GB2193720A - Monoclonal antibody - Google Patents

Abstract

Papaya peptidases A and B are separated from a crude latex extract of the unripe Carica papaya fruit on an anionic ion-exchange resin, whereas previous methods have used cationic resins. The resulting enzymes are sufficiently pure to allow highly specific antibodies to be prepared. Two hybridoma cell lines secreting such monoclonal antibodies have been deposited. The antibodies can be used to detect levels of the enzyme in food and drink. The enzymes themselves are also useful medically, for degrading slipped discs.

Description

SPECIFICATION Monoclonal antibody This invention relates to monoclonal antibodies.

Protease enzymes of the papain family have been commercially extracted from the unripe fruit of Carica papaya for a considerable time.

Commercial extraction of the protease enzymes results in a mixture of the enzymes, while more recent chromatographic techniques allow for a greater purification. However considerable confusion has built up in literature on protease enzymes in the papain family due to difficulties in separating and identifying the individual enzymes.

Papain is the best known of the enzymes and the easiest to isolate, but the enzyme present in greatest quantity in the family seems to be chymopapain (Jansen and Balls (1941)). Chymopapain is left in the residual supernatant after ammonium sulphate precipitation of papain. The discovery that chymopapain was heterogeneous (Kimmel & Smith, 1954) has lead to a long running confusion about the nature of chymopapain.

Cayle et al (1961) fractionated chymopapain into two forms A and B which both have either Glu or Tyr as their N-terminal residues (Kunimitsu & Yasunobu, 1967, 1970). Initially it was calculated, (Kunimitsu & Yasunobu, 1967, 1970), that chymopapain A had a relative molecular mass of 34.5K and chymopapain B one of 36.4K. However later the mass of chymopapain B was thought to be 28K rather than 36.4K and the definition relied more on specific activity with synthetic substrates or thiol group determination.

Dispute continued on the number of chymopapains with the identification of another form, chymopapain S (Khan & Polgar, 1983). This form of the enzyme had a molecular mass of 24700 but the difference between chymopapain S and chymopapain A has been questioned (Brocklehurst et al., 1984). It was acknowledged that there is difficulty in classifying the chymopapain B (perhaps 3 isoenzymes) and chymopapain A enzymes (Polgar, 1984).

In a recent paper the separation of crude papaya latex was described using fast protein liquid chromatography, which is a process that provides for improved and rapid separation of enzymes and proteins that are difficult to resolve, (Buttle & Barrett, 1984). Chymopapain was the major peak and papain avery minor component. The molecular mass of the chymopapain was claimed to be 28K but in a later paper using the FPLC method to separate the proteases it was assumed that the relative molecular mass was 25K (Zucker et al., 1985). The isoelectric points of papain, 8.75, and chymopapain, 10.1 are sufficiently different to give reasonable separation using acidic polyacrylamide gel electrophoresis as an additional identification step.

Schack (1967) separated a very basic protein (isoelectric point of 11.1) which he called papaya peptidase A but which is now known by general agreement (Barrett & Buttle, 1985) as papaya protinrase 3 or as papaya proteinase A.

It has been shown that the active site of papaya proteinase A is different from that of papain (Baines & Brocklehurst, 1982).

Some recent work using FPLC (Buttle & Barrett, 1984 and Zucker et al., 1985) has raised some new and important questions concerning the hydrodynamic properties of the enzymes in the papain family. In particular the percentage of papain detected appeared to be very low and the main peaks of separation, although easily reproducible, were heterogeneous on both "native" and SDS (sodium dodecyl sulphate) polyacrylamide gels using an acid buffer system.

It is the opinion of Barrett & Buttle that the chymopapain group should be considered as one enzyme with multiple chromatographic forms (Barrett & Buttle 1985); this is opposed by Brocklehurst et al. (1985). The data of Zucker et al. (1985) indicates three proteases all with relative molecular masses of 25K.

Brocklehurst and Salih (1983) claim that there are four enzymes in the papaya proteinase family which do not correspond to either papain or papaya proteinase.

The inadequacies of previous separation techniques, which could not identify all the enzymes, or isolate them in significant quantities in a sufficiently pure form, and the confusion in the classification of the papain family, as evidenced in the above discussion, has presented a very major obstacle in the way of developing immunological techniques for detecting the protease enzymes in the papain family. The production of monoclonal antibodies is a particularly desirable immunological technique, which comprises fusing antibody producing cells, derived from an animal primed with a specific antigen, with myeloma cells in order to produce hybridoma cells each producing a particular species of antibody. A hybridoma cell producing the desired monoclonal antibody is selected and the antibody may be used for various purposes where the particular antigen is of interest.

