THAb was detected by radioimmunoprecipitation technique described in previous researches,21 using anti‐human IgM‐Agarose (Sigma‐Aldrich) or protein G (Merck KGAA, Darmstadt, Germany). In brief, 500 μl of serum was incubated with 0.5 μCi 125I‐T3 or 125I‐T4 (Beijing Fury Runze Biotechnology Co., Ltd) for 60 min at 23 C. Twenty microliters of this mixture was then incubated with 150 μl anti‐human IgM‐Agarose or protein G, both prediluted 1:10 with saline containing bovine serum albumin (BSA) (Sigma) at a final concentration of 0.5%. After 24‐h incubation at 4℃, tubes were centrifuged at 2,000× g for 1 min and the supernatant was aspirated. Finally, the radioactivity of immunoprecipitation was detected. The percentage of THAb was equal to total radioactivity divided by immunoprecipitation radioactivity.
Anti human igm agarose
Manufactured by Merck Group
Sourced in Germany
Anti-human IgM-Agarose is a solid-phase adsorbent material used in laboratory procedures. It is composed of agarose beads coupled with antibodies specific to human immunoglobulin M (IgM). This product can be used for the detection, isolation, or purification of human IgM from biological samples.
Automatically generated - may contain errors
3 protocols using anti human igm agarose
1
Radioimmunoassay for Thyroid Hormone Antibodies
2
Depleting IgG and IgM from Human Serum
3
Glycan Microarray Analysis of IgM-Depleted Sera
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Anti-human IgM agarose (Sigma A9935) was used for IgM depletion and Sepharose 4B (Sigma, 4B200) was used as control resin. Both resins were equilibrated on column cartridges, washed with 5× PBS buffer and then mixed with equal column volume of PBS buffer. For IgM depletion assays, 110 uL Anti-human IgM agarose solution was mixed with 110 uL cord or maternal serum samples (4× dilution, in PBS buffer containing 3% BSA, 1% HSA and 0.05% Tween 20, pH 7.4). In the control assays, 110 uL Sepharose 4B was mixed with 110 uL diluted sera. These solutions were incubated at room temperature for 1.5 h with gentle agitation (150 rpm). The resins were centrifuged at 10,000×g for 5 min, and the serum supernatant (∼130 μL/sample) was collected for antibody profiling the glycan microarray. Both cord and maternal sera were treated using the same method.
Each sample was evaluated on a glycan microarray in duplicate as described above with the following differences: the volume of serum samples was 60 μL/well, the slides were scanned with a GenePix 4000B fluorescent scanner (Molecular Devices), and fluorescent intensity for each array component was quantified using GenePix Pro 7.0 software (Molecular Devices). Since these samples were evaluated at a dilution of 1:8, any signals lower than 900 RFU were adjusted to 900 (floor).
Each sample was evaluated on a glycan microarray in duplicate as described above with the following differences: the volume of serum samples was 60 μL/well, the slides were scanned with a GenePix 4000B fluorescent scanner (Molecular Devices), and fluorescent intensity for each array component was quantified using GenePix Pro 7.0 software (Molecular Devices). Since these samples were evaluated at a dilution of 1:8, any signals lower than 900 RFU were adjusted to 900 (floor).
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