Immunofluorescence detection of CPAF in chlamydia-infected cells was carried out as described previously 6 7 . In brief, HeLa cell monolayer was infected with Chlamydia trachomatis L2 for 30 h. The monolayer, after it was fixed with paraformaldehyde (Sigma-Aldrich) and permeabilized with Saponin (Sigma-Aldrich), was costained with Hoechst 32258 (blue), anti-MOMP antibody MC22 (probed with an FITC-conjugated, mouse IgG3-specific secondary antibody), and anti-CPAFn antibody 54b (probed with a Cy3-conjugated, mouse IgG1-specific secondary antibody). Images were acquired individually for each stain in gray using a Cooker digital camera connected to an AX70 Olympus microscope, and the single-color images were merged in frame into the triple-color image using the software Image Pro.
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IgG3
IgG3
IgG3 (Immunoglobulin G3) is a subclass of the IgG antibody family, which plays a crucial role in the human immune response.
This antibody isotype is characterized by its distinct structural and functional properties, including enhanced effector functions and the ability to activate the complement system.
IgG3 is involved in the neutralization of pathogens, opsonization, and the facilitation of antibody-dependent cell-mediated cytotoxicity.
Researcherss studying IgG3 can utilize the PubCompare.ai platform to optimize their research protocols, identify the best methods for enhanced reproducibility and accuracy, and experience the future of scientific discovery.
This antibody isotype is characterized by its distinct structural and functional properties, including enhanced effector functions and the ability to activate the complement system.
IgG3 is involved in the neutralization of pathogens, opsonization, and the facilitation of antibody-dependent cell-mediated cytotoxicity.
Researcherss studying IgG3 can utilize the PubCompare.ai platform to optimize their research protocols, identify the best methods for enhanced reproducibility and accuracy, and experience the future of scientific discovery.
Most cited protocols related to «IgG3»
Antibodies, Anti-Idiotypic
Cells
Chlamydia
Chlamydia trachomatis
Chlorpropamide-Alcohol Flushing
Fingers
Fluorescein-5-isothiocyanate
Fluorescent Antibody Technique
HeLa Cells
hoechst 32258
IgG1
IgG3
Immunoglobulins
Microscopy
Mus
paraform
Reading Frames
Saponin
Stains
Microtitre plates (Immulon 4HBX, Thermo) were coated with recombinant MSP-119.GST (to which antibodies are predominantly of the IgG1 subclass [36 (link)]) or MSP-2.GST (to which antibodies are predominantly IgG3 [36 (link)]) and blocked with 1% (w/v) skimmed milk powder. Samples were assayed as described previously[36 (link),37 (link)] except that coating antigens, test samples and secondary antibody conjugate were each added in a total volume of 50 μl per well. Ten microliters of the antibody-containing eluate of each spot were added to individual wells of the coated and blocked microtitre plate together with 40 μl blocking buffer to give a final concentration of 1:1,000 with respect to the corresponding plasma sample. Each plate included a five-fold dilution series (1:50 to 1:156,250 final dilutions) of a standard African hyperimmune plasma pool. Bound antibodies were detected with either rabbit anti-human-IgG -HRP (Dako, Ely, UK), or sheep-anti-human IgG1 or IgG3-HRPconjugates (The Binding Site, Birmigham, UK) secondary antibodies and developed with o-phenylenediamine-H2O2.
A titration curve was fitted to the ODs obtained for the standard plasma dilutions by least squares minimisation using a three variable sigmoid model and the solver add-in in Excel (Microsoft), assuming an arbitrary value of 1000 Units/ml of antibody against each antigen in the standard pool. OD values for the spot extracts were converted to units/ml using this fitted curve.
Recoveries for blood spots were estimated as follows (full details in Additional file1 ): serum or plasma ODs were converted to concentrations as above, the concentrations were multiplied by a recovery factor and then converted back to 'corrected' ODs – the ODs which would have been obtained if the serum or plasma had been more dilute. The value of the recovery factor was then optimized by weighted least squares minimisation comparing the actual ODs for the blood spots and the corrected OD values for serum or plasma, using the solver add-in in Excel™.
