Quantification Of Immunoglobulin Research Articles

A linear-amplification VDJ-seq technique for quantification of immunoglobulin and T cell receptor diversity.

The V(D)J recombination is essential for generating a highly diverse repertoire of antigen receptors expressed on T and B lymphocytes. Here, we developed a linear-amplification VDJ-seq technique for quantifying V(D)J recombination of antigen receptor genes. This technique takes advantage of linear amplification using in vitro transcription and reverse transcription to avoid bias generated by the PCR amplification of low copy number of target DNA. The unrearranged alleles are removed by in vitro cleavage with the CRISPR-Cas9 system. The linear-amplification VDJ-seq assay was applied in quantification of the Vκ-Jκ recombination of the mouse Igκ gene with Jκ capture primers. The Jκ genes were detected in 95.86% of clean reads with more than half containing the Vκ gene, indicating high specificity of capturing and amplification. We also applied this approach to quantify the usage of Jα within the Trav12 gene family of the Tcra gene.

Bimodal determination of immunoglobulin E by fluorometry and ICP-MS by using platinum nanoclusters as a label in an immunoassay.

The authors describe the use of platinum nanoclusters (PtNCs) as bimodal labels in a competitive immunoassay for immunoglobulin E (IgE). Both fluorometry and inductively coupled plasma - mass spectrometry (ICP-MS) are used. Optimization of the PtNCs synthesis using lipoic acid as ligand was carried out. The time for synthesis and the effect of NaOH added to the PtNCs precursor mixture was optimized with the aim to obtain PtNCs with strong red fluorescence and low size dispersity. Maximal fluorescence was obtained at excitation/emission wavelengths of 455/620nm. The average diameter (1.5nm) and crystal structure (face-centered cubic structure) of the PtNCs were determined by HR-TEM. It was calculated that each PtNC contains 116 Pt atoms at average. Labelling of the antibody (Ab) against IgE with PtNCs was optimized in terms of recognition capabilities and fluorescence intensity. A molar ratio (Ab:PtNCs) of 1:11 is found to be best. A competitive immunoassay for IgE was developed and detection was carried out by using both ICP-MS (by measuring 195Pt) and fluorometry. The limit of detection (LOD) of the fluoroimmunoassay is 0.6ngmL-1 of IgE. The LOD of the ICP-MS method is as low as 0.08ngmL-1. The method was evaluated by analyzing four (spiked) serum samples by ICP-MS. No sample pretreatment excepting dilution is needed. Results compared favorably with those obtained by a commercial ELISA kit. Graphical abstract Schematic representation of the bimodal quantification (fluorescence and ICP-MS) of immunoglobulin E (Ig E) in human serum using antibody against human Ig E, labelled with several platinum nanoclusters (NCs) as immunoprobe. Elemental mass spectrometry (MS) allows high amplification of the signal because of the high number of platinum atoms per nanocluster (~116 Pt/NC).

Hypertension: An immune related disorder?

Hypertension is a multifactorial disorder with serious complications and unknown etiology. Among potential contributors, immune dysregulation has been also proposed. The study population included 61 consecutive hypertensive patients and 55 healthy individuals of similar age and sex distribution. All study participants underwent a thorough evaluation for subclinical atherosclerosis. A full immunological profile including quantification of immunoglobulins (IgG, IgM, IgA) and lymphocyte subpopulations was also obtained. Immunoglobulin levels IgG, IgA and IgM and complement factor C3 were found to be significantly increased in the hypertensive compared to the HC group while a statistically significant decrease in peripheral blood CD3+, CD4+ and CD8+ in hypertensive patients versus controls was detected. These findings might support a putative involvement of altered cellular and humoral immune responses in the pathogenesis of hypertension, implying a promising role for immunomodulatory strategies, already implemented in the treatment of autoimmune diseases, in the future management of hypertension.

