WO2001094948A1

WO2001094948A1 - Elisa for baculovirus

Info

Publication number: WO2001094948A1
Application number: PCT/GB2001/002412
Authority: WO
Inventors: Paul Henry Hissey
Original assignee: Glaxo Group Limited
Priority date: 2000-06-02
Filing date: 2001-05-31
Publication date: 2001-12-13
Also published as: GB0013561D0; AU2001260473A1

Abstract

The present invention provides a method of determining the presence, absence or titre of baculovirus particles in a sample, the method comprising detecting the binding of a primary antibody which recognises an epitope present in a baculovirus particle to the baculovirus particle, if present in the sample, thereby determining the presence, absence or titre of baculovirus particles in a sample.

Description

E ISA FOR BACULOVIRUS

Field of the invention

The invention relates to the use of an antibody in a method of determining the presence, absence or titre of baculovirus particles in a sample. Preferably, this is done by means of an ELISA (enzyme-linked immunoadsorbent assay).

Background of the invention

Baculovirus is a large DNA virus that has been adapted for use as an expression vector. The sequence encoding the polypeptide of interest is inserted into the baculovirus vector. The baculovirus expression vector is then used to infect insect cells. The polypeptide of interest is expressed in the infected cells and then recovered from the culture. Currently, the presence, absence or titre of baculovirus particles in a culture is determined by plaque formation assays. In plaque formation assays a sample from the culture is plated onto a lawn of insect cells, followed by incubation for an appropriate period of time (normally at least a day) and then determining the number of viral plaques formed. The titre of extracellular baculovirus particles is determined by first removing the cells from the sample of culture and then performing the plaque formation assay using the supernatant.

Kitts and Green (Anal. Biochem. 1999, 268 (2): 173-8) have developed a modified plaque formation assay. In this assay samples from the culture of infected cells are plated onto a lawn of cells, the cells are incubated for two days and then foci of baculovirus infection are identified using antibodies to stain infected cells expressing baculovirus proteins. The number of foci are then counted by light microscopy in order to determine the titre of baculovirus present in the original sample. Although more rapid than a conventional plaque formation assay this assay still requires several days to determine the titre of baculovirus in a culture.

All plaque formation assays for determining baculovirus titre take at least a day, sometimes several days, to give results and by this time the titre of baculovirus in the culture will usually have changed substantially and unpredictably. This effectively removes any advantage that could have been gained from knowing the levels of baculovirus in the culture at the time the sample was taken. Current, plaque-based, techniques are thus unsatisfactory for assays where "real time" measurements are required.

A more rapid method for determining baculovirus titres that would allow the determination of the titre of baculovirus in a culture in "real time", before it has substantially changed, would be an advantage. Such a method would allow feed and harvest regimes for the culture to be altered accordingly in order to maximise the productivity and efficiency of the culture.

Summary of the invention

The present invention involves GP64 (Glycoprotein 64), a viral envelope glycoprotein, present as a trimer on the surface envelope of baculovirus particles. GP64 is involved in baculovirus attachment to insect cells, pH-activated membrane fusion during endocytosis of a baculovirus particle and in viral assembly.

The inventors have shown that an antibody can be used to detect the presence, absence or titre of baculovirus particles in a sample. More specifically the inventors have shown that an ELISA (enzyme-linked immunoadsorbent assay) can be used to determine the presence, absence or titre of baculovirus particles in a sample.

Using plaque formation assays quantification of the titre of baculovirus in a culture can only be achieved at least a day, sometimes several days, after the sample has been taken. This effectively removes any advantage that could have been gained from knowing the levels of baculovirus in the culture at the time the sample was taken. It is in this area that the assay of the invention is most useful. The assay of the invention enables the speedy and easy quantification of baculovirus titres in samples from a culture. The assay is most useful for rapidly gaining an idea of the level of baculovirus growth.

One advantage of the assay of the invention is its ability to follow baculovirus infection within a culture on an hour by hour basis. Typically, the assay allows determination of the baculovirus titre in a culture within two to three hours. This allows the determination of times of peak infection, and thus productivity, at the time they are actually occurring rather than days later. In this way feed and harvest regimes can be optimized accordingly to increase the output and efficiency of the culture.

Using an antibody specific for GP64 (Glycoprotein 64) the inventors have developed a sandwich ELISA capable of determining the presence, absence or titre of baculovirus particles in a sample.