A titration curve was fitted to the ODs obtained for the standard plasma dilutions by least squares minimisation using a three variable sigmoid model and the solver add-in in Excel (Microsoft), assuming an arbitrary value of 1000 Units/ml of antibody against each antigen in the standard pool. OD values for the spot extracts were converted to units/ml using this fitted curve.
Recoveries for blood spots were estimated as follows (full details in Additional file
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1,2-diaminobenzene
anti-IgG
Antibodies
Antigens
Binding Sites
BLOOD
Buffers
Exanthema
factor A
Homo sapiens
IgG1
IgG3
Immunoglobulins
Milk, Cow's
Negroid Races
Peroxide, Hydrogen
Plasma
Powder
Rabbits
Serum
Sheep
Sigmoid Colon
Technique, Dilution
Titrimetry
Genomic DNA was isolated from B cells, either resting or cultured with LPS for 4 d. Cell pellets were incubated with proteinase K (0.5 mg/ml), RNaseA (100 μg/ml), and SDS (0.5%) in STE (0.1 M NaCl, 20 mM Tris, 1 mM EDTA) for 2 h at 37°C, followed by 3–4 extractions with phenol/chloroform (1:1) and precipitation with 0.3 M sodium acetate, pH 7, and ethanol. DNA was wound out on glass rods and resuspended in TE, pH 8. The germline Sγ3 segment was amplified by PCR from resting purified B cells from WT(129 × B6) mice for comparison to Sμ-Sγ3 junctions from cells induced to switch to IgG3. Expand HiFidelity Taq polymerase (Roche Laboratories) was used with the following primers: g3–1 (5′CAGGCTAAGATGGATG- CTACAGGG-3′) (MUSIGHANA 404–427) and g3–2 (5′TAC- CCTGACCCAGGAGCTGCATAAC-3′) (MUSIGHANA 2603–2628) to amplify the 2.22-kb fragment of germline Sγ3. Sμ-Sγ3 junctions were amplified by PCR using Expand Long Template Taq polymerase (Roche Laboratories) and the primers μ3-H3 (5′AACAAGCTTGGCTTAACCGAGATGAGCC-3′) and g3–2 (above). The germline Sμ sequence was deduced by comparing the sequences of a large number of Sμ-Sγ3 junctions from wild-type mice. For the sequence analyses, the wild-type sequences from the corresponding littermates were used.
B-Lymphocytes
Cells
Chloroform
Edetic Acid
Endopeptidase K
Ethanol
Genome
Germ Line
IgG3
Mice, 129 Strain
Mus
Oligonucleotide Primers
Pellets, Drug
Phenols
Rod Photoreceptors
Sequence Analysis
Sodium Acetate
Sodium Chloride
Taq Polymerase
Tromethamine
Wounds
Alexa 350
Anti-Antibodies
anti-IgD
anti-IgM
Antibodies
Antibodies, Anti-Idiotypic
Be1 Cells
Biotin
Bromodeoxyuridine
CD44 protein, human
Cells
Collagenase
CY5.5 cyanine dye
Cytokinesis
Deoxyribonucleases
Flow Cytometry
Fluorescein-5-isothiocyanate
Formaldehyde
IgG1
IgG2B
IgG3
Integrin alpha4beta1
lumiliximab
Lymphocyte Count
Medical Devices
Mus
NT5E protein, human
Saponin
Serum
SPN protein, human
Streptavidin
TNFRSF13B protein, human
Trees
Fig. S1 depicts analyses of lymphocyte development in Vav2ko and Vav3ko mice. Flow cytometry analyses of thymocytes, splenocytes, LN, and BM cells from WT mice (WT), Vav2ko (Vav2), or Vav3ko (Vav3) mice are shown. Fig. S2 shows the strategy for generation of Vav3-deficient mice. Southern blotting analyses of targeted ES cells used to generate chimeric mice and their offspring and Western blotting analyses of Vav3ko splenocyte lysates with Vav3-specific antibodies showing no full-length or truncated Vav3 protein present are also shown. Fig. S3 presents analyses of humoral TD responses in Vav3-deficient mice. Concentrations of TNP-specific IgM, IgG1, IgG2b, and IgG3 antibodies analyzed at various time points post-primary or secondary immunization with TNP-conjugated keyhole hemagglutinin protein (TNP-KLH) are shown. Fig. S4 presents analyses of cytokine production by Vav-deficient T cells showing defective IL-2 and IFNγ production. Figs. S1–4 are available online at http://www.jem.org/cgi/content/full/jem.20030874/DC1 .
