This protocol describes the detection of protein antigens using a “sandwich” Enzyme Linked Immunosorbent Assay (ELISA) technique, in which an antigen is sandwiched between two different antibodies. The principle by which this ELISA technique operates is shown below:

• Immobilization of capture antibody.
• Binding of antigen to immobilized antibody.
• Binding of second antibody, linked to an enzyme, to bound antigen (formation of immune complex).
• Detection of immune complex using appropriate enzyme substrate.

This protocol uses Goat Anti-Rabbit IgG (GAR) as capture antibody, Rabbit Anti-Mouse IgG (RAM) as antigen and Goat Anti-Rabbit Horseradish Peroxidase conjugate (GAR-POD) or Goat Anti-Rabbit Alkaline Phosphatase conjugate (GAR-AP) in an immune “sandwich” assay. Concentrations of immune reagents listed in these methods are specific for this set of reagents. Immune reagents from other suppliers may be used; however, specific activities will be different. Therefore, working dilutions must be adjusted for desired sensitivity.

Immunoassay systems using other reagents should be optimized for antibody concentrations, blocking conditions, immune incubation times, wash conditions and choice of enzyme/substrate combination.

Membranes
Biodyne or Immunodyne ABC Membranes

General Reagents

• Phosphate-buffered Saline pH 7.2 (PBS) containing 150mM NaCl, 40mM Na2 HPO4 and 8mM NaH2PO4
• Casein blocking solution: 0.5% Hammersten Grade Casein (Gallard Schlessinger Cat. No. 44020) (w/v) in PBS
• Wash solution: 0.1% Triton X-100 (w/v) in PBS
  

Immune Reagents

• Goat Anti-Rabbit IgG (Sigma Chemicals Cat. No. R-3128)
• Rabbit Anti-Mouse IgG (Sigma Chemicals Cat. No. M-9637)
• Goat Anti-Rabbit IgG conjugated to Alkaline Phosphatase (Sigma Chemicals Cat. No. A-7650)

Enzyme Detection Reagents

• Nitro blue tetrazolium (NBT) (Sigma Chemical Co., Cat. No. N-6876) 75mg/ml in 70% (v/v) di-methyl formamide
• 5-Bromo-4-chloro-3-indolyl Phosphate (BCIP) (Sigma Chemical Co., Cat. No. B-8503) 50mg/ml in di-methyl formamide Buffer: 0.1M
• Tris-HCL (pH 7.5), 0.1M NaCl, 50mM MgCl2 Working Substrate solution is prepared as follows: 33µl NBT, 25µl BCIP in 7.5ml buffer. For optimal results use 0.25ml substrate /cm² membrane. Solution should be prepared freshly.

Preparation of Membrane
Note: Gloves should be worn at all times to prevent membrane contamination by fingerprint residue.

• Remove the roll of membrane from package, cut to desired dimensions. (In this assay ICM wide strips are used.) Replace the roll of membrane in package. If Immunodyne ABC Membrane is used, the unused portion of the roll should be replaced in the mylar bag containing the supplied desiccant removal of excess air from the bag prior to sealing (e.g. by tape or heat seal) and storage at room temperature or placement in vacuum desiccator is recommended.
• 1cm wide strips of membrane are used for testing. These strips are subdivided into 1cm segments by lines drawn with a pencil.
  Note: It is important to number the test strips with a pencil to ensure that they can be identified during testing. Numbers should be applied to area not covered by spot.
• Load: Dilute capture antibody in PBS. Other buffers at neutral pH may be used with Biodyne membranes. Buffers containing amino groups such as Tris should not be used for immobilization on Immunodyne ABC Membranes. Note: Place the membrane strips on a non-absorbent surface. Spot load 1µL spots containing 100pg, 1ng, 10ng,100ng, and 1µg of capture antibody onto squares of the membrane strip.
• Air-dry the membrane for 5 minutes. Note: Two to five minutes are sufficient for complete binding. Longer times may be used. In certain systems, highest sensitivities have been achieved when the membrane was blocked immediately after spot loading.

Immunodetection
Each of the following steps is performed in a 100mm petri dish containing 10mL of solution to ensure adequate interaction between membrane strips and reagents. (Plastic bags, boxes or tubes on a roller mixer may also be used to perform incubation steps.) Up to 5 membrane strips, 1 x 5cm each, may be incubated in one petri dish. Make sure that all strips are completely immersed beneath the surface of the liquid. Gently agitate samples during all incubation steps on an orbital shaker.

• Block: Place the membrane strips in a petri dish with 0.5% casein/PBS blocking solution. Place the petri dish on an orbital shaker and gently agitate for 30 minutes. Note: At this point, the membrane strips may be dried at 37°C for 10 minutes, or air-dried at 20-30°C for >30 minutes and stored.
• Antigen: Place the membrane strips into two clean petri dishes with RAM antigen diluted in PBS to 1µg/mL. Use 10mL of diluted antigen per petri dish. Gently agitate the membrane strips on an orbital shaker for 30 minutes.
• Wash: Replace antigen solution in petri dishes with 10mL wash solution per dish. Wash membrane strips in 2x changes of wash solution, 5 minutes per wash. Gently agitate membrane strips on orbital shaker during wash steps.
• Rinse: Replace wash solution in petri dishes with 10mL of PBS. Gently agitate membrane strips on orbital shaker for 1 minute.
• Second antibody-conjugate: Replace the PBS solution in the petri dishes with 10mL of Goat Anti-Rabbit IgG-alkaline Phosphatase (GAR-AP) diluted 1/1000 in Blocking Solution.
• Wash: Repeat wash procedure listed above.
• Rinse: Replace wash solution in petri dishes with two changes of deionized or distilled water. Gently agitate membrane strips on orbital shaker, 1 minute per change of rinse solution. (Substrate buffer may also be used for this rinse step.)

Detection Methods

• Prepare substrate solution immediately prior to use.
• After performing previous steps, replace water in petri dishes with 7.5mL per dish of substrate solution. Gently agitate membrane strips on orbital shaker for 5 minutes.
• The reaction may be stopped by removing color development solution and adding deionized water. 
• Color intensity of the spots may be measured with a reflectometer. If a permanent record is required the strips should be photographed or scanned shortly after development. Color intensity may fade with time.
• As an alternative Peroxidase conjugates may also be used with appropriate Substrates i.e. 4CN, DAB or TMB. These may result in different sensitivities and would therefore require re-optimization.