Team:USTC CHINA/Notebook/Protocols/ELISA

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<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a></div></div>
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<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a>&gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/ELISA">protocols ELISA</a></div></div>
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<h1>Gel Extraction</h1>
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<h1>ELISA</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>ELISA
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Protocol
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A. Antigen Coating
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1. Excise gel slice containing DNA fragment of interest.
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1. Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer (coating buffer), e.g. human IgG (0.025mg/ml) in coating buffer.
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2. Add 3×sample volume of Buffer DE-A.
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2. Pipette 0.2 ml of the above solution to each well of the microtiter plate.
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Incubate at 75° C for 15-20 min or until gel melts completely.
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3. Incubate at 37 0C for 30 min.
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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4. Remove the coating solution. Wash three times with PBS-T (0.2 ml/well).
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<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />
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5. Blocking step: 0.5% BSA-PBS (0.2ml/well) at 370C for 30 min (an additional blocking step may be required to block non-specific binding).
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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B. Primary Antibody Reaction
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1. Dilute the primary (first) antibodies in PBS-T, e.g. Rabbit-anti-human IgG antiserum (different dilutions of antiserum, 1:400~1:51200).  
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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2. Add 0.2 ml of the diluted first antibody to each well. Negative control should be included (No first Ab.).  
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Repeat wash with Buffer W2
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3. Incubate at 37 0C for 1 hour.
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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4. Wash three times with PBS-T (0.2 ml/well).
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C. Application of Secondary Antibody
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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1. Dilute the peroxidase conjugated secondary antibody in PBS-T, e.g.  
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Goat-anti-rabbit IgG-HRP (1:20,000). Add 0.2 ml of this solution to each well.
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2. Incubate at 37 0C for 30 min. 
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3. Wash three times with PBS-T (0.2 ml/well).
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D. Substrate Preparation
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1. During the last incubation and immediately before use, dissolve  
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o-Phenylenediamine in Citric acid-sodium hydrogen phosphate buffer, add 30% H2O2 before use.
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E. Development
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1. Add 0.2 ml of the freshly prepared substrate to each well.
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2. Orange-yellow color should develop in positive wells after 5 minutes.
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3. Stop reaction with 50 l per well of preferred stopping reagent and read at 490nm in a microplate reader. 
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REAGENTS
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1. Coating buffer (Carbonate-Bicarbonate buffer, pH 9.6) :    Na2CO3  0.15g, NaHCO3  0.293g,  add H2O to 100ml (pH 9.6). 
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2. Phosphate buffered saline (PBS):    NaCl  8g,   KCl    0.2g,   KH2PO4  0.24g,Na2HPO4. 12 H2O  2.9g,  add H2O to 1000ml (pH 7.4). 
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3. Washing buffer (PBS-T):  PBS + 0.05% Tween 20.  
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4. First antibody: Rabbit-ani-human IgG antiserum.
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5. Peroxidase conjugated secondary antibody: Goat-anti-rabbit IgG-HRP (1:20,000). 
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6. Substrate: substrate buffer(fresh): 
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0.1M Citric acid (2.1g/100ml), 6.1ml;
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0.2M Na2HPO4. 12 H2O(7.163g/100ml), 6.4ml;  add H2O 12.5ml, 
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dissolve 8mg o-Phenylenediamine, and add 40l of 30% H2O2 before use. 
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7. Stopping reagent: 2M H2SO4
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Revision as of 14:37, 25 September 2013

ELISA

ELISA A. Antigen Coating 1. Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer (coating buffer), e.g. human IgG (0.025mg/ml) in coating buffer. 2. Pipette 0.2 ml of the above solution to each well of the microtiter plate. 3. Incubate at 37 0C for 30 min. 4. Remove the coating solution. Wash three times with PBS-T (0.2 ml/well). 5. Blocking step: 0.5% BSA-PBS (0.2ml/well) at 370C for 30 min (an additional blocking step may be required to block non-specific binding). B. Primary Antibody Reaction 1. Dilute the primary (first) antibodies in PBS-T, e.g. Rabbit-anti-human IgG antiserum (different dilutions of antiserum, 1:400~1:51200). 2. Add 0.2 ml of the diluted first antibody to each well. Negative control should be included (No first Ab.). 3. Incubate at 37 0C for 1 hour. 4. Wash three times with PBS-T (0.2 ml/well). C. Application of Secondary Antibody 1. Dilute the peroxidase conjugated secondary antibody in PBS-T, e.g. Goat-anti-rabbit IgG-HRP (1:20,000). Add 0.2 ml of this solution to each well. 2. Incubate at 37 0C for 30 min. 3. Wash three times with PBS-T (0.2 ml/well). D. Substrate Preparation 1. During the last incubation and immediately before use, dissolve o-Phenylenediamine in Citric acid-sodium hydrogen phosphate buffer, add 30% H2O2 before use. E. Development 1. Add 0.2 ml of the freshly prepared substrate to each well. 2. Orange-yellow color should develop in positive wells after 5 minutes. 3. Stop reaction with 50 l per well of preferred stopping reagent and read at 490nm in a microplate reader. REAGENTS 1. Coating buffer (Carbonate-Bicarbonate buffer, pH 9.6) : Na2CO3 0.15g, NaHCO3 0.293g, add H2O to 100ml (pH 9.6). 2. Phosphate buffered saline (PBS): NaCl 8g, KCl 0.2g, KH2PO4 0.24g,Na2HPO4. 12 H2O 2.9g, add H2O to 1000ml (pH 7.4). 3. Washing buffer (PBS-T): PBS + 0.05% Tween 20. 4. First antibody: Rabbit-ani-human IgG antiserum. 5. Peroxidase conjugated secondary antibody: Goat-anti-rabbit IgG-HRP (1:20,000). 6. Substrate: substrate buffer(fresh): 0.1M Citric acid (2.1g/100ml), 6.1ml; 0.2M Na2HPO4. 12 H2O(7.163g/100ml), 6.4ml; add H2O 12.5ml, dissolve 8mg o-Phenylenediamine, and add 40l of 30% H2O2 before use. 7. Stopping reagent: 2M H2SO4

notebook

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