Development of a polyclonal antibody for detection and sensitive quantification of immunoglobulin M-like antibody in Pangasius hypophthalmus plasma

Immunoglobulins (Igs) are an essential defense mechanism in fish, yet it is difficult to reliably quantify these components because a lack of standardization in methodology, and limited commercially available reagents. In this study, a polyclonal antibody was developed for the specific detection and quantification of IgM-like immunoglobulin in the plasma of striped catfish (Pangasius hypophthalmus). The detection probe was prepared by initial isolation and characterization of the IgM-like isotype from the fish species. An SDS-PAGE analysis of the purified striped catfish antibody revealed a heavy and a light chain with molecular weights of 70 and 25 kDa, respectively, and thus an estimated size of at least 760 kDa for the tetrameric form of this antibody isotype. This fish antibody was then utilized for immunizing New Zealand white rabbits, followed by affinity chromatography and biotinylation of the rabbit anti-pangasius Ig. The polyclonal rabbit anti-pangasius antibody was strongly reactive towards fish of the genus Pangasius, and had a weaker degree of reactivity towards two members of the genus Ictalurus. The polyclonal antibody was utilized for developing a sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of plasma levels of the IgM-like molecule in striped catfish. The reference interval (RI) for this immune analyte had a range of 1.2–7.6 mg mL−1, and a median value of 3.3 mg mL−1. The ELISA methodology presented here, although not novel, it is new for fish. Implementation of these methods will help in efforts to reliably quantify the levels of total immunoglobulin in other species of fish, without the background measurements often seen with other fish detection antibodies. Knowledge of the immunoglobulin titers is also relevant, as it is the first report of the RI for this immunological analyte in striped catfish cultured in recirculating aquaculture systems. The normal physiology findings of this study will aid in monitoring changes in the health status of striped catfish, when cultured in different systems, and when the animals undergo different types of stress events (e.g. vaccination, biological insult, chemical exposure).

Immunocrit, serum amino acid concentrations and growth performance in light and heavy piglets depending on sow\u2019s farrowing system

BackgroundThe conventional farrowing crate is criticised due to the limited mobility of sows during farrowing and lactation. The present study aims to investigate the effects of three different farrowing systems on the performance of suckling neonates on the basis of immunocrit (IC; a quantification of immunoglobulins), serum amino acid (AA) concentrations and growth performance.MethodsFrom a total of 149 sows placed in three housing systems (farrowing crate – FC, loose housing – LH, group housing – GH), 18 sows and their respective litters, formed the basis for a two-factorial study design (farrowing system and body weight (BW) of neonates). Therefore, also blood samples of two light (1.0–1.4 kg) and two heavy (≥ 1.4 kg) piglets were taken within 48 h post natum (p.n.) and on the day of weaning (day 26) to determine the immunocrit (IC; a quantification of immunoglobulins) and levels of serum AAs.ResultsThe IC (FC: 0.148a, LH: 0.153a, GH: 0.117b) as well as serum levels of arginine, leucine, lysine, proline and threonine within 48 h p.n. were significantly lower in GH. Additionally, in general, these piglets showed (except for the first week of life) the lowest average daily weight gain. On the day of weaning, piglets in GH had the lowest levels of arginine (in mg/dL; FC: 3.68a, LH: 3.40ab, GH: 2.94b) and threonine (in mg/dL; FC: 3.59a, LH: 3.02ab, GH: 2.49b). The concentrations of leucine, lysine, proline and valine at this time were significantly lower in LH.ConclusionThe observed significant lower IC indicates a lower Ig intake of piglets in the tested GH. No significant differences regarding the IC and AA levels within 48 h p.n. of the piglets in FC and LH could be seen. In principle, differences at weaning in AA levels were rather small, although the body weight of GH piglets at weaning was lower. Therefore, further research needs to clarify whether there are medium-term effects on health and performance.

