The antibody profile of subjects was determined using in-house ELISA. We primarily investigated both the neutralizing (anti-receptor binding domain (RBD-IgG) and anti-Spike (S1-IgG) and non-neutralizing antibodies (anti-N-IgG) in patients’ serum using our in-house ELISA method.44 ,45 (link) Briefly, commercially obtained RBD, S1, and N proteins (Sino Biological, China) were coated on ELISA plates (ExtraGene, USA). After blocking, diluted (1:100) serum samples were applied, and SARS-CoV-2 specific human IgG, IgM, and IgA were detected using HRP tagged goat anti-human IgG (The Native Antigen, UK), goat anti-human IgM (Abcam, USA), and goat anti-human polyclonal IgA (Sigma-Aldrich, USA). Furthermore, we investigated antibody dynamics against S1+S2 (Sino Biological, China) using the similar method. Results were obtained by a microplate reader (Thermo Scientific, USA) at 450 nm.
Goat anti human igm
Manufactured by Abcam
Sourced in United Kingdom
Goat anti-human IgM is a secondary antibody that binds to human immunoglobulin M (IgM) antibodies. It is used for the detection and quantification of human IgM in various immunoassays and research applications.
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4 protocols using goat anti human igm
1
Antibody profiling of SARS-CoV-2 infection
2
Antibody Isoforms Detection by ELISA
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Two antibody isoforms (IgG and IgM) in human serum were assessed using a commercially available BLV indirect gp51 ELISA kit (JNC, Tokyo, Japan), according to the manufacturer’s instructions. Human serum specimens, diluted in 1:50 with the sample dilution buffer in the kit, were used as primary antibodies, followed by HRP-conjugated goat anti-human IgG (diluted as 1/1000) (Abcam PIC.) or goat anti-human IgM (diluted as 1/10,000) (Abcam PIC.) the secondary antibody. All samples were run triplicate. During each assay, to check the validity of the experiment procedure, we used two controls, as indicated in ELISA for anti-BLV p24 antibody.
3
Immunoblotting of Proteins from 2-DE Gels
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Proteins on 2-DE gels were transferred onto nitrocellulose membrane (Immun-Blot, 0.2 μm) using a dry transfer technique (iBlot, Dry Blotting System; Invitrogen, Carlsbad, CA) for 10 min at a constant current of 25 mA. The membrane was blocked for 1h in PBS containing 1.5% skim milk. The membranes were incubated with goat serum at a dilution of 1:1000 in PBS containing 1.5% skim milk for 1h, and then washed three times in PBS. Next, a secondary antibody of donkey anti-goat IgG or IgM conjugated with horseradish peroxidase (abcam, Cambridge, MA) was added for immunoglobulin G or M detection. Both of the secondary antibodies were diluted with 1/1000. Protein spots were visualized using a chromogenic substrate, 3, 3ʹ-diaminobenzidine (DAB) solution. The human sera used in this study were treated similarly to the goat sera analysis except for the use of a goat anti-human IgG secondary antibody (Promega, Madison, WI) with 1/1000 dilution and goat anti-human IgM (abcam, Cambridge, MA) with 1/1000 dilution.
4
Lateral Flow Immunoassay Strip for SARS-CoV-2 Detection
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Each LFIA strip consists
of a nitrocellulose membrane (GE Healthcare, Chicago, IL), a sample
pad, a test line conjugate pad containing SARS-CoV-2-N protein conjugated
to 150 nm carpoxyl gold nanoshells, a control area conjugate pad containing
normal rabbit control IgG conjugated to 150 nm carpoxyl gold nanoshells,
and an absorbent pad. The nitrocellulose membrane (NCM) contains two
manually applied areas using a 0.5–10 μL pipette: a test
line area that has goat anti-human IgM or goat anti-human IgG antibodies
(Abcam, U.K.) and a control area that contains mouse anti-rabbit antibodies
(Abcam, U.K.). After placing the NCM on top of the backing card (DCNovations),
all reagents were applied at 0.25 μg/5 mm wide test strip onto
the NCM and allowed to dry at RT for 1 h. Then, the conjugate pads,
chopped glass with a binder (Ahlstrom-Munksjo), and the CytoSep layer
(Ahlstrom-Munksjo) were layered at the NCM first end, as illustrated
inFigure 1 . The sample
pad, chopped glass with a binder (Ahlstrom-Munksjo), was then layered
on top of all of the components at this end, and adhesive tape was
placed at the joint between the control area conjugate pad and the
NCM (Figure 1 ). The
strip also contains the NCM second-end cotton absorbent pad (Ahlstrom-Munksjo).
of a nitrocellulose membrane (GE Healthcare, Chicago, IL), a sample
pad, a test line conjugate pad containing SARS-CoV-2-N protein conjugated
to 150 nm carpoxyl gold nanoshells, a control area conjugate pad containing
normal rabbit control IgG conjugated to 150 nm carpoxyl gold nanoshells,
and an absorbent pad. The nitrocellulose membrane (NCM) contains two
manually applied areas using a 0.5–10 μL pipette: a test
line area that has goat anti-human IgM or goat anti-human IgG antibodies
(Abcam, U.K.) and a control area that contains mouse anti-rabbit antibodies
(Abcam, U.K.). After placing the NCM on top of the backing card (DCNovations),
all reagents were applied at 0.25 μg/5 mm wide test strip onto
the NCM and allowed to dry at RT for 1 h. Then, the conjugate pads,
chopped glass with a binder (Ahlstrom-Munksjo), and the CytoSep layer
(Ahlstrom-Munksjo) were layered at the NCM first end, as illustrated
in
pad, chopped glass with a binder (Ahlstrom-Munksjo), was then layered
on top of all of the components at this end, and adhesive tape was
placed at the joint between the control area conjugate pad and the
NCM (
strip also contains the NCM second-end cotton absorbent pad (Ahlstrom-Munksjo).
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