and improve the health of people. In this work, by using ultra bright fluorescent nanospheres (FNs) and portable lateral flow test strip (LFTS), a point-of-care and ultrasensitive sensing method has been developed for the detection of tetanus antibodies in human serum. This assay works quite well for tetanus antibodies in the concentration range from 0.0002 to 0.0220 IU/mL with a low detection limit of 0.00011 IU/mL, which is 100-fold lower than conventional gold-based LFTSs. The high sensitivity makes this method suitable for use to detect the low-abundant target in real samples. Besides, this cost-effective FN-based LFTS assay possesses good selectivity, high accuracy, and satisfactory reliability, which holds great potential as a robust candidate for routine medical diagnosis and rapid home testing. Graphical abstract.

Proteomics of Bordetella pertussis whole-cell and acellular vaccines.

OBJECTIVES: Bordetella pertussis is the etiological agent of whooping cough, a bacterial infection of especially children, which may be fatal without treatment. In frame of studies to investigate putative effects of vaccination on host-pathogen interaction and clonal distribution of strains, in addition to Corynebacterium diphtheriae and Clostridium tetani toxoid vaccines, also whole-cell and acellular pertussis vaccines were analyzed by mass spectrometry. DATA DESCRIPTION: LC-MS/MS spectra were generated and analyzed using B. pertussis genome data and proteins present in whole-cell and acellular pertussis vaccines were identified. Subcellular localization of proteins and presence of signal peptides was determined bioinformatically.

Safety and immunogenicity of tetanus/diphtheria vaccination in patients with rheumatic diseases-a prospective multi-centre cohort study.

OBJECTIVES: We aimed to assess the safety and immunogenicity of a diphtheria/tetanus vaccine booster dose in three different patient groups with rheumatic diseases on a variety of immunosuppressive/immunomodulatory medications compared with healthy controls (HCs). METHODS: We conducted a multi-centre prospective cohort study in Switzerland. We enrolled patients with RA, axial SpA/PsA, vasculitis (Behçet's disease, ANCA-associated vasculitis) and HCs. Diphtheria/tetanus vaccination was administered according to the Swiss vaccination recommendations. Blood samples were drawn before vaccination, and 1 month and 3 months afterwards. Antibody concentrations against vaccine antigens were measured by ELISA. Immunogenicity was compared between patient and medication groups. A mixed model was applied for multivariate analysis. Missing data were dealt with using multiple imputation. RESULTS: Between January 2014 and December 2015, we enrolled 284 patients with rheumatic diseases (131 RA, 114 SpA/PsA, 39 vasculitis) and 253 HCs. Of the patients, 89% were on immunosuppressive/immunomodulatory medication. Three months post-vaccination 100% of HCs vs 98% of patients were protected against tetanus and 84% vs 73% against diphtheria. HCs and SpA/PsA patients had significantly higher responses than RA and vasculitis patients. Assessing underlying diseases and medications in a multivariate model, rituximab was the only factor negatively influencing tetanus immunogenicity, whereas only MTX treatment had a negative influence on diphtheria antibody responses. No vaccine-related serious adverse events were recorded. CONCLUSION: Diphtheria/tetanus booster vaccination was safe. Tetanus vaccination was immunogenic; the diphtheria component was less immunogenic. Vaccine responses were blunted by rituximab and MTX. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov, Identifier: NCT01947465.

Skewed B cell receptor repertoire and reduced antibody avidity in patients with DOCK8 deficiency.

DOCK8 immunodeficiency syndrome (DIDS) is a combined immunodeficiency characterized by recurrent viral infections, severe atopy and early onset malignancy. Immunological abnormalities include lymphopenia, CD8+ T-cell cytoskeleton dysfunction, defective B cell memory and variable serum immunoglobulin levels. Here, we analyse the B cell receptor repertoire (BCR) characteristics and antibody avidity of four DIDS patients, attempt to understand the dysregulated humoral immunity in DIDS patients with a normal antibody titre and suggest a scientific basis for intravenous immunoglobulin (IVIG) replacement therapy for these patients. We analysed BCR characteristics, including somatic hypermutation (SHM) frequency, using deep sequencing of multiplex PCR products derived from BCR heavy chain CDR3 regions from DIDS patients and controls. The antibody avidity of human tetanus and hemophilus influenza B antibodies was determined by ELISA using thiocyanate elution. IVIG replacement treatment and infection conditions were investigated retrospectively. We found skewing of the BCR repertoire and decreased antibody avidity in patients with DIDS. DIDS patients had fewer negatively charged amino acids than healthy controls. The SHM frequency of the IGHV3 gene was lower in patients with DIDS. Patients received regular IVIG therapy, resulting in fewer and less severe infections. We conclude that although IgG levels are normal in most DIDS patients, IVIG replacement therapy is still necessary.

Validation of monoplex assays detecting antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19 for incorporation into Multiplex Serology.

Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual's infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Multiplex Serology is a suspension bead array-based high-throughput methodology for simultaneous measurement of antibodies against multiple pathogens in a single reaction vessel, thus economizing sample volume, measurement time, and costs. We developed and validated bead-based pathogen-specific Monoplex Serology assays, i.e. assays including only antigens for the respective pathogen, to detect antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19. The developed assays expand the portfolio of existing pathogen-specific bead-based serology assays and can be efficiently incorporated into larger Multiplex Serology panels. The newly developed Monoplex Serology assays consist of only one antigen per infectious agent, expressed as Glutathione S-transferase-fusion proteins in E. coli. Specificity, sensitivity and Cohen's kappa statistics in comparison with routine clinical diagnostic assays were calculated for serum dilutions 1:100 and 1:1000. All pathogen-specific assays were successfully validated at both serum dilutions with the exception of rubella Monoplex Serology which showed impaired sensitivity (57.6%) at dilution 1:1000. Specificities of successfully validated Monoplex Serology assays ranged from 85.6% to 100.0% (median: 91.7%), and sensitivities from 81.3% to 95.8% (median: 90.9%); agreement with the reference assays ranged from substantial to almost perfect (kappa: 0.66-0.86, median: 0.78). Statistical performance and slim assay design enable efficient incorporation of the developed assays into Multiplex Serology.

Serum anti-tetanus and measles antibody titres in Ugandan children aged 4 months to 6 years: implications for vaccine programme.

To study the antibody response to tetanus toxoid and measles by age following vaccination in children aged 4 months to 6 years in Entebbe, Uganda. Serum samples were obtained from 113 children aged 4-15 months, at the Mother-Child Health Clinic (MCHC), Entebbe Hospital and from 203 of the 206 children aged between 12 and 75 months recruited through the Outpatients Department (OPD). Antibodies to measles were quantified by plaque reduction neutralisation test (PRNT) and with Siemens IgG EIA. VaccZyme IgG EIA was used to quantify anti-tetanus antibodies. Sera from 96 of 113 (85.0%) children attending the MCHC contained Measles PRNT titres below the protective level (120 mIU/ml). Sera from 24 of 203 (11.8%) children attending the OPD contained PRNT titres 0.15 IU/ml by EIA, a level considered protective. The overall concentration of anti-tetanus antibody was sixfold higher in children under 12 months compared with the older children, with geometric mean concentrations of 3.15 IU/ml and 0.49 IU/ml, respectively. For each doubling in age between 4 and 64 months, the anti-tetanus antibody concentration declined by 50%. As time since the administration of the third DTP vaccination doubled, anti-tetanus antibody concentration declined by 39%. The low measles antibody prevalence in the children presenting at the MCHC is consistent with the current measles epidemiology in Uganda, where a significant number of measles cases occur in children under 1 year of age and earlier vaccination may be indicated. The consistent fall in anti-tetanus antibody titre over time following vaccination supports the need for further vaccine boosters at age 4-5 years as recommended by the WHO.

A lateral flow assay for the determination of human tetanus antibody in whole blood by using gold nanoparticle labeled tetanus antigen.

