TIPO DE CAMELIDOS SUDAMERICANOS

The use of the light scattering technique for the study of the interactions between antigen and antibodies is reviewed here, with the intention to highlight the specific requirements that the light scattering technique must meet for such applications.

3.1.2.1 The Immunoprecipitin Reaction

The combination of antigen and antibody is a reversible reaction which results in the formation of immune complexes. The reaction kinetics are complicated, due to the fact that a protein possesses many antigenic determinants and an antiserum may represent a heterogeneity of antibodies, as well as to the existing variation in the degree of reactivity between antigenic sites and antibodies (Price et a l, 1983). Of particular interest is the fact that a protein acts as a polyvalent antigen and will react with its antibodies to yield a precipitated complex. This represents the immunoprecipitin reaction. The amount of precipitate is dependent on the ratio of antibody to antigen concentration. The general form of the immunoprecipitin reaction is shown in figure 1.2.

There are thought to be at least two distinct stages to this reaction (Price et a l, 1983). The first stage is a primary reaction involving binding o f antigen to antibody. The antigen is regarded as being polyvalent while most people regard the antibody as being bivalent. The second stage of the reaction involves the formation of the aggregate and is the portion of the reaction that can be observed by the formation of the precipitate. Some authors regard this phase of the reaction as being in two stages, involving first lattice formation, followed by aggregation to form a precipitate. The

Chapter 5. Development o f a DLS-Based Assay_________________________________ 73 shape of the immunoprecipitin curve, antibody titre and avidity play a major part in the design of analytical systems for the quantitation of specific proteins by immunoprécipitation. Equivalence is regarded as the point where maximum immunoprecipitate is observed.

The major determining factor in the primary reaction between antigen and antibody is the mole ratio of antigen to antibody. The pH and ionic strength of the reaction environment have a limited effect on the primary reaction, although the nature of the ionic species can have a marked effect.

In the zone of antibody excess, complexes are of constant size and their number is proportional to the antigen concentration (Whicher and Blow, 1980). Primary interaction gives rise to simple, soluble, binary^ antibody antigen complexes, which, in the presence of adequate free antibody, form a stable lattice, resulting in the production of large complexes. In time, aggregation of these complexes occurs, giving rise to a visible precipitate (Whicher and Blow, 1980). In the antigen excess zone, complexes are solubilised by free antigen, they become smaller and consequently scatter less light. It is thus clear that a certain amount of light scattering may result from two possible antigen concentrations. It is therefore essential that all light scattering assays utilise the antibody excess zone of the precipitin reaction.

The measurement of scatter is best performed in dilute solutions, where absorption and reflection is minimal. Under these conditions the relationship between the concentration of scattering particles and the amount of light scattered is almost linear over a wide range of concentration (Kusnetz and Mansberg, 1978). These conditions are easy to achieve in immunochemical assays as low concentrations of antigen can be obtained by dilution.

3.1.2.2 Parameters Affectins Lisht Scatterins Immunoassay

The main factors to be considered when establishing a light scattering immunoassay (Whicher and Blow, 1980) are the correct antigen-antibody ratio, the assay sensitivity and range, the time course of the reaction, the need for polymer enhancement, the suitability of the antiserum and the economy o f antiserum consumption.

For a particular antigen, the shape of the immunoprecipitin curve varies considerably depending on the type of antibody, its animal of origin and whether polymer enhancement is used. It is essential for the antigen-antibody ratio to be on the antibody excess side of the curve, as far as reasonably possible from the equivalence point. To establish the correct antigen-antibody ratio, a series of immunoprecipitin curves can be produced for a wide range of antigen dilutions at different antibody concentrations. It is noted that small proteins tend to go into antigen excess more readily than larger proteins (Whicher and Blow, 1980).

The sensitivity of a light scattering immunoassay is limited by the presence of inherent light scattering material present in the assayed sample and also to some extent by the difficult}^ of completely removing dust and particulate matter from the inner surfaces of cuvettes. Increasing the instrument sensitivity may not improve assay sensitivity, as the major problem is to resolve small scattering signals due to the immune complexes against a high background signal. Amplification results only in an increased signal size and does not alter the ratio of background to test scatter (Price et a l, 1983).

