Turbidimetry and Nephelometry

Measurement of Precipitation by Light Scattering Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance. Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution. Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes.

Precipitation/Flocculation When soluble antibody binds to soluble antigen (sensitization) there will come a point where lattice formation will occur resulting in precipitation occurring resulting in a visible reaction These immune complexes have fallen out of solution. The Ab at the bottom in the illustration at right is still in the soluble phase.

Turbidimetry Measures turbidity or cloudiness of a solution by measuring the amount of light PASSING THROUGH the solution. Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness. The more cloudy the solution, the less light can pass through.

Turbidimetry The amount of substance being quantitated is calculated based on the results obtained on standards (aka calibrators) and controls. Calibrator is a substance of an EXACT amount, i.e. 50 mg/dL, used to create standard curve. Controls are substances similar to patient samples and have a range, i.e., 43-56 mg/dL Turbidimetry is very simple but not very sensitive.

Standard Curve

Nephelometry Widely used in clinical laboratories because it is relatively easily automated. Based on the principle that a dilute suspension of small particles will scatter light passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (usually about 70 or 75 degrees).

Nephelometry Can detect either antigen or antibody. Endpoint tests all Ag/Ab reaction to go to completion. If complexes get to large will fall out of solution. Causes falsely decreased results Kinetic tests add antigen and antibody then measure at a specific time. Rate of formation must be known. Calculate concentration based on standards.

Nephelometry Measures SCATTERED light bouncing off antigen-antibody complexes. The more light that is scattered the higher the concentration.

Turbidimetry versus Nephelometry Turbidimetry – measures light which PASSES through. Nephelometry – measures light which is SCATTERED.

Turbidity Measure of suspended particles in water – can include inorganic (sediments) as well as organic (bacteria, plankton). High turbidity reduces light transmission (by both absorption and scattering), i.e. larger light attenuation coefficient (K). Can measure with secchi disk (secchi depth, cm) Can measure by weight (total suspended solids (TSS, mg/L) Can measure with turbidimeter = nephelometer (NTU)

NTU Nephelometric Turbidity Units (NTU) Nephelometer comes from the Greek word for cloud, nephos, detector mirror A more popular term for this instrument in water quality testing is a turbidimeter. However, there can be differences between models of turbidimeters, depending upon the arrangement (geometry) of the source beam and the detector. A nephelometric turbidimeter always monitors light reflected off the particles and not attenuation due to cloudiness. In the United States environmental monitoring the turbidity standard unit is called Nephelometric Turbidity Units (NTU)

Turbidity standards Formazine based (carcinogenic) – typically 4000NTU stock diluted. Need to test against NIST standards. Precipitates unless stirred constantly. AMCO beads – non-toxic. Long shelf life. More expensive. Slightly different scattering properties.

Some NTU numbers DI or RO water (approx 0 NTU) Tapwater (<0.3NTU) Surface waters clear (<5NTU) Surface waters visibly turbid (10-50NTU) Silted waters from erosion (up to 1000NTU) Problems for fish/organisms (>50-100NTU) Protect benthic seagrasses (<5-10NTU)

Light Limitation is the Principal Determinant of SAV Distribution Water Quality drives light limitation Secchi depth (m) or attenuation coefficient (Kd) in 1/m Light-limitation depends on water quality Light is absorbed (a) and scattered (b) by water+TSS+color+chl a From mathematical models of light absorption and scattering a direct link between water quality and light available to SAV can be calculated. TSS

Calibration Always check expiration dates. Make Formazin solutions fresh EACH time – check against a standard in turbidimeter before use. Always check 0 NTU first (DI water) Always use a standard higher than max value expected (e.g. 10 NTU or 100 NTU) ALWAYS check for AIR BUBBLES – this is the major source of error or bad data.

Turbidimetry and nephelometry principle: measurement of immune complexes Ag-Ab amounts in the Ab excess reaction in liquid phase in cuvette evaluation: Ag concentration is proportional to - production speed of complexes (kinetic system) - turbidity of complexes („end point“)

Turbidimetry precipitation in solution measurement of light extraction (precipitate absorption) standard curve Turbidimetry extraction measurement laser/quartz lamp photodetector cuvette

Nephelometry precipitation in solution measurement of scattered light standard curve Scattered light detector Nephelometry Scattered light measurement

Usage of turbidimetry and nephelometry measurement of serum proteins‘ concentration (immunoglobulins, acute-phase proteins, complement components C3, C4, transferrin, albumin,…) rapid, fully-automated techniques for large quantity of samples

Measuring Water Clarity Turbidity is a measure of the degree to which the water looses its transparency due to the presence of suspended particulates.  A turbidity measurement could be used to provide an estimation of the TSS (Total Suspended Solids)

Causes of Turbidity There are various parameters influencing the cloudiness of the water. Some of these are:         Phytoplankton         Sediments from erosion        Resuspended sediments from the bottom (frequently stir up by bottom feeders like carp)   Waste discharge    Algae growth  Urban runoff 

Ways to Measure Turbidity Turbidity is measured in NTU: Nephelometric Turbidity Units. The instrument used for measuring it is called nephelometer, colorimeter or turbidimeter, which measures the intensity of light scattered at 90 degrees as a beam of light passes through a water sample. In lakes or bays, turbidity is measured with a secchi disk

Turbidity Values? Turbidity is measured in NTUs and sometimes FTUs (Forel Turbidity Units) No suspended solids present is a value of O NTU, while drinking water should be no more than 5 NTU