The authors describe a lateral flow assay (LFA) for the antibody against the infectious bacterium Clostridium tetani. Gold nanoparticles (AuNPs) were linked to tetanus antigen and are captured in the test line via the formation of a sandwich structure composed of AuNP-labeled tetanus antigen, tetanus antibody, and tetanus antigen. This leads to the formation of a characteristic red line due to the accumulation of AuNPs. The formation of the color line allows for a highly sensitive and selective detection of tetanus antibody, both with bare eyes and by smartphone-based quantitative analysis. This assay offers a wide detection range from 0 to 0.5 IU·mL-1 and has a linear relationship from 0.01 to 0.1 IU·mL-1 with an experimental detection limit of 0.01 IU·mL-1. This assay is simple, fast, inexpensive and highly selective. When applied to the detection of tetanus antibody in spiked whole blood, it provided reliable results that compared well to those obtained with a commercial ELISA kit. Graphical abstract ᅟ.

A randomized, open-label clinical trial to evaluate immunogenicity and safety of an indigenously developed DTwP-Hib tetravalent combination vaccine (Easyfour®-TT) with Quadrovax® in Indian infants.

An open-label, randomized, multi-center trial was conducted to compare the immunogenicity and safety of an indigenously developed tetravalent DTwP-Hib vaccine, Easyfour®-TT with a commercially available vaccine, Quadrovax®. A total of 244 infants in good health, aged 6-10 weeks, were randomized in a 1:1 allocation to receive three doses of the test or comparator vaccine. Immunogenicity of the vaccines was determined by measuring the baseline and post-vaccination antibody response against the vaccine antigens; safety was evaluated in terms of local and systemic reactions (solicited and unsolicited) reported during the trial. Similar levels of seroprotection/seroresponse were achieved, 4 weeks after receiving 3 doses of Easyfour®-TT and Quadrovax®, and the antibody response of Easyfour®-TT was found non-inferior to Quadrovax®, against all four vaccine antigens. Both vaccines were well tolerated and had similar reactogenicity profiles, with a significantly lower occurrence of local (redness at injection site) and systemic reactions (irritability post-vaccination) with Easyfour®-TT vaccine as compared to Quadrovax® (p < 0.05). All adverse events resolved completely with no sequelae. All through the study, only one serious adverse event was observed that completely resolved upon treatment and was deemed unrelated to the vaccine administered. This study demonstrated that Easyfour®-TT vaccine was safe and immunogenic. CLINICAL TRIAL REGISTRATION NUMBER: CTRI/2014/12/005326 (registered with the Clinical Trial Registry of India (CTRI)).

Is there any difference in tetanus IgG levels of diabetic patientswith respect to the presence of foot ulcers?

BACKGROUND/AIM: The aim of this study was to reveal the tetanus immunization status of diabetic patients and to determine whether diabetic patients with foot ulcers have different TIG levels. MATERIALS AND METHODS: A cross-sectional study was designed that included diabetic patients with foot ulcers (n = 30) and diabetic patients without ulcers (n = 30). The groups were compared for serum TIG levels along with total serum protein, albumin, C-reactive protein (CRP), and total immunoglobulin G (Ig G). RESULTS: For diabetic patients without foot ulcers, 17 of 30 (56.6%) patients were found to have nonprotective TIG levels whereas for diabetic patients with foot ulcers, 28 of 30 (93.3%) patients were found to have nonprotective TIG levels. The mean value of TIG for diabetic patients without foot ulcers was 0.345 ± 0.281 IU/mL and for diabetic patients with foot ulcers the mean TIG value was 0.055 ± 0.033 IU/mL. Statistically significant differences were observed in TIG (P = 0.008), total protein (P < 0.001), albumin (P < 0.001), and CRP levels (P < 0.001) between the two groups. CONCLUSION: The majority of the diabetic patients had low TIG levels and they were significantly lower in diabetic patients with ulcers. A booster dose of tetanus vaccine should be considered for diabetic patients with and without diabetic foot ulcers.

Protection against Staphylococcus aureus and tetanus infections by a combined vaccine containing SasA and TeNT­Hc in mice.

In developing countries, trauma patients and neonates are vulnerable to Staphylococcus aureus (S. aureus) and Clostridium tetani infections. It has been suggested that a combined vaccine against the two infections may be a reliable and cost­effective strategy. Previous studies have indicated that the S. aureus surface protein A (SasA) and the C fragment of tetanus neurotoxin (TeNT­Hc) may be suitable candidates for a vaccine against S. aureus and tetanus infections, respectively. In the present study, mice were immunized with a combined vaccine containing SasA and TeNT­Hc, which induced a robust immune response to both antigens, and mutual interference between SasA and TeNT­Hc was not observed. In the S.aureus challenge model, the combined vaccine fully protected BALB/c mice against lethal intraperitoneal challenges with 3x109 colony­forming units of a methicillin­resistant S. aureus USA300 strain. In the TeNT challenge model, the combined vaccine conferred complete protection against a lethal dose of (2x103) xLD50 tetanus toxin. These results implied that SasA and TeNT­Hc promising components for a combined vaccine against S. aureus and tetanus infections.

Adjuvants for Clostridium tetani and Clostridium diphtheriae vaccines updating.

It's known that diphtheria and tetanus are a contagious lethal diseases over the years, they caused by pathogenic microbes corynebacterium diphtheria and Clostridium tetani, respectively. The diseases result from the production of bacterial toxin. Vaccination with bacterial toxoid vaccines adsorbed on particulates adjuvants still are the best way to prevent this epidemic diseases from spread. The particulate vaccines have been shown to be more efficient than soluble one for the induction of the immune responses. Nanoparticles can be engineered to enhance the immune responses. As well known the immune response to inactivate killed and subunit vaccine enhances by alum adjuvants. The adjuvants examined and tested after reducing its size to particle size, thus mimic size of viruses which is considered smallest units can derive the immune system. The major issue is minimizing the adjuvant particles, to gain insight of resulting immunity types and impact on immune response. The adjuvant effect of micro/nanoparticles appears to largely be a consequence of their uptake into antigen presenting cells.

Effect of Tdap when administered before, with or after the 13-valent pneumococcal conjugate vaccine (coadministered with the quadrivalent meningococcal conjugate vaccine) in adults: A randomised controlled trial.

Sequential or co-administration of vaccines has potential to alter the immune response to any of the antigens. Existing literature suggests that prior immunisation of tetanus/diphtheria-containing vaccines can either enhance or suppress immune response to conjugate pneumococcal or meningococcal vaccines. We examined this interaction among adult Australian travellers before attending the Hajj pilgrimage 2014. We also investigated tolerability of these vaccines separately and concomitantly. We randomly assigned each participant to one of three vaccination schedules. Group A received adult tetanus, diphtheria and acellular pertussis vaccine (Tdap) 3-4weeks before receiving CRM197-conjugated 13-valent pneumococcal vaccine (PCV13) and CRM197-conjugated quadrivalent meningococcal vaccine (MCV4). Group B received all three vaccines on one day. Group C received PCV13 and MCV4 3-4weeks before Tdap. Blood samples collected at baseline, each vaccination visit and 3-4weeks after vaccination were tested using the pneumococcal opsonophagocytic assay (OPA) and by ELISA for diphtheria and tetanus antibodies. Funding for meningococcal serology was not available. Participants completed symptom diaries after each vaccination. A total of 111 participants aged 18-64 (median 40) years were recruited. No statistically significant difference was detected across the three groups in achieving OPA titre ⩾1:8 post vaccination. However, compared to other groups, Group A had a statistically significant lower number of subjects achieving ⩾4-fold rise in serotype 3, and also significantly lower geometric mean titres (GMTs) to six (of 13) pneumococcal serotypes (3, 5, 18C, 4, 19A and 9V). Group C (given prior PCV13 and MVC4) had statistically significant higher pre-Tdap geometric mean concentration (GMC) of anti-diphtheria IgG; however, there was no difference across the three groups following Tdap. Anti-tetanus IgG GMCs were similar across the groups before and after Tdap. No serious adverse events were reported. In conclusion, Tdap vaccination 3-4weeks before concomitant administration of PCV13 and MCV4 significantly reduced the antibody response to six of the 13 pneumococcal serotypes in adults. The trial is registered at the Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12613000536763.

