Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay

Schnabel, Christiane L.; Babasyan, Susanna; Freer, Heather; Wagner, Bettina

doi:10.1016/j.vetimm.2017.04.001

Cited by 11 publications

(7 citation statements)

References 30 publications

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“…As relative standards, serum of pre-screened sera from three horses with high tetanus toxoid (TT)-binding serum antibody titers was pooled. From this serum pool immunoglobulins (Ig) were purified with Protein G as previously described ( 35 , 36 ). Briefly, serum was diluted in sodium phosphate buffer and Ig allowed to bind to a HiTrap Protein G column (Cytvia, Freiburg, Germany) on an ÄKTA pure FPLC instrument (Cytvia).…”

Section: Methodsmentioning

confidence: 99%

CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses

et al. 2022

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Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. T cells were analyzed by multicolor flow cytometry of PBMC after re-stimulation with TT in vitro. Reactive T helper (Th) cells were characterized by increased frequencies of CD4+CD154+ lymphocytes in in vitro TT-re-stimulated PBMC on day 14 after vaccination of the horses compared to pre-vaccination. The majority of all CD154+ cells after TT re-stimulation were CD4+ Th cells, but CD154 was also induced on CD4- cells albeit in lower frequencies. CD154+CD4+ Th cells were enriched in cytokine-expressing cells compared to CD154-CD4+ Th cells. Similar to the CD4+CD154+ frequencies, CD4+IL-4+, CD4+IFN-γ+ and CD4+TNF-α+ were increased after vaccination, but IL-4+ increased later than IFN-γ+ and CD4+TNF-α+, which already exceeded pre-vaccination frequencies on day 7. CD4+CD154+ frequencies correlated positively with those of CD4+IL-4+ (Th2) on day 14, and negatively with CD4+IFN-γ+ induction on day 7, but did not correlate with CD4+TNF-α+ frequencies or TT-specific antibody concentrations. CD154 appears to be a useful marker of antigen-reactive equine Th cells in combination with cytokine expression. The T cell analyses established here with TT can be applied to other antigens relevant for infections or allergies of horses and in horse models for translational research.

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Section: Methodsmentioning

confidence: 99%

CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses

et al. 2022

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“… A Jackson ImmunoResearch #108–035-003; B ( 59 ), C ( 60 , 61 ), D ( 62 ), E ( 63 ), F ( 64 , 65 ). G All det.…”

Section: Methodsmentioning

confidence: 99%

“…Total contents of Ig isotypes were quantified by bead-based assays as previously described for serum (62,64,66). Briefly, IgM,…”

Section: Total Immunoglobulin Isotype Contents In Balf and Serummentioning

confidence: 99%

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma

Jentsch,

Keilhaue,

Wagner

et al. 2024

Front. Immunol.

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IntroductionEquine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF).MethodsSerum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A. fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays.ResultsMEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig.DiscussionA. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis.

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“…Total contents of Ig isotypes were quantified by bead-based assays as previously described for serum (62,64,66). Briefly, IgM,…”

Section: Total Immunoglobulin Isotype Contents In Balf and Serummentioning

confidence: 99%

“…IgG1, IgG3/5, IgG4/7, and IgG6 were quantified in a multiplexed assay in undiluted BALF or 1:50,000 diluted serum, with isotypespecific monoclonal antibodies coupled to different beads (compare detection antibodies in Table 3) and detection by polyclonal biotinylated goat-anti-horse-Ig (H+L) (62). IgA and IgE were quantified in undiluted BALF or 1:10 diluted serum using pairs of monoclonal antibodies coupled to the beads and biotinylated for detection (64,66). After incubation with R-phycoerythrinconjugated streptavidin (Invitrogen, Carlsbad, CA, USA), the total content of each isotype was quantified as median fluorescent intensity (MFI) on a BioPlex200 instrument (Bio-Rad).…”

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Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay

Cited by 11 publications

References 30 publications

CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses

CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma

DataSheet_1.pdf

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