Endocab igm

Manufactured by Hycult Biotech

Sourced in Netherlands, United States

EndoCAb IgM is a laboratory assay kit developed by Hycult Biotech to measure the levels of anti-endotoxin core antibodies (EndoCAb) of the IgM class in human serum or plasma samples. The kit provides a quantitative determination of EndoCAb IgM concentrations, which can be used as a marker of endotoxin exposure and response.

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EndoCAb (8 citations)

8 protocols using endocab igm

Measuring Microbial Translocation Markers

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Plasma sCD14 and EndoCAb IgM were used to measure the level of microbial translocation in our study (28 (link)). Lipopolysaccharide (LPS) directly stimulates sCD14 production. When LPS enters the cycle, EndoCAb IgM clears LPS by binding to it, and EndoCAb IgM titres are reduced (28 (link)). High levels of sCD14 and low levels of EndoCAb IgM are considered to be the increase of microbial translocation. sCD14 (R&D) and EndoCAb IgM (Hycult Biotech) in plasma were detected by an enzyme-linked immunosorbent assay. All experimental procedures were performed according to the manufacturer’s protocols.

Zhang Y., Xie Z., Zhou J., Li Y., Ning C., Su Q., Ye L., Ai S., Lai J., Pan P., Liu N., Liao Y., Su Q., Li Z., Liang H., Cui P, & Huang J. (2023). The altered metabolites contributed by dysbiosis of gut microbiota are associated with microbial translocation and immune activation during HIV infection. Frontiers in Immunology, 13, 1020822.

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Biomarkers of Microbial Translocation, Inflammation, and Intestinal Damage in Clostridioides difficile Infection

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For each subject, blood samples were collected at T0 (ie, before therapy or within 24 hours of the onset of CDI-specific therapy) and T1 (within 48 hours of the end of CDI-specific therapy), respectively.
In subjects who received FMT after a 4-day course of high-dose oral vancomycin, T0 and T1 were defined as immediately before and 10 days after the FMT procedure, respectively, in accordance with the institutional protocol on blood collection in patients undergoing FMT.
Markers of microbial translocation (lipopolysacharide-binding protein, Hycult Biotech; EndoCab IgM, Hycult Biotech), inflammation (IL-6, R&D Systems), and intestinal damage (intestinal fatty acid binding protein [I-FABP], R&D Systems) were evaluated using enzyme-linked immunosorbent assays in plasma. Briefly, after collection, blood was immediately centrifuged at 2000 rpm for 10 minutes and further frozen at –80°C until the assays were performed. LBP was expressed as ng/mL (sample diluted 1:2000), EndoCab IgM as MU/mL (sample diluted 1:100), IL-6 as pg/mL (sample undiluted), and I-FABP as pg/mL (sample diluted 1:5). Samples from healthy nonhospitalized subjects (controls, n = 15) matched for age and sex were also included and collected once.

Oliva A., Aversano L., De Angelis M., Mascellino M.T., Miele M.C., Morelli S., Battaglia R., Iera J., Bruno G., Corazziari E.S., Ciardi M.R., Venditti M., Mastroianni C.M, & Vullo V. (2019). Persistent Systemic Microbial Translocation, Inflammation, and Intestinal Damage During Clostridioides difficile Infection. Open Forum Infectious Diseases, 7(1), ofz507.

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Biomarker Measurement Protocols in Clinical Research

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EndoCAb IgG, EndoCAb IgM, and EndoCAb IgA were measured using direct ELISA kits procured from Hycult Biotech. LBP and LL-37 were measured using sandwich ELISA kits procured from Hycult Biotech (Pennsylvania, USA). sCD14 and FABP2 were measured using sandwich ELISA kits from R&D systems (Minneapolis, USA). CXCL16 was analyzed using a sandwich ELISA kit procured from Thermo Scientific (Frederick, MD, USA). Galectin-3 was measured using sandwich ELISA kit (Abcam, MA, USA). Total IgG, IgA, and IgM were measured using sandwich ELISA kits procured from Invitrogen (Carlsbad, CA, USA). The samples were diluted in appropriate buffers, which contains HeteroBlock (Omega Biologicals, Bozeman, MT, USA) to block non-specific antibodies which may interfere with the assay. All the analyses were performed blinded to clinical status with the exception of galectin-3 and total immunoglobulins IgG, IgA, and IgM.

Ayyappan P., Harms R.Z., Buckner J.H, & Sarvetnick N.E. (2019). Coordinated Induction of Antimicrobial Response Factors in Systemic Lupus Erythematosus. Frontiers in Immunology, 10, 658.

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Quantification of Immune Biomarkers

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sCD14 and LBP were measured using sandwich ELISA kits procured from R&D systems (Minneapolis, USA) and Hycult Biotech (Pennsylvania, USA), respectively. EndoCAb IgG, EndoCAb IgA, and EndoCAb IgM were measured using direct ELISA kits procured from Hycult Biotech. CXCL16 was analyzed using a sandwich ELISA kit procured from Thermo Scientific (Frederick, MD, USA). Lysozyme levels were measured using sandwich ELISA kit procured from MBL (Massachusetts, USA). Total IgG, IgA, and IgM were measured using sandwich ELISA kits procured from Invitrogen (Carlsbad, CA, USA). To block non-specific antibodies that may interfere with the assay, the samples were diluted in appropriate buffers, which contained 50 μg/ml of HeteroBlock (Omega Biologicals, Bozeman, MT, USA) and kept for 30 minutes before adding into the ELISA plate. All the analyses were performed blinded to case/control and clinical status. In order to maintain the test quality and reproducibility, an internal control was included in all the assays and the coefficient of variation (CV) of replicates was set at ≤ 10%.