Evaluation of the concordance of three methods for the quantification of total immunoglobulin G with the sum of the individuals immunoglobulin g subclasses

Evaluation of the concordance of three methods for the quantification of total immunoglobulin G with the sum of the individuals immunoglobulin g subclasses

Systemic Vasculitis and High IgE Level in a Patient with LPS-Responsive Beige-Like Anchor (LRBA) Deficiency

Background: Mutations in LPS-responsive and beige-like anchor (LRBA) gene in patients were firstly described to associate with a syndrome of immune deficiency and autoimmunity in 2012. However, there was still no LRBA deficient patient reported in China. Objectives: We present a Chinese patient with heterozygous LRBA gene mutations with his clinical, immunological and genetic features. Methods: Patient’s clinical data was collected and analyzed. Laboratory results included lymphocyte subsets analysis and immunoglobulin quantification. Targeted gene capture followed by next-generation sequencing was used to identify the gene mutations, and flow cytometry assay was used to analyze B cell immunophenotyping of this patient. Results: The patient mainly suffered from recurrent respiratory tract infections, EBV-associated lymphoproliferative disease and systemic vasculitis. Heterozygous LRBA gene mutations were identified in the patient, which were inherited from his parents, respectively. B cell immunophenotyping revealed that he had decreased total B cells, bone marrow progenitor B cell subsets (HSC, CLP, Pro-B and Pre-B cells), immature B cells, non-switched memory B cells, switched memory B cells and B1 cell subsets. Besides, he had extremely high level of IgE. Conclusions: Herein, we firstly report a patient with heterozygous LRBA gene mutations in China. LRBA might play an important role in B cell development.

Effects of plant features on symptoms and airway inflammation in compost workers followed over 18 months

This study investigated the plant features associated with increased irritation symptoms and levels of inflammation markers among compost workers (CWs). Ninety CWs were followed over 18 months, using questionnaires on respiratory symptoms, fractional exhaled nitric oxide measurements, spirometry, a methacholine bronchial challenge test, and quantification of specific immunoglobulins E (IgE) and G. CWs in plants processing the highest quantities of waste exhibited more airway irritation symptoms. So did the CWs in partially and fully indoor plants as compared to those in plants entirely outdoors. Working in sewage sludge versus green waste plants and having a high level of exposure were associated with higher levels of different IgE. The duration of employment decreased the FEV1 by 16 ml per year. Working in an indoor plant is linked to symptoms and inflammation markers in CWs.

Quantification of Immunoglobulin G against Trypanosoma cruzi in Individuals with Chronic Chagas Disease Treated with Nifurtimox and Evaluated in Prolonged Follow-Up.

In the indeterminate chronic period of Chagas disease (ChD) the treatment has not been conclusive, because the serological negativization requires many years. This study aims to evaluate the efficacy of nifurtimox (NF) in the treatment of chronic ChD in prolonged follow-up by serological techniques of indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) IgG comparing 2 groups of patients, treated and non treated. Mann-Whitney test was performed for ELISA and IFA, with significant difference between the groups (P<0.05). IgG levels were lower in individuals treated compared with untreated patients, indicating chemotherapeutic efficacy in prolonged follow-up.

Multiplex Assay for Quantification of Acute Phase Proteins and Immunoglobulin A in Dried Blood Spots.

Inflammation is the first line defense mechanism against infection, tissue damage, or cancer development. However, inappropriate inflammatory response may also trigger diseases. The quantification of inflammatory proteins is essential to distinguish between harmful and beneficial immune response. Currently used immunoanalytical assays may suffer specificity issues due to antigen-antibody interaction and possible cross-reactivity of antibody with other protein species. In addition, immunoanalytical assays typically require invasive blood sampling and additional logistics; they are relatively costly and highly challenging to multiplex. We present a multiplex assay based on selected reaction monitoring (SRM) for quantification of seven acute-phase proteins (i.e., SAA1, SAA2-isoform1, SAA4, CRP, A1AT-isoform1, A1AG1, A1AG2) and the adaptive immunity effector IGHA1 in dried blood spots. This type of sample is readily available from all human subjects including newborns. The study utilizes proteotypic isotopically labeled peptides with trypsin-cleavable tag and presents optimized and reproducible workflow and several important practical remarks regarding quantitative SRM assays development. The panel of inflammatory proteins was quantified with sequence specificity capable to differentiate protein isoforms with intra- and interday precision (<16.4% coefficient of variation (CV) and <14.3% CV, respectively). Quantitative results were correlated with immuno-nephelometric assay (typically greater than 0.9 Pearson's R).