The present application is directed towards overcoming the above mentioned problems.

Proteinases separated according to the invention may be used in the food and drink industry, for example to preserve canned beer, and medically to degrade unwanted tissue, for example a so-called "slipped disc".

Alternatively, antibodies to the separated enzymes may be used to antagonise or detect the enzyme(s) in question. Monoclonal antibodies are generally preferred for such purposes, although sufficiently pure polyclonal antibodies may be adqeuate and would often offer cost savings. The antibodies may be presented as part of a diagnostic kit based on well-known immunoassay techniques, such as those described in Chard: "An Introduction to Radioimmunoassay and Related Techniques", Elsevier 1982. Enzyme-linked immunosorbant assay (ELISA) techniques are particularly preferred, for example using horseradish peroxidase and an appropriate dye.

One aspect of the present invention provides a monoclonal antibody produced by a murine derived cell line, the antibody being capable of specifically binding to a protease enzyme of the papain family.

The accompanying drawings provide: Figure 1 is a graph of the results from Example 1 which shows the presence of protein as detected by U.V. light absorption (280nm) and which also- shows the NaCI concentrations used in Example 1; Figure 2 is a graph of the results from Example 2 which shows the presence of protein as detected by U.V. light absorption (280nm) and which also shows the NaCI concentrations used in Example 2; Figure 3 is a graph of the results from Example 3 which shows the presence of protein as detected by U.V. light absorption (280nm) and which also shows the NaCI concentrations used in Example 3; Figure 4 shows a representation of the separation of fractions from Example 1 as described in Example 4; Figure 5 is a graph of the results of isoelectric focussing as described in Example 6; and Figure 6 is a representation of the separation of fragments of casein as described in Example 7.

Analysis of spray-dried latex Spray-dried papaya latex, produced from Carica papaya, (suppled by Powell and Scholefield, Liverpool, L7 3JG, UK) is dissolved in a small volume (10 ml) of pH 10.8, 20mM buffer which is prepared by adding 1.91 ml of 1 ,3-diaminopropanq to 1.1 litre of distilled water and adjusting the pH with HCI. This small volume of buffer containing the spraydried latex is dialysed against 5 dm3 of the same buffer for approximately 16 hours. The spray-dried latex is then applied to a mono-Q anion exchange column (HR 515 capacity 1 ml supplied by Pharmacia) attached to a Pharmacia Fast Protein Liquid Chromatography apparatus (also supplied by Pharmacia); the proteins are then eluted by using increasing concentrations of NaCI in the 1,3-diaminopropane pH 10.8, 20mM buffer.Concentrations which have been used are O.OM increasing to 0.035M NaCI followed by a period of 0.075 NaCI and then finally an increasing gradient from 0.035M to 0.2M NaCI. A second gradient has been used, which is O.OM increasing to 0.2M NaCI, increasing at 0.00235M Na Cl/min. The eluted protein is collected in fractions and the degree of U.V. light absorption at 280 nm is recorded for each fraction.

The Fast Protein Liquid Chromatography on the mono Q column gives a reproducible pattern of protein elution.

Care must be taken to ensure that all the non-protein material is dialysed away before chromatography. Incubation with diathiothreitol does not alter the pattern obtained. All the peaks shown have activity towards HPA (Hyde powder azure) and fi-casein. Relatively the more protein eluted the more the activity.

Example 1 The buffer of 1,3-diaminopropane pH 10.8, 20 mM was used to elute the proteins from the mono Q column contained NaCI whose concentration was increased at 6mM/min from 0.0 to a final concentration of 200 mM. 1g of spray-dried latex was dissolved in 5 ml of the 1,3-diaminopropane buffer and after dialysis 100 ul were added to the column. The spraydried latex varied in protein composition but it was normally found to be about 40% protein.

Fig. 1 shows the 280 nm U.V. light absorption peaks which represent the presence of eluted protein in each fraction. Separation of a papain sample (supplied by Sigma Ltd) shows two main peaks corresponding to peaks 4 and 5.

Example 2 The same procedure was followed as in Example 1 except that four fold amounts of protein were added to the mono Q column.

Fig. 2 shows the protein- detected in each fraction by U.V. light absorption.

Example 3 The same procedure was followed as in Example 1 except that a rate of 2.35 mM/min NaCI increase was used and 500 ,ul of 50 mg spray-dried latex per ml of the 1,3-diaminopropane buffer is used.

Fig. 3 shows the protein detected in each fraction by U.V. light absorption.

Example 4 The protein containing fractions are subjected to polyacrylamide gel electrophoresis and isoelectric focussing in polyacrylamide gel as described below.

