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Elisa plate reader

Manufactured by Gene Tech
Sourced in China, Hong Kong

The ELISA plate reader is a laboratory instrument used to measure the absorbance or fluorescence of samples in a microplate format. It is a core piece of equipment in enzyme-linked immunosorbent assay (ELISA) procedures, providing accurate and reproducible data for quantitative analysis of various analytes such as proteins, hormones, and antibodies.

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7 protocols using elisa plate reader

1

SARS-CoV-2 S1 Protein ELISA Assay

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The SARS-CoV-2 (Wuhan-Hu-1 strain) S1 protein (Sanyou Biopharmaceuticals Co., Ltd., Shanghai, China) was added at a concentration of 0.1 μg/100 μL/well to 96-well ELISA plates (Corning, NY, USA). Subsequently, serum was added to the wells using 2-fold multiplicative dilution and then incubated at 37 °C for 1 h. Following this, the wells underwent five washes using PBST before 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher, Waltham, MA, USA) was applied to each well and incubated again at 37 °C for a duration of 40 min. During the serum IgG subtype detection experiments, the antibodies were replaced with horseradish peroxidase-conjugated goat anti-mouse IgG1 and horseradish peroxidase-conjugated goat anti-mouse IgG2a (Abcam, Cambridge, MA, USA). The plate was then washed five times with PBST buffer. Then, 100 μL of TMB substrate (3,3′,5,5′-tetramethylbenzidine) was added for 10 min to allow for color development. Finally, 50 μL of ELISA stop solution (Solarbio, Beijing, China) was added to terminate color development. The results were read using an ELISA plate reader (Gene Company, Hong Kong, China) at a wavelength of 450 nm. The terminal dilution criterion was met when the OD value in the sample wells exceeded 2.1 times that of the blank wells.
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2

SARS-CoV-2 Spike Protein ELISA Assay

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An ELISA assay assessing binding antibody levels against the wild-type SARS-CoV-2 was conducted using the wild-type Spike protein. ELISA plates (Corning Costar; Corning, NY, USA) were coated with S protein (Sanyou Biopharmaceuticals Co., Shanghai, China) at a concentration of 5 μg/well and incubated overnight at 4 °C. During the experiment, ELISA plates were blocked with 5% BSA-phosphate-buffered saline (PBS), incubated with serially diluted serum samples, and visualized by reaction with an HRP-conjugated antibody (Abcam, Waltham, MA, USA) and TMB substrate (Solarbio, Beijing, China), using previously described methods [16 (link)]. The absorbance of each well at 450 nm was measured using an ELISA plate reader (Gene Company, Beijing, China). Antibody serum samples yielding OD values at least 2.1-fold higher than the negative control at a test sample dilution of 1:400 were considered positive. Endpoint titers (ETs) were defined as the highest serum dilutions yielding positive OD values. GMTs were calculated as the geometric mean of the ETs of positive serum samples in each group. ELISA assays assessing binding antibody levels against variant strains of SARS-CoV-2 (Alpha, Beta, Gamma, Delta, and Omicron) were conducted using commercial ELISA kits (ACRO Biosystems Co., Beijing, China) according to the manufacturer’s instructions.
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3

Evaluating miR-9 Effects on Angiogenesis

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VECs transfected with miR-9 mimic or miR-9 control were added to the upper chamber of the Transwell apparatus. After incubation for 12 h, the upper chamber was fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (sigma) for 10 min in the dark and photographed by the phase-contrast microscopy. Finally, the crystal violet was dissolved in 300 μl of 33% acetic acid (sigma) and the absorbance of the solution was measured by an ELISA plate reader (Gene Company Limited, HK, China). To investigate the effect of the miR-9 on the angiogenic activity of VECs in vitro, we performed a tube formation assay. Ninety-six-well culture dishes were coated with 50 μl of matrigel matrix (BD company) and incubated for 30 min at 37 °C. VECs transfected with miR-9 mimic or miR-9 control were seeded onto the solidified gels at a density of 2 × 105 cells/well in 50 μl of culture medium. After incubation for 12 h, the total tube-like structures were photographed by phase-contrast microscopy (× 100) and counted. The above experiments were repeated three times.
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4

