Home LMS THMG155 – Assays and Colormetrics

THMG155 – Assays and Colormetrics


In this episode, Mike and Bob review a few “old school” technologies that don’t use batteries. They still have a place in our arsenal, though!

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Complete Show Notes

5:05 What are Biological Immunoassay Indicators and How Do They Work?

  • Biological immunoassay indicators are simply a series of tests that pick up the presence of a substance
  • Their high degree of specificity results from the use of antibodies and purified antigens as reagents
  • An antigen is basically anything inside a biological system that shouldn’t be there – not just chemicals, but complex proteins and other organisms
  • An antibody is a protein or molecule within a body or a cell
  • One part of the immune system creates the antibody and sticks it to a foreign substance – once the antibody is where it shouldn’t be, the immune system starts getting rid of any substance the antibody is attached to

7:40 What is a Handheld Assay Test?

  • Immunochromatography is used primarily for biological warfare agents
  • We’ve been able to synthetically create antigens that bind to specific proteins on certain organisms – handheld assay tests take advantage of that
  • These tests start off with a sample going onto a sample delivery pad – this is the first step in filtering out things within the sample that aren’t necessary
  • As the sample sits in the staging area, it’s slowly brought up to the conjugate release pad – this is the part of the detector that contains the antibodies, as well as a dye that allows us to see them visually
  • At this point, the sample starts to move up the sample pad across a nitrocellulose membrane – this membrane has areas that are able to capture and hold the antibodies
  • These areas are the little lines we see on many of the tests – if a line shows up, we have a winner!

10:10 What are the Capabilities of Handheld Assay Tests?

  • Used extensively in lab settings and hospital settings for performing a wide variety of tests
  • Much more limited in our world, but used to identify many of the major threats we see
  • This test gives both qualitative and quantitative results, which is very helpful for our purposes

12:05 What are the Limiting Factors of Handheld Assay Tests?

  • These tests are very protein specific, so you need to perform a separate test for each one of the items you’re looking for
  • Time can also be a factor – some tests require at least a few minutes for the magic to happen
  • It’s important to wait a minute or two in both directions – if you wait too long, you can get a false positive because the antibodies will start moving around the test strip and bind to the collection point
  • A matrix effect can also cause you to see both results at once – occurs when there is a product in your sample that prevents the antibodies from sticking to the target protein
  • You can avoid this by doing a 1:10 dilution factor and re-running the test – ideas is to dilute enough to reduce the effect of the non-binding agent
  • Be sure to always check your control line – without a control line, you might have something in your sample that’s producing bad results
  • You can also get a false positive if the substance you’re testing for is similar to what you’re testing – antibody sees a protein on an organism, but doesn’t know it’s not the organism you’re looking for
  • Another false positive is referred to as the hook effect, which is what happens when too much of an antigen is added to the assay test

18:35 What are Chemical Agent Monitors (CAMs) and How Do They Work?

  • Comprised of a variety of colorimetric indicators – includes colorimetric detector tubes, indicating papers (pH paper and meters), reagents, and test strips
  • All of these tests use a chemical reaction to create a change in something’s (a medium’s) color – this is intentionally vague
  • Types of colorimetric indicators:
    • pH paper
      • Used when we’re testing for acids and bases – color changes accordingly
      • Some high-quality papers even indicate the specific number of the acid or base
      • Basics are blue, while acids are red
      • You’ll see a wide variety of pH papers on the market – some are very simple, while others are more complicated and specific
      • There’s always plenty of room for human error when you’re using pH paper – judging colors with the human eye can be problematic, especially since everyone sees color differently
      • pH paper strips can also get bleached out before we get a chance to read them
      • Also not great when you’re dealing with dyed or dirty products since the strips will just turn brown
    • Colorimetric tubes
      • Colored cigarette-shaped object with sand, glass screens, cotton, etc.
      • Break off the tip, put it into your pump, and pull an air sample based on the directions on the tube
      • Air sample goes through a series of reagents that are typically solid
      • Tube then shows you the color changes as the concentration intensifies based on the gradients on the glass (or other directions)
      • Widely used in industry – pull a specific chemical to make sure you’re not dealing with any other chemicals
      • Very chemical-specific, so they help you verify the readings on your meters
      • Cross-sensitivities aren’t really an issue with colorimetric tubes, which is very helpful
      • This method can be expensive since you have to test each substance individually – this also means you need a lot of them
    • M8 paper
      • Military version of pH paper for chemical warfare agents
      • Dip paper in sample – colorimetric change occurs and you then compare it against three potential agents
      • Largely used to detect nerve agents and blister agents
      • Lots of cross sensitivities to deal with, which can be problematic
    • Chip sets
      • Also referred to as CMS kits (i.e. Dräger)
      • Puts colorimetric tubes onto 10 or more plastic rectangles – electronic system reads the barcode on the chip and tells you exactly what to do
      • Very simple and effective, but it doesn’t always work – it’s important to be careful when loading the chip into the electronic system
      • Doesn’t require you to pump the colorimetric tubes, which is a big positive because it saves you time and effort
    • Test strips
      • Examples include Spilfyter strips, water finding strips, and chemical finding strips
      • Work similarly to pH paper – take multiple samples and drop it across 5 or 6 tests
      • Provides information on hydrocarbons, pH, oxidizers, nitrates, etc.
      • Also includes water-finding paste, hydrocarbon-finding paste, dyes, KI paper, F paper, etc.
      • Limiting factors are similar to what we deal with when using pH paper
    • pH reagents
      • Dip reagent into water, add red and yellow dye to create a graph indicator, overlay colors, and get your pH reading
      • These are generally much more specific than a general pH strip and are often used when maintaining pools
      • Limiting factors are similar to what we deal with when using pH paper
    • M256A1 chemical agent detector kits
      • Canadian-made kit used for the detection of blister, blood, and nerve agents
      • Kit contains 12 vapor samplers, M8 detector paper, and instruction cards
      • Vapor samplers are sealed in individual plastic laminated foil envelopes, used once, and then discarded
      • Each vapor sampler holds five test components used in an analysis:
        • Glass ampoules
        • Hinged heater assembly
        • Three test spots
        • Hinged protective strip
        • Lewisite-detecting tablet with a running tab
      • The two glass heater ampoules are used with the heater assembly to detect the blister agents
      • There are three labeled test spots on each sampler – each one is a different shape and made from a different material for a specific analysis
      • The blister agent test spot is square and made of chromatographic media
      • The blood agent test spot is round and made of a glass fiber filter
      • The nerve agent spot is star-shaped and made from filter paper
      • Expected color changes are printed above each test spot and take around 15-20 minutes to occur
      • A protective strip is hinged to the sample over the blood and nerve agent test spots to protect them from accidental contamination
      • The Lewisite detector tab is covered by a protective plastic pull-tab that must be removed before use
      • False readings may occur under high temperatures and when exposed to petroleum products
      • Capable of detecting chemical warfare agents in small amounts
      • This test is very difficult to do properly in one shot – takes a lot of practice and training to get it right
      • Cross-contamination is also a problem when using M256A1 chemical agent detector kits
  • Colorimetric methods are seldom used as part of an integrated system for detecting and identifying chemicals – instead, they’re often used independently or to confirm a preexisting analysis
  • Colorimetric tubes and pH paper comprises about 27% of the chemical detection equipment that’s used

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