Antibodies
Cells
Chimera
Cytokine
Figs
Flow Cytometry
Hemagglutinin
IgG1
IgG2B
IgG3
Interferon Type II
Lymphocyte
Mus
Proteins
Secondary Immunization
Southern Blotting
T-Lymphocyte
Thymocyte
trinitrophenyl keyhole limpet hemocyanin
Western Blot
Most recents protocols related to «IgG3»
First, the 96-well plates were coated with 100 µL of B. abortus A19 LPS (100 μg/mL) and incubated overnight at 4°C. Then, the plates were washed with PBST 3 times, and 200 μL of blocking buffer (5% skim milk in PBST) was added to each well and incubated at 37°C for 2 h. After blocking, sera from each mouse were added to the corresponding wells and serially diluted with dilution buffer (10% blocking buffer) and incubated at 37°C for 1 h. The plates were washed and 100 μL of HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b, or IgG3 antibody (Abcam, Cambridge, MA, United States) (1:15,000) was added to each well and incubated at 37°C for 1 h. After washing, the Soluble TMB Kit (CWBio, Beijing, China) was used for color development, and the absorption at a wavelength of 450 nm was measured using a microplate spectrophotometer.
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anti-IgG
Buffers
Fetuses, Aborted
Goat
IgG1
IgG2A
IgG2B
IgG3
Immunoglobulins
Mice, Laboratory
Milk, Cow's
Serum
Technique, Dilution
Complement proteins FH (#341274), FB (#341262), C3b (#204860), C3 (#204885), factor I (FI) (#341280), C1q (#204876), goat anti-FB (#341272) and goat anti-FH (#341276) antiserum were obtained from Merck (Darmstadt, Germany; obtained via Merck Life Science Kft., Budapest, Hungary). FD (#A409), properdin (#A412), mouse anti-C5b-9 (#A239), anti-FB (Bb) (#A227), anti-FB (Ba) (#A225) and C3a EIA kit (#A032) were from Quidel (obtained via Biomedica, Budapest, Hungary). Purified human IgG (#I2511), human serum albumin (#A3782), human alpha-1 antitrypsin (#SRP6312), antibodies against C1q (#234390), human IgG (#A6029), IgM (#A0420), IgA (#A0295), IgG1 (clone HP-6001, #I9388), IgG2 (clone HP-6002, #I9513), IgG3 (clone HP-6050, #I7260), IgG4 (clone HP-6025, #I7385), IgGκ (clone KP-53, #K4377), IgGλ (clone HP-6054, #L6522), and avidin-HRP (#A7419) were purchased from Merck. Mouse IgG1 (clone MOPC-21, #BZ-400101) was obtained from BioLegend (San Diego, CA). MBL (#9086-MB) and biotin-conjugated goat anti-MBL (#BAF2307) were from R&D Systems (Minneapolis, MN), HRP-conjugated goat anti-mouse (#P0447), rabbit anti-goat (#P0449) and swine anti-rabbit (#P0217) antibodies were from DAKO (Hamburg, Germany). Goat anti-C3 F(ab’)2 (#55062) and HRP-conjugated goat anti-C3 F(ab’)2 (#55237) were from MP Biomedicals (Solon, OH).
