scholarly journals Characterization of an Antibody Depletion Assay for Analysis of Bactericidal Antibody Specificity

2009 ◽  
Vol 16 (12) ◽  
pp. 1789-1795 ◽  
Author(s):  
Wendell D. Zollinger ◽  
Elizabeth E. Moran ◽  
Deborah H. Schmiel
Keyword(s):  
Protective Immunity ◽  
Vaccine Development ◽  
Solid Phase ◽  
Soluble Antigen ◽  
Inhibition Assay ◽  
Direct Inhibition ◽  
Test Mixture ◽  
Bactericidal Antibody ◽  
Soluble Immune Complexes

ABSTRACT Serum bactericidal antibodies are important for protection against systemic Neisseria meningitidis infections. Consequently, identifying the specific targets of bactericidal antibodies is important for understanding protective immunity to meningococcal disease and for vaccine development and evaluation. We have developed a new assay that can be used to investigate the specificity of serum bactericidal antibodies. Prior to testing for bactericidal activity, antibodies specific for a given antigen or group of antigens are depleted from a serum sample by incubation with the antigen(s) bound to the wells of a 96-well microplate. A dilution series of the antigen is bound to the plate to assess the effectiveness of the antigen in removing the bactericidal antibodies. Removal of antibodies with solid-phase antigen prior to bactericidal testing avoids depletion of complement by soluble immune complexes that can form when soluble antigen is present in the bactericidal test mixture (direct inhibition). The parameters associated with this assay are investigated and compared with those associated with a direct-inhibition assay. The bactericidal depletion assay can be an effective tool for studying the specificity of serum bactericidal antibodies.

scholarly journals The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 257
Author(s):  
Rachel Tanner ◽  
Emily Hoogkamer ◽  
Julia Bitencourt ◽  
Andrew White ◽  
Charelle Boot ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Protective Immunity ◽  
Vaccine Development ◽  
Autologous Serum ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Vaccine Candidates ◽  
Mycobacterial Growth

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

scholarly journals The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 257
Author(s):  
Rachel Tanner ◽  
Emily Hoogkamer ◽  
Julia Bitencourt ◽  
Andrew White ◽  
Charelle Boot ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Protective Immunity ◽  
Vaccine Development ◽  
Autologous Serum ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Vaccine Candidates ◽  
Mycobacterial Growth

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

TEM characterization of Au/Polysilicon interactions

1990 ◽  
Vol 48 (4) ◽  
pp. 650-651
Author(s):  
N. David Theodore ◽  
Leslie H. Allen ◽  
C. Barry Carter ◽  
James W. Mayer
Keyword(s):  
Solid Phase ◽  
Single Crystal Silicon ◽  
Chemical Vapor ◽  
Electrical Contacts ◽  
Silicon Substrates ◽  
Crystal Silicon ◽  
Transmission Electron ◽  
Polysilicon Films ◽  
The Stability

Metal/polysilicon investigations contribute to an understanding of issues relevant to the stability of electrical contacts in semiconductor devices. These investigations also contribute to an understanding of Si lateral solid-phase epitactic growth. Metals such as Au, Al and Ag form eutectics with Si. reactions in these metal/polysilicon systems lead to the formation of large-grain silicon. Of these systems, the Al/polysilicon system has been most extensively studied. In this study, the behavior upon thermal annealing of Au/polysilicon bilayers is investigated using cross-section transmission electron microscopy (XTEM). The unique feature of this system is that silicon grain-growth occurs at particularly low temperatures ∽300°C).Gold/polysilicon bilayers were fabricated on thermally oxidized single-crystal silicon substrates. Lowpressure chemical vapor deposition (LPCVD) at 620°C was used to obtain 100 to 400 nm polysilicon films. The surface of the polysilicon was cleaned with a buffered hydrofluoric acid solution. Gold was then thermally evaporated onto the samples.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

Immunochemical Characterization of Setaria cervi Microfilarial Antigens Using Novel Antibodies