Immunoglobulinopathies in patients with angioimmunoblastic T-cell lymphoma.

The aim of the study was to characterize quantitative and qualitative immunoglobulinopathies in patients with AITL at the onset of the disease. 55 patients with newly diagnosed AITL were enrolled in the study, the male/female ratio was 30/25; median age was 61 (29-81) years. Diagnosis was based on standard WHO criteria. Immunochemical studies of blood serum included serum protein electrophoresis/immunofixation, nephelometric quantification of total immunoglobulins, serum free light chain assay. Quantitative and qualitative immunoglobulinopathies were determined in 49 (89,1%) of 55 pts. Quantitative immunoglobulinopathies were revealed in 47 (85.5%) of 55 cases, qualitative - in 14 (25,5%). Combination quantitative and qualitative immunoglobulinopathies was observed in 12 (21,8%) of 55 pts. The detected immunoglobulinopathies were divided into 4 groups: polyclonal hypergammaglobulinaemia, hypogammaglobulinaemia, oligoclonal gammapathy, and monoclonal gammapathy. Polyclonal hypergammaglobulinaemia was marked in 41 (74.5%) of 55 pts, elevated level of IgG was determined in 27 (49,15%) of 55 cases, IgM - in 18 (32,7%) and IgA - in 21 (38.2%). Interestingly, polyclonal IgE hypergammaglobulinaemia was detected in 12 (48,0%) of 25 cases of performed studies. Hypogammaglobulinaemia was detected in 8 (14,5%) of 55 cases. Oligoclonal gammapathy was determined in 4 (7.3%) of 55 pts. Monoclonal gammapathy was revealed in 11 (20,0%) of 55 cases. The amount of monoclonal immunoglobulin varied from 2.6 to 14.1 g/l. Monoclonal immunoglobulin Gk was detected in 5 of 11 pts, Gλ - in 2, Mλ - in 2, Mk - in 2. Monoclonal gammapathy was accompanied by polyclonal hypergammaglobulinaemia in 9 of 11 cases, hypogammaglobulinaemia - in 2. Quantitative and qualitative immunoglobulinopathies are observed in most patients at the onset of AITL. Quantitative abnormalities were determined more often than qualitative. Monoclonal gammapathy can be a manifestation of lymphoproliferation and other concomitant disorders. The prognostic value of immunochemical parameters is still unclear and requires dynamic observation and study.

Comparación de dos métodos para la cuantificación en suero de subclases de inmunoglobulinas

Background and aimThe quantification of serum immunoglobulins, and particularly of IgG and IgG subclasses, is of interest for the diagnosis of numerous diseases. The usual detection methods provide different results according to the analyser used. The aim of this study was to compare the results obtained with two different analysers in the measurement of the concentration of IgG and IgG subclasses. Material and methodsA total of 116 serum samples, regardless of the clinical diagnosis of the patients to whom the samples belonged, were analysed. The analyses were performed on a BNII® System (Siemens Healthcare GmbH, Germany) and Optilite® system (The Binding Site Group Ltd., Birmingham). ResultsThe correlation between total IgG concentration (mg/dl) and the sum of the individual IgG subclasses detected was higher using the Optilite® analyser (0.976 vs. 0.866). The percentage of agreement between assays ranged from 43% to 71%, with the lower limit being for the IgG3 agreement. An absence of the usual IgG subclass physiological proportion (IgG1>IgG2>IgG3>IgG4) was detected using BNII®. These findings were a due to the significantly lower proportion of IgG3 obtained by BNII® compared to Optilite® (P<.001), whereas the IgG4 concentration was not significantly different between analysers (P=.117). ConclusionsDifferences between the results obtained with the two different methods suggest that they should not be interchangeable, and that each clinical laboratory should only use one type of analyser. The reference ranges should be standardised according to the results obtained.