The ELISA thus allows the determination of the presence, absence or titre of extracellular baculovirus particles. In order to achieve this a sample from the baculovirus culture is taken and the insect cells are removed by centrifugation. The number of baculovirus particles in the supernatant is then determined.

The ELISA of the invention typically measures the amount of GP64 protein present in the sample. The inventors have shown that the amount of GP64 correlates with the number of baculovirus particles present in the sample. The assay is also capable of detecting GP64 that is not associated with a virus. However, usually cells have been removed from the sample for analysis and the amount of GP64 that is not associated with virus in such samples is low.

Baculovirus cultures may contain defective interfering particles (DIPs). DIPs are baculovirus particles that lack an essential part of the baculovirus genome and cannot replicate without the help of a second baculovirus. The ELISA of the invention measures the total number of baculovirus particles present in a sample, including DIPs. However, this does not detract from the accuracy of the assay of the invention. This is because the Inventors have shown that there is a correlation between the titre of baculovirus particles determined using the ELISA of the invention and the titre determined using a plaque formation assay (which, by definition only measures replication competent virus). Therefore the ELISA of the invention is capable of giving a measure of the number of replication competent viral particles in a sample. Accordingly, the invention provides an antibody-based system for the determination of the presence, absen e or titre of baculovirus particles in a sample.

More particularly the invention provides a method of determining the presence, absence or titre of baculovirus particles in a sample, the method comprising detecting the binding of a primary antibody which recognises an epitope present in a baculovirus particle to the baculovirus particle, if present in the sample, thereby determining the presence, absence or titre of baculovirus particles in a sample. Typically, in the method according to he invention the titre of baculovirus particles, the titre of extracellular baculovirus particles present or the titre of intracellular baculovirus particles present is determined.

The epitope recognised in the method of the invention may be an epitope of GP64 (Glycoprotein 64). Preferably the binding of the primary antibody to the baculovirus particle is detected using a secondary antibody which recognises an epitope present in the baculovirus particle or protein.

In a preferred embodiment in the method of the invention the determination is carried out using an ELISA (enzyme-linked immunoadsorbent assay). Preferably the method of the invention comprises:

(i) contacting the sample with the primary antibody to obtain a baculovirus particle, if present in the sample, bound to the primary antibody;

(ii) contacting the primary antibody-bound baculovirus particle with the secondary antibody to obtain a primary antibody/baculovirus/secondary antibody complex; and

(iii) detecting the primary antibody/baculovirus/secondary antibody complex, if present, and thereby determining the presence, absence or titre of a baculovirus particle in the sample.

Typically the detection step (iii) is carried out by: (a) contacting a substrate with an enzyme bound to the secondary antibody; or

(b) binding the secondary antibody to an enzyme and contacting with a substrate, and measuring the amount of product formed by the reaction of enzyme and substrate, thereby determining the presence, absence or titre of a baculovirus particle in the sample.

In part (b), preferably the binding of the enzyme to the secondary antibody is through the interaction of streptavidin and biotin. In one embodiment of the invention the primary and secondary antibodies both recognise the GP64 antigen. Preferably the enzyme used in the method of the invention is horseradish peroxidase.

Typically, the secondary antibody is at a concentration in the range of 40 to 0.010 :g/ml, preferably 20 to 0.010 :g/ml, more preferably 1 to 0.010 :g/ml, even more preferably 0.100 to 0.010 :g/ml or 0.080 to 0.040 :g/ml The sample analysed in the method of the invention is typically: an aliquot of a culture of baculovirus infected insect cells; an aliquot of a culture of lysed baculovirus infected insect cells; or a suspension of baculovirus. Preferably the method of the invention takes under 24 hours, preferably under 12 hours and more preferably under 5 hours to determine the presence, absence or titre of baculovirus particles in a sample. The invention further provides an ELISA method for determining the presence, absence or titre of baculovirus particles in a sample, the ELISA comprising detecting the binding of a primary antibody which recognises an epitope present in a baculovirus protein to the baculovirus protein, if present in the sample, thereby determining the presence, absence or titre of baculovirus particles in a sample.

