ELISA: A Step By Step Method Guide

Step by Step ELISA Guide on SciGine

ELISA: A Step By Step Method Guide

ELISA Biological Method Overview

ELISA is the common acronym for Enzyme-Linked-Immunosorbent Assay. It’s a quick plate based technique for detecting an antigen from a solution. This antigen could be a peptide, protein, antibody, or small molecule. In general, for an ELISA, an antigen is first immobilized on a surface (Step 1 below). Next, an antibody specific to the antigen is flowed over the surface (Step 2). This antibody, is also attached to a chemiluminescence-related enzyme. Treatment with the chemiluminescent substrate facilitates detection of the antibody and the antigen (Step 3). Take a look at these pictures to get an overview of the strategy:

ELISA Steps - SciGine Biological Methods

Types of ELISAs

There are a few different types of ELISA assays but they all follow the basic strategy outlined above.  Essentially, one can choose how to immobilize the antigen on the surface and how the antigen is  detected via the antibody.

  1. Direct Assay: In this method, the antigen is immobilized to the surface and detected directly via an  antibody that’s bound to a chemiluminescent enzyme. (Same as above)
  2. Indirect Assay: In this method, the detecting antibody doesn’t have the chemiluminescent  enzyme. So, another antibody must bind to the first antibody to facilitate detection.
  3. Sandwich Assay: The most common type of ELISA. In this assay, a “capture” antibody is first  immobilized to the substrate. Then antigen is flowed over it so that it gets immobilized to the  surface along with the capture antibody. Finally the detection antibody is flowed over the  substrate and it binds the antigen. This detection antibody may be directly conjugated to the  chemiluminescent enzyme (just like a direct assay) or another antibody may be needed (just like  the indirect assay).

Types of ELISA Assays - SciGine

A Complete Sandwich ELISA protocol

Materials for ELISA

96 well polystyrene plate

Plate shaker

Pipettes

Coating buffer

0.2 M sodium carbonate/bicarbonate buffer, pH 9.4

Wash buffer

0.1 M phosphate, 0.15 M sodium chloride, pH 7.2 with 0.05% Tween 20

Blocking buffer

2% w/v Bovine Serum Albumin in Wash Buffer

Diluent buffer

2% w/v BSA in Wash buffer or a more appropriate buffer such as cell culture media

Stop buffer

2 M Sulfuric Acid

Capture Antibody Solution

15 ug/ml antibody in coating buffer

Detection Antibody Solution

10 ug/ml in (20% Diluent buffer/80% Wash Buffer)

Enzyme Conjugated Antibody Solution

200 ng/ml in (20% Diluent buffer/80% Wash Buffer)

HRP Substrate

TMB (3,3′,5,5′-tetramethylbenzidine). 1 mg/ml. Usually commercially available as a solution.

Step-By-Step Method for ELISA

  1. Prepare a standard curve with your antigen in Diluent Buffer spanning a wide range of concentrations from 0 pg/ml to 3 times your maximum expected antigen concentration (3000 pg/ml approximately)
  2. Dilute the capture antibody to 15 ug/ml and have enough for 100 ul/well
  3. Add the capture antibody to the polystyrene plate, cover, and incubate at room temp. for 2 hours
  4. Remove the solution from each well and add in wash buffer (200 ul per well). Shake for 5 minutes. Repeat 3-5 times.
  5. Add 200 ul of blocking buffer per well, cover and incubate at room temperature for 1 hour (or overnight at 4 oC).
  6. Prepare the samples and standards such that you have 100 ul per well
  7. Remove the wash buffer and add in your sample + standard antigens into different wells. Cover and incubate at room temperature for 1 hour
  8. Repeat Step 4  to wash the plate
  9. Add 100 ul of the Detection Antibody per well. Incubate at room temperature for 1 hour.
  10. Repeat Step 4 to wash the plate
  11. Add 100 ul of the Enzyme conjugated Antibody to each well and incubate for 1 hour at r.t.
  12. Repeat Step 4 to wash the plate (2 times). We need to make sure the plate is very clean and any non-specific binding is minimized.
  13. Add 100 ul of the HRP substrate solution (1 mg/ml TMB)
  14. Incubate until blue (usually about 10 minutes at room temperature)
  15. Add 100 ul of Stop Buffer. This should make the solution yellow.
  16. Measure using a plate reader at 450 nm absorbance.

Notes on this ELISA method

      Note 1. Your standard curve needs to span beyond your antigen concentration because you need to determine the exact amount of your antigen within the linear range of the standard curve. If necessary, dilute your antigen solution down to a point where it is within your standard range.
      Note 2. Concentration of antibodies used will need to be optimized. It is highly likely that you will need to dilute each of the antibodies down rather than increase their concentration because these are at the upper ranges of the necessary concentration.

Tools for Scholarly Search & Research in Biology

Efficient scholarly research helps to reduce unnecessary experiments

Scholarly Research reduces unnecessary experimentation

Nobody wants to reinvent the wheel. Look at all of the pain staking work that goes into re-testing previous experiments and developing methods that have already been known for decades. By taking our time to define our problems, hypothesize our solutions,and search through literature prior to executing on our plans, we can dramatically improve the effectiveness of our research. In fact, people dedicate entire chunks of their lives to being good at searching and testing! (Hint…it’s called a PhD).