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Antibodies
avidin-horseradish peroxidase complex
Biotin
Clone Cells
Complement Membrane Attack Complex
Complement System Proteins
Fibrinogen
Goat
Homo sapiens
IgG1
IgG2
IgG3
IgG4
Immune Sera
Mus
Properdin
Rabbits
SERPINA1 protein, human
Serum Albumin, Human
Solon
Sus scrofa
Autoantibodies were detected using ELISA method, as described by Józsi and Uzonyi (36 (link)). Briefly, FH, FB, C3b, FI in 5 µg/ml, MBL and C1q in 2 µg/ml and HSA or alpha-1 antitrypsin as negative controls were immobilized on microtiter plates. After blocking with 5% BSA in DPBS-0.1% Tween-20, serum samples were added in DPBS-0.1% Tween-20 at a dilution of 1:50 for 1 hour at RT. To detect autoantibodies against solid phase C3 convertase of the alternative pathway (C3bBbP), the convertase was built up (see below) and incubated with serum samples diluted 1:50. The bound autoantibodies were detected with HRP-conjugated goat anti-human IgG, anti-human IgA or anti-human IgM. Color reaction was developed by adding TMB solution (BioLegend). After stopping with H2SO4, absorbance was measured at 450 nm and at 620 nm as reference wavelength. To detect anti-MBL autoantibodies, serum samples were diluted in DPBS containing 10 mM EDTA. In the case of detecting anti-C1q autoantibodies, serum samples were diluted in DPBS containing 1 M NaCl to avoid interaction of C1q globular heads with IgG Fc parts. Samples were accepted as positive if the mean OD value for the investigated protein was at least two-fold greater than the mean OD value for the negative control protein alpha-1 antitrypsin. The isotypes of the autoantibodies were determined using monoclonal antibodies specific for IgG1, IgG2, IgG3, IgG4, IgGκ and IgGλ.
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anti-IgA
anti-IgG
anti-IgM
Autoantibodies
Complement C3 Convertase, Alternative Pathway
Edetic Acid
Enzyme-Linked Immunosorbent Assay
Goat
Head
Homo sapiens
IgG1
IgG2
IgG3
IgG4
Immunoglobulin Isotypes
Monoclonal Antibodies
Proteins
SERPINA1 protein, human
Serum
Sodium Chloride
Technique, Dilution
Tween 20
For isolation of IPF and control MPCs, primary IPF mesenchymal cells were labeled with mouse anti–human SSEA4 antibody conjugated to Alexa Fluor 647 (clone MC-813-70; catalog 560796; BD Biosciences) and mouse anti–human CD44 conjugated to FITC (clone IM7; catalog 103006; BioLegend). Cells were sorted on a FACS Aria Cell Sorter (BD Biosciences). Cells that were SSEA4+ and CD44+ (relative to mouse IgG3 κ isotype control conjugated to Alexa Fluor 647 and mouse IgM κ isotype control conjugated to FITC, respectively) (clone J606, catalog 560803 [BD Biosciences] and catalog 402207 [BioLegend]) were collected. To generate sufficient numbers of MPCs for the in vivo mouse studies, SSEA4+CD44+ cells were expanded by culture in DMEM + 10% FCS for 7 days prior to use. The resulting MPC cultures were reanalyzed for SSEA4 expression by FACS analysis and for colony formation in vitro. In total, 97% of day 7 MPCs were SSEA4+ and formed colonies in methylcellulose indicating retention of progenitor self-renewal properties during in vitro expansion.