Typically, in the ELISA method the epitope is an epitope of GP64. Preferably the ELISA is the ELISA method wherein the binding of the primary antibody to the baculovirus protein is detected using a secondary antibody which recognises an epitope present in the baculovirus protein. Preferably the ELISA method comprises:

(i) contacting the sample with the primary antibody to obtain a baculovirus protein, if present in the sample, bound to the primary antibody; (ii) contacting the primary antibody-bound baculovirus protein with the secondary antibody to obtain a primary antibody/baculovirus protein/secondary antibody complex; and

(iii) detecting the primary antibody/baculovirus protein/secondary antibody complex, if present, and thereby determining the presence, absence or titre of a baculovirus. The invention further provides a method of producing recombinant polypeptide in an insect cell culture infected with recombinant baculovirus capable of expressing said polypeptide, which method comprises:

(i) culturing the infected cells; (ii) carrying out a method according to the invention in order to determine the feed and/ or harvest regime for the culture;

(iii) feeding and/ or harvesting the culture on the basis of the information thus obtained; and optionally

(iv) recovering said polypeptide. The invention also provides a kit for carrying out a method according to the invention, which kit comprises:

(i) a primary antibody suitable for use in the invention; and . optionally

(ii) means for determining the presence, absence or titre of baculovirus particles in a sample.

In the kit detection means (ii) may comprise a secondary antibody and optionally other means for carrying out the method of the invention.

Brief Description of the Drawings

Figure 1 shows the results of the ELISA detailed in Example 3. The graph shows the the absorbance for ELISA reactions with no primary antibody (zero primary), no secondary antibody, (zero secondary), no virus (zero virus) or with all the necessary reagents.

Figure 2 shows the results of the ELISA detailed in Example 4. Figure 2 (a) shows the absorbance measured in reactions with or without virus at the different concentrations of secondary antibody titrated. Figure 2(b) shows the ratio of these two values graphically.

Figure 3 shows the results obtained in Example 6. The graph shows the change in absorbance against baculovirus titre.

Figure 4 shows the results obtained in Example 6. The absorbance measured is plotted against the concentration of baculovirus particles. Figure 4(a) shows the results obtained using a secondary antibody concentration of 0.225 :g/ml. Figure 4(b) shows the results obtained using a secondary antibody concentration of 0.045 :g/ml.

Figure 5 shows the results obtained in Example 6. Figure 5 shows the baculovirus titres in a bioreactor over 62 days measured using the ELISA of the invention. Figure 5(a) shows the individual results obtained. Figure 5(b) shows the average of the results obtained.

Figure 6 shows the results obtained in Example 7. Figure 6 shows the change in baculovirus titre over time in a bioreactor measured using the ELISA of the invention and a rapid plaque forming assay.

Detailed description of the invention

The present invention provides an antibody-based method of determining the presence, absence or titre of baculovirus particles in a sample.

Samples for analysis

Any suitable sample may be assayed. Typically the sample is an aliquot, or is derived from an aliquot, of a culture of insect cells infected with baculovirus. In one embodiment of the invention the culture of insect cells is being grown in a receptacle or container such as a flask, fermentation vessel or bioreactor. The receptacle may be fed and harvested continuously or discontinuously. Alternatively, the sample for analysis may be a suspension of baculovirus particles that does not contain cells or may be a sample of isolated cells.

The sample is intended to be representative of the culture from which it is derived, such that judgements can be made about baculovirus growth in the culture from the results of the assay of the invention. Values which may be determined

The method of the invention may be used to determine whether or not baculovirus particles are present in the sample. Thus, it can be used qualitatively, to determine the presence or absence of baculovirus particles. Preferably, though it is used quantitatively, usually to determine the titre of baculovirus particles in a sample.

The method of the invention may be used to determine the optimal feed and harvest regime for a culture. The method may be used to optimise the productivity of a culture i.e. the amount of baculovirus which is obtainable from a culture per unit of money spent obtaining it. The optimal time of harvest will typically be the time at which the titre of extracellular baculovirus is at its highest. The optimal time for feed will typically be at 24 to 48 hour intervals, preferably at 24 hour intervals.

Typically, the method of the invention is used to determine the titre of baculovirus particles in a sample. The titre of baculovirus particles is a measure of the number of baculoviral particles per unit volume in the sample (i.e. a measure of concentration).

The invention provides a method of determining in a sample the total number of baculovirus particles present, the number of extracellular baculovirus present or the number of intracellular baculovirus present.