SARS-CoV-2 S1 Protein ELISA Detection

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The S1 protein (Sanyou Biopharmaceuticals Co., Ltd., Shanghai, China) of the SARS-CoV-2 Wuhan-Hu-1 strain was utilized to coat 96-well ELISA plates (Corning, NY, USA) at a concentration of 0.1 μg/well and incubated at 4 °C overnight. Then, the plates were blocked with 1% BSA–phosphate-buffered saline (PBS) and visualized with horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher, USA) and TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Solarbio, Beijing, China) according to previously described methods [26 (link)]. The reaction was evaluated at 450 nm by an ELISA plate reader (Gene Company, Hong Kong, China), and the S1-specific IgG titers were determined by end titration utilizing the reciprocal of the lowest serum dilution that produced an OD value 2.1-fold greater than that in the prebleed.
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5

ELISA for SARS-CoV-2 Antibody Detection

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ELISA was conducted with antibodies against the S and N proteins. S and N proteins (Sanyou Biopharmaceuticals Co.) were used to coat the ELISA plates (Corning Costar; Corning, NY, USA) at a concentration of 5 μg/well, and the plates were incubated overnight at 4°C. The plates were blocked with 5% BSA, incubated with serum samples, and incubated with an HRP-conjugated antibody (Abcam). The immune complexes were visualized using TMB substrate (Solarbio) as described previously [21 (link)]. The absorbance at 450 nm was measured using an ELISA plate reader (Gene Company, Beijing, China). The antibody serum samples that yielded OD values at least 2.1-fold higher than that of the negative control at a test sample dilution of 1:400 were considered positive. The endpoint titer (ET) was defined as the highest serum dilution that yielded a positive OD value. The GMT was calculated as the geometric mean of the ETs of the positive serum samples in each group.
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6

Transwell Assay for Cell Migration

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In this experiment, 5 × 104 EA.hy926 cells were added to the upper chamber of the Transwell apparatus and supplemented with 180 μL of DMEM and 20 μL of 1% BSA, and the lower chamber was filled with 500 μL of DMEM-LG complete medium with or without BMSCS supernatant (100 μL). After incubation for 12 h, the upper chamber was removed and the cells were fixed with 4% paraformaldehyde for 30 min. The lower surface of the filter membrane was stained with 0.1% crystal violet for 10 min in the dark. After the membrane was thoroughly washed, it was observed with a digital camera and light microscope. The experiment was repeated three times. Finally, the crystal violet was dissolved in 300 μL of 33% acetic acid and the absorbance of the solution was measured at 573 nm with an ELISA plate reader (Gene Company Limited, HK, China).
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7

ELISA for SARS-CoV-2 Antibody Detection

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ELISAs were conducted with antibodies against the S protein, the N protein and virions that were developed by this institute. S/N protein (Sanyou Biopharmaceuticals Co., Ltd., Shanghai, China) and purified viral antigen were used to coat 96-well ELISA plates (Corning, NY, USA) at a concentration of 5 μg/well, and the plates were incubated at 4 °C overnight. The plates were then blocked with 5% BSA and incubated with serum samples, and immune complexes were visualized using an HRP-conjugated antibody (Abcam, MA, USA) and TMB substrate (Solarbio, Beijing, China) as described in a previous report [13] (link). The absorbance of each well at 450 nm was measured using an ELISA plate reader (Gene Company, Beijing, China). The antibody serum samples that yielded OD values at least 2.1-fold higher than that of the negative control at a test sample dilution of 1:400 were defined as positive. The endpoint titer (ET) was defined as the highest serum dilution that yielded a positive OD value. The GMT was calculated as the geometric mean of the ETs of the positive serum samples in each group. For neutralizing antibodies, seroconversion was defined as seropositivity after immunization in previously seronegative subjects.
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