The time course of the immunoreaction depends on the rate at which antigen- antibody complexes are formed. The latter is a function of the titre and affinity of the antibody and on the nature of the antigen. It is important to control the incubation time precisely, especially if an assay has poor plateau characteristics, otherwise assay precision will suffer.

Polyethylene glycol has been used in the assay of serum samples to reduce the solubility of proteins by exclusion of water and thus to enhance the rate of the immunoprecipitin reaction and the sensitivity of the reaction (Hellsing, 1978). PEG 6000 was found to be the most effective polymer and it has been reported that at concentrations of 20-40 g/1, PEG increases the sensitivity or slope of the precipitin curve when in antibody excess, as well as reducing the time to reach equilibrium. PEG extends the antibody excess side of the immunoprecipitin curve, driving equivalence towards higher antigen concentrations (Hellsing et a l, 1977; Hellsing 1978). On the other hand, by decreasing protein solubility, PEG increases sample blanks due to increasing inherent light scattering material. Ways to overcome the problem is to measure blanks in polymer containing buffer, remove precipitable proteins before the assay, or use high sample dilutions.

Chapter 3. Development o f a DLS-Based Assay_________________________________ 75 Antiserum characteristics are important in the assay performance. The titre dictates the dilution of antiserum to be used, and the affinity defines the speed of reaction and the slope of the reference curve. The use of monoclonal antibodies against specific antigens has a potential to improve greatly the accuracy of immunochemical methods, as the system recognises only one antigenic determinant of a polyvalent antigen. The density of the antigenic determinant affects the sensitivity of the method. The presence of a single bivalent antibody against a single antigenic determinant reduces the possibility of lattice formation with polyvalent antigens.

Finally, the reproducibility of reagents is another aspect for the successful application of immunoprécipitation techniques. Especially if the assay is to be used in a process environment, calibration may be necessary for each analytical run and it should be demonstrated what degree of reproducibility exists in terms of the calibration curve parameters.

3.1.2.3 Lisht Scatterins Immunoassay Applications

Most of light scattering immunoassays reported have been performed in serum samples or purified proteins. The possibility of enhancing the sensitivity of light scattering detection systems to enable lower concentrations of protein and small molecular weight haptens to be measured has been investigated.

In the case of low molecular weight haptens, the antibody-hapten complexes are not of sufficient size to be detected. A modified technique is used where the molecular size of the hapten-antibody is increased, by coupling the hapten to latex particles (Grange et a l, 1977; Marchand et a l, 1992; Borque et a l, 1992).

Cohen and Benedek (1976) have measured agglutination of antigen coated latex spheres when antibody was added to the particle suspension by measuring changes in diffusion coefficient with laser light scattering. DLS has been shown to offer a 100- fold increase in sensitivity compared to conventional assays. The same principle of the latex immunoassay has been used in the inhibition mode (von Schulthess et a l,

19766), where antigen is measured by the blocking effect on antibodies which act as the agglutinator for latex particles coated with antigen. A 1000-fold increase in sensitivity is reported with this method. It compares in sensitivity with radioimmunoassay but uses no radiochemicals and can be performed rapidly with no

prior separation of bound and unbound antigen with volumes as low as 1 pi (von

Schulthess et a l, 1976a and 19766).

Another approach that has been used by Cambiaso et al. (1977) is to measure the agglutination of latex particles coated with antibody or antigen using particle counting techniques, with instruments that were designed for counting red blood cells. The principle is the reduction of the total number of particles when they are agglutinated.

Sittampalam and Wilson (1984) have demonstrated the possibility to monitor the growth of immunoprecipitate with time and successfully used the rate of change of scatter intensity to establish the amount of reactive antigen in an assayed sample. Savory et a l (1974) have used stopped flow nephelometry to study the rate o f IgG- anti IgG complexe formation paving the way for discrete nephelometric systems.

Chapter 3. Development o f a DLS-Based Assay_________________________________ 77 3.2 Results

In this section, the experiments leading to the development o f a laser light scattering assay for monitoring virus-like particles in clarified yeast cell homogenate are presented.

It is worth mentioning that the term untransformed homogenate used here refers to homogenate of cells not expressing VLPs, whereas the transformed cell line has been genetically altered to produce the particles (sections 1.1.2.1, 2.1.2 and figure

1.1).

The light scattering experiments were performed as described in section 2.4.