The presence, absence or titre of extracellular baculovirus particles in a sample may be determined by removing the insect cells from the sample before measurement of the amount of baculovirus. Typically the cells are removed by centrifugation and the presence, absence or titre of baculovirus particles in the supernatant determined.

The presence, absence or titre of intracellular baculovirus particles present in a culture may be determined by isolating the cells from an aliquot of a culture, lysing the cells and then determining the number of baculovirus particles released from the cells. The presence, absence or titre of baculovirus particles present in the whole of a sample (i.e. the sum of the number of intracellular and extracellular baculovirus particles) may be determined by lysing the cells present in an aliquot of culture, without isolating them first, and then determining the number of baculovirus particles present in the whole sample. Alternatively, the presence, absence or total amount of baculovirus particles present in the whole of a sample can be determined by measuring the amount of intracellular and extracellular particles separately and then adding them. Typically the invention may comprise:

(i) contacting a sample with a primary antibody to obtain a baculovirus particle, if present in the sample, bound to the primary antibody;

(ii) contacting the primary antibody-bound baculovirus particle with a secondary antibody to obtain a primary antibody/baculovirus/secondary antibody complex; and

Preferably, the primary antibody/baculovirus/secondary antibody complex will be detected, if present by:

(a) contacting a substrate with an enzyme bound to the secondary antibody (typically the enzyme will be biotinylated and the antibody conjugated with streptavidin with the enzyme being bound to the antibody through the interaction of streptavidin and biotin); or (b) binding the secondary antibody to an enzyme and contacting with a substrate, and measuring the amount of product formed by the reaction of enzyme and substrate in order to determine the presence, absence or titre of baculovirus particles in the sample.

Antibodies

Any suitable antibody may be used. The antibodies utilised in the invention may be polyclonal or monoclonal. The antibodies may be mono- specific or bi-specific. Antibody fragments which comprise an antigen binding site may also be used in the method of the invention. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies.

The primary antibody used in the invention recognises an epitope present in a baculovirus particle or protein. Typically, the primary antibody recognises an epitope on the surface of a baculovirus particle. In a preferred embodiment of the invention the primary antibody recognises an epitope present in the baculovirus glycoprotein GP64. A baculovirus protein is typically a protein encoded by the baculovirus or baculovirus vector or which is present in the baculovirus particle. In one embodiment the antibody binds to the foreign protein expressed by the baculovirus which may be present on the surface of the baculovirus particle. Preferably, however, the antibody recognises a protein which is present in the unaltered baculovirus before the introduction of the sequence encoding the foreign protein.

Preferably, any suitable antibody against GP64 may be used in the method of the invention. The antibody may be a monoclonal antibody, typically a mouse monoclonal antibody. The mouse monoclonal anti-GP64 antibody used by Kitts and Green (Anal. Biochem. 1999, 268 (2): 173-8) in the rapid plaque formation assay can be used.

The primary and secondary antibodies may recognise different epitopes present in the same molecule. In a preferred embodiment of the invention the secondary antibody binds to GP64. The primary and secondary antibodies may recognise the same or a different epitope on GP64. Alternatively the secondary antibody may recognise the primary antibody.

In one embodiment of the invention the primary and/or secondary antibodies may recognise an epitope present on a baculovirus particle but do not recognise any of the individual components of the baculovirus particle before assembly. Such antibodies are particularly useful for determining the titre of baculovirus in samples containing or derived from lysed cells.

Detection of binding of the primary antibody to baculovirus

Typically, the binding of the primary antibody to a baculovirus particle is detected using a label. Any suitable label may be used. The label may be an enzyme, fluorochrome, radionucleotide or coloured compound. The label may be conjugated with or bind specifically to the primary antibody or alternatively to a secondary antibody which recognises the primary antibody or the baculovirus particle or protein.

The secondary antibody may be directly conjugated to the enzyme. In another embodiment of the invention the enzyme and secondary antibody are not conjugated but bind to each other via a specific interaction. The secondary antibody may be biotinylated and the enzyme conjugated with streptavidin.

In a preferred embodiment of the invention the enzyme is a horseradish peroxidase and the substrate is preferably tetramethylbenzidine.

