Dot Blot Assay Research Articles

Effect of Molecular Chaperone on the Soluble Expression of Recombinant Fab Fragment in E. coli

Molecular chaperones are folding modulators that play a pivotal role in conformational quality control of proteome and assist proper protein folding upon translation. In this study, the effects of cytoplasmic chaperones on the production of anti-TNF-α Fab antibody in Escherichia coli were investigated. To increase the solubility of anti-TNF-α Fab, different chaperone plasmids including pG-KJE8 (GroELS and DnaKJE), pGro7 (GroELS), pKJE7 (DnaKJE), pG-Tf2 (GroELS and TF) and pTf16 (TF) co-expressed in BL21 cells containing pET-22b-Fab construct were used. The solubility of recombinant anti-TNF-α Fab was analyzed by SDS-PAGE. The reactivity of the recombinant protein was tested by ELISA and dot blot assays. SDS-PAGE analysis of the expressed Fab revealed that chaperone coexpression increased the solubility of the Fab antibody. Among the chaperone sets co expressed with Fab construct, the dnaK/dnaJ/grpE chaperones showed the highest effect on soluble expression with producing about 23.2% soluble Fab antibody. ELISA test results showed, the pKJE7 chaperone led to an approximately sevenfold increase in the activity of produced Fab fragment. Dot blot test results revealed reactivity of recombinant Fab to both transmembrane and soluble form of TNF-α. Our results demonstrated the significant advantage of molecular chaperones in improvement of the soluble expression of Fab antibody in E. coli.

Wound fluid enhances cancer cell proliferation via activation of STAT3 signal pathway in vitro.

Wound healing begins immediately after surgery with a modification of the microenvironment via a well‑orchestrated interaction between cells, cytokines and growth factors. Some of these growth factors and cytokines have mitogenic effects on cancer cells, which may lead to enhanced cancer cell proliferation and early metastatic events. The present study aimed to investigate the effects of wound fluid (WF) on the head and neck squamous carcinoma cell lines FaDu and HLaC78 in vitro. WF was harvested from 7 patients who had undergone a planned neck dissection. The presence of cytokines and growth factors was evaluated with the dot blot assay. Proliferation and cell viability were investigated via MTT assay and Ki-67 staining. Cell invasion was measured via tree‑dimensional invasion assay. Western blotting was used to investigate STAT 3 activation. WF contained several cytokines and growth factors responsible for pro‑ and anti‑inflammation, chemotaxis, proliferation and angiogenesis. The proliferation effect of WF on FaDu and HLaC78 was concentration dependent. Media with 40% WF resulted in the highest proliferation effect. FaDu and HLaC78 exhibited enhanced motility after cultivation with 40% WF compared with cultivation with expansion medium. Cultivating cancer cells with WF had no advantageous effect on cell viability after the paclitaxel treatment. Western blot analysis revealed enhanced activation of the STAT3 signaling pathway by WF in both FaDu and HLaC78. In conclusion, surgery leads to excessive release of mitogenic factors. The contact of non‑resected cancer cells and these factors may have a negative impact on patient outcome. Future investigations should specifically focus on the inhibition of mitogenic factors following cancer surgery in order to prevent early metastasis and cancer recurrence.

Metformin sensitizes endometrial cancer cells to progestin by targeting TET1 to downregulate glyoxalase I expression

