Assay Types
Assay Types:
You could spend the rest of your life studying the physics, equations, and theories behind common lab measuring techniques. We’re going to touch on the basics of a few important ones and bullet point a few important concepts to give you a solid general understanding.
Immunoassays:
Labels for immunoassays can be enzymes, chemiluminescent groups, and fluorescent groups along with other less common techniques.
Competitive (Simultaneous):
In this assay all reactants are added together simultaneously. Labeled antigen and unlabeled patient antigen compete to bind with the antibody. The avidity of the labeled and unlabeled antigen must be the same for this assay to work. The amount of labeled antigen bound to antibody is inversely proportional to the concentration of the unlabeled antigen.
Competitive (Sequential):
In this assay unlabeled patient antigen is mixed with excess antibody first and the reaction reaches equilibrium. Next the labeled antigen is added and allowed to reach equilibrium. The labeled antigen attached to antibody is measured and inversely proportional to the amount of unlabeled patient antigen. Since the assay is sequential it allows more unlabeled patient antigen to bind which allows for smaller amounts of antigen to be detected (a lower detection limit).
Noncompetitive (Sandwich):
In this assay a capture antibody binds to the patient antigen. There is a wash step, then another labeled detection antibody is added and binds to patient antigen. Unbound antibody is washed away and the amount of detection antibody is measured and is directly proportional to the amount of antigen present. The protein or molecule of interest is sandwiched between the capture antibody and the detection antibody.
Noncompetitive assays can also be simultaneous or sequential. In the simultaneous reaction, the patient antigen reacts simultaneously with the capture and labeled antibodies. If the patient antigen is high enough, it can reduce the number of complexes formed and produce a falsely low result known as a hook effect. In a hook effect, the assay response drops off at high concentrations of analyte. (insert picture of hook effect). The hook effect can effect multiple types of immunoassays.
Enzyme linked immunosorbent assay (ELISA):
ELISA is a type of immunoassay that follows the principles outlined above and uses an enzyme for quantification.
Direct vs Indirect ELISA:
In direct ELISA, the detection antibody sticks directly to the molecule of interest. In indirect ELISA, the secondary detection antibody sticks indirectly to the molecule of interest.
Chemiluminescence:
Chemiluminescence, bioluminescence, and electrochemiluminescence are types of luminescence where the excitation state is caused by a chemical, biochemical, or electrochemical reaction and not light. They are used in immunoassays and DNA probe assay systems.
Chemiluminescence is emission of light induced by a chemical reaction. Typically an antibody will bind to the patient antigen of interest, and then another antibody that will cause the luminescence is added to bind to the first antibody. The luminescence is measured and proportional to the amount of antigen in the sample. This is a very common method in the clinical lab.
Electrochemiluminescence:
Electrochemiluminescence is similar to chemiluminescence except the chemical reaction is catalyzed by an electrode instead of a chemical.
Fluorescence:
The basics of fluorescence as a measuring tool is there is an excitation source (usually some form of UV light) that is sent toward a sample. The light passes through a monochromator and emission slit before entering the sample. The sample will absorb the light, reach an excited state, and re-remit the light. The re-emitted light goes through another monochromator and emission slit before being measured using a detector and a computer. The amount of light can be used to quantify the amount of a particular molecule or compound in a sample.
An important concept to understand is after absorption by a molecule, the re-emitted light will have less energy than it originally took in. The wavelength of the re-emitted (emission) light will be longer than the initial excitation light because longer wavelengths have less energy.
A good way to think about fluorescent energy is to use the analogy of a chain reaction car crash. If somebody rear ends the car in front of them and then that car hits the car in front of them, the second collision will have less force than the first collision. A certain amount of energy from the first collision is being absorbed into the car that won’t be transferred into the next collision.
Fluorescent Polarization:
Polarization simply means light is only being let through in one orientation. For example, light from the sun comes at all different directions and orientations, it’s not polarized. Polarized filters (like the ones on your sunglasses) will filter light so only one orientation passes through.
In fluorescent polarization, a fluorescent tag (eg fluorescein) bound to a biochemical molecule will reemit polarized light in the same wavelength it received it as long as the rotational relaxation is slower than the fluorescent decay time. Small molecules will have a faster rotational relaxation time and exhibit depolarized light; however, if a macromolecule such as an antibody is attached to the small molecule the rotational relaxation will be slower than the fluorescent decay time and exhibit polarized light which can be quantified.
To clarify, imagine a molecule absorbs polarized UV light and it is re-emitting that light toward a polarized gradient in the same orientation it received the polarized UV light. The light needs to go through the first gradient, the molecule, and the second gradient before the molecule rotates. If the molecule is spinning too quickly the light won’t travel through the polarized gradient because the rotational relaxation is faster than the fluorescent decay time; however if that same molecule is bound to an antibody it will spin much slower and allow the light to pass through. Specific antibodies can be used to measure targeted molecules using this technique.
Nephelometry and Turbidimetry:
Nephelometry and turbidimetry are analytical techniques used to measure scattered light. Light scattering occurs when radiant energy passing through a solution encounters a molecule in an elastic collision which results in light being scattered in all directions. Unlike fluorescence emission, the wavelength of the scattered light is the same as that of the incident light. Nephelometry and turbidimetry are used to measure immune complexes and drugs. Turbidimetry specifically measures a decrease in transmitted light (how turbid the sample is).