In this blog post, I wanted to discuss the tools that are available to help Biologists and biochemists perform effective research. In particular I wanted to focus on biology-related search engines and their development/features since their inception.

A list of tools for biologists to efficiently perform their scholarly searches

  1. Scigine: A search engine for methods in biology, biochemistry, pharmaceutics, and clinical science. Provides step-by-step methods along with the ability to take “notes“, modify methods, and view them on a phone/tablet while performing experiments. Users can also share their notes with colleagues with the click of a button. (~600,000+ methods)
  2. Protocols.io: A “git-hub” for biology with the ability to “fork” methods and modify them. Also includes the ability to form groups with others to share methods online.  (~700 methods)
  3. Protocols-online: A collection of methods aggregated from multiple authors along with forums to ask questions and trending jobs for biologists and biochemists. (~1000 methods)
  4. Vadlo: Or “Fig”, hones google in to specific biology-related websites by providing a custom search text box. It is possible to use this search feature to broadly find methods, presentations, and articles that would normally be found through google.
  5.  DOAJ: The directory of open access journals provides access to over 1.9 million journal articles in biology, biochemistry, and related fields. In case your library does not provide access to journal articles, the DOAJ is a solid resource that is freely available.
  6. PubMed: From the website – “PubMed comprises more than 26 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.” This is the go-to site for scholarly research in biology and related fields.
  7. Microsoft Research Tools: A collection of online tools for genomics and bioinformatics provided by the Microsoft Biology Initiative.
  8. PubAlert: Automatic weekly or daily digests of PubMed searches for your keywords. This is a great tool for keeping up with the latest research in your field. Example, setting up an alert for “Actin” will provide you with an email digest of the most recent articles with “Actin” in the title or abstract.

Hopefully, this blog post provides a good “lay of the land”.

Enjoy the list of tools!

Karthik

SciGine: Inception of A Biology Methods Search Engine

Biology and Biochemistry Methods Search EngineDoing a PhD is about the pursuit of knowledge

The typical biology PhD student spends 5+ years in a PhD program. If you were to follow a student, you might start in the morning by watching him/her looking through papers to check what latest research might be out there related to their topic. Have they been scooped? Is there contradictory research out there that they now have to mention in their next research paper? What new information can they glean from their PubMed searches to understand their research challenges better and inch their way forward with their existing hypothesis?

After performing broad searches on their field of specialization, they would then narrow down their focus on their task at hand. How do I get to my next publication? How can I prove that my protein is being expressed? Is it being degraded by proteases? What kind of data will I need to prove my hypothesis? Really, completing a PhD is all about answering questions and pursuing knowledge — ie performing Biology and Biochemistry ReSearch.  But, the tools that are typically used in this search are not efficient or complete. Academic research, even though it is considered at the edge of knowledge and understanding, moves at a snail’s pace because it lacks the right tools.

 

Improving research efficiency in Biology by using free tools like SciGine

This was the reason that SciGine was created. By using the SciGine search engine, I was hoping that future generations of Biologists and Biochemists would be able to more efficiently tackle their research. The website is set up such that any one can use it by going to SciGine and typing in the search box. The next page shows up results similar to how PubMed and Google organize results as individual biology methods with their search query highlighted along with the author name and time/year of publication. Clicking on an individual method then takes the user to a split screen view consisting of the method steps on the left and a notes section on the right.

I realized that many times, the methods we use for research are amalgamations from multiple articles. So why not design a user interface that allows visitors to take notes while viewing multiple methods easily? On a typical method such as Western Blotting for Kidney Amino-Acid Oxidase this makes method development very simple. I can search for multiple western blot methods using the Browse or Search functionality and then take notes on what exactly I want to do with a protein of interest to me on he right side of the screen. With over 1000+ results from a simple “Western Blot” search, I’m amazed at how much this would have helped me during my PhD.

Saving Biology Methods online for ease-of-access later in lab

Beyond simply taking notes, users can save their methods and protocols and keep a running list of them as part of their Online Methods Notebook. This page is for editing their user profile but also can be used as an online lab notebook. I can envision users using it to keep their day-to-day methods and results online or just to keep a general repository of the methods that they tried out. It’s especially useful to keep this information online because a user can, for example, share their immunoblot method with a lab mate easily and also view their method in lab via a phone or tablet while performing their experiment. There are also option to keep methods private (un-searchable) or public (searchable by everyone) so that important information doesn’t get into the wrong hands.

The ability to upload complete methods online also has the advantage of making it easy to write publications later on. Too often, in a publication, details of their methods are lacking.  By keeping track of what materials were used (especially their part numbers and vendors), the references that were combined to make a method, and how each material was used step-by-step, writing a “Materials and Methods” section for a publication will be a breeze. I want researchers to be able to determine if a chemical from Sigma Aldrich vs. Fisher Scientific was the culprit when it came to their bad data. SciGine will, I expect, improve the quality of research produced by it’s users.

Eventually, I expect there to be more viewers asking and answering questions specific to the methods that they find on SciGine. However, several other resources exist which can help with questions for now. In particular Research Gate and Protocols Online have active communities on their forums that make it easy to get questions answered.

Thank you for reading about the inception of SciGine and how it can be used to advance your Biology and Biochemistry research. Look forward to more posts in the future!

Sincerely,

Karthik