Indirect Agglutination Test

Recent years have heralded an increase in the use of clinical diagnostic methods involving immunological procedures because they are specific and have high sensitivity. Of the many heterogeneous and homogeneous immunological assay methods available, those based on the agglutination of latex particles continue to be widely used in biology and medicine for the detection of small quantities of an antibody or antigen in a fluid test sample. Some advantages of these assays are that the procedures are simple, widely applicable, and nonhazardous, and test results are obtained in a very short time. The agglutination reaction involves in vitro aggregation of microscopic carrier particles (usually of polymeric nature, referred to as latex). This aggregation is mediated by the specific reaction between antibodies and antigens, one of which is immobilized on the surface of the latex particles to enhance the sensitivity and extend the point of equivalence. In one format, a fluid containing the ligand of interest is introduced into a suspension of the sensitized carrier particles, and the presence of agglutination is noted as indicative of the ligand. The degree of agglutination plotted as a function of the agglutinant concentration follows a bell-shaped curve similar to that for precipitin. The agglutination reaction may be used in several different modes to detect an antigen or antibody (the ligand of interest), and each has its own limitations and applications:

1. A direct latex agglutination test for the detection of the presence of an antigen or hapten in a biological sample. The biological sample is mixed with a suspension containing antibodies against that antigen bound to latex particles (Fig. 1). If antigen is present in the sample it will react with the antibodies to form an aggregate. If no antigen is present in the sample the mixture will keep its appearance as a smooth suspension. This method is

Indirect Agglutination Test
FIG. 1 Latex immunoagglutination assay: antibody-coated particles agglutinated by antigen molecules (direct test); antigen-coated particles agglutinated by antibody molecules (indirect test).

applicable to the detection of polyvalent antigens, e.g., proteins and microorganisms.

2. An indirect latex agglutination test for the detection of an antibody in a biological sample. This works based on similar principles whereby antigens of the antibody corresponding are bound to latex particles (Fig. 1). This approach is applicable to mono- and polyvalent antigens, e.g., drugs, steroid hormones, and proteins.

3. An agglutination inhibition mode using antigen immobilized particles. A fixed quantity of antibody is mixed with a dilution of the test sample containing the ligand of interest. This reaction mixture is then combined with the antigen immobilized carrier particles. The degree to which the ligand of interest (antigen) in the test sample inhibits the aggregation of the carrier particles that would otherwise have occurred, indicates the concentration of ligand present in the sample.

4. An agglutination inhibition mode with antibody immobilized particles. A fixed quantity of antigen is mixed with a dilution of the test sample containing the ligand of interest (a specific antibody) which inactivates a portion of the antigen. This reaction mixture is then combined with the antibody-immobilized carrier particles. The degree to which the ligand (antibody) present in the test sample inhibits the aggregation of carrier particles, in comparison to that which would otherwise have occurred, indicates the concentration of antibody present.

Latex immunoagglutination assay was first described in 1956 by Singer and Plotz [1] and applied to rheumatoid factor. One can realize the importance of this kind of assays when perusing the specialized literature. In the last decade alone more than 400 publications in medicine and veterinary journals reported the use of latex immunoagglutination assays as analysis or research tools. The popularity of this diagnostic technology is illustrated by the fact that in 1992 there were over 200 commercial reagents available employing this approach to detect infectious diseases from "strep throat" to AIDS [2]. These include bacterial, fungal, parasitic, rickettsial, and viral diseases. The tests are also useful for cancer detection and for identification of many other substances (hormones, drugs, serum proteins, etc). The most familiar application of latex immunoas-says is the pregnancy determination. In this procedure, a suspension of latex particles covered by human chorionic gonadotropin (HCG) is mixed with a drop of urine. New latex applications and technologies are still being devised and applied to new analytes.

Immunoglobulins are bifunctional molecules that not only bind to antigens but also initiate a number of other biological phenomena such as complement activation and histamine release by mast cells (activities in which the antibody acts as a directing agent). These two kinds of functional activities are localized to different portions of the molecule: the antigen binding activity to the Fab and the biological activities to the Fc portion of the molecule. Structurally they have a tetrameric arrangement of pairs of identical light and heavy polypeptide chains held together by non-covalent forces and usually by interchain disulfide bridges. Each chain consists of a number of loops or domains of more or less constant size. The N-terminal domain of each chain has greater variation in amino acid sequence than the other regions, and it is this factor that imparts the specificity to the molecule. There are five types of heavy chains, which distinguish the class of immunoglobulins IgM, IgG, IgD, IgA, and IgE, and two types of light chains. Of these, immunoglobulin G (IgG) is the most abundant and its structural characteristics are better understood.

