Latex aggluination: is a method for rpaidly measuring a substance to be analyzed, by visually or optically detecting a degreer of latex-particle-allutination caused by a reaction of the substance to be analyzed with the antigen or antibody immobilized on the latex particles. When an antigen and its corresponding antibody come into contact, they combine to form a complex which is less soluble. However, the particle size is so small that certain carriers must be employed to aid visualization. Fisk (US 3088875) discose the preparation and use of a polymerized styrene latex as a carrier. Optimum results are acheived when polystyrene latex is employed in a particle size in the range from 0.15 to 0.25 micron (um).
Agglutination of latex micro-particles is used in the diagnosis of various infections, and to detect disease markers or chemical compounds in specimens of body fluids. Test-card latex particle agglutination (LPA) is usually read by eye as a qualitative indicator in medical diagnostic laboratories, although quantitative assays that rely on turbidimetric methods can determine analyte concentration.
–Immobilization of the antigen or antibody:
Sjoholm (US 4,061,466) discloses immobilized biologically active substances comrpising microporus, spherical particles in gel form and in the form of a 3D network. The particles which have sizes below 10 um and an average diameter in the range of from 0.5 to 4 um, preferbly 1m are capable of exeriting biological activity against substances. The particles can be prepared by dispersion polymerisation.
Sharma (US 4,360,358) discloses a method for prearing an immunologically active agent such as an antibody or antigen coated solid phase such as a coated tube or a polymer latex for solid phase immuno assays.
Nowinski (US 4,752,638) also discloses antigens, antibodies, receptors, and ligands bound to organic monomers either directly by chemical reaciton or indirectly by chemical spacer arms, and these conjugates may be polymerizedwith non-derivatized monomers to form polymers.
1. Physical adsorption:
2. Covalent binding: via a functional group located on the surface of the latex particles such as an amino group, a carboxyl group, a mercapto group, a hydroxyl group, an aldehyde group or an epoxy group can also be used.
3. Emulsion polymerization:
In carrying out an emulsion polymerization, an initiator (also referred to in the art as a catalyst) is preferably empolyed at a concentration sufficient to initiate the polymerization reaction. Illustrative initiators include hydrogen peroxide. (US 2009/0264585).
–Miniemulsion polymerization: is a technique to produce a variety of latexes ranging from simple polystyrene particles to well defined nanocapsultes. The particles are performed as oil droplets with a size of 50-500 nm by hsearing a system containing oil, water, a surfactant, and a highly water insoluble compound, the so-called hydrophobe. The surfactant stablizes the droplets against collisions, and mass exchange (Ostwalk ripening) between the droplets is suppressed by the use of the hydrophboe. Marie discloses the polymerication of styrene using a typical miniemulsion procedure with chitosan as stabilizer, producing highly monodisperse particles (Marie, Biomacromolecules 2002, 3, 475-481).
(i) Mini-Emulsion polymerization in presence of antibody/angigen: which is carried out in the presence of the antigen or antibody at low temperature in the synthesis of the latex partciles is another way of immobilzing an antigen or antibody on the latex particles. The antigen or antibody is introduced inot the surface of the latex particles when the synthesis is completed and a result, the removal of the unbound antigne or antibody from the latex particles by washing can be avoided. Because the antigen or antibody is introduced into the surface, the denaturation due to steric hindrance by the binding does not occur and thus a reaciton with a sample in which a nonspecific reaction is observed when conventional latex particles are used can be avoided.
Sawai (UA patent applicaiton No: 12/445863) teaches a suspension of latex particles haivng an antigen or an antibody specific for a substance to be analyzed introduced into the surface of the latex particles, produced by polymerizing a monomer in the presence of the antigen or antibody to thereby synthesize the latex particles. The miniemulsion polymerization is carried using a monomer (e.g., styrene, acrylic acid, methacrylic acid, acrylic acid esters), a radical polymerization initiator, such as a redox initiator, (e.g., combination of ascorbic acid and H2O2; a redox initiator is preferable because the polymerization reaction can be at low temrpature), a peroxide initiator, (e.g., benzoyl peroxide (TPO), di-t-butyl peroxide (DBPO) and amonium peroxide) a persulfate initiator or an azo-initiator, (e.g., azobisisobutyronitirle (AIBN) an emulsifier, such as a surfactant, (e.g., polymeric surfactant have a polyethylene glycol chain) and a hydrophobe such hexadecane or polystyrene, in the presence of the antigen or antibody to thereby produce the polymer particles.