Progestins has been used widely for endometrial cancer (EC) patients. However, long term use of high dose progestin often lead to progestin resistance. Our previous studies have demonstrated that metformin reversed progestin resistance through the downregulation of the expression of glyoxalase I (GLOI) in type I endometrial cancer. Recent studies have demonstrated the role of Ten-eleven translocation 1 (TET1) in endometrial cancer, but the physiological role of TET1 in GLOI-mediated progestin resistance has been poorly addressed. Immunohistochemistry was used to detect the expression of TET1, GLOI and 5hmC in various endometrium. Western blot was carried out to analyses TET1 and GLOI expression with different treatment. Cell counting kit-8 was used to evaluate cell proliferation after various treatment. Dot blot assay and HMeDIP assay were performed to detect global hydroxmethylation levels and hydroxymethylation levels in GLOI gene respectively. In current study, we found that metformin effectively sensitized progestin in endometrial cancer cell lines through the down regulation of the expression of TET1 and GLOI. Interestingly, the exogenous increase of TET1 expression enhanced total 5hmC level and hydroxymethylation modification in glyoxalase I promoter region. This effect was abated by metformin treatment. Moreover, the expression profile of TET1 and glyoxalase I in various endometrial tissue parallelized with 5hmC level. Therefore, this finding suggests that metformin sensitized progestin in endometrial cancer through the TET1-5hmC-GLOI signaling pathway.

Polymorphisms of α-Globin Genes Compromise Polymerase Chain Reaction-Based α-Thalassemia Genotyping in Three Chinese Families

In practice, gap-polymerase chain reaction (gap-PCR) and reversed dot-blot are the two most frequently used molecular diagnostic methods for α-thalassemia (α-thal) genotyping. Here, we describe three Chinese individuals from three unrelated families in whom a polymorphism on the α-globin gene cluster led to diagnostic pitfalls. During general molecular diagnosis of thalassemia, three individuals with unexplained results were found. Blood or chorionic villus samples were collected from these three individuals and their family members. Hematological investigations and genetic tests were performed. In Family 1, a polymorphism of HBA2: c.301-24delinsCTCGGCC at the annealing site of the forward primer used in the PCR-reverse dot-blot assay was identified, leading to allele drop-out during the PCR amplification process. In Family 2, a synonymous mutation of C>T substitution at codon 125 of the α2 gene (HBA2: c.376C>T) was identified, leading to the failure of PCR-reversed dot-blot for the HBA2: c.377T>C (Hb Quong Sze or Hb QS) mutation. In Family 3, the size of the PCR fragment from the α2-globin allele carrying the HBA2: c.-771_-428del mutation was smaller and nearly equal to the size of the fragment corresponding to the –α4.2 (leftward) deletion; we also found that the HBA2: c.-771_-428del mutation was linked to a known HBA1: c.-673A>G mutation in this family. In conclusion, diagnostic errors may be caused by technical pitfalls or inherent properties of the DNA sample. All logical steps should be taken to monitor and thus preclude such events.

Fructose-1,6-bisphosphate aldolase is involved in Mycoplasma bovis colonization as a fibronectin-binding adhesin

Mycoplasma bovis is a common pathogenic microorganism of cattle and represents an important hazard on the cattle industry. Adherence to host cells is a significant component of mycoplasma-pathogenesis research. Fibronectin (Fn), an extracellular matrix protein, is a common host cell factor that can interact with the adhesions of pathogens. The aims of this study were to investigate the Fn-binding properties of M. bovis fructose-1,6-bisphosphate aldolase (FBA) and evaluate its role as a cell adhesion factor during mycoplasma colonization. The fba (MBOV_RS00435) gene of M. bovis was cloned and expressed, with the resulting recombinant protein used to prepare rabbit polyclonal antibodies. The purified recombinant FBA (rFBA) was shown to have fructose bisphosphate aldolase activity. Western blot indicated that FBA was an antigenically conserved protein in several M. bovis strains. Western blot combined with immunofluorescent assay (IFA) revealed that FBA was dual-localized to both cytoplasm and membrane in M. bovis. IFA showed that rFBA was able to adhere to embryonic bovine lung (EBL) cells. Meanwhile, an adhesion inhibition assay demonstrated that anti-rFBA antibodies could significantly block the adhesion of M. bovis to EBL cells. Moreover, a dose-dependent binding of rFBA to Fn was found by dot blotting and enzyme-linked immunosorbent assays. Together these results provided evidence that FBA is a surface-localized and antigenic protein of M. bovis, suggesting that it may function as a virulence determinant through interacting with host Fn.

