AlphaLISA analysis for detecting anti-FIRs autoantibodies (IgG) was performed as described previously [23 (link)]. Briefly, AlphaLISA was performed in 384-well microtiter plates (white opaque OptiPlate™, Perkin Elmer, Waltham, MA, USA) containing 2.5 μL of 1:100-diluted serum and 2.5 μL of GST-fusion antigen proteins (10 μg/mL) in AlphaLISA buffer (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL Dextran 500, and 0.05% Proclin 300). The reaction mixture was incubated at room temperature for 6–8 h, mixed with anti-human IgG-conjugated acceptor beads (2.5 μL at 40 μg/mL), and glutathione-conjugated donor beads (2.5 μL at 40 μg/mL), and then incubated for seven days at room temperature in the dark. The chemical emission was read on an EnSpire Alpha microplate reader (PerkinElmer). Specific reactions were calculated by subtracting Alpha values of GST control from the values of GST-fusion proteins. The list of esophageal cancer patients and the results of their anti-FIRΔexon2s autoantibodies with other tumor markers are indicated (Supplementary Table 4 ). The cut off value of anti-FIRΔexon2 autoantibodies (IgG) was 3,171 counts indicated by +2 S.D. of mean of healthy group (Table 1 and Supplementary Table 5 ).
Enspire alpha microplate reader
Manufactured by PerkinElmer
The EnSpire Alpha microplate reader is a versatile laboratory instrument designed for sensitive and accurate detection of various assays in microplate formats. It is capable of performing a range of detection modes, including absorbance, fluorescence, and luminescence, to support a diverse set of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
13 protocols using enspire alpha microplate reader
1
AlphaLISA Assay for Anti-FIRΔexon2 Autoantibodies
2
AlphaLISA Assay for Serum Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AlphaLISA was performed in 384‐well microtiter plates (white opaque OptiPlate™, Perkin Elmer) containing either 2.5 µL 1:100‐diluted serum with 2.5 µL GST or GST‐LRPAP1 protein (10 µg/mL) in AlphaLISA buffer (25 mmol/L HEPES, pH 7.4, 0.1% casein, 0.5% Triton X‐100, 1 mg/mL dextran‐500, and 0.05% ProClin‐300). The reaction mixture was incubated at room temperature for 6‐8 hours, following which anti‐human IgG–conjugated acceptor beads (2.5 µL at 40 µg/mL) and glutathione‐conjugated donor beads (2.5 µL at 40 µg/mL) were added and incubated prior to another incubation at room temperature in the dark for 1‐14 days. Chemical emissions were read on an EnSpire Alpha microplate reader (PerkinElmer), as previously described.19 , 20 , 21 , 22 , 23 , 24 , 25 Specific reactions were calculated by subtracting the alpha counts of the GST control from the counts of GST‐fusion proteins.
3
Measuring Anti-ING1 Antibody Levels with AlphaLISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using 384-well microtiter plates (white opaque OptiPlate, PerkinElmer, Waltham, MA) containing 2.5 μL 1/100-diluted sera and 2.5 μL GST-ING1 or control GST proteins (10 µg/mL), or biotinylated peptides (400 ng/mL) in AlphaLISA buffer containing 25 mM HEPES (pH 7.4), 0.1% casein, 0.5% Triton X-100, 1 mg/mL dextran-500, and 0.05% Proclin-300. We incubated the reaction mixtures at room temperature for 6 to 8 h, and then incubated them at room temperature in the dark for 7 to 21 days with anti-human IgG-conjugated acceptor beads (2.5 µL at 40 µg/mL) and glutathione-conjugated or streptavidin-conjugated donor beads (2.5 µL at 40 µg/mL). After many AlphaLISA trials, we concluded that an incubation time of 7 to 21 days was most suitable to obtain specific, stable, low background, and reproducible results. We read the Alpha photon counts in microtiter plates using an EnSpire Alpha microplate reader (PerkinElmer) as described previously [8 (link), 9 (link), 12 (link)–14 (link)]. Specific anti-ING1 antibody levels were obtained by subtracting the Alpha counts for the GST and buffer controls from those for the GST-ING1 protein and ING1 peptide, respectively.