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Alexa Fluor 647
CD44 protein, human
Cells
Clone Cells
Fluorescein-5-isothiocyanate
Homo sapiens
IgG3
Immunoglobulin Isotypes
Immunoglobulins
isolation
Mesenchymal Stem Cells
Methylcellulose
Mus
NRG1 protein, human
Retention (Psychology)
stage-specific embryonic antigen-4
Standardized ELISAs were used to quantify serum RH5.1–specific IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, and IgM responses in vaccinees. Nunc MaxiSorp flat-bottom ELISA plates (44-2404-21, Invitrogen) were coated overnight with 5 μg/mL of RH5.1 protein in PBS. Plates were washed with washing buffer composed of PBS containing 0.05% TWEEN 20 (P1379, Sigma-Aldrich) and blocked with 100 μL of Blocker Casein in PBS (37582, Thermo Fisher Scientific). After removing blocking buffer, standard curve and internal controls were created in casein using a pool of high-titre volunteer plasma, specific for each isotype or subclass being tested, and 50 μL of each dilution was added to the plate in duplicate. Test samples were diluted in casein to a minimum dilution of 1:50, and 50 μL was added in triplicate. Plates were incubated for 2 hours at 37°C and washed in washing buffer. An alkaline phosphatase–conjugated secondary antibody from Southern Biotech was diluted at the manufacturer’s recommended minimum dilution for ELISA in casein. The antibody used was dependent on the isotype or subclass being assayed and were as follows: mouse anti–human IgG1 Fc-AP (catalog 9054-04), IgG2 Fc-AP (catalog 9060-04), IgG3 hinge-AP (catalog 9210-04), IgG4 Fc-AP (catalog 9200-04), IgA1-AP (catalog 9130-04), and IgA2-AP (catalog 9140-04) and goat anti–human IgA-AP (catalog 2050-04) and IgM-AP (catalog 2020-04). In total, 50 μL of the secondary antibody dilution was added to each well of the plate and incubated for 1 hour at 37°C. Plates were developed using PNPP alkaline phosphatase substrate (N2765, Sigma-Aldrich) for 1–4 hours at 37°C. Optical density at 405 nm was measured using an ELx808 absorbance reader (BioTek) until the internal control reached an OD405 of 1. The reciprocal of the internal control dilution giving an OD405 of 1 was used to assign an AU value of the standard. Gen5 ELISA software v3.04 (BioTek) was used to convert the OD405 of test samples into AU values by interpolating from the linear range of the standard curve fitted to a 4-parameter logistics model. Any samples with an OD405 below the linear range of the standard curve at the minimum dilution tested were assigned a minimum AU value according to the lower limit of quantification of the assay.
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4-aminophenylphosphate
Alkaline Phosphatase
anti-IgA
ATP8A2 protein, human
Biological Assay
Buffers
Caseins
Enzyme-Linked Immunosorbent Assay
Goat
Homo sapiens
IgA1
IgA2
IgG1
IgG2
IgG3
IgG4
Immunoglobulin Isotypes
Immunoglobulins
Mus
Plasma
Serum
Technique, Dilution
Tween 20
Vision
Voluntary Workers
Top products related to «IgG3»
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IgG2b is a type of immunoglobulin G (IgG) subclass that is commonly used in laboratory research and applications. It functions as an antibody, playing a role in the immune system's response to foreign substances. IgG2b has specific structural and functional characteristics that make it a valuable tool for various experimental and analytical purposes.
Sourced in United States
IgG2c is a type of immunoglobulin G (IgG) antibody. It is a laboratory product that can be used for various research and analytical applications involving the detection and quantification of IgG2c in biological samples.
Sourced in United States
IgG2a is an immunoglobulin subclass that functions as an antibody. It is produced by plasma B cells and plays a role in the adaptive immune response.
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Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood plasma. It is a protein that serves as a stabilizer and blocking agent in various biochemical and immunological applications. BSA is widely used to maintain the activity and solubility of enzymes, proteins, and other biomolecules in experimental settings.
Sourced in United States
IgG2a is a type of immunoglobulin G (IgG) antibody. It is a component of the adaptive immune system and plays a role in the humoral immune response. IgG2a is involved in the neutralization of toxins and viruses, as well as the activation of the complement system.
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The FACSCalibur is a flow cytometry system designed for multi-parameter analysis of cells and other particles. It features a blue (488 nm) and a red (635 nm) laser for excitation of fluorescent dyes. The instrument is capable of detecting forward scatter, side scatter, and up to four fluorescent parameters simultaneously.