In the main immunoagglutination assays, the particles employed to adsorb the antibody or the antigen are latex particles rather than another kind of solid support (e.g., sheep or human erythrocytes, metal sols, etc.). This is due to the following factors: (1) Uniform size particles can be synthesized with diameter in the range 50-10,000 nm. The monodispersity is an important property for detection of immunoagglutination by light scattering techniques. (2) A wide selection of functional groups can be incorporated onto the latex surface to bind proteins covalently or to achieve colloidal stability. (3) Biological molecules adsorb strongly to the hydrophobic surface of latex particles.

In the recent past, numerous researchers have done extensive work in an attempt to optimize the multiple variables affecting the reproducibility, detection limit, analytical range, sensitivity, and reliability of latex immunoassays. Some of the parameters that must be taken into account are the size of the particles used to immobilized the biological molecule, the surface charge density and hydrophilic-hydrophobic nature of the particle surface, the means of attachment of the biological molecule to the latex, the experimental conditions for immobilization and agglutination, and the optical method for detecting the extent of immunoaggregate formation [3].

The attachment of molecules to latex particles can be achieved through physical adsorption or covalent coupling. Polymer engineering has facilitated the synthesis of latex particles with surface reactive groups that enable covalent coupling of protein molecules to the particle. In addition, spacer groups may be introduced between the particle surface and the immunoproteins. The spacer groups are thought to permit a degree of freedom to the reagent moiety separating it from the particle surface, thereby lending enhanced specificity.

Apart from some visual methods for detecting qualitatively the agglutination of sensitized particles, there exist optical techniques to quantify the agglutination. The most important ones are turbidimetry, nephelometry, angular aniso-tropy, and photon correlation spectroscopy (light scattering measurements). The require particle size is different for qualitative (bigger particles) than for quantitative methods (smaller particles). In general, for visual slide agglutination the particle diameter range is 0.2-0.9 pm, whereas for light scattering immunoassays the diameter range is 0.01-0.3 pm.

The major problem of particle-based assays, which require careful attention, is the nonspecific agglutination. The presence of this nonspecific agglutination has been one of the main reasons why, for a long time, latex immunoagglutination tests were considered to be semiquantitative at best [4]. Nonspecific agglutination can be caused by a variety of factors:

(1) Many body fluids, such as serum, often contain other undefined substances in addition to the particular analyte of interest. Such substances can cause or inhibit agglutination. The mechanisms by which they interfere are poorly understood, and no particular causative agent or set of conditions is responsible for these effects. Moreover, interferences of these types cannot be corrected by comparison of the assay results with a similar assay using a sample not containing the analyte in question as a blank sample because the blank may not be truly representative of the serum under test. As a result, much time and effort has been expended in the search of eliminating nonspecific interferences. Some methods of reducing nonspecific interferences in latex immunoagglutina-

tion assays are as follows: massive dilution of the test sample; addition of detergents; covering of the bare surface of the sensitized particles with inactive proteins; rigorous pretreatment of the test sample including heat treatment for 30 minutes at 56°C; and enzymatic treatment with proteases reaction. These procedures are time consuming and can carry with them the undesirable effect of drastically reducing the potential sensitivity and accuracy of the immunoassay as a result of the required manipulations.

(2) Sometimes nonspecific agglutination occurs by a bridging mechanism. This mechanism assumes that the biomolecule attached to the particle has chains or loops extending to the dispersion medium sufficiently far to encounter another particle, provoking the unspecific linking of the two particles. The agglutination by bridging phenomena is important at low degrees of coverage. This process can be eliminated using an inactive protein to cover the free surface of antibody-coated particle.

(3) After the protein coating procedure, the latex particles show low colloidal stability and the aggregation occurs at pH and ionic strength values reproducing the physiological conditions. This self-aggregation process is undesirable. The difficulty in keeping the protein-coated particle system colloidally stable is the main reason that half of all latex immunoagglutination testing is unsuccessful.

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Responses

  • Fiona
    What are carrier particles agglutination tests?
    2 years ago
  • stefan
    How is indirect latex agglutination dilution reported?
    9 months ago

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