2019 ◽  
Vol 19 (14) ◽  
pp. 1263-1274 ◽  
Author(s):  
Anuradha Kalani ◽  
Komal Kalani ◽  
Poonam Chaturvedi ◽  
Pankaj Chaturvedi
Keyword(s):  
Adult Worm ◽  
Diagnostic Tests ◽  
Protective Immunity ◽  
Complex Nature ◽  
Immune Recognition ◽  
Setaria Cervi ◽  
Crossed Immunoelectrophoresis ◽  
Study Results ◽  
Immune Rabbit

Background:Filariasis affects millions of people in tropical and subtropical regions of the world and is caused by nematode roundworm. In order to develop a vaccine and specific diagnostic tests, it is important to characterize different stages of the filarial worms. Microfilariae (Mf) stage of the roundworm is found in host’s blood or lymph vessels and can be important not only for developing better immunodiagnostics but also for understanding immune recognition and its relevance to immunepathogenesis and protective immunity.Objective:The present study aimed to immunocharacterize Mf and adult worm antigens that could be helpful in future diagnostic tests.Method:Four different immune sera against Setaria cervi intact live, intact live with adjuvant, intact glutaraldehyde fixed with adjuvant and total somatic Mf were prepared and used for the immunocharacterization of Mf antigens.Results:Our study results suggest that compared to fixed intact Mf, live intact Mf are more immunogenic, as the immune sera generated against intact live Mf showed high ELISA reactivity with Setaria cervi Mf and adult worm antigens. All the four immune sera IgG fractions had surface specificity as determined through considerable ELISA reactivity with S. cervi intact Mf. When tested under native conditions (immunoelectrophoresis and crossed immunoelectrophoresis), all the four immune rabbit sera were able to detect antigens of S. cervi Mf and adult stages.Conclusion:These results can be useful in detailed understanding of the complex nature of the Mf and adult antigens, which are prerequisites in the development of vaccine and more specific diagnostic tests.

scholarly journals Characterization of Volatile Organic Compounds in Mango Ginger (Curcuma amada Roxb.) from Myanmar

Metabolites ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 21
Author(s):  
Yanhang Chen ◽  
Musavvara Kh. Shukurova ◽  
Yonathan Asikin ◽  
Miyako Kusano ◽  
Kazuo N. Watanabe
Keyword(s):  
Solid Phase ◽  
Species Variation ◽  
Mango Ginger ◽  
Food Industries ◽  
Curcuma Amada ◽  
Alternative Medicines ◽  
Flight Mass Spectrometry ◽  
Volatile Organic ◽  
Loss Of Appetite

Curcuma amada Roxb. (Zingiberaceae), commonly known as mango ginger because its rhizome and foliar parts have a similar aroma to mango. The rhizome has been widely used in food industries and alternative medicines to treat a variety of internal diseases such as cough, bronchitis, indigestion, colic, loss of appetite, hiccups, and constipation. The composition of the volatile constituents in a fresh rhizome of C. amada is not reported in detail. The present study aimed to screen and characterize the composition of volatile organic compound (VOC) in a fresh rhizome of three C. amada (ZO45, ZO89, and ZO114) and one C. longa (ZO138) accessions originated from Myanmar. The analysis was carried out by means of headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-time-of-flight-mass spectrometry (GC-TOF-MS). As a result, 122 VOCs were tentatively identified from the extracted 373 mass spectra. The following compounds were the ten most highly abundant and broadly present ones: ar-turmerone, α-zingiberene, α-santalene, (E)-γ-atlantone, cuparene, β-bisabolene, teresantalol, β-sesquiphellandrene, trans-α-bergamotene, γ-curcumene. The intensity of ar-turmerone, the sesquiterpene which is mainly characterized in C. longa essential oil (up to 15.5–27.5%), was significantly higher in C. amada accession ZO89 (15.707 ± 5.78a) compared to C. longa accession ZO138 (0.300 ± 0.08b). Cis-α-bergamotene was not detected in two C. amada accessions ZO45 and ZO89. The study revealed between-species variation regarding identified VOCs in the fresh rhizome of C. amada and C. longa.

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