To protect yourself from any possible hazards associated with this task wear eye protection. You should also wear latex, nitrile, or vinyl gloves and a lab coat with long sleeves. To protect your legs and feet wear closed shoes and long trowsers. Do not wear sandles, shorts or a short skirt. Wash your hands before eating and when leaving the laboratory. You should review the MSDS for any chemical used in this procedure. In case of a spill with a toxic chemical remove all contaminated clothing and wash affected areas with copious quantities of water. Check location of the nearest safety shower. Eyes should be washed copiously for 15 minutes.
When preparing 0.6 N Hydrochloric acid solution wear goggles that form a seal and nitrile gloves. Always add the acid to the water, not vice versa.
This protocol is based on the Maine Biotechnology Services ELISA protococol for SeM.
| Reagent | Amount | Separate SOP? | Ready |
| Coating buffer (0.15 M PBS, pH 7.6) | 1 liter | Yes | ____ |
| Capture Antibody - MAB212 diluted in coating buffer (2 ug/mL) | 10 mL | Yes | ____ |
| Wash Buffer (0.15 M PBS, 0.05% tween 20) | 1 liter | Yes | ____ |
| Blocking Buffer (1% Non Fat Dried Milk (NFDM) in 0.15 M PBS) | 100 ml | Yes | ____ |
| Sample: phagelysin digested sample, variable dilution profiles, 50 uL per well, | as needed | Yes | ____ |
| Detector Antibody MAB211-biotin diluted in blocking buffer (0.5 ug/mL) | 10 mL | Yes | ____ |
| Tracer Streptavidin Horse Radish Peroxidase (HRP) diluted in wash buffer (1:10,000) | 10 mL | Yes | ____ |
| Substrate, which is turned blue by HRP, TMBW (undiluted - single step reagent) | 10 mL | No | ____ |
| Stop solution which causes blue to yellow color change (0.6 N Hydrochloric acid) | 10 ml | No | ____ |
| Material | |||
| ELISA Plates | ____ | ||
| Plate washer | ____ | ||
| Multichannel pipette | ____ | ||
| Unichannel pipettes | ____ | ||
| Pipette tips | ____ | ||
| Reagent tubes | ____ | ||
| Reagent boats | ____ |
Comments:
| Label Plates | ____ |
| Dispense 50 ul of 2 ug/ml MAB212 capture antibody to all wells** | ____ |
| Cover and refridgerate at 4 C overnight | ____ |
**Use Multichannel pipette
Comments:
| Wash # 1with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Flick then blot dry | ____ |
Comments:
| Prepare blocking buffer (1 g NFDM in 100 mL PBS) | ____ |
| Label expiration date 1 week from date of preparation | ____ |
| Dispense 300 ul of Blocking Buffer (1% Non Fat Dried Milk in 0.15 M PBS) to all wells** | ____ |
| Refridgerate any excess blocking buffer | ____ |
| Incubate plates at room temperature for 1 hour or overnight at 4 C | ____ |
**Use Multichannel pipette
Comments:
| Wash # 1with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Flick then blot dry | ____ |
Comments:
| Prepare phagelysin digested samples/controls in dilution tubes, label and record on summary sheet | ____ |
| Dispense 50 ul of 0.15 M PBS to all wells | ____ |
| Add 50 ul (or 10 ul) of samples to rows in column 1* | ____ |
| Transfer 50 ul (or 10 ul) to column 2** | ____ |
| Repeat transfer through row 12** | ____ |
| Discard the last 50 ul (or 10 ul) from row 12 | ____ |
| Incubate plates with rotation at 37 C for 30 minutes or at room temp for 1 hour | ____ |
| During this incubation, prepare a 0.5 ug/ml solution of the detector antibody MAB211P-biotin, in blocking buffer (1% NFDM in 0.15 M PBS; see separate SOP) | ____ |
*Use single channel pipette
**Use Multichannel pipette, mixing 8 X before each transfer
Comments:
| Wash # 1with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Flick then blot dry | ____ |
Comments:
| Pipet 50 ul well of the detector antibody (MAB211P-biotin) to each well** | ____ |
| Incubate at 37C for 30 minutes or 1 hour at RT, with rotation if possible. | ____ |
| During this incubation, prepare a 1:10,000 dilution of the Streptavidin HRP in wash buffer. See separate SOP | ____ |
**Use Multichannel pipette
Comments:
| Wash # 1 with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Flick then blot dry | ____ |
Comments:
| Pipet 50 ul of the Streptavidin HRP at 1:10,000 to each well** | ____ |
| Incubate at 37C for 30 minutes or 1 hour at RT, with rotation if possible. | ____ |
**Use Multichannel pipette
Comments:
| Wash # 1 with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Wash # 2 with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Wash # 3 with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Wash # 4 with automatic plate washer (0.15 M PBS, 0.05% tween 20), fill 300 uL | ____ |
| Flick then blot dry | ____ |
Comments:
| Add 50 ul of the TMB to each well on plate ** | ____ |
| Start timer for 10 minutes and cover plate** | ____ |
| After 10 minutes, stop the enzyme reaction by adding 50 ul of 0.6N HCL | ____ |
| Read the plate at A(450 nm) | ____ |
**Use Multichannel pipette
Comments: