ESQUILA

The next stage aimed to the quantification of VLPs in clarified transformed yeast cell homogenate. The basis of the light scattering assay was the use of antibodies to recognize VLPs selectively, and facilitate VLP detection and discrimination from contaminant particles (mainly cell debris) in yeast homogenate. DLS was employed to relate the signal change due to the antibody-VLP immunoreaction to VLP concentration in the sample.

The preliminary experiment described in section 3.2.3.1 involved the use o f IgA antibody. As mentioned in section 3.2.2, experiments showed that the most appropriate antibody for the purposes of the light scattering immunoassay would be

the IgG, so later work presented in sections 3.2.3.2 and 3.2.3.3 involved the use of IgG type anti-VLP antibody.

3.2.3.1 Clarified Homosenate Dilution Series

This was a preliminary experiment to investigate whether the addition of anti- VLP antibody in a sample of transformed yeast homogenate would result in a size change detectable by DLS due the antibody binding on the particles. It involved assaying samples of a fixed concentration of antibody added to a variable concentration of transformed homogenate.

Expérimentation: Yeast cell homogenate (62.5 gdcw/1) was prepared as described in section 2.2.6. Sufficient amount of yeast cell homogenate was defrosted and was spun at 12000xg for 5 min in a microcentrifuge to remove whole cells and large cell wall fragments. IgA anti-VLP antibody aliquots were thawed from -70®C to room temperature before use.

Homogenate supernatant was diluted with phosphate buffer (O.IM KH2PO4, pH

7.0, filtered through 0.2 pm filter) to create dilutions from 0.01 to 1 (homogenate volume before/after dilution). 200 pi homogenate of appropriate dilution was sized before and after the addition of 20 pi IgA anti-VLP antibody (0.6 mg/ml). The mean size for each sample was the average o f five measurements. Size change due to antibody binding was evaluated by subtracting the average mean size after antibody addition from the equivalent parameter for the sample after antibody addition. The scattering angle was 90°, the sample time 10 ps, the temperature 20°C.

Results: Figure 3.6 shows the change in size evaluated by DLS due to the binding of antibodies to the particles.

This result showed a dependence o f size increase measured by DLS to the concentration of homogenate in the sample analysed (Tsoka et a l, 1995), giving positive indication that further experiments could link the size change due to the specific recognition of VLPs from antibodies with the concentration of VLP, so that the analyte could be monitored against a background of contaminant cell debris particles in homogenate.

Chapter 3. Development o f a DLS-Based Assay_________________________________ 81 3.2.3.2 Sizin2 Varvin2 Amounts o f Purified Virus-like Particles with a Fixed Amount

o f Antibody

To investigate the effect of the VLP-antibody reaction on the light scattering signal, varying amounts of pure VLPs were assayed by DLS before and after the addition of the same amount of IgG antibody, in the absence o f contaminating substances.

Expérimentation: 2.5 to 30 pi of VLP (0.92 mg/ml) were diluted with phosphate buffer (O.IM KH2PO4, pH 7.0, filtered through 0.2 pm filter) to a final volume of

500 pi, so that the final VLP concentration in the sample ranged from 4.6 to 55.2 pg/ml. Each sample was sized before and after the addition o f 7 pi of IgG anti-VLP antibody (1.74 mg/ml) as described in section 2.4. The angle was 90° and the temperature 20 ± 1.5°C.

Results: For simplicity, only one of the particle size distributions corresponding to the 18.4 pg/ml VLP sample is presented graphically here. Figure 3.7 shows the intensity and number based size distribution of this sample before the addition of antibody . The distribution based on the intensity o f light scattered by each size class showed the presence of some aggregated VLPs, while the distribution based on the number of particles in each size class is given to show that these aggregates formed a very small part of the population. The mean size for a VLP molecule was 77 nm as both types of distribution indicated.

Figure 3.8 shows the size distribution of the same sample after the addition of 24 pg/ml of IgG antibody. A size increase was observed which is equal to 32.8 nm (approximately double the IgG diameter) suggesting that at this antibody to VLP ratio VLP particles were almost fully coated by the antibody molecules.

The size increase for all the analysed samples of VLP (4.6 to 55.2 pg/ml) after the addition of the antibody was plotted against the antibody to VLP ratio in each sample in figure 3.9 (for antibody to VLP ratio calculation see section 3.3.3). The plot resembled the immunoprecipitin curve, clearly indicating the antibody excess, equivalence and antigen excess region of the immunoreaction. A maximum size increase of 38 nm (approximately double the IgG diameter) indicated monolayer coverage of VLPs by the antibody molecules as the interaction mechanism.