Transient expression of etanercept therapeutic protein in tobacco (Nicotiana tabacum L.).

Etanercept is a recombinant fusion protein of TNFR2 with the Fc portion of human IgG1. Etanercept, an anti-TNF drug, treats autoimmune diseases and improves patients' health. The main goal of the present study was to investigate the possibility of expressing recombinant protein of etanercept in a plant system. For this aim, first a modified version of pCAMBIA1305.1 plasmid with a new multiple cloning site and signal sequence of KDEL for protein secretion was constructed (pCAMBIA1305.1-linker). Then etanercept gene was cloned into the linker fragment of pCAMBIA1305.1-linker vector. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. To evaluate the transient expression of the gene, agroinfiltrated tobacco leaves were inoculated with Agrobacterium tumefaciens containing etanercept gene cassette. The recombinant etanercept protein was examined by dot blot and ELISA assays. Our results using anti-human IgG HRP-conjugated antibody confirmed a high level expression of etanercept gene in the tobacco leaves.

TuMV as an efficient transient vector for expressing heterologous proteins in Nicotiana tabacum and N. benthamiana

Nowadays the production of recombinant proteins such as drugs and commercial protein compounds in plants is called molecular farming. It has some benefits such as fast and large quantity production of recombinant proteins with low cost. In this research, the green fluorescent protein (GFP) was transiently expressed in two tobacco species via turnip mosaic virus (TuMV) derived vector, a virus which can infect a wide range of plant species. Florescence microscopy results indicated that TuMV could infect tobacco plants and accumulate GFP protein in plant leaves. In addition, RT-PCR, Dot-Blot and ELISA assays demonstrated the recombinant gene transcription, translation and stability. This is the first report of using TuMV-based viral vectors for producing recombinant proteins in tobacco. Optimized TuMV-based viral vectors could be used for producing recombinant proteins in tobacco.

One-step multiplex RT-PCR for simultaneous detection of four viroids from hop (Humulus lupulus L.)

Hop (Humulus lupulus L.) plants are hosts to several viroids; some of them can be highly aggressive, and their infection can manifest as complete plant dieback. Since molecular detection of multiple viroids can be time consuming and cost inefficient, a reliable one-step multiplex RT-PCR (mRT-PCR) was developed to detect simultaneously all four viroids infecting hops: Hop latent viroid (HLVd), Hop stunt viroid (HSVd), Apple fruit crinkle viroid (AFCVd) and Citrus bark cracking viroid (CBCVd). Several primer pairs were tested on different viroid variants from hops, citruses and grapevines, and from among them, specific primer pairs for detection of hop viroids were selected and confirmed in a single-tube assay. To improve mRT-PCR reliability and validate its effectiveness, nad5 and DRH1 genes were included as an internal control. The specificities of single and mRT-PCR assays for all four viroids were comparable. The sensitivity of mRT-PCR was compared with that of dot-blot hybridization and single RT-PCR assay on biolistically infected hop plants. The results show mRT-PCR to be more sensitive than the dot-blot and slightly less sensitive than the single RT-PCR assay. Furthermore, mRT-PCR was validated using field samples and a group of 135 hop plants, which are used in the certification scheme for planting material propagation, and the method proved to be robust, rapid and simple. Additionally, this approach can be applicable to similar methods of systematic surveys of emerging diseases and epidemiological studies.

Anthraquinone Derivative Reduces Tau Oligomer Progression by Inhibiting Cysteine-Cysteine Interaction.