Use of a standard suspension of baculovirus and controls

In an especially preferred embodiment of the invention a standard containing a known number of baculovirus particles is used. Typically, standard samples containing known amounts of virus will be analysed at the same time, under the same conditions and preferably on the same plate as the samples containing unknown amounts of baculovirus.

Typically the standard containing a known number of baculovirus particles is used to obtain a standard curve from which the number of baculovirus particles present in other samples can be determined. A standard curve may be prepared each time an ELISA is performed.

Preferably several samples from the same culture will be analysed for each time point analysed and an average value of the baculovirus titre calculated.

Use of serial dilutions and controls

In one embodiment of the invention serial dilutions of a sample will be analysed. Typically doubling dilutions or tenfold dilutions will be used.

In a further embodiment of the invention control reactions where one or more reagent is omitted may also be performed. The controls will typically lack primary antibody, baculovirus or secondary antibody.

ELISA based assays

In an especially preferred embodiment of the invention the method for determining the presence, absence or titre of baculovirus comprises an ELISA. Typically, the ELISA will comprise of the following:

- a multi-well plate is coated by incubating with an anti GP64 antibody;

- the plate is washed several times; - the sample to be analysed is added and after incubation for an appropriate period of time the plate is washed to remove any unbound baculovirus particles;

- a biotinylated anti-GP64 antibody is added and after incubation for an appropriate period of time the plate is washed; - an enzyme conjugated with streptavidin is added and after incubation for an appropriate period of time the plate is washed;

- the substrate for the enzyme is added and the plate incubated; and

- the enzyme is inactivated and the change in absorbance is measured in order to determine the presence, absence or titre of baculovirus particles.

In one embodiment the ELISA may be used to detect the presence or absence of a baculovirus protein on the surface of an infected cell or the isolated baculovirus protein itself in order to determine the presence, absence or titre of baculovirus particles in a sample. Such an embodiment may also be used to detect the presence, absence or number of infected cells in a sample.

Preferably absorbance at an appropriate wavelength is measured to determine the amount of the product formed by the action of the enzyme on its substrate. In one embodiment asorbance at 450nm is typically measured in order to determine the amount of substrate that has been converted by the enzyme to product.

Typically the ELISA is carried out in a multi-well plate. The multi-well plate may be a 12, 48, 96, 192 or 384 well plate or may have more wells. In a preferred embodiment of the invention the plate is a 96 well plate. The steps of the invention may be carried out at room temperature or at a lower temperature, for example 4°C. The incubations may be between 10 minutes and 12 hours, preferably between 10 minutes and two hours and more preferably between twenty minutes and an hour Typically wells of the ELISA plate will be coated with the primary antibody. In a preferred embodiment of the invention the wells are coated by incubation with the primary antibody at a concentration of between 1 to 100 :g/ml, preferably between 10 and 50 :g/ml and more preferably at a concentration of 20 :g/ml.

In a preferred embodiment of the invention a blocking agent is used in the ELISA. The blocking agent reduces non-specific binding of proteins to other proteins or to the ELISA plate. In one embodiment of the invention the blocking agent is bovine serum album at between 0.1 and 5% concentration, more preferably between 0.5 and 2.5 % concentration and even more preferably at 2% concentration. The blocking agent may be added at the same time as the secondary antibody, enzyme and substrate.

After incubation with the primary antibody, secondary antibody, or enzyme the ELISA plate is washed. Typically the plate is washed between 1 and 7 times more preferably between 3 and 5 times. Preferably the plate is washed with water or PBS (phosphate buffered saline). Typically the wash buffer contains detergents such as Tween 20 at 0.01 to 0.05%.

Any of the parameters in the method of the invention may be varied in order to determine their optimal values for the accurate measurement of baculovirus titres.

Kits

The invention also provides a kit for determining the presence, absence or titre of baculovirus. Typically, the kit comprises a primary antibody according to the invention and instructions for detecting the binding of the primary antibody to a baculovirus particle. In addition to the primary antibody the kit may comprise a secondary antibody, enzyme or the enzyme substrate. In a preferred embodiment of the invention the kit comprises the primary antibody and the reagents necessary to carry out the ELISA of the invention.

The following Examples illustrate the invention: Examples

Example 1

A mouse monoclonal anti-GP64 antibody was used. The anti-GP64 antibody was biotinylated according to the method of Ed Harlow and David Lane (Antibodies: a laboratory manual, Cold Spring Harbor Laboratory Press, 1988). The biotinylation method used is described below.