3,23,3 Spikins Purified Virus-Like Particles in Untransformed Homo2enate

The aim here was to generate a calibration method to relate the DLS measurement of homogenate samples containing varying amounts of VLPs to VLP concentration.

Samples o f increasing VLP to particulate contaminant ratio were prepared by spiking increasing amount of VLP to a constant volume o f untransformed homogenate. Each sample was assayed by DLS before and after the addition of a fixed amount of antibody. The levels of VLP spiked in untransformed homogenate were the same as the ones analysed in the absence of contaminants in the previous section.

Expérimentation: Untransformed homogenate (42 gdcw/1) was clarified by borax treatment as described in section 2.7. Volume equal to 0-30 |xl of purified VLP (0.92 mg/ml) was spiked in 100 pi untransformed cell homogenate and the volume was adjusted to 500 pi with phosphate buffer (O.IM KH2PO4, pH 7.0, filtered through

a 0.2 pm filter), so that the final VLP concentration ranged from 4.6 to 55.2 pg/ml as shown in table 3.2.

Antibody dosing was based on Brookman et a l (1995) where for 25 pg of VLPs, maximum coverage (and so maximum size increase) was observed after the addition of 10 pg of IgG antibody.

Each sample was analysed by DLS before and after the addition of 7 pi of monoclonal antibody BB2 (IgG type, 1.74 mg/ml). Final antibody concentration in each sample was 24 pg/ml. The measurement procedure consisted o f five consecutive measurements of 100 s duration for each sample. The size distributions corresponding to each measurement were converted to a histogram, were averaged and the error bars were set equal to the standard deviation o f the individual measurements, as described in section 2.4. The angle was 90° and the temperature 20± 1.5°C.

SDS-PAGE was carried out for the same samples after dynamic light scattering to analyse the soluble protein content of each sample. A 12% polyacrylamide gel was used (section 2.6). Samples 1 to 8 (table 3.2) were TCA precipitated after DLS

analysis. The pelletted sample was resuspended in 100 pi sample buffer of which 10 pi were loaded on the gel (lanes 3 to 11, figure 3.14).

Chapter 3. Development o f a DLS-Based Assay_________________________________ 83 Table 3.2: Composition o f Samples in Calibration Experiment fo r

Measuring VLPs in Clarified Untransformed Homogenate

untransformed homogenate (pi) VLP spiked w phosphate buffer (pi) Final VLP conc. (pg/ml) sample 1 100 0 400 0 sample 2 100 2.5 397.5 4.6 sample 3 100 5 395 9.2 sample 4 100 10 390 18.4 sample 5 100 15 385 27.6 sample 6 100 20 380 36.8 sample 7 100 30 370 55.2

5 of molecular weight marker (high molecular weight standard mixture, Sigma) showed a range of molecular weights from 29,000 to 205,000 (lane 1). For a VLP marker, 5 of pure VLP solution (0.7 mg/ml) were mixed with 5 pi sample buffer and loaded on the gel (lane 2).

Results: The size distributions of the homogenate samples based on the intensity of light scattered by each size class of particles before and after the addition of antibody are shown in figures 3.10 and 3.11 respectively. The distributions based on the number of particles in each size class are given in figures 3.12 and 3,13 (before and after antibody respectively).

A photograph of the SDS-PAGE gel is shown in figure 3.14. This gave an indication of the soluble protein components for each sample and it was clearly seen that the intensity of the VLP band was increasing in comparison to the background of contaminant soluble protein. What was also important was that the untransformed homogenate analysed (lane 3) showed no trace o f p i -381 proving that only the spiked VLP existed in the samples analysed by DLS.

The size distributions before the addition of the antibody (figures 3.10 and 3.12) clearly showed that the VLP added appeared in the size range below 100 nm. As the VLP to cell particulate ratio increased, the first peak o f the distribution shifted to

lower mean diameter values due to the increasing contribution of the VLP particles to the light scattering signal.

By comparing the size distributions after the addition of the antibody (figures 3.11 and 3.13) with the ones before antibody addition, it was seen that the lower size peak of the distribution increased in mean diameter, an effect which was attributed to the increase in size due to antibody binding on VLPs.

Further manipulation of these data to extract quantitative information of the immunoreaction and develop a calibration curve for VLP monitoring is presented in the discussion (section 3.3.4).

Chapter 3. Development o f a DLS-Based Assay_________________________________ ^

3.3 Discussion of Results

The results that were presented in section 3.2 are discussed here, with the intention to highlight the steps towards understanding the interaction of VLPs with the corresponding antibodies, so that the immunoreaction would provide the framework for a quantitative assay.