Tau protein is a natively unfolded protein whose primary role is to participate in axonal transport closely associated with microtubules. Neurodegenerative disorders including Alzheimer's disease and Tauopathies involved tau protein that is found hyperphosphorylated in vivo; then, tau is detached from microtubules to form toxic aggregates or oligomers, which have a deleterious effect on membranes, triggering an inflammatory response. Considering finding tau inhibitors, we isolated two compounds in the ethyl acetate extract from Xanthoria ectaneoides (Nyl.) Zahlbr; ergosterol peroxide (1) and a new anthraquinone (2). We established the structure through spectroscopic data and biogenic considerations, and we named it “2‐hydroxy‐3‐((8‐hydroxy‐3‐methoxy‐6‐methylanthraquinonyl)oxy)propanoic acid”. This new anthraquinone was evaluated as a tau inhibitor by ThT fluorescence, dot blot assays and total internal reflection fluorescence microscopy. Our results strongly suggest that this anthraquinone remodels soluble oligomers and diminishes β‐sheet content. Moreover, through the fluorescence labeling of cysteine inside of the microtubule‐binding domain (4R), we showed that this anthraquinone could reduce the oligomers progression by inhibiting cysteine interactions.

Prevalence of the crpP gene conferring decreased ciprofloxacin susceptibility in enterobacterial clinical isolates from Mexican hospitals.

This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants. A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain. The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli. These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.

Values of Hematological Indicators in the Screening of α-Thalassemia in Fujian Area of China

To analyze the genotypes and the hematological phenotypic characteristics of α-thalassemia in different areas of Fujian and to evaluate the values of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin (Hb), RBC distribution width/red blood cell (RDW/RBC) for screening α-thalassemia in this area. The Gap-PCR assay was applied for detecting 3 common deletional mutations of patients with α-thalassemia, and the reverse dot-blot (RDB) assay was adopted to detect the foci of 3 common non-deletional gene mutations.Then,the hematological parameters of individuals with α-thalassemia were analyzed. Finally, the optimal cut-off value in hematological indexes for screening α-thalassemia were determined by the ROC curve. Altogether 16 types of gene mutations were found in 772 patients with α-thalassemia. Among them, the -SEA/αα deletion mutation was the most common which was observed in 521 cases(67.49%). Compared with the control group, the differences in MCV, MCH, and Hb were statistically significant between the patients of the same sex but no same type. In male groups, the RDW/RBC ratio was statistically significant in individuals of light type and HbH disease as compared with the healthy control group. But in female groups, the statistical different of RDW/RBC ratio was found between only HbH disease group and control group. MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L, with the highest specificity and the highest sensitivity, were the best cut-off points for screening α-thalassemia in the laboratory. Due to the difference of regional heterogeneity and hospital equipment environment, the different laboratories need to establish cut-off value for screening α-thalassemia suitable for its local region. In future, our laboratory can use MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L for value for clinical screening, of α-thalassemia.

Natural alkaloid harmine promotes degradation of alpha-synuclein via PKA-mediated ubiquitin-proteasome system activation

BackgroundParkinson's disease (PD) is an age-dependent progressive movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Accumulation of <alpha>-synuclein (<alpha>-syn) positive protein aggregates in the substantia nigra is a pathological hallmark of PD, indicating that protein turnover defect is implicated in PD pathogenesis. PurposeThis study aims to identify neuroprotective compounds which can alleviate the accumulation of <alpha>-syn in neuronal cells and dissect the underlying mechanisms. MethodsHigh throughput screening was performed by dot blot assay. The degradation of different forms of <alpha>-syn by candidate compounds were assessed by western blot. The autophagy lysosome pathway and ubiquitin-proteasome system were examined to dissect the degradation pathway. The UPS activity was assessed by cellular UPS substrates degradation assay and biochemical proteasome activity assay. Q-PCR was performed to test the mRNA level of different proteasome subunits. Furthermore, Neuroprotective effect of candidate compound was tested by LDH assay and PI staining. ResultsThrough the high throughput screening, harmine was identified as a potent <alpha>-syn lowering compound. The time-dependent and dose-dependent effects of harmine on the degradation of different forms of <alpha>-syn were further confirmed. Harmine could dramatically promote the degradation of UPS substrates GFP-CL1, Ub-R-GFP and Ub-G76V-GFP, and activate cellular proteasome activity. Mechanistically, harmine dramatically enhanced PKA phosphorylation to enhance proteasome subunit PSMD1 expression. PKA inhibitor blocked the effects of harmine in activating UPS, up regulating PSMD1 and promoting <alpha>-syn degradation, indicating that harmine enhances UPS function via PKA activation. Moreover, harmine efficiently rescued cell death induced by over-expression of <alpha>-syn, via UPS-dependent manner. ConclusionHarmine, as a new proteasome enhancer, may have potential to be developed into therapeutic agent against neurodegenerative diseases associated with UPS dysfunction and aberrant proteins accumulation.