1. N-hydroxysuccinimide biotin was mixed with dimethyl sulphoxide (DMSO) to give a mixture of lOmg/ml of the biotin ester in DMSO.

2. The anti-GP64 antibody was dialysed in borate buffer (0.2M boric acid and 0.05 sodium tetraborate mixed to give a solution of pH 8.8) for 1 hour;

3. The anti-GP64 antibody was made up to a concentration of 1-3 mg/ml in borate buffer; 4. The biotin ester was added to the antibody solution at a ratio of 200μg of ester per mg of antibody present (if a higher level of biotinylation of the antibody was required the amount of ester used was increased).

5. The biotinylation reaction was stopped by adding Tris at lOOmM concentration in the reaction mixture (other reagents that contain reactive amino groups can be used to neutralise remaining active groups on the biotin ester);

6. The reaction mixture was dialysed with PBS (phosphate buffered saline) for an extended period to remove any unbound biotin ester; and

7. The anti-GP64 antibody was aliquoted in suitable volumes and stored at -20°C. Before use the anti-GP64 antibody was diluted as appropriate in 2% Bovine

Serum Albumin (BSA) PBS (phosphate buffered saline).

Example 2 General ELISA protocol

The following ELISA protocol was used in experiments.

1. 50μl of unbiotinylated anti-GP64 antibody at a concentration of 20

:g/ml (diluted in carbonate coating buffer pH 9.6) was added to each well of a 96 well plate. The plate was then incubated for 1 hour at room temperature. 2. The plate was washed 3 times with PBS.

3. The plate was blocked by incubation with 2% BSA PBS (300μl per well) for 1 hour at room temperature.

4. The plate was washed 5 times with PBS. 5. 50μl of an appropriate baculovirus suspension or sample was added to the wells. The plate was incubated for 1 hour at room temperature. The plate was washed 5 times with PBS.

The secondary antibody, biotinylated anti-GP64 antibody diluted in 2% BSA PBS at an appropriate concentration, was added. The plate was incubated at room temperature for 30 minutes.

The plate was washed 5 times with PBS.

50μl of streptavidin/horseradish peroxidase at 1/100 dilution (in 2% BSA PBS) was added. The plate was incubated at room temperature for 30 minutes. The plate was washed 5 times with PBS. The substrate (tetramethyl benzidine liquid substrate obtained from Sigma) was added and the plate left for approximately 10 minutes.

The reaction was stopped by addition of 25μl of 3M sulphuric acid. The absorbance at 450nm was measured using the SoftMax Pro system. Control reactions with no primary antibody, secondary antibody, or baculovirus were normally performed with every ELISA. Where an antibody or the baculovirus was omitted, the reagent was replaced with an equivalent volume of 2% PBS-BSA.

Example 3

The ELISA was performed using a secondary antibody concentration of 1.77:g/ml and a standard suspension of baculovirus particles containing 1.3 x 10 pfu/ml. Reactions were performed with either all the necessary reagents or without primary antibody, secondary antibody or baculovirus. The results obtained are shown in Figure 1.

Example 4 The ELISA was repeated using the conditions given in Example 3 but the concentration of secondary antibody was titrated in order to determine the optimal concentration of secondary antibody at which the ratio of absorbance with baculovirus (specific signal) and without baculovirus (non- specific signal) is highest.

Secondary antibody concentrations of 0.28 :g/ml to 9:g/ml were used. The results obtained are shown in Figures 2(a) and (b). Figure 2(a) shows the absorbance values for each secondary antibody concentration in reactions with and without baculovirus. Figure 2(b) shows the ratio of these values for each antibody concentration. The results show that the secondary antibody concentration which gave the lowest signal to noise ratio was 2.25 :g/mf. This concentration of secondary antibody gives a high level of specific signal and is suitable for use in the ELISA. Increasing the antibody concentration beyond 2.25 :g/ml gives a higher specific signal, but significantly increases the non-specific signal.

Example 5

The ELISA was repeated on two separate occasions in order to determine the reproducibility of the results obtained. A secondary antibody concentration of 2.25 :g/ml was used and samples with varying titres of baculovirus particles analysed.