Multiple myeloma associated with IgA lambda gammopathy and multiple myeloma oncogene 1 in a Yorkshire terrier

An eight-year-old spayed female Yorkshire terrier was presented with a one-month history of conspicuous weight-bearing lameness in the right hindlimb, mild anorexia, intermittent vomiting and marked polydipsia and polyuria. Radiographs revealed circular radiolucent foci of variable size in the skeleton. Haematological and serum biochemistry examination revealed mild leucopoenia with severe neutropaenia, mild non-regenerative anaemia, moderate thrombocytopoenia, moderate hyperglobulinaemia, mild hypoalbuminaemia, mild azotaemia and moderate hypercalcaemia. Quantification of serum immunoglobulins revealed elevated IgA and IgG. Serum protein electrophoresis showed a broad appearance with a β-region spike. Plasma cells accounted for 7.6% of the cells in the bone marrow. Serum immunofixation electrophoresis (IFE) revealed IgA lambda gammopathy. Immunohistochemistry in the bone marrow was diffusely positive for multiple myeloma oncogene 1 (MUM-1) and CD20. To our knowledge, this is first case report of multiple myeloma associated with IgA lambda gammopathy confirmed via IFE and immunohistochemical expression of MUM-1 in a dog.

Recombinant Spider Silk as Mediator for One‐Step, Chemical‐Free Surface Biofunctionalization

AbstractA unique strategy for effective, versatile, and facile surface biofunctionalization employing a recombinant spider silk protein genetically functionalized with the antibody‐binding Z domain (Z‐4RepCT) is reported. It is demonstrated that Z‐silk can be applied to a variety of materials and platform designs as a truly one‐step and chemical‐free surface modification that site specifically captures antibodies while simultaneously reducing nonspecific adsorption. As a model surface, SiO2 is used to optimize and characterize Z‐silk performance compared to the Z domain immobilized by a standard silanization method. First, Z‐silk adsorption is investigated and verified its biofunctionality in a long‐term stability experiment. To assess the binding capacity and protein–protein interaction stability of Z‐silk, the coating is used to capture human antibodies in various assay formats. An eightfold higher binding capacity and 40‐fold lower detection limit are obtained in the immunofluorescence assay, and the complex stability of captured antibodies is shown to be improved by a factor of 20. Applicability of Z‐silk to functionalize microfluidic devices is demonstrated by antibody detection in an electrokinetic microcapillary biosensor. To test Z‐silk for biomarker applications, real‐time detection and quantification of human immunoglobulin G are performed in a plasma sample and C1q capture from human serum using an anti‐C1q antibody.

A case report of combination of asthma and allergic rhinitis caused by local household sensitization with a brief analysis of literature

Currently diagnosis of allergic diseases is based largely on results of skin tests and laboratory methods of quantification of total and specific immunoglobulins E in serum in addition to clinical and instrumental data. However, in recent years it has been showed that systemic atopy is not always registered with these methods and specific immunoglobulins E may not have diagnostic concentrations in serum that makes it difficult to diagnose allergic diseases. The article presents a clinical case of combination of asthma and allergic rhinitis caused by local IgE production with normal level of serum I-4, reduced levels of IgG1, IgG2 and serum and secretory IgA in child aged 3 years.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Comparison of single radial immunodiffusion and ELISA for the quantification of immunoglobulin G in bovine colostrum, milk and calf sera