A novel tandem repeat cloning technique for creation of multiple short peptide repeats to differentiate closely related antigens

Antibody cross-reactivity is a problem often associated with closely related antigens. This study was aimed to develop a method enabling differentiation of closely related toxins based on antigen designing strategy. The method involves identification of disparate amino acids (AA) confined to target antigen in comparison with two or more closely related antigens, their assembly into a DNA oligomer and further cloning as six tandem repeats (TR) using restriction and ligation strategy into a desired vector and finally generation of antigen specific antibodies. The practical utility of this method was demonstrated by generating and testing the specificity of polyclonal antibodies against staphylococcal enterotoxin C (SEC). Cross-reactivity is a problem often associated with SEC in immunoassays due to its amino acid sequence identity with staphylococcal enterotoxin B (SEB) (40–60%). To circumvent the same, the above-mentioned strategy was applied. Unique AA of SEC (36 AA) in comparison to SEB were selected, reassembled and with deduced corresponding nucleotides, an oligomer of 117 bases was designed. Using primers with restriction overhangs, three constructs were created each with two repeats using a common restriction site. The resulting three constructs were sequentially cloned into alternating restriction sites of pRSET A vector in directional orientation, expressed in E. coli for rTR/SEC protein which was used to generate specific polyclonal antibodies against SEC. Specificity was compared with antibody raised against whole SEC recombinant protein using Western blot and dot blot assays. High specificity was achieved through the developed strategy signifying its possible application to address cross-reactivity problem associated with closely related antigens.

Surface Plasmon Enhanced Light Scattering Biosensing: Size Dependence on the Gold Nanoparticle Tag.

Surface plasmon enhanced light scattering (SP-LS) is a powerful new sensing SPR modality that yields excellent sensitivity in sandwich immunoassay using spherical gold nanoparticle (AuNP) tags. Towards further improving the performance of SP-LS, we systematically investigated the AuNP size effect. Simulation results indicated an AuNP size-dependent scattered power, and predicted the optimized AuNPs sizes (i.e., 100 and 130 nm) that afford extremely high signal enhancement in SP-LS. The maximum scattered power from a 130 nm AuNP is about 1700-fold higher than that obtained from a 17 nm AuNP. Experimentally, a bio-conjugation protocol was developed by coating the AuNPs with mixture of low and high molecular weight PEG molecules. Optimal IgG antibody bioconjugation conditions were identified using physicochemical characterization and a model dot-blot assay. Aggregation prevented the use of the larger AuNPs in SP-LS experiments. As predicted by simulation, AuNPs with diameters of 50 and 64 nm yielded significantly higher SP-LS signal enhancement in comparison to the smaller particles. Finally, we demonstrated the feasibility of a two-step SP-LS protocol based on a gold enhancement step, aimed at enlarging 36 nm AuNPs tags. This study provides a blue-print for the further development of SP-LS biosensing and its translation in the bioanalytical field.