4
Optimizing AlphaLISA Assay Conditions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AlphaLISA was performed in 384-well microtiter plates (white opaque OptiPlate™, Perkin Elmer, Waltham, MA, USA), containing either 2.5 µL of 1:100-diluted serum with 2.5 µL of GST or GST-PCK1 proteins (10 µg/mL) in AlphaLISA buffer [25 mM N-(2-hydroxyethyl) piperazine-Nʹ-2-ethane sulfonic acid, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL dextran-500, and 0.05% ProClin-300]. We incubated the reaction mixture at room temperature for 6–8 h, and then added anti-human IgG-conjugated acceptor beads (2.5 µL at 40 µg/mL) and glutathione-conjugated donor beads (2.5 µL at 40 µg/mL). The mixture was then incubated at room temperature in the dark for 7–21 days. We measured the chemical emissions using an EnSpire Alpha microplate reader (PerkinElmer), as described previously [8 (link), 9 (link), 17 (link), 18 (link)]. We calculated the specific reactions by subtracting the emission photon counts of the GST control from the counts of the GST-fused PCK1 protein.
AlphaLISA is a novel, recently developed method. After examining suitable AlphaLISA conditions in this study, we concluded that incubation for 7–21 days is the best option to obtain specific antigen-Ab reaction as well as to reduce background noise.
AlphaLISA is a novel, recently developed method. After examining suitable AlphaLISA conditions in this study, we concluded that incubation for 7–21 days is the best option to obtain specific antigen-Ab reaction as well as to reduce background noise.
5
AlphaLISA Immunoassay for Serum Antibody Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum antibody levels of candidate antigens between the patient group and HD group were compared using the AlphaLISA® immunoassay. Briefly, 2.5 μl of serum diluted 1:100 with AlphaLISA® ImmunoAssay Buffer (Perkins Elmer, Waltham, MA, USA) and 2.5 μl GST or GST-fusion protein (10 μg/mL) was placed in a 384-well microtiter plates (Perkin Elmer). The mixture was incubated for at least 3 h at room temperature. Then, anti-human IgG Acceptor Beads (2.5 μl of 40 μg/ml) and Glutathione Donor Beads (2.5 μl of 40 μg/ml) were added, followed by a 14-day incubation. The plate was read using EnSpire Alpha microplate reader (Perkin Elmer). Serum antibody levels against the GST-fusion proteins were determined by subtracting the alpha counts for GST protein from the alpha counts for the GST-fusion proteins.
6
AlphaLISA for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) was performed using 384-well microtiter plates (white opaque OptiPlateTM, Perkin Elmer, Waltham, MA) containing 2.5 μl of 1:100 diluted sera and 2.5 μl of GST or GST-fusion proteins (10 μg/ml) in AlphaLISA buffer (25 mM HEPES (pH 7.4), 0.1% casein, 0.5% Triton X-100, 1 mg/ml dextran-500, and 0.05% Proclin-300). The reaction mixture was incubated at room temperature for 6-8 h. Next, anti-human IgG-conjugated acceptor beads (2.5 μl of 40 μg/ml) and glutathione-conjugated donor beads (2.5 μl of 40 μg/ml) were added and incubated further for 7 days at room temperature in the dark. The chemical emission was examind using EnSpire Alpha microplate reader (Perkin Elmer). Specific reactions were calculated by subtracting Alpha values of GST control from the values of GST-fusion proteins.
7
Quantification of PCSK9 Antibodies via AlphaLISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The levels of serum PCSK9-Abs and -protein were measured using an AlphaLISA for PCSK9. AlphaLISA was conducted with 384-well microtiter plates (white opaque OptiPlate™, PerkinElmer) containing 2.5 μL of 1/100-diluted sera and 2.5 μL of GST or GST-fusion proteins (10 μg/mL) in AlphaLISA buffer (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100 of 1 mg/mL dextran-500, and 0.05% Proclin-300) according to the manufacturer’s instructions (PerkinElmer, http://www.perkinelmer.com/lab-solutions/resources/docs/GDE_ELISA-to-AlphaLISA.pdf ). The reaction mixture was incubated at room temperature for 6–8 h, after which anti-human IgG-conjugated acceptor beads (2.5 μL of 40 μg/mL) and glutathione-conjugated donor beads (2.5 μL of 40 μg/mL) were added, and the mixture was incubated for a further 7–21 days at room temperature in the dark. Chemical emission at 607–623 nm (Alpha photon count), which represents the antigen–antibody binding level, was read on an EnSpire Alpha microplate reader (PerkinElmer). Specific reactions were estimated by subtracting the alpha values of the GST control from the values of the GST-PCSK9 proteins. These methods were precisely described before1 (link)–3 (link).The PCSK9-Abs titers are measured in alpha photon counts and have no units.