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IgG2b is an immunoglobulin subclass that plays a role in the immune system. It is a component of the antibody response. IgG2b has core functions related to immune system processes, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
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The LPS laboratory equipment is a high-precision device used for various applications in scientific research and laboratory settings. It is designed to accurately measure and monitor specific parameters essential for various experimental procedures. The core function of the LPS is to provide reliable and consistent data collection, ensuring the integrity of research results. No further details or interpretations can be provided while maintaining an unbiased and factual approach.
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The FACSCanto II is a flow cytometer instrument designed for multi-parameter analysis of single cells. It features a solid-state diode laser and up to four fluorescence detectors for simultaneous measurement of multiple cellular parameters.
Sourced in Denmark, United States, United Kingdom
ELISA (Enzyme-Linked Immunosorbent Assay) plates are a type of laboratory equipment used for performing enzyme-linked immunosorbent assays. These plates typically consist of a flat surface with multiple wells, allowing for the simultaneous analysis of multiple samples or conditions. The wells are designed to facilitate the binding of specific proteins or other molecules to the plate surface, enabling the detection and quantification of target analytes in the samples.
More about "IgG3"
Immunoglobulin G3 (IgG3) is a crucial subtype of the IgG antibody family, playing a vital role in the human immune response.
This antibody isotype is characterized by its distinct structural and functional properties, including enhanced effector functions and the ability to activate the complement system.
IgG3 is involved in the neutralization of pathogens, opsonization, and the facilitation of antibody-dependent cell-mediated cytotoxicity (ADCC).
Researchers studying IgG3 can leverage the power of the PubCompare.ai platform to optimize their research protocols and identify the best methods for enhanced reproducibility and accuracy.
This AI-driven platform compares protocols from literature, pre-prints, and patents, helping researchers locate the ideal IgG3 research procedures with ease and confidence.
In addition to IgG3, researchers may also be interested in exploring other IgG subtypes, such as IgG2b, IgG2c, and IgG2a.
These antibody isotypes exhibit distinct functional properties and can play important roles in various immune responses.
Furthermore, researchers may utilize bovine serum albumin (BSA) as a blocking agent in IgG3 assays, such as flow cytometry using a FACSCalibur or FACSCanto II instrument, or in enzyme-linked immunosorbent assays (ELISAs) on ELISA plates.
Understanding the role of IgG3 and its interactions with other immune components, like lipopolysaccharide (LPS), can provide valuable insights into the complexities of the human immune system.
By optimizing their research protocols and leveraging the insights gained from the PubCompare.ai platform, researchers can advance their understanding of IgG3 and its implications in various disease states and therapeutic interventions.
This antibody isotype is characterized by its distinct structural and functional properties, including enhanced effector functions and the ability to activate the complement system.
IgG3 is involved in the neutralization of pathogens, opsonization, and the facilitation of antibody-dependent cell-mediated cytotoxicity (ADCC).
Researchers studying IgG3 can leverage the power of the PubCompare.ai platform to optimize their research protocols and identify the best methods for enhanced reproducibility and accuracy.
This AI-driven platform compares protocols from literature, pre-prints, and patents, helping researchers locate the ideal IgG3 research procedures with ease and confidence.
In addition to IgG3, researchers may also be interested in exploring other IgG subtypes, such as IgG2b, IgG2c, and IgG2a.
These antibody isotypes exhibit distinct functional properties and can play important roles in various immune responses.
Furthermore, researchers may utilize bovine serum albumin (BSA) as a blocking agent in IgG3 assays, such as flow cytometry using a FACSCalibur or FACSCanto II instrument, or in enzyme-linked immunosorbent assays (ELISAs) on ELISA plates.
Understanding the role of IgG3 and its interactions with other immune components, like lipopolysaccharide (LPS), can provide valuable insights into the complexities of the human immune system.
By optimizing their research protocols and leveraging the insights gained from the PubCompare.ai platform, researchers can advance their understanding of IgG3 and its implications in various disease states and therapeutic interventions.