Immunohistochemical localization of GnRH-immunoreactive cell bodies and fibers in the nerve ganglion of Perinereis aibuhitensis (Annelida: Polychaeta)

The gonadotropin-releasing hormone (GnRH) gene sequence has been identified in an annelid polychaete marine worm using continual genome sequencing. The distribution of GnRH immunoreactive (ir) cell bodies and fibers in the nerve ganglion of the clam worm Perinereis aibuhitensis (Polychaeta) was examined by immunohistochemistry using a newly produced rabbit polyclonal antibody raised against the marine worm GnRH (mwGnRH). The specificity of the antibody was confirmed by dot blot assay. The antibody cross-reacted with mwGnRH, but not with other forms of GnRH such as octopus GnRH, tunicate GnRH-I, II, owl limpet GnRH, and lamprey GnRH-II. In P. aibuhitensis, mwGnRH-ir cell bodies were detected in the nuclei 15–22, the caudal part of the cerebral ganglion. Furthermore, mwGnRH-ir fibers were mainly observed in the optic neuropil, but mwGnRH-ir fibers were also detected in the central neuropil region, the subpharyngeal ganglion, and the ventral nerve cord. These results indicate that mwGnRH is synthesized in the cerebral ganglion, is transported through the subpharyngeal ganglion and the ventral nerve cord, and functions either as a neurotransmitter or neuromodulator.

A comparison of antioxidant and Fourier Transform Infrared Spectroscopy (FTIR) analysis on extracts of Syzygium guineenses (Myrtaceae)

Background: Syzygium guineenses, (the most common and abundant specie in Nigeria) is a medicinal plant used by traditional practitioners in northern Nigeria for a variety of healing purposes. Objective: The main objective of this project was to carry out a comparison of antioxidant activities and Fourier Transform Infrared Spectrophotometric (FTIR) analysis on both methanol and hexane leaf extracts of S. guineenses. Methods: Phytochemical screening, Semi-quantitative DPPH (1,1-diphenyl-2-picrylhydrazyl)- dot blot assay and FTIR analysis were performed on both extracts to determine antioxidant activity and identify the functional groups present. Results: Phytochemicals tested for, were observed to be more prominent in the methanol extract than hexane. The in vitro antioxidant assay also revealed a more intense yellow colour of inhibition in methanol extract than the hexane extract. The FTIR spectra revealed different characteristic peak values with various functional compounds in both extracts. The methanol extract displayed major peaks of absorption at 3341 cm-1 (-OH) for alcohol, 1736 cm-1 (C=O) carbonyl group, 1161.83 cm-1, 1036.49 cm-1 (C-O) of esters. Other absorption bands like 1452.25 cm-1 and 1612.20 cm-1 for alkenes were present in both extracts. Conclusion: This result shows that the methanol extract of S. guineenseshas a higher potential of phytochemicals, antioxidants and functional groups than the hexane extract.

Immunoassay detection of fly artifacts produced by several species of necrophagous flies following feeding on human blood

Foraging behavior of necrophagous flies commonly leads to distortion of human bloodstains and production of artifacts that confound reconstruction efforts at crime scenes. Currently there is no reliable method for detection of fly-derived stains or distinction of the artifacts from human bloodstains. To overcome these deficiencies, a confirmatory test was developed based on immunological detection of cathepsin D found in digestive fluids of Musca domestica and Protophormia terraenovae. Anti-serum (anti-md3 serum) was generated toward a 17-amino acid synthetic peptide based upon predicted antigenic amino acid sequences for the propeptide and mature enzyme of cathepsin D proteinase from larvae of M. domestica. The serum was used to test the hypothesis that digestive artifacts produced by an array of necrophagous flies associated with human decomposition could be detected with the immunoassay. Anti-md3 serum was able to bind artifacts from 27 species of flies representing 9 families. The antiserum reacted with both regurgitate and defecatory stains, but not transfer patterns. Stains from 4 fly species displayed no reactivity with anti-serum in dot blot assays. Anti-md3 serum did not bind to either human or bovine blood stains on filter paper. However, when both types of blood were spiked with synthetic md3 peptide the antiserum was able to bind. Dot blot assays displayed positive reactions with stains produced from larvae and teneral adults of Sarcophaga bullata, and with artifacts as old as 7-years after deposition. These observations indicate that the immunoassay permits distinction of artifacts from a wide range of species from human bloodstains, from multiple development stages, and from artifacts that remain at crime scenes for many months to years after deposition. Further work is needed to determine whether the detection of fly artifacts using the antiserum is suitable for non-laboratory conditions.