8
Measuring Serum Antibody Levels by AlphaLISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) was performed, as described previously [27 (link), 28 (link), 29 , 30 , 34 , 35 , 36 , 37 ]. The assay was performed in 384-well microtiter plates (white opaque OptiPlate™, Perkin Elmer, Waltham, MA) containing 2.5 μl of sera (1:100 dilution) and 10 μg/ml GST or GST-fusion proteins in 2.5 μl of AlphaLISA buffer [25 mM HEPES (pH 7.4), 0.1% casein, 0.5% Triton X-100, 1 mg/ml dextran-500, and 0.05% proclin-300]. After incubating the reaction mix at room temperature for 6–8 h, anti-human IgG-conjugated acceptor beads (40 μg/ml in 2.5 μl) and glutathione-conjugated donor beads (40 μg/ml in 2.5 μl) were added and the samples were incubated for seven days at room temperature in the dark. Chemiluminescence was examined using an EnSpire Alpha microplate reader (Perkin Elmer). Specific reactions were calculated by subtracting the Alpha photon counts of GST controls from those of the GST-fusion proteins.
9
AlphaLISA Assay for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AlphaLISA was performed in 384-well microtiter plates (white opaque OptiPlate™; PerkinElmer, Waltham, MA) containing either 2.5 μL of 1:100 diluted serum with 2.5 μL of GST or GST–DIDO1 protein (10 μg/mL) or biotinylated peptides (bDIDO1-297, bFOXJ2-426, and bCPSF2-607; 400 ng/mL) in AlphaLISA buffer (25 mM HEPES, pH 7.4; 0.1% casein, 0.5% Triton X-100, 1 mg/mL Dextran 500, and 0.05% ProClin 300). The reaction mixture was incubated at room temperature for 6–8 h, after which antihuman IgG-conjugated acceptor beads (2.5 μL at 40 μg/mL) and glutathione- or streptavidin-conjugated donor beads (2.5 μL at 40 μg/mL) were added and incubated, followed by another incubation at room temperature in the dark for 1–14 days. Chemical emissions were read on an EnSpire Alpha microplate reader (PerkinElmer) as described previously [32 (link)–38 (link), 40 (link)–43 (link), 48 , 51 (link)]. Specific reactions were calculated by subtracting the Alpha counts of GST control and buffer control without antigenic peptides from the counts of GST-fusion proteins and biotinylated peptides, respectively.
10
Quantifying Serum Antibodies Using AlphaLISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AlphaLISA was used to quantify the serum antibodies against the purified proteins. The α-luminescent photon counts represent the serum antibody levels (35 (link), 40 (link)). The AlphaLISA assay was performed in 384-well microtiter plates (white opaque OptiPlate; PerkinElmer, Waltham, MA, USA). Each well contained 2.5 μL serum (1:100 dilution) and 2.5 μL GST or GST fusion protein (10 μg/mL) in AlphaLISA buffer (25 mM HEPES [pH 7.4], 0.1% (w/v) casein, 0.5% (w/v) Triton X-100, 1 mg/mL Dextran-500, and 0.05% (w/v) Proclin-300). The mixture was then incubated at 25°C for 8 h. Then 2.5 μL of 40 μg/mL anti-human IgG-conjugated acceptor beads and 2.5 μL of 40 μg/mL glutathione-conjugated donor beads were added and the mixture was incubated in the dark at 25°C for 7–21 d. Chemical emission was measured in an EnSpire Alpha microplate reader (PerkinElmer) as previously described. The reactions were calculated by subtracting the Alpha counts for the GST control from those for the GST fusion proteins.
The levels of serum squamous cell carcinoma antigen (SCC-Ag) (41 (link)) and p53 antibody (p53-Abs) (42 (link)) were evaluated as previously described. The serum SCC-Ag and p53-Abs cutoff values were 1.5 ng/mL and 1.3 IU/mL, respectively.
The levels of serum squamous cell carcinoma antigen (SCC-Ag) (41 (link)) and p53 antibody (p53-Abs) (42 (link)) were evaluated as previously described. The serum SCC-Ag and p53-Abs cutoff values were 1.5 ng/mL and 1.3 IU/mL, respectively.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!