Tetanus neurotoxin HCC protein commits T cells to IFN-γ producing cells.

A protective response against tetanus toxin and toxoid demands efficient specific T cell and B cell responses. Tetanus neurotoxin (TeNT), a 150 kDa polypeptide, is the main cause of tetanus disease. TeNT consists of two structurally distinct chains, a 50 kDa N-terminal light (L) and a 100 kDa C-terminal heavy (H) chain. C-terminal heavy (H) chain (fragment C) has two sub-domains named as proximal HCN and carboxy sub-domain or HCC. Beside neural binding property, HCC has been recently found as an immunodominant module of TeNT. In the present study, we investigated the effects of recombinant HCC (rHCC) on the expression of lineage specific transcription factors and secretion of a panel of functional cytokines including IFN-γ, IL-4, and IL-17 from purified human T cells. Our results revealed that T-bet transcript level, as TH1 specific transcription factor, was significantly increased in the cells treated with 10 and 20 µg/ml of rHCC following 48 h treatment(p<0.05). Treated purified human T cells with rHCC showed significant increase in IFN-γ mRNA level and cytokine secretion, but not IL-4 and IL-17, following 48 h treatment. In conclusion, our results showed that treatment of T cells with r HCC resulted in development of Th1 lineage phenotype, which might lead to a specific and protective antibody mediated response against tetanus toxin.

Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques.

Several studies have been performed to develop effective neutralizing monoclonal antibodies. The Epstein-Barr virus (EBV) can efficiently immortalize B-cells to establish lymphoblastoid cell lines (LCL) and so it has been used extensively for transformation of B-cells to produce and secrete immunoglobulin. The present study addressed the effect of TLR7/8 agonist (R848), feeder cells layer and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) cell separation methods on the transformation efficiency of antibody-producing memory B-cells. For these studies, the antigen used for analyses of antibody formation was the tetanus neurotoxin (TeNT) derived from Clostridium tetani. The results here showed that employing an HFFF.PI6 feeder cell layer, R848 agonist and FACS-mediated purification of memory B-cells led to increased transformation efficiency. Altogether, the effects of the R848 and the feeder cells provided an efficient method for EBV transformation of human B-cells. Moreover, there was an advantage in using FACS sorting of B-cells over the MACS method in the context of EBV transformation and immortalization of precursors of antigen-specific B-cells.

An experimental design approach to optimize an amperometric immunoassay on a screen printed electrode for Clostridium tetani antibody determination.

An immunoassay for the determination of anti-tetani antibodies has been developed using a screen printed electrode (SPE) as solid support for toxoid (antigen) immobilization. The assay was performed in guinea pig serum. The immunoreaction and the subsequent amperometric detection occurred directly onto the SPE surface. The assay consisted of spiking the anti-tetani sample directly onto the toxoid modified SPE, and then a second antibody, i.e. a HRP-labeled anti-immunoglobulin G, was deposited onto the biosensor. Subsequent amperometric detection was realized by spiking 10 µL of a hydroquinone (HQ) solution into 40 µL of buffer solution containing hydrogen peroxide. An experimental design approach was implemented for the optimization of the immunoassay. The variables of interest, such as bovine serum albumin (BSA) concentration, incubation times and labeled antibody dilution, were optimized with the aid of the response surface methodology using a circumscribed central composite design (CCCD). It was observed that two factors exhibited the greatest impact on the response, i.e. the anti-tetani incubation time and the dilution factor of the labeled antibody. It was discovered that in order to maximize the response, the dilution factor should be small, while the anti-tetani antibody incubation time should be long. The BSA concentration and the HRP-anti-IgG incubation had very limited influence. Under the optimized conditions, the immunoassay had a limit of detection of 0.011 IU/mL and a limit of quantification of 0.012 IU/mL. These values were below the protective human antibody limit of 0.06 IU/mL.