ABSTRACTThe overall objective was to compare immunoglobulin G (IgG) concentrations measured by single radial immunodiffusion (sRID) and ELISA-based methods in samples of bovine colostrum and transition milk from contrasting breed types (Limousin × Friesian (n = 10) and Holstein (n = 10)). Jugular blood samples were collected at 48 h post-birth from beef (n = 10) and dairy (n = 10) calves and sera harvested subsequent to colostrum consumption. Absolute colostrum IgG values determined by ELISA showed poor agreement with mean (SD) IgG values measured using sRID, fixed bias (sRID – ELISA) was 31.89 (±9.84) mg/mL; having wide limits of agreement (12.61–51.17) and a low concordance coefficient (0.26). The agreement between ELISA and sRID when measuring serum IgG was greater than that of colostrum, fixed bias (sRID – ELISA) was 12.36 (±6.60) mg/mL; having narrower limits of agreement (−0.58 to 25.30) and serum IgG concentrations had a greater concordance coefficient (0.44) between samples. Calf sera IgG measured using the indirect zinc sulphate turbidity test showed a strong correlation with the sRID and ELISA methods (P < .001), R2 = 0.78 and R2 = 0.77 respectively. Overall, the ELISA and sRID methodologies had a strong positive association with almost a twofold (1.8) difference between values; therefore, they provide diverse absolute values of IgG concentration.

Immunoassay for Human IgG Using Antibody-functionalized Silver Nanoparticles.

A simple colorimetric immunoassay for quantification of human immunoglobulin G (hIgG) is herein described. The assay is based on the aggregation inhibition of silver nanoparticles (AgNP) functionalized with hIgG antibody (anti-hIgG) on the surface. The aggregation is measured in terms of attenuance values ratio at 400 and 530 nm (A400/A530). A linear response between A400/A530 and hIgG concentration is observed in the range 25 - 200 ng mL-1, and the detection limit is estimated as 11 ng mL-1 hIgG.

Multiple myeloma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.

These Clinical Practice Guidelines are endorsed by the Japanese Society of Medical Oncology (JSMO) incidence and epidemiology Multiple myeloma (MM) accounts for 1% of all cancers and ∼10% of all haematological malignancies. The incidence in Europe is 4.5–6.0/100 000/year with a median age at diagnosis of between 65 and 70 years; the mortality is 4.1/100 000/year. Almost all patients with MM evolve from an asymptomatic pre-malignant stage termed monoclonal gammopathy of undetermined significance (MGUS). MGUS progresses to MM at a rate of 1% per year. In some patients, an intermediate asymptomatic but more advanced pre-malignant stage termed smouldering (or indolent) multiple myeloma (SMM) can be recognised. SMM progresses to myeloma at a rate of 10% per year over the first 5 years following diagnosis, 3% per year over the following 5 years and 1.5% per year thereafter [1]. diagnosis Diagnosis of MM should be based on the following tests: [1] -Detection and evaluation of the monoclonal (M-) component by serum and/or urine protein electrophoresis (concentrate of 24 h urine collection); nephelometric quantification of IgG, IgA and IgM immunoglobulins; characterisation of the heavy and light chains by immunofixation; and serum-free light-chain (FLC) measurement; -Evaluation of bone marrow (BM) plasma cell infiltration: BM aspiration and/or biopsies are the standard options to evaluate the number and characteristics. Moreover, the BM sample should be used for cytogenetic/fluorescence in situ hybridization (FISH) studies and also has the potential for immunophenotypic and molecular investigations; -Evaluation of lytic bone lesions: a radiological skeletal bone survey, including spine, pelvis, skull, humeri and femurs is necessary. A magnetic resonance imaging (MRI) or computed tomography (CT) scan may be needed to evaluate symptomatic bony sites, even if the skeletal survey is negative and the patient has symptoms suggesting bone lesions. Moreover, MRI provides greater detail and is recommended whenever spinal cord compression is suspected. Fluorodeoxyglucose positron emission tomography is currently under evaluation but should not be systematically used; -Complete blood cell count, with differential serum creatinine and calcium level.

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

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse.Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account.IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum.The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses.