Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody.

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

Comparative Investigation of Methods for Analysis of SARS-CoV-2-Spike-Specific Antisera.

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen-antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera-as in cases of non-ARDS sera-combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen-antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.

Identification of Common CD8+ T Cell Epitopes from Lassa Fever Survivors in Nigeria and Sierra Leone.

Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.

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.

Identification of canine IgG and its subclasses, IgG1, IgG2, IgG3 and IgG4, by immunofixation and commercially available antisera.

Immunofixation is a diagnostic and research tool used for characterizing the electrophoretic location of immunoglobulin fractions in serum and urine. Commercially available polyclonal antisera which discriminate two IgG subclasses (IgG1 and IgG2) are available and commonly used. More recently, four IgG subclasses have been defined in the dog based on cDNA data. Archived serum from 16 dogs with naturally occurring monoclonal or biclonal gammopathies were characterized using routine serum protein electrophoresis, routine immunofixation and LCMS/MS as 3 IgA, 3 IgM, 2 IgG2, 7 IgG3 and 2 IgG4 heavy chain predominant cases. Immunofixation reactivity of a panel of commercially available antisera to these cases was characterized. The anti-human IgG antisera was the only tested antisera which bound all canine IgG restricted bands without also labelling IgA or IgM heavy chains or light chains. The tested polyclonal antisera labeled as reacting with canine IgG2 bound canine IgG2, IgG3, IgA and IgM and may label IgG1. The tested polyclonal antisera labeled as reacting with canine IgG1 bound the canine IgG4 bands but not those identified as IgA, IgM, IgG2 or IgG3 and likely did not bind IgG1. This data suggests that commercially available polyclonal IgG1 antisera (Bethyl A40 - 120A and Bio-Rad AHP947) can be used to positively but possibly not selectively identify canine IgG4 by immunofixation.

Detection of fly artifacts from four species of necrophagous flies on household materials using immunoassays.

An immunoassay was previously developed as a technique to improve methods for detection and analysis of fly artifacts found at crime scenes. The dot blot assay utilized a polyclonal antiserum (anti-md3) based on a unique digestive cathepsin D found in cyclorrhaphous Diptera. In this study, artifacts produced by adults of Calliphora vicina, Cynomya cadaverina, Sarcophaga bullata, and Protophormia terraenovae were examined using the immunoassay to determine if insect-derived stains could be distinguished from a range of human body fluid stains. A lift technique was developed which permitted transfer of fly artifacts from test materials to filter paper for dot blot analyses. All species readily deposited artifacts on all test household materials regardless of diet consumed. Despite differences in texture and porosity of the household materials, artifacts of all species transferred to the filter paper. With all fly species, anti-md3 serum bound to artifacts produced after feeding on semen, blood, feces, urine, and saliva. By contrast, anti-md3 serum did not react with any of the human fluids tested, nor with any of the lifts from household materials not exposed to flies. There was no evidence of false positives with any of the fly species tested, regardless of diet consumed. There was also no indication of false negatives with any of the dot blot assays. These observations suggest that immunoassays using anti-md3 serum performed on a simple lift of suspected fly artifacts can be used effectively as a confirmatory assay to distinguish fly regurgitate and fecal stains from human body fluids.

Concept of sample-specific correction of immunoassay results for precise and accurate IgG quantification in horse plasma.

The hyperimmune horse plasma (HHP), prepared through active immunisation of horses with an antigen of interest, is the most common starting material for antitoxin (animal antibody-based therapeutics) production. Precise IgG quantification in plasma is a prerequisite for accurate estimation of the purification process efficiency. Although immunoglobulins from HHP have been purified for over a century, there is still no in vitro method for precise and accurate determination of IgG content in HHP. For this reason, the purification process efficiency has been assessed by antibody activity measurements, mostly performed in vivo. Here we describe the development of a precise and accurate in vitro immunoassay for IgG quantification in HHP. We showed and highlighted that any difference in composition of IgG population between the standard and the sample, with respect to both IgG subclass distribution and antigen-specific IgG content, leads to inaccurate IgG quantification. We demonstrated that caprylic acid precipitation as the method for IgG isolation from horse plasma renders the composition of IgG population unchanged. This very efficient, fast, simple and inexpensive method was used to prepare internal, sample-specific reference IgG for each plasma sample, which was tested simultaneously to a respective plasma sample. Deviation of IgG quantity determined by ELISA for each sample-specific reference from its nominal value was used for correction of the results of respective plasma sample, which led to accurate and precise IgG quantification as shown by method validation. The here presented novel concept of sample-specific correction of immunoassay results could be widely applicable and easily introduced in different immunoassays for more accurate and precise plasma IgG quantification.

Evaluation of an OV-16 IgG4 Enzyme-Linked Immunosorbent Assay in Humans and Its Application to Determine the Dynamics of Antibody Responses in a Non-Human Primate Model of Onchocerca volvulus Infection.

Onchocerciasis is a neglected parasitic disease targeted for elimination. Current World Health Organization guidelines for elimination include monitoring antibody responses to the recombinant Onchocerca volvulus antigen OV-16 in children to demonstrate the absence of transmission. We report the performance characteristics of a modified OV-16 enzyme-linked immunosorbent assay (ELISA) and describe anti-OV-16 responses in serum samples from laboratory-inoculated nonhuman primates (NHPs) in relation to microfilariae (mf) in skin snip biopsies. This OV-16 IgG4 ELISA had sensitivity and specificity of 88.2% and 99.7%, respectively, as determined by receiver operator characteristic analysis using a serum panel of 110 positive and 287 negative samples from people infected with other filariae or other parasitic infections. Anti-OV-16 responses in inoculated NHP (N = 9) were evaluated at quarterly intervals for IgM and the four IgG subclasses. Enzyme-linked immunosorbent assay results showed a well-defined IgG4 reactivity pattern and moderate IgG1 antibody responses. Meanwhile, the reactivity by IgG2, IgG3, or IgM did not show a clear pattern. Temporal evolution of IgG4 reactivity was evaluated through monthly testing, showing that NHPs developed anti-OV-16 IgG4 on average at 15 months postinoculation (range: 10-18 months). The average time to detectable mf was also 15 months (range: 11-25). The OV-16 ELISA used in this study was robust and allowed the detection of IgG4 responses, which were observed only among animals with detectable mf (N = 5), four of which showed declines in antibody responses once mf cleared. These findings also confirmed that the most informative antibody subclass responses to OV-16 are IgG4.

Development and evaluation of polyclonal antisera for detection of the IgM heavy chain of multiple fish species.

Antibodies are widely considered to be essential tools for detection of immune responses in various fish species. Here we produced the peptide polyclonal antisera (anti-fish IgH-1 and anti-fish IgH-2) to detect IgM of various fish species. The peptides were designed based on the conserved sequence of the fish immunoglobulin heavy chains of seven fish species (Japanese flounder, seabream, yellowtail, carp, rainbow trout, hybrid sturgeon and banded houndshark). By Western blotting, anti-fish IgH-1 antiserum detected the IgMs of all fish species except banded houndshark. Anti-fish IgH-2 antiserum clearly reacted with the IgMs of only three of the fish species (seabream, yellowtail and rainbow trout). Attempts to use the antisera to measure fish antibody titer by ELISA were unsuccessful. These results demonstrate that anti-fish IgH-1 peptide polyclonal antiserum is a potentially applicable tool for detecting immunoglobulins in various fish species by Western blotting.

Indirect Immunometric ELISA.

This assay facilitates the immunometric determination of assay-associated reagents including the capture and detection antibodies as well as the analyte (i.e., antigen or antibody). It can precede the development of a sandwich enzyme-linked immunosorbent assay (ELISA) in which optimal antibody concentrations are applied for the quantitative measurement of the antigen. This protocol describes the materials and equipment required for the measurement of chromogenic substrate development; however, it can be adapted for use with chemiluminescent- and fluorescent-labeled reporters.

Agua del grifo versus suero salino estéril en la limpieza de heridas agudas y crónicas / Tap water vs. sterile saline for cleaning acute and chronic wounds

Introducción: El uso de agua del grifo como agente en la limpieza de las heridas está siendo usado de forma frecuente en la práctica clínica, especialmente en el seno de la atención comunitaria. Objetivo: Comprobar si hay diferencias en la tasa de infección y en la de cicatrización de la herida, cuando se limpian con agua del grifo o con solución salina estéril. Metodología: Tipo de estudio: ensayo clínico controlado y aleatorizado doble ciego. Los sujetos fueron reclutados de las unidades de enfermería comunitaria del área de un hospital local en Hong Kong (China). Se incluyeron personas mayores de 18 años que tuvieran una herida aguda o crónica y que fueran tratados en dicho centro. Los pacientes fueron asignados aleatoriamente a uno de los 2 grupos, limpieza de la herida con agua del grifo (grupo experimental) o solución salina estéril (grupo de control). Para el cegamiento se prepararon 100ml de agua de grifo o de suero fisiológico exactamente iguales y sin etiqueta. La evaluación de la herida fue realizada en cada visita a la casa, y la evaluación del tamaño de la herida se llevó a cabo una vez por semana. Las medidas de resultado fueron: la aparición de una infección y la cicatrización de heridas. El periodo de seguimiento fue de 6 semanas. Resultados principales: Veintidós sujetos (11 sujetos en cada grupo) con 30 heridas han participado en el estudio. Del total, 19 eran heridas crónicas y 11 heridas agudas (siendo mayor la proporción de heridas crónicas en el grupo experimental), 16 heridas fueron tratadas con de agua del grifo en la limpieza y 14 fueron asignados al azar para el tratamiento con solución salina estéril. El análisis no reveló ninguna diferencia significativa entre los grupos experimentales y de control en las tasas de infección de la herida (p=0,49), ni en la cicatrización de heridas, medido por el descenso del tamaño de la herida (p=1,00), ni en la presencia de tejido de granulación-epitelización (p=0,23). Conclusiones: Los hallazgos del estudio indican que el agua del grifo es una alternativa segura a la solución salina estéril para la limpieza de la herida en un entorno comunitario

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Rice-produced MSP142 of Plasmodium falciparum elicits antibodies that inhibit parasite growth in vitro.

Many malaria antigens contain multiple disulphide bonds involved in the formation of inhibitory B-cell epitopes. Producing properly folded malaria antigens in sufficient quantities for vaccination is often a challenge. The 42-kDa fragment of Plasmodium falciparum merozoite surface protein 1 (MSP142 ) is such a kind of malaria antigen. In this study, we investigated the expression of MSP142 in a rice system (9522, a cultivar of Oryza sativa ssp. japonica), which was used as a bioreactor for protein production. The MSP142 gene was synthesized according to rice-preferred codons and transformed into rice plants via an Agrobacterium-mediated method. The recombinant antigen was efficiently expressed in rice seeds with a level up to 1.56% of total soluble protein and was recognized by both the conformational monoclonal antibody 5.2 (mAb5.2) and the pooled sera of P. falciparum malaria patients. Rabbits were immunized intramuscularly with the purified MSP142 formulated with Freund's adjuvant. High antibody titres against MSP142 were elicited. The rabbit immune sera reacted well with the native protein of P. falciparum parasite and strongly inhibited the in vitro growth of blood-stage P. falciparum parasites, demonstrating that transgenic rice can become an efficient bioreactor for the production of malaria vaccine antigens.

Levels of anti-fructose-modified HSA antibodies correlate with disease status in diabetic subjects.

OBJECTIVE: This study aimed to assess the changes induced in HSA upon fructose-modification and to use the modified protein as an antigen for studying the presence of antibodies in diabetic patients. Further, magnitude of oxidative stress was also assessed. METHODS: HSA was modified with fructose, changes induced were studied by DSC measurements and near-UV CD. The binding characteristics of antibodies in the sera of diabetes patients to native and modified-HSA was assessed by ELISA and band shift assay. The oxidative stress in these patients was studied by carbonyl content estimation, FRAP assay and TBARS determination RESULTS: DSC revealed that fructose modified-HSA was more thermostable than its native form. Changes in tertiary structure of fructose-modified HSA were seen in near-UV CD. Patient studies showed that fructose-modified HSA acts as a potent immunogen compared to its native form and the levels of antibodies against fructose-modified HSA served as a parameter for tracking the glycemic control and oxidative stress parameters (carbonyl content, FRAP value and MDA level) in diabetic patients. CONCLUSIONS: Fructose-modification of HSA causes perturbations in its structure and function, thereby, making the protein antigenic besides decreasing its antioxidant capacity. This study suggests that fructose-modified-HSA is an important contributor in diabetic pathophysiology.

Identification of Lactobacillus proteins with different recognition patterns between immune rabbit sera and nonimmune mice or human sera.

BACKGROUND: The genus Lactobacillus belongs to a large heterogeneous group of low G + C Gram-positive anaerobic bacteria, which are frequently used as probiotics. The health-beneficial effects, in particular the immunomodulation effect, of probiotics depend on the strain and dose used. Strain variations may be related to diversity of the cell surface architecture of bacteria and the ability to express specific antigens or secrete compounds. The use of Lactobacillus as probiotic requires a comprehensive understanding of its effect on host immune system. To evaluate the potential immunoreactive properties of proteins isolated from four Lactobacillus strains: L. johnsonii 142 and L. johnsonii 151, L. rhamnosus LOCK 0900 and L. casei LOCK 0919, the polyclonal sera obtained from mouse and human have been tested as well as with sera from rabbits immunized with whole lactobacilli cells. RESULTS: The reactivity of isolated proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and sequenced, in particular the fractions were identified as phosphoglycerate kinase (L. johnsonii 142), glyceraldehyde 3-phosphate dehydrogenase (L. johnosnii 142, L. rhamnosus LOCK 0900), hypothetic protein JDM1_1307 (L. johnsonii 151) and fructose/tagatose-bisphosphate-aldolase (L. casei LOCK 0919). CONCLUSION: The different prevalence of reactions against tested antigens in rabbit, mouse and human sera may indicate significant differences in immune system and commensal cross-talk in these groups. The identification of immunoreactive lactobacilli proteins opens the possibility to use them as an antigens for development of vaccines.

Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol.

D-Ribitol, a five-carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27-30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid-Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol-KLH-Sepharose CL-6B resulted in pure ribitol-specific antibodies (~45-50 µg/mL). The affinity constant of ribitol antibodies was found to be 2.9 × 10(7) M(-1) by non-competitive ELISA. Ribitol antibodies showed 100% specificity towards ribitol, ~800% cross-reactivity towards riboflavin, 10-15% cross-reactivity with sorbitol, xylitol and mannitol, and 5-7% cross-reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4%) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.

Estudo do perfil microbiológico da Seção de Envasamento e Acondicionamento de Soros e Vacinas desenvolvidos no Instituto Butantan

Diversos são os controles que devem ser realizados pela indústria farmacêutica para garantir a qualidade do ambiente onde são fabricados os medicamentos. Este trabalho contemplou alguns pontos do controle ambiental da Seção de Envasamento e Acondicionamento de soros e vacinas desenvolvidos no Instituto Butantan, como os pontos críticos de amostragem, isolamento dos microrganismos presentes nesses pontos, a identificação microbiológica por métodos macro e microscópicos e técnicas de biologia molecular e as possíveis fontes contaminação, de acordo com as características do microrganismo. Os gêneros de bactérias mais frequentes associados à amostragem das áreas de produção foram Staphylococcus spp., Micrococcus luteus e Bacillus spp. Os pontos de amostragem com maior frequência de microrganismos isolados foram nos monitoramentos de operadores e de partículas viáveis em operação. A sanitização das áreas limpas é um aspecto importante na fabricação de produtos estéreis. Assim, a implantação de um rodízio de sanitizantes pode ser mais efetiva por ampliar o espectro de ação destes em diferentes espécies de microrganismos.

Estudo do perfil microbiológico e monitoramento ambiental da Seção de Plasmas Hiperimunes e do Serviço de Formulação de Soros e Vacinas do Instituto Butantan

Diversos são os controles que devem ser realizados pela indústria farmacêuticapara garantir a qualidade do ambiente onde são fabricados os medicamentos.Este trabalho contemplou alguns pontos do controle ambiental da Seção dePlasmas Hiperimunes e do Serviço de Formulação de soros e vacinas, comoos pontos críticos de amostragem, isolamento dos microrganismos presentesnesses pontos e a identificação desta microbiota por métodos macro emicroscópicos e técnicas de biologia molecular. O gênero de bactéria maisfrequente associado à amostragem das áreas de produção foi Staphylococcusspp., seguido de Bacillus spp. e Micrococcus luteus. Os pontos de amostragemcom maior frequência de microrganismos isolados foram nos monitoramentosde operadores e de partículas viáveis em operação.