Table 1 shows the results obtained. The differences between the sets of results obtained on the two occasions are small. These differences probably reflect minor changes in incubation times on the two occasions. Variation in the time of incubation with the substrate is most likely to have caused these differences as small variations in this incubation time can result in large differences in absorption between two experiments.

In order to avoid differences due to variations in incubation times samples and standard-curve titrations should be carried out at the same time, using the same reagents and preferably on the same plate.

Table 1

Example 6

The ELISA was repeated using the same conditions used for the ELISA in Example 5 but with different titres of baculovirus. The results obtained are shown in Figure 3. Figure 3 shows that at higher concentrations of baculovirus particles i.e. at 1 x 10⁸ pfu/ml the absorbance actually begins to drop. This drop in absorption is probably due to the product formed by the action of horseradish peroxidase precipitating and dropping out of solution. To avoid the precipitation of the product the use of lower concentrations of biotinylated secondary antibody was investigated. Using a lower secondary antibody concentration should mean less product is formed and hence precipitation should be less likely to occur.

Titrations measuring change in absorbance against concentration of baculovirus were performed using secondary antibody concentrations of 0.225μg/ml and 0.045μg/ml. The results obtained are shown in Figure 4 (a) and (b). Figure 4 (a) and (b) show that use of the two lower secondary antibody concentrations gives standard curves without the drop in absorbance found using higher secondary antibody concentrations. It is possible to use different standard curves (by using differing antibody concentrations) to give more accurate estimations for the highest or lowest baculovirus titres. In this way the assay can be tailored to enable it's range to be increased. Using a secondary antibody concentration of 0.225μg/ml still gave a rapid development of the product and it was decided that secondary antibody concentrations of 0.045μg/ml and 0.075μg/ml would be used in the ELISA. Using these dilutions it was possible to achieve r values of around 0.998 or 0.999 using a 4-parameter fit calculated using the Molecular Devices "Soft Max" programme, indicating that the fit of the standard curve was very accurate. Thus extrapolation of the baculovirus titres from the standard curve should be more accurate

Example 7

Using the conditions determined in Example 6 the ELISA was used to give a reading on the level of baculovirus particles in a bioreactor at different time points.

Plates were set up using 50μl of sample from days 6 to 62, and with titrations of known baculovirus stock, from 1.0 x 10⁸ to 3 x 10⁵ pfu/ml in half log dilutions. The standards were used to construct a standard curve using a 4-parameter fit as described in Example 6. The equation obtained was then used to calculate the baculovirus titres in the samples from the bioreactor. The ELISA was performed a number of times in order to test the reproducibility of the results.

Figure 5 (a) shows that each set of ELISAs gave similar results, with only slight variations in the values of the absolute titres that were determined. Figure 5 (a) gives the average values for all the ELISAs and hence the final results. The results show a well-conserved profile of baculovirus levels over the 62 days of the run. Although there was slight variation in absolute baculovirus titre as determined by the ELISA for each time point, the overall pattern of baculovirus levels remains remarkably similar throughout the ELISAs that were carried out.

Example 8

To illustrate that the ELISA technique of the invention can be used to reliably determine baculovirus titres it was compared to the rapid plaque assay technique for determining viral titres of Kitts and Green (Anal. Biochem. 1999, 268(2): 173-8).

The results obtained are shown in Figure 6. The values for viral titre obtained using the ELISA differ from those obtained from the rapid plaque forming assay by approximately a factor of two for most samples. The rapid titre method requires the operator to multiply the final result obtained in foci-forming units/ml by a factor of two to give the result in plaque-forming units/ml. This conversion factor was determined empirically by Kitts and Green who devised the rapid plaque forming assay. Therefore if the same logic is applied to the results obtained using the ELISA, a result is obtained which is very close to that found by the rapid plaque forming method.

Even ignoring the possibility of using a conversion factor it can be seen that the trend shown by the rapid plaque forming assay method is similar to that shown by the ELISA method. The ELISA therefore is a valid method for the quick determination of baculovirus titre. The rapid plaque assay technique measures the number of replication competent baculovirus particles in a sample. It does not detect any defective interfering particles present. If there were a large number of defective interfering particles present in the samples it would be expected that the results obtained using the ELISA would be higher than those for the rapid plaque forming assay. As this is not the case it appears that defective interfering particles do not represent a significant problem.

Claims

1. A method of determining the presence, absence or titre of baculovirus particles in a sample, the method comprising detecting the binding of a primary antibody which recognises an epitope present in a baculovirus particle to the baculovirus particle, if present in the sample, thereby determining the presence, absence or titre of baculovirus particles in a sample.

2. A method according to claim 1 wherein the titre of baculovirus particles, the titre of extracellular baculovirus particles present or the titre of intracellular baculovirus particles present is determined.

3. A method according to claim 1 or 2 wherein the epitope is an epitope of GP64 (Glycoprotein 64).

4. A method according to any one of the preceding claims wherein the binding of the primary antibody to the baculovirus particle is detected using a secondary antibody which recognises an epitope present in the baculovirus particle or protein.

5. A method according to any one of the preceding claims wherein the determination is carried out using an ELISA (enzyme-linked immunoadsorbent assay).

6. A method according to claim 4 or 5 which comprises:

(i) contacting the sample with the primary antibody to obtain a baculovirus particle, if present in the sample, bound to the primary antibody; (ii) contacting the primary antibody-bound baculovirus particle with the secondary antibody to obtain a primary antibody/baculovirus/secondary antibody complex; and

7. A method according to claim 6 wherein detection step (iii) is carried out by:

(a) contacting a substrate with an enzyme bound to the secondary antibody; or (b) binding the secondary antibody to an enzyme and contacting with a substrate, and measuring the amount of product formed by the reaction of enzyme and substrate, thereby determining the presence, absence or titre of a baculovirus particle in the sample.

8. A method according to claim 7, part (b), wherein the binding of the enzyme to the secondary antibody is through the interaction of streptavidin and biotin.

9. A method according to any one of claims 6 to 8 wherein the primary and secondary antibodies both recognise the GP64 antigen.

10. A method according to any one of claims 5 to 9 wherein the enzyme is horseradish peroxidase.

11. A method according to anyone of claims 4 to 10 wherein the secondary antibody is at a concentration in the range of 40 to 0.010 :g/ml, preferably 20 to 0.010 :g/ml, more preferably 1 to 0.010 :g/ml, even more preferably

0.100 to 0.010 :g/ml or 0.080 to 0.040 :g/ml

12. A method according to claim 1 wherein the sample is:

- an aliquot of a culture of baculovirus infected insect cells;

- an aliquot of a culture of lysed baculovirus infected insect cells; or - a suspension of baculovirus.

13. A method according to any one of the preceding claims which takes under 24 hours, preferably under 12 hours and more preferably under 5 hours to determine the presence, absence or titre of baculovirus particles in a sample.

14. An ELISA method for determining the presence, absence or titre of baculovirus particles in a sample, the ELISA comprising detecting the binding of a primary antibody which recognises an epitope present in a baculovirus protein to the baculovirus protein, if present in the sample, thereby determining the presence, absence or titre of baculovirus particles in a sample.

15. A method according to claim 14 wherein the epitope is an epitope of GP64.

16. A method according to claim 14 or 15 wherein the binding of the primary antibody to the baculovirus protein is detected using a secondary antibody which recognises an epitope present in the baculovirus protein.

17. A method according to any one of claims 14 to 16 which comprises: (i) contacting the sample with the primary antibody to obtain a baculovirus protein, if present in the sample, bound to the primary antibody;

(ii) contacting the primary antibody-bound baculovirus protein with the secondary antibody to obtain a primary antibody/baculovirus protein/secondary antibody complex; and

(iii) detecting the primary antibody/baculovirus protein/secondary antibody complex.if present, and thereby determining the presence, absence or titre of a baculovirus.

18. A method of producing recombinant polypeptide in an insect cell culture infected with recombinant baculovirus capable of expressing said polypeptide, which method comprises:

(i) culturing the infected cells;

(ii) carrying out a method according to any one of claims 1 to 13 in order to determine the feed and/ or harvest regime for the culture; (iii) feeding and/ or harvesting the culture on the basis of the information thus obtained; and optionally (iv) recovering said polypeptide.

19. A kit for carrying out a method according to any one of claims 1 to 13, which kit comprises: (i) a primary antibody as defined in claim 1 , 2 or 3; and optionally

20. A kit according to claim 15 wherein detection means (ii) comprise a secondary antibody as defined in claim 4 and optionally other means for carrying out the method, as defined in any one of claims 5 to 10.