Monoclonal Antibodies Against the Capsular Polysaccharides A, C, Y, W, and X of Neisseria meningitidis: A Platform for the Quality Control of Meningococcal Vaccines.

Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.

Comparative performance of TCBS and TSA for the enumeration of trh+ Vibrio parahaemolyticus by direct colony hybridization

Vibrio parahaemolyticus is one of the important foodborne pathogens is of public health concern due to the emergence of pandemic strains causing disease outbreaks worldwide. We evaluated the DNA based colony hybridization technique for the detection and enumeration of total and pathogenic V. parahaemolyticus from the bivalve shellfish, clam using non-radioactive, enzyme-labeled probe targeting the tlh and trh genes, respectively. The digoxigenin (DIG) labeled probes designed in this study showed 100% specificity by dot blot assay. Colony hybridization using DIG probes was performed using both non-selective, trypticase soy agar (TSA) and the selective medium, thiosulfate citrate bile salts sucrose (TCBS) agar. Of 32 clam samples analyzed, 71.88% had>10,000 V. parahaemolyticus cells/g in TSA whereas it was 18.75% in case of TCBS. All the samples showed the presence of total V. parahaemolyticus in TSA and 97% in the case of TCBS. Interestingly, results of the trh+V. parahaemolyticus samples were quite high while using TCBS plates (62.5%) as compared to TSA (43.75%). However, the cell numbers obtained from TSA were higher than from TCBS. Several yellow colonies on TCBS turned out to be V. parahaemolyticus using colony hybridization, which was further confirmed by PCR and sucrose utilization test. Colony hybridization using DIG-labeled probe was found to be highly sensitive and could differentiate and enumerate pathogenic and non-pathogenic strains of V. parahaemolyticus. Since traditional methods are not only labor-intensive and time-consuming but also less sensitive, colony hybridization using DIG-labeled probes would be a useful alternative for the enumeration of V. parahaemolyticus in naturally contaminated seafood.

Association between bisphenol A diglycidyl ether-specific IgG in serum and food sensitization in young children

BackgroundRecent studies have reported that endocrine-disrupting compound (EDC) exposure is related to food sensitization. Bisphenol A diglycidyl ether (BADGE) is one of the most widespread EDCs and its biological effects are considered to be greater on children than on adults. This study investigated the relationship between serum BADGE-specific immunoglobulin G (IgG) concentrations and food sensitization in young children by measuring food-specific IgE levels.MethodsIn total, 98 young children (59 boys and 39 girls; median age: 7 months; 25th and 75th percentile ages: 6 and 8 months, respectively) were enrolled. Blood samples were collected twice from all children (median sampling interval: 6 months; 25th and 75th percentile: 5 and 7 months). Food sensitization was evaluated based on food-specific IgE titers (egg white, milk, and wheat), which were determined using the capsulated hydrophilic carrier polymer-radioallergosorbent test. Furthermore, a dot-blotting assay for BADGE-specific IgG and quantitative reverse-transcriptase PCR for IL-6, IL-8, IL-10, and COX-2 mRNA expression were conducted.ResultsBADGE-specific IgG was detected in 20% of study subjects. A significant association was observed between the presence of BADGE-specific IgG and elevated wheat-specific IgE levels (OR = 3.56; 95% CI 1.13–11.2; P = 0.031). This relationship was particularly strong in girls (OR = 9.46; 95% CI 1.01–89.0; P = 0.049). A slight but non-significant association was noted between the presence of BADGE-specific IgG and elevated milk-specific IgE levels (OR = 2.77; 95% CI 0.93–8.22; P = 0.067). The expression of IL-6 mRNA among children with BADGE-specific IgG tended to increase, along with wheat-specific IgE levels.ConclusionBADGE exposure might enhance food sensitization in early childhood. Therefore, this should be strictly regulated, especially in younger children.