Ayyappan P., Harms R.Z., Seifert J.A., Bemis E.A., Feser M.L., Deane K.D., Demoruelle M.K., Mikuls T.R., Holers V.M, & Sarvetnick N.E. (2020). Heightened Levels of Antimicrobial Response Factors in Patients With Rheumatoid Arthritis. Frontiers in Immunology, 11, 427.

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COVID-19 Biomarker Profiling of Blood Samples

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For each subject, whole blood samples were collected at the moment of (T0) and after 7 days (T7) from the diagnosis of COVID-19, respectively. Ten milliliters of whole blood were collected by venipuncture in Vacutainer tubes containing ethylene-diamine-tetra-acetic acid (EDTA) (BD Biosciences, San Jose, CA) at each study visit. Plasma was immediately separated by centrifugation at 2000 rpm for 10 minutes and further stored at –80°C until the assays were performed.
Markers of MT [LPB (Lipopolysacharide Binding Protein, RayBiotech); EndoCab IgM, Hycult Biotech] and ID [I-FABP (Intestinal Fatty Acid Binding Protein), R&D Systems] were evaluated using enzyme-linked immunosorbent assays in plasma, according to manufacturers’ instructions.
LBP was expressed as ng/mL (sample diluted 1:1000), EndoCab-IgM as MU/mL (sample diluted 1:50) and I-FABP as pg/mL (sample diluted 1:5). Samples from healthy non-hospitalized subjects were also included and collected once.

Oliva A., Miele M.C., Di Timoteo F., De Angelis M., Mauro V., Aronica R., Al Ismail D., Ceccarelli G., Pinacchio C., d’Ettorre G., Mascellino M.T, & Mastroianni C.M. (2021). Persistent Systemic Microbial Translocation and Intestinal Damage During Coronavirus Disease-19. Frontiers in Immunology, 12, 708149.

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Intestinal Permeability and Endotoxemia

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I-FABP is a surrogate marker of enterocyte damage and was analysed in serum with a Quantikine human ELISA kit (R&D Systems, Inc. Minneapolis, MS, USA), with inter-assay CV ≤ 10% across the assay working range of 3.6–1000 pg/mL. Systemic LPS concentrations are used as a surrogate marker of GI distress, with increases suggesting greater disruptions in GI integrity. Serum LPS was measured by CusaBio ELISA kit (CusaBio Technology LLC, Houston, TX, USA), with inter-assay CV of ≤ 12% across the measurement range of 6.3–400 pg/mL. Anti-LPS antibodies were measured with a commercially available ELISA kit (HK504, EndoCAb® IgM, Hycult Biotech, Uden, Netherlands); CV for this analysis was ≤ 7.2%.

Taylor G., Leonard A., Tang J.C., Dunn R., Fraser W.D., Virgilio N., Prawitt J., Stevenson E, & Clifford T. (2022). The effects of collagen peptides on exercise-induced gastrointestinal stress: a randomized, controlled trial. European Journal of Nutrition, 62(2), 1027-1039.

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Quantification of Immune Markers in Plasma

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Immediately after sample collection, plasma and peripheral blood mononuclear cells (PBMCs) were separated from whole blood and stored at −80 °C for batch assays. T cell population assessments and plasma HIV-1 RNA load assays were performed by using flow cytometry and Cobas Amplicor (Roche, USA) at the clinical diagnostic laboratory of the SPHCC. Customized kits (MILLIPLEX® MAP; Merck Millipore, Germany) were used to quantify 38 cytokines in the plasma samples (Supplementary Table 1). In addition, enzyme-linked immunosorbent assays (ELISAs) were performed to quantify plasma markers for microbial translocation, including endotoxin core immunoglobulin M (EndoCAb IgM, Hycult Biotech, The Netherlands), lipopolysaccharide binding protein (LBP, Hycult Biotech, The Netherlands), soluble CD14 (sCD14, R&D Systems, USA), soluble CD163 (sCD163, R&D Systems, USA), and intestinal fatty acid-binding protein (I-FABP, R&D Systems, USA). All assays were performed following the manufacturer’s instructions. Measured values below the limit of detection were considered zero. For values beyond the detection range, the value of the upper limit was assigned.

Ji Y., Zhang F., Zhang R., Shen Y., Liu L., Wang J., Yang J., Tang Q., Xun J., Qi T., Wang Z., Song W., Tang Y., Chen J, & Lu H. (2018). Changes in intestinal microbiota in HIV-1-infected subjects following cART initiation: influence of CD4+ T cell count. Emerging Microbes & Infections, 7, 113.

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Gut Microbiota Translocation Assay

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We used commercially available ELISA to detect gut microbiota translocation indicators, namely EndoCAb IgG (Hycult Biotech, Uden, The Netherlands), sCD14 (R&D Systems, Inc., Minneapolis, Minnesota, USA), and EndoCAb IgM (Hycult Biotech) in the plasma of individuals. We conducted the experiments according to the standardized protocols provided by the manufacturers.

Zhou J., Yang Y., Xie Z., Lu D., Huang J., Lan L., Guo B., Yang X., Wang Q., Li Z., Zhang Y., Yang X., Ai S., Liu N., Cui P., Liang H., Ye L, & Huang J. (2023). Dysbiosis of gut microbiota and metabolites during AIDS: implications for CD4+ T cell reduction and immune activation. AIDS (London, England), 38(5), 633-644.

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