Detailed Diagram of a Sandwich ELISA protocol

Come back next week where I will  post some information about Western Blocking procedures to help all life science researchers. If you have a request for different topic during the next few weeks please let me know.

Hi again, we are very sorry for our absence but, we are going to start writing and providing technical information for researches throughout the world. If you have a question or would like to start a topic please let us know.

This weeks topic is a sandwich ELISA protocol to use with our Leinco Technologies ELISA development kits or for general purposes. Leinco Technologies validates many of their products using ELISA (Enzyme-linked immunosorbent assay) methods.  This protocol provides an initial set of conditions; however, further optimization may be required on an individual basis.

STEP 1:

Coating the Plate with Capture Antibody

1.  Dilute unlabeled capture antibody to a final concentration of 1-10 μg/mL using PBS or carbonate/bicarbonate buffer (pH7.4).

2.  Transfer 50-100 µL per well to ELISA microplate (Nunc Maxisorp Prod.#442404).

3.  Seal the plate and incubate overnight at 4°C.

4.  Bring the plate to room temperature and flick off the capture antibody solution.

5.  Wash the plate three times with PBS/Tween by using a squirt bottle, multi-channel pipettor or automatic plate washer.

6.  Tap the plate on paper towels after the last wash to remove residual wash solution.

Step 2:

Blocking the Plate

7.  Make a 1x blocking solution consisting of BSA in PBS azide and add 200 ul/well

8.  Seal the plate and incubate at 37°C for 2 hr or at 4°C overnight.

Step 3:

Add Antigen Coating Sample

Dilute samples and standards to desired concentrations using Blocking buffer.

9.  Add appropriate amount of diluted samples to each well.

10.  Seal plate and incubate for 2-4 hrs at RT or overnight at 4°C.

11.  Wash the plate three times with PBS/Tween and blot on paper towels after last wash.

Step 4:

Adding Detection Antibody

12. Dilute the biotin-labeled detection antibody to 0.25-2 µg/mL in Blocking buffer and add 100 µL to each well.

13.  Seal the plate and incubate for 1-2 hr at RT.

14.  Wash the plate three times with PBS/Tween and blot on paper towels after last wash.

Step 5:

Adding UltraAvidin-Horseradish Peroxidase (Av-HRP)

15.  Dilute the Av-HRP conjugate to its optimal concentration in Blocking buffer (typically 1/500-1/2000).

16.  Add 100 μL per well, seal the plate and incubate for 30 min at RT.

17.  Wash the plate five times with PBS/Tween and blot on paper towels after last wash.

Step 6:

Detection

18.  Add 100 µL of TMB Microwell Substrate (Leinco Prod. #T118) solution to each well (substrate solution should be at RT prior to use).

19.  Once a soluble blue reaction product develops, the plate can be read at 370 nm or in the range of 620 nm to 650 nm. The absorbance values of the sample should be monitored so that a linear curve can be plotted. For increased sensitivity, add 50 μl of a stop solution such as 1.0 N HCl or 1.0 N sulfuric acid to create a soluble bright yellow reaction product. After stopping the enzymatic reaction the plate should be read at 450 nm. Estimated incubation times for substrate range from 20 to 30 minutes.

Solutions You Need:

Note: Do not use sodium azide in any buffers or solutions, as sodium azide inactivates the horseradish-peroxidase enzyme.  All solutions should be at ambient temperature prior to use.

PBS: Dilute 10xPBS to 1xPBS in sterile water.

Wash Buffer: 0.05% Tween-20 in PBS

Block Buffer: 1% BSA in PBS *

Diluent: 0.05% Tween-20, 0.1% BSA in PBS *

* Sterile filter and store at 4°C for up to 1 week.

Thanks and see you again next week

I would like to suggest that world class scientists conducting In vivo and In vitro functional research with monoclonal antibodies and recombinant proteins consider purity specifications as important criteria for selecting a suitable  protein reagent. It’s my opinion that researchers do not have readily available at their disposal, monoclonal antibodies and recombinant proteins that meet the purity levels required to generate reliable data from functional research studies.  I believe such data is desired by world class scientists, whether conducting basic life science research or for development of protein therapeutics.

Through our close collaborations with you, our academic and corporate partners, we have determined that certain critical purity specifications should be considered for validation of authentic functional formulation antibodies and recombinant proteins. Important purity specifications for functional applications include minimization or complete removal of antibody aggregates, endotoxins, leached protein A, DNA, RNA, and the selection of a final formulation buffer that is neutral to physiological functions within test animals and stabilizes the protein’s biological activity and format.

There are nearly 200 monoclonal antibodies (constituting 20% of the protein therapeutics) in human clinical trials. 1

A robust purification process is required to achieve homogenous monomeric monoclonal antibody preparations. For monoclonal antibodies, it is important to develop a final buffer formulation and storage conditions that maintain the original monomeric form of the protein following purification.  Potential effects of antibody aggregates include hypersensitivity or anaphylaxis, changes in biological activity, organ failure, activation of cellular receptors intending to neutralize and diminished overall efficacy of the antibody. 

Antibodies can undergo covalent and non-covalent associations that are highly dependent on formulation including pH, ionic strength and excipients. 1 Multiple intra-domain and inter-domain linkages can lead to disulfide bonds causing heterogeneity and aggregation. 1 Based on our extensive development and manufacturing experience, I would like to suggest that the following factors affecting aggregation be studied to assure stability of your antibody for functional applications at a very early stage: protein structure, freeze rates, storage temperature, freeze thaw cycles, storage containers, size of containers, buffer formulation, pH and photo stability.

Endotoxins are complex bacterial toxins composed of proteins, lipids, and polysaccharides, which are released upon lyses of gram negative (-) bacteria that may have entered the manufacturing process. Endotoxins, also called lipopolysaccharides (LPS), are major contaminants found in commercially available proteins, antibodies or biologically active substances, which often complicate study of the biological effects of the main protein ingredient. The presence of small amounts of endotoxin in recombinant proteins and antibody preparations can cause side effects in host animals such as endotoxin shock, tissue injury, and even death. Due to these reactions, it is essential to remove endotoxins from drugs, injectables, and other biological and pharmaceutical products.  In conclusion, endotoxin levels must be considered when developing potential protein therapeutics, conducting preclinical trials and manufacturing medical devices that come in contact with blood, lymph or cerebrospinal fluid. 2, 3, 4

The presence of Protein A in preclinical or therapeutic monoclonal antibody preparations due to leaching during Protein A chromatography has been linked with toxicity, anaphylaxis, antibody aggregation and interference with immunoassays in host animals and humans.  Sensitive ELISA assays have been developed to detect sub nanogram levels of leached protein A. 5, 6  Purification procedures must be developed to minimize protein A leaching. Considerations such as temperature, column washing and matrix regeneration should be considered to reduce protein A contamination to levels < 10 ng/mg of antibody.  

We typically use a two step chromatography method to remove trace amounts of genomic DNA and RNA to assure purity criteria is met for In vivo functional formulation monoclonal antibodies.  Once the purification method is established, formulation screening is initiated early in the development phase before establishing the dose for animals.  Formulation is an important factor to eliminate degradation from monomer to aggregates especially when protein concentrations are often high due to dosage requirements.   

1. Addressing Stability of Biological Drugs. Genetic Engineering & Biotechnology News. Vol. 25, No. 6 Mar 15, 2005

2. Transfusion and Infusion Assemblies and Similar Medical Devices. Chapter <161>. USP 31, p. 124. United States Pharmacopeial Convention. Rockville, MD, 2007.

3. Bacterial Endotoxins Test. Chapter <85>. USP 31, pp. 98-102. United States Pharmacopeial Convention, Rockville, MD, 2007.

4. Methods of Endotoxin Removal from Biological Preparations a Review.  J Pharm Pharmaceut Sci 10 (3): 388-404, 2007 

5. The measurement of staphylococcal protein A by ELISA in immunoglobulin preparations. Biologicals Oct, 19 (4) 271-279, 1991

6.  Purification and Analysis of Recombinant Proteins a Book. By Ramnath Seetharam, Satish K. Sharma Science 321 Pages 1991