The process can be carried out via convention miniemulsion polymerization (for example, M. Antonietti, K. Landfester, Prog. Polym. Sci., 2002, 27, 689-757 and J.M.Asua, Prog. Polym. Sci., 2002, 27, 1283-1346) except that an antigen or antibody coesists in the polymerication reaction. Conventional mimiemulsion polymerication can comrpise the steps of mixing a monomer, a readical polymerication initiator, and emulsifier and a hydrophobe to prepare a mixture, shearing the mixture, and heating the sheared mixture to the polymerication initiation temperature to thus carry out polymerication. In minimeulsion polymerication, after a monomer for polymerication is mixed with an emulsifier, a shearing step is carried out by, for example, supersonic radiation, to disrupt the monomer by a shearing force and form monomer dropets coated with the emulsifier. The monomer droplets are polymerized by ehaitng the mixture to the polymericaiton initiation temerpature of the radical polymerication initiator to obtain the polymer particles.
Magnetic particle agglutination: is where magnetic particles instead of say latex particles are used in the agglutination. In a sanswich method, a substanced is trapped with a first antibody immobilized on the magnetic particles, the magentic particles are then washed to remove unreacted substances and a second antibody labeled with a signal generating substance such as an enzyme or a fluorescent agent to thereby perofrom the analysis is added.
Ozaki (US 2005/0065288) discloses a process for producing particles for diagnostic reagent which comrpises a step of polymerizing a polymerizable monomer in the presence of a matrix particle having a magnetic substances layer containg at least one of Fe2O2 and Fe3O4 on the surface.
–Carboxylated magnetic polymer latexes: are an important class for the biomedical applications. They are generally prepared by copolymerication of hydrophobic monomer (usually styrene) with carboxylic acid monomers (such as acrylic acid, methacrylic acid) in the presence of magnetite particles (Zheng, J. Magentisms and Magnetic Materials 293, 2005, 199-205).
Competitive Immunoglutination:
–Inhibition Mode
Cohen (US 4,851,329) discloses agglutination reactions performed in inhibition modes. In one embodiment, antigen-coated spheres and antibody are mixed with a test sample containing an antigen. The degree to which the antigen in the test sample inhibits the aggregation of the carrier particles, that would otherwise have occurred, indicates the concentration of antigen present. In a separate embodiment, Cohen teaches that the agglutination reaciton is performed where the antibody is coated onto to spheres and antigen is mixed with a test sample containing antibody. The degree to which the antibody present in the sample inhibits the aggregation of the carrier particles, which would otherwise have occurred, indicates the concentration of antibody present.
Mochida (US 4,332, 788) also teaches a method for determining an antigen or antibody in a sample by causing an antigen-antibody reaction among a sensitized carrier particle which is formed by binding an antigen or antibody to a carrier composed of particles, a substance such as an antigen or antibody to be determined and an agglutinating agent which is an antigen or antibody in wich the antigen or antibody to be determined inhibits the immunochemical agglutination reaction of the insoluble carrier particles with the agglutinating agent.
Kojima (US 12/438035) disclose a method for determination of an antigen or an antibody against the antigen in a sample by mixing the same with a set of reagents, the first member of the set being an antibody and the seonc member being an antigen supported on microparticles wherein a decrease in agglutination indicates that antigen is present in the sample. In a separate embodiment, Kojima teaches that the antibody is supported on microparticles and that a decrease in agglutination indiates that the antibody is present in the sample and wherein an increase in agglutation indicates that antigen is present in the sample.