Detecting Immunoagglutination A Visual Observation

The light scattered from a monodisperse suspension of particles makes the latex look milky in appearance. If the suspension of particles is aggregated by some process then the microspheres clump together to produce a coarse granular suspension that resembles curdled milk in appearance. Many of the latex agglutination tests developed are performed manually and the agglutination is detected by visual observation. In these tests larger particles of several hundred nanometers have to be used, the most common size being 0.8 pm. It has been established that about 100 clumps must be seen to determine agglutination and that these clumps must be about 50 pm to be seen by eye [100]. For a particle size

FIG. 13 Immunoreactivity of IgG anti-CRP (■) and F(ab')2 anti-CRP (•) molecules physically (open symbol) and covalently (closed symbol) adsorbed on latex particles. The immunoreactivity is measured by nephelometric monitoring of the immunoaggrega-tion reaction with human CRP for 10 min. Both IgG-latex conjugates have the same protein coverage. Also, both F(ab')2-latex conjugates have the same protein coverage.

FIG. 13 Immunoreactivity of IgG anti-CRP (■) and F(ab')2 anti-CRP (•) molecules physically (open symbol) and covalently (closed symbol) adsorbed on latex particles. The immunoreactivity is measured by nephelometric monitoring of the immunoaggrega-tion reaction with human CRP for 10 min. Both IgG-latex conjugates have the same protein coverage. Also, both F(ab')2-latex conjugates have the same protein coverage.

of 0.8 pm, about 105 latex particles is required to make one visible aggregate, and about 107 particles is needed to determine agglutination in a given test. Based on these calculations and assuming that about 10 bonds are required per particle to hold them together, Bangs has evaluated the sensitivity of such a manual test to be in the order of picograms [100]. Although quite useful in the laboratory and cheap due to the absence of equipment needs, the major limitation of these manual assays is that the only quantitation possible is to use serial dilutions of the test fluid and to look for the disappearance of immunoagglutination.

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