Slabs of polyacrylamide (14 x 16cm x 1.5mm) are prepared according to the method of Maurer (1971). The separation gels used are normally 15% acrylamide at pH 4.3, the spacer gel is 3% at pH 6.7 and the number of slots for samples is adjusted to be either 5, 10, or 20. Buffer in both upper and lower electrode chambers is ss-alanine/a- cetic acid at pH 4.3. The separation is towards the cathode and pyronine Y is used as a tracking dye. Gels are stained with 0.005% Coomassie blue R250 dissolved in 25% iso propanol and 10% acetic acid, gels are destained using hot 10% acetic acid. The gels can be restained with a silver stain (Switzer et awl., 1979).Separation gels at 15% are used without a spacer but with 0.1 h SDS in the gel for determining relative molecular mass according to a standard procedure. The pH of the gels is 8.3 and the upper and lower buffer chamber contain tris/glycine buffer at pH 8.3 with SDS at 0.1%. Separation is towards the anode and standards are lysozyme (14.3K), ss lactoglobulin (18.4K), trypsinogen (24.0K) and pepsin (34.7K).

Slabs of polyacrylamide (12x25cmx3mm) are prepared from a mixture of 5% acrylamide and pH 9-11 ampholytes. Standards used in the gel are cytochrome c (pl 10.25), trypsinogen (pl 9.3) and lentii lectin (Pl 8.65). The pH is determined in the gel after electro-focussing for 2 hours by using an electrode with a flat base. After the pH profile is determined the gels are stained with Coomassie blue R250.

Fig. 4 shows results from the separation of fractions from the mono 0 column, using 15% acrylamide gel pH 4.3, towards the anode using pyronine Y as marker. Buffer was ss-alani- ne/acetic acid at pH 4.3. Current was 10mA/track.

The apex fractions from each peak (as shown in Fig. 1) were electrophoresed on acid gels and these are shown in Fig. 4. Peak 5 travelled the least far on acidic electrophoresis and this was the same distance as the major peak from the commercial papain (supplied by Sigma Ltd) so a relative molecular mass of about 23K was indicated from SDS electrophoresis (papain is known to have a molecular weight of 23.406K). Papain was always contaminated by other material because, as the concentration of NaCI increased, subsequent to peak 5 a large amount of the eluted material was papain accompanied by protein from peak 4.

Analysis of peak 1 in Fig. 1 showed that this material had a relative molecular mass of 28K and was always totally excluded from the column. This peak was homogeneous and on an acidic gel ran after papain (Fig. 4 Band A) as the next slowest protein (Fig. 2, Band B).

The second peak eluted from the mono 0 column contained a heterogeneous mixture of all peaks, this could be reduced by extensive dialysis before injection but never totally removed. The third peak was also homogeneous, like peak 1, and ran fastest on acidic gels, the relative molecular mass was 24-25K (Band D). The two largest peaks both contained a doublet of protein bands on acidic gels and gave an Mr of 25K (Band C).

When the chromatographic separation was heavily overloaded as required in Example 2 and shown in Fig. 2 a papain peak was produced which was homogeneous on acidic gels but a large proportion of papain was also associated with other material.

The best separation was achieved by using the shallower linear gradient of NaCI in 1,3diaminopropane as required in Example 3 and shown in Fig. 3. The first peak, fraction 28, is the second slowest protein on acidic gels and was homogeneous with a relative molecular mass of 28K by SDS-electrophoresis. While the majority of the second peak (fraction 30) travelled fastest of all the proteins from this separation on acidic gels, it was slightly contaminated by material from fraction 28. Fraction 45 was the faster of the two proteins shown in Fig. 2 Band C, and fraction 62-64 contained the slower of these bands. The relative molecular masses were, as mentioned above, between 24K and 25K.

Fig. 5 shows results from the isoelectric focussing of the protease fractions over the pH range 7.8-11.4. 100 ul of sample is placed on paper wicks just 2 cm from the anode.

Bands of protein are visualised with Coomassie blue R250.

Data displayed in Fig. 5 shows that the range of pl values obtainable using the pH 9-11 ampholytes was 7.8-11.4. The numbers on the graph indicate fractions from Fig.

4. The protein from fraction 28 had a pl-of 11-11.1, as did the proteins from fraction 30.

Fraction 45 had a pl of 10.7 and fractions 62-64 had pls of 10.3-10.4, two clear bands being visible. Fraction 80 had a pl of 8.7-8.9.

As well as measurements by a flat pH electrode, small samples of the gel were extracted with water and the pH measured. Cytochrome c and lentil lectin were used as standard calibration proteins.

N-terminal analysis of the proteins from Mono Q separation The indentity of the N