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Protocols

Sandwich ELISA Using Two Primary Antibodies

Required Reagents:
Capture Antibody (preferably affinity purified)
Standard
HRP-Conjugated Primary Antibody
Coating Buffer, 0.05 M Carbonate-Bicarbonate, pH 9.6
Wash Solution, 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0
Blocking (Postcoat) Solution, 50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0
Sample/Conjugate Diluent, 50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0
Enzyme Substrate, TMB
Stop Solution, 0.18 M H2SO4 or other appropriate solution
High Protein Binding Microtiter Plate (i.e. Nunc C bottom Immunoplate 96 well, 446612)

Preparation of Reagents:
Bethyl Laboratories ELISA Accessory Kit is strongly recommended for best results.

Coating Buffer
3.7 g Sodium Bicarbonate (NaHCO3)
0.64 g Sodium Carbonate (Na2CO3)
1 L of distilled water

Tris Buffered Saline (TBS)
6.06 g Tris Base
8.2 g NaCl
6.0 ml 6 M HCl
1 L of distilled water

pH should be 7.2 to 7.8, conductivity should be 14,000 to 16,000

Wash Solution
1 L of TBS
5 ml of 10% Tween 20

Blocking (Postcoat) Solution
1 L of TBS
10 g BSA

Sample/Conjugate Diluent
1 L of TBS
10 g of BSA
5 ml of 10% Tween 20

0.18 M H2SO4
Stock is 18 M
10 ml of Stock solution
1 L distilled water

Procedure:

(Perform all steps at room temperature.)

Coat with Capture Antibody

  1. Determine the number of single wells needed. Standards, samples, blanks and/or controls should be analyzed in duplicate.
  2. Dilute capture antibody to a concentration of 2 – 10 mcg/ml in Coating Buffer. Transfer 100 mcl to each well.
  3. Incubate coated plate for 60 minutes.
  4. After incubation, aspirate the Capture Antibody solution from each well.
  5. Wash each well with Wash Solution as follows:
    1. Fill each well with Wash Solution
    2. Remove Wash Solution by aspiration
    3. Repeat for a total of 3 washes.

Blocking (Postcoat)

  1. Add 200 mcl of Blocking (Postcoat) Solution to each well.
  2. Incubate 30 minutes.
  3. After incubation, remove the Blocking (Postcoat) Solution and wash each well three times.

Standards and Samples

  1. Dilute the standards in Sample Diluent according to desired concentration.
  2. Dilute the samples, based on the expected concentration of the analyte, to fall within the concentration range of the standards.
  3. Transfer 100 mcl of standard or sample to assigned wells.
  4. Incubate plate 60 minutes.
  5. After incubation, remove samples and standards and wash each well 3 times.

HRP-Conjugated Primary Antibody

  1. Dilute the HRP Conjugate in Conjugate Diluent.
  2. Transfer 100 mcl to each well.
  3. Incubate 60 minutes.
  4. After incubation, remove HRP Conjugate and wash each well 5 times.

Enzyme Substrate Reaction

  1. Prepare the substrate solution according to the manufacturer's recommendation. TMB is highly recommended but OPD or ABTS can be used.
  2. Transfer 100 mcl of substrate solution to each well.
  3. Incubate plate 5 - 30 minutes.
  4. To stop the TMB reaction, apply 100 mcl of 0.18 M H2SO4 to each well. If using another substrate, use the stop solution recommended by manufacturer.
  5. Using a microtiter plate reader, read the plate at the appropriate wavelength for the substrate. (450 nm for TMB)

Calculation of Results

  1. Average the duplicate readings from each standard, control, and sample.
  2. Subtract the zero reading from each averaged value above.
  3. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. Other curve fits may also be used.
  4. A standard curve should be generated for each set of sample.

Troubleshooting

Problem: Low absorbance
Incorrect dilutions or pipetting errors
Improper incubation times
Improper preparation of the TMB substrate.
Wrong filter on microtiter reader. Wavelength should be 450 nm for TMB, 490 nm for OPD, or 405 nm for ABTS.
Reagents are contaminated or expired.
Incorrect reagents used.

Problem: High Absorbance
Cross contamination from other samples or positive control
Incorrect dilutions or pipetting errors
Improper washing
Wrong filter on microtiter reader. Wavelength should be 450 nm for TMB, 490 nm for OPD, or 405 nm for ABTS.
Contaminated buffers or enzyme substrate
Improper incubation times
Reagents are contaminated or expired.

Problem: Poor Duplicates
Poor mixing of specimens
Incorrect dilutions or pipetting errors
Technical error
Inconsistency in following ELISA protocol
Inefficient washing

Problem: All wells are positive
Contaminated buffers or enzyme substrate
Incorrect dilutions or pipetting errors
Kit materials or reagents are contaminated or expired.
Inefficient washing

Problem: All wells are negative
Procedure not followed correctly
Contaminated buffers or enzyme substrate
Contaminated Conjugate
Kit materials or reagents are contaminated or expired.

Technical Hints
When preparing coating buffer from the gel capsule, break the capsule apart and pour ingredients into water. Do not place gel capsule into water. The gelatin from the capsule interferes with the binding of the coating antibody to the plate.
Capture antibody diluted with coating buffer should be added to wells immediately.
Coated (covered) plates are stable overnight at 4oC.
Check all buffers for contamination and expiration. When trouble shooting, it may be helpful to start with all new buffers. Make buffers in new or properly cleaned vessels.
Sodium Azide should not be added to any of the buffers.
Dilutions should be made shortly before application and immediately applied to appropriate wells.
Wash buffer should be aspirated from wells. Pouring/Dumping wash buffer from wells may lead to cross contamination.
Excess antibody/analyte should be wiped from pipettes tips when making dilutions.
Incubation time of the Enzyme Substrate will depend on the substrate used and the intensity of the color change. The high standard should have an O.D. reading of about 2.0 and the O.D. reading of the low standard should be above background.
Stop solution should be added to the plate in the same order as the Enzyme Substrate.