When light strikes particles suspended in liquid or air, it can be absorbed, reflected, or scattered.
Turbidimetry - measurement of light transmitted through a suspension of particles. Often uses a spectrophotometer as a detector.
Nephelometry - A direct method of measuring light scattered by particles suspended in solution. The use of a sensitive detector is required
Immunonephelometry and Immunoturbidimetry
The determination of soluble antigen concentration by nephelometric or turbidemetric methods involves an antibody and antigen reaction to form insoluble complexes is called immunonephelometry or immunoturbidimetry respectively.
For specific proteins, immunonephelometry has a greater detection range and better sensitivity than immunoturbidimetry.
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1854 Tyndall: all materials scatter light to some extent - even pure liquids and dust-free gases are very slightly turbid.
1871 Rayleigh: developed light scattering theory for small particles
When monochromatic light of intensity Io and wavelength 8 falls on a small particle, light - being electromagnetic radiation, forces the electron clouds of the molecules within the particle to be displaced and oscillate synchronously with the electric field of the incident light.Thus an oscillating dipole is induced in the particle by the incident light.The magnitude of this dipole moment is proportional to the electric field of the incident light.The proportionality constant between the magnitude of the dipole moment and the electric field strength of the light is the polarisability of the electron cloud surrounding the particle.The oscillating electron clouds serve as a secondary source of radiation re-radiating the light in all directions in space.The re-radiated or scattered light has the same wavelength as the incident light and involves no net loss in radiant power - only the direction of propagation is affected.According to Rayleigh’s formula, the total intensity of scattered light is proportional to the number of particles as well is their size and shape.The ratio of light scattered forward to light scattered backward at any pair of supplementary angles centred on 90E is known as the dissymmetry ratio (equal to 1 for small particles)Rayleigh scattering theory applies to the scattering of light by small particles whose dimensions are much smaller than the incident wavelength eg < 8/10.Different theoretical treatment (eg Debye and Mie theory) is required for larger particles since the angular scatter distribution is asymmetrical - more scattered forward than backward. The percentage of light scattered forward increases as particle size increases. Debye and Mie further extended Rayleighs work.
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Turbidimeter: Any spectrophotometer can be used.Most spectrophotometers have low optical noise and can accomplish the task of measuring turbidity effectively.
Nephelometer: The proportion of light scattered in nephelometry is small and the technique requires high-intensity light sources and sophisticated amplification systems.
The mehods Nephelometer employs can be choosed as:
- Kinetic
- Endpoint or
- Fixed time
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CRM470 as WHO reference preparations are formally adopted by the WHO expert committee on biological standardisation and used in assigning values.
Assigned values for 15 plasma proteins, More may follow: Albumin, Caeruloplasmin, haptoglobin, transferrin, transthyretin, IgG, IgA, IgM, CRP, C3, C4, A1AG, α1AT, α1CT, α2 macroglobulin.
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They are a combination of latex agglutination and nephelometry.
Small latex particles are coated with antibody, antigen, or hapten. These latex particles are used in a direct nephelometric assay or in a nephelometric inhibition immunoassay. Particles used vary in size from several nm to several hundred um. There are 4 basic assay systems:
- Ab coupled to particle which is the direct agglutination assay(PETIA)
- Ag coupled to particle and the Ab remains in the solution phase ie inhibition of agglutination formation (PETINIA)
- Dual particle approach where both Ag and Ab are coupled to particles
- Measurement of unagglutinated particle ie particle counting (PACIA)
Acronyms for particle enhanced assays
- LIA Latex ImmunoAssay
- PETIA Particle Enhanced Turbidimetric ImmunoAssay
- PETINIA Particle Enhanced Turbidimetric Inhibition ImmunoAssay
- PACIA PArticle Counting ImmunoAssay
- NIA Nephelometric ImmunoAssay
- NIIA Nephelometric Inhibition Immuno-Assay
- HIA Haemagglutination ImmunoAssay
- HIIA Haemagglutination Inhibition immunoamay
- RPA Reverse Passive Agglutination
- SPIA Sol-Particle ImmunoAssay
History of Particle enhanced light scattering immunoassay
- 1925 Freund demonstrated that soluble proteins adsorbed onto the surfaces of small particles (He used tubercle bacilli) would agglutinate when mixed with antisera raised against the protein
- 1920’s synthetic particles produced
- 1940’s Cannon and Marshall demonstrated usefulness of this approach to evaluate the titre of antisera
- 1943 Calveti studied characteristics of collodion particles to enable specific antibody mediated aggregations to occur
- 1948 Rose Waaler test for Rheumatoid Arthritis using RBC’s from sheep
- 1950’s Boyden first agglutination inhibition test for HCG used as a pregnancy test (Used tannic acid to pretreat RBC’s)
- 1952 reliable and precise production of particles of narrow size distribution
- 1956 Singer & Plotz.Classic paper on use of Latex particles – avoided non-specific agglutination incurred when using rbc’s due to the myriad cell surface antigens.
- 1960’s Particle based assays gain in popularity (faster) usually in a slide test format and visual inspection
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(List some regular analyses of specific proteins)
- α1 microglobulin
- α1 acid glycoprotein
- α1 antitrypsinAlbumin
- α2 macroglobulin
- Apoliprotein A
- Apolipoprotein B
- ASOT (Antistreptolysin titre)
- β2 microglobulin
- C3, C4
- Caeruloplasmin
- CRP
- Haptoglobulin
- IgA, IgG,IgM
- Urine Immunoglobulin
- λ chain, κ light chain
- Microalbumin
- Prealbumin
- Properdin factor B
- RF
- Transferrin
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