latex agglutination

1)What is the principle behind Latex Agglutination?

When a sample containing the specific antigen (or antibody) is mixed with an antibody (or antigen) which is coated on the surface of latex particles (sensitized latex), agglutination is observed. he interaction between a particulate antigen and an antibody results in visible clumping called agglutination. Antibodies that produce such reactions are known as agglutinins. Agglutination reactions are similar in principle to precipitation reactions; they depend on the cross linking of polyvalent antigens. When the antigen is an erythrocyte it is called hemagglutination. All antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly good agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a good agglutinating antibody. Occasionally, it is observed that when the concentration of antibody is high (i.e. lower dilutions), there is no agglutination and then, as the sample is diluted, agglutination occurs. The lack of agglutination at high concentrations of antibodies which is called the prozone effect is due to antibody excess resulting in very small complexes that do not clump to form visible agglutination.

2)What is agglutination inhibition test?

    If the antibody is incubated with antigen prior to mixing with latex, agglutination is inhibited; this is because free antibodies are not available for agglutination.In agglutination inhibition , the absence of agglutination is diagnostic of antigen, provides a high sensitive assay for small quantities of antigen. for eg : Home pregnancy kits includes latex particle coated with human chorionic gonadotropin ( HCG) and antibody to HCG. HCG is a glycoprotein hormone secreted by developing placenta shortly after fertilization. The addition of urine from a pregnant women, which contains HCG , inhibits agglutination of latex particles when the anti-HCG antibody is added; thus the absence of agglutination indicates pregnancy.

3)Why is glycine-saline buffer used to reconstitute the antigen and antiserum?

It is an amino acid, one of the building blocks of proteins. Also can play a role of osmoprotectant, helping organisms to withstand osmotic stress. pKa value of glycine is 9.78, hence the effective pH range for glycine is 8.8 and 10.6.So the basisiyy will suit for reconstituting the antigen and antiserum.  

4)Why are latex beads used for coating the antigens? Can we use anything else? Why? Which would be a better option?

1.      List the conditions under which you would get a positive result in the reaction and the factors responsible for it.

2.      Suppose you do the reaction and get a negative result where you think you should observe a positive reaction. List at least 5 factors which you would consider for trouble-shooting the experiment.

3.      Suppose you do the reaction and get a positive result where you think you should observe a negative reaction because you know that the antiserum is not against that antigen. List at least 3 valid scientific reasons for such an observation.

4.      How can you test and justify if the antigen has been coated on the latex beads? (Other than testing it with the positive reaction by addition of antiserum)

5.      What is the role of blocking buffer in the experiment?

6.      Can you think of at least 5 applications for latex agglutination in diagnostics, labs, industry or research?