Tmb substrate

Manufactured by Tiangen Biotech

Sourced in China

TMB substrate is a commonly used chromogenic substrate in various immunoassays, such as enzyme-linked immunosorbent assay (ELISA). It is a colorless solution that, when catalyzed by the enzyme horseradish peroxidase (HRP), produces a blue-colored product. The intensity of the color change is proportional to the amount of target analyte present in the sample, making it a useful tool for quantitative and qualitative analysis.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (2 mentions) Hrp conjugated goat anti mouse igg antibody, by Merck Group (1 mentions) Nonfat dry milk, by BD (1 mentions)

Close spelling variants of this product

TMB (3,3′,5,5′-tetramethylbenzidine) substrate Tetramethylbenzidine (TMB) substrate TMB microwell peroxidase substrate system TMB substrate solution Soluble TMB Substrate Solution

7 protocols using tmb substrate

Mapping Antibody Epitope in H. parasuis

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In order to verify whether MAb 1B3 can recognize the corresponding epitope in H. parasuis, eight peptides were synthesized with the linkage of the irrelevant peptide “HHHHHHHHHHHHHHH” as the peptide control (Table 1). Then, the reactivity of the polypeptide with MAb 1B3 or the murine antisera was performed by ELISA.
In order to confirm the mapped epitope recognized by MAb 1B3 in H. parasuis, a competitive inhibition ELISA was performed. The 96-well plates were coated with 10 µg/well whole-cell lysates of H. parasuis HS80 strain in 0.1 M NaHCO₃ (pH 8.6) at 4°C overnight, and then blocked with 5% skimmed milk diluted in PBS for 1 h at 37°C. Serial dilutions of synthetic peptides were pre-incubated separately with MAb 1B3 (0.5 µg/mL) for 1 h at 37°C. The antibody-peptide mixture was incubated for 20 min at 37°C. The HRP-conjugated goat anti-mouse antibody (1∶5000) was added for 1 h after the plates were washed five times. TMB substrate (Tian Gen, China) was added for colorimetric detection. The results were analyzed using a spectrophotometer at an absorbance of 450 nm.

Zheng N., Chai Z., Fu F., Jiang F., Wang X., Zhang X., Wang Z, & Li X. (2014). Identification of a Novel Haemophilus parasuis-Specific B Cell Epitope Using Monoclonal Antibody against the OppA Protein. PLoS ONE, 9(1), e84516.

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ELISA for H. parasuis Antibody

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Plates were coated with 0.4 µg/well of H. parasuis lysates diluted in carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight. The coated plates were blocked with 5% (w/v) skim milk in PBST (1× PBS with 0.05% Tween 20) and then incubated with the supernatant of hybridoma culture and HRP-conjugated goat anti-mouse IgG antibody (Sigma, USA) for 1 h at 37°C. TMB substrate (Tian Gen, China) was added for colorimetric detection. The results were analyzed using a spectrophotometer at an absorbance of 450 nm.

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ELISA for Parasitic Infection Diagnosis

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Ten μg/ml rTs-TTPA in 100 μl CBS (carbonate buffer solution) was coated on an EIA-ELISA plate at 4 °C overnight. After the encapsulation solution was discarded, it was blocked with 200 μl 5% skim milk at 37 °C for 1 h, and then PBST (phosphate buffer saline with 2% Tween-20) was used for three 1-min washes. After patting the liquid dry, serum collected from pigs on different days post-infection with 10,000 T. spiralis or serum infected with Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis, diluted with a blocking solution (1:100), was added for the binding reaction. After the same washing and patting, HRP-goat-anti-pig secondary antibodies (Abcam, Cambridge, UK) in a blocking solution (1:5000) were incubated at 37 °C for 45 min. After washing and termination, TMB substrate (Tiangen, Beijing, China) was added to detect absorbance at 450 nm (Biotek, Vermont, USA).

Li C., Li C., Xu F., Wang H., Jin X., Zhang Y., Liu X., Wang R., You X., Liu M., Bai X, & Yang Y. (2023). Identification of antigens in the Trichinella spiralis extracellular vesicles for serological detection of early stage infection in swine. Parasites & Vectors, 16, 387.

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Mxra8-Fc Binding Assay for CHIKV

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A fragment of cDNA encoding the Mouse-Mxra8 extracellular domain (residues 23–336, GenBank accession number NM_024263.4) or Human-Mxra8 extracellular domain (residues 24–337, GenBank accession number NM_032348.3) was appended with a TEV enzyme site and a human IgG1 Fc at the C-terminus as well as the IL-2 signal peptide at the N-terminus in pcDNA 3.4 expression vectors and transiently transfected into HEK293 cells followed by media collection and purification using protein A sepharose. The Mxra8-Fc binding assay was adapted from the previously described [27 (link)]. MaxiSorp ELISA plates (Corning) were coated with 2 µg/mL anti-mouse CHKV E2 monoclonal antibodies in CBS (pH9.6) overnight at 4 °C. The wells were washed four times with PBS and blocked with 4% BSA(Sigma-Aldrich) for 1 h at room temperature (RT). CHIKV virions (1 µg/ml) were diluted and added for 1 h at RT. After washing, MoNb-2E8 and MoNb-3C5 or Mouse Mxra8-Fc fusion protein (all at 10 µg/ml) were incubated for 30 min. Plates were washed and Human Mxra8-Fc (tenfold serial dilutions) were added to the plates and incubated for 1 h at RT. Plates were washed again and incubated with secondary rabbit anti-human IgG-Fc (1:5000, Bioss). After washing, the plates were developed with TMB substrate (TIANGEN) and 2 N H₂SO₄. Absorbance was measured at 450 nm.

Li Q., Zhang F., Lu Y., Hu H., Wang J., Guo C., Deng Q., Liao C., Wu Q., Hu T., Chen Z, & Lu J. (2022). Highly potent multivalent VHH antibodies against Chikungunya isolated from an alpaca naïve phage display library. Journal of Nanobiotechnology, 20, 231.

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Enzyme-Linked Immunosorbent Assay for Autoantibody Detection

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Flat-bottom culture dishes were coated with the recombinant target protein (2 µg/ml; diluted with 50 mM carbonate buffer, pH 9.6) and were incubated in a humidified chamber at 4°C overnight. After washing three times with PBS with 0.05% (vol/vol) Tween 20 (pH 7.4; PBST), the protein in the plate was blocked with 300 µl 2% BSA (diluted in PBS) in each well at room temperature for 3 h. Plates were rinsed with PBST three times, and serum (1:100, diluted in PBS containing 2% BSA) was added to the wells and cultured at room temperature for 90 min. Subsequently, the protein was washed six times and co-incubated with goat anti-human IgG conjugated with horseradish peroxidase (1:2,500, cat. no. W4031, Promega Corporation) diluted in PBS containing 2% BSA (100 µl/well) at room temperature for 1 h. The plates were rinsed with PBST six times, and the antibodies were measured using TMB Substrate (Tiangen Biotech Co., Ltd.). The entire process was terminated by the addition of 50 µl 2M H₂SO₄. The absorbance was detected using a multi-scan microplate reader (Metertech 960, Metertech Inc.) at 450 nm. The serum from 8 healthy subjects was pooled and used as the control group. The auto-antibody levels were measured using the binding index (BI), which was calculated as follows: BI=(OD of the sample-OD of the blank group)/(OD of the control group-OD of the blank group) (32 (link)).

Hao S., Zhang Y., Hua L., Xie N., Xiao N., Wang H., Fu R, & Shao Z. (2020). Antibodies specific to ferritin light chain polypeptide are frequently detected in patients with immune-related pancytopenia. Molecular Medicine Reports, 22(3), 2012-2020.

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IgG Antibody Quantification by ELISA

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IgG titers of immune sera were determined by enzyme-linked immunosorbent assay (ELISA) as described [85 (link)]. Briefly, ATCC43816 was grown to OD₆₀₀ 0.1 before being pelleted and resuspended in the same volume of PBS. Each well of 96-well plates was coated with 100 μl bacterial suspension, and incubated overnight at 4°C, and blocked with 200 μl 5% non-fat dry milk (BD, USA). Mouse serum was serially diluted in PBS and added to appropriate wells incubating at 37°C for 2 h. Each well was washed three times with PBS, and bound antibody was detected with alkaline phosphatase-conjugated goat anti-mouse IgG (1:2,000, EasyBio, Beijing, China) at 37°C for 1h before addition of the TMB substrate (TIANGEN, Beijing, China). The optical absorbance was measured at a 450 nm wavelength using the BioTek Synergy H1 microplate reader (BioTek, Germany).

Huang X., Li X., An H., Wang J., Ding M., Wang L., Li L., Ji Q., Qu F., Wang H., Xu Y., Lu X., He Y, & Zhang J.R. (2022). Capsule type defines the capability of Klebsiella pneumoniae in evading Kupffer cell capture in the liver. PLoS Pathogens, 18(8), e1010693.

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Chikungunya E2 Glycoprotein Epitope Mapping

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A pool of 10-mer peptides with 5 amino acid overlap spanning the Chikungunya/SL-CK1 E2 glycoprotein was generated by chemistry to a purity of 90% (GL Biochem). All peptides were provided as a lyophilized powder, reconstituted in dimethylsulfoxide (DMSO) to a concentration of 1 mg/mL, and stored at − 80 °C. Each peptide was coated at 1ug/mL in CBS (pH9.6) overnight at 4 °C and then blocked with 5% BSA(Sigma-Aldrich) in PBST. An irrelevant SARS-CoV-2 peptide was used as the negative control. The binding of the coated peptide was characterized by incubation with 2 µg/mL antibodies. Mouse monoclonal anti-6 × His tag HRP-conjugated (Abcam) was used at 1:5000 dilution in the blocking buffer, and further absorbance measurements of the enzymatic reaction in TMB substrate (TIANGEN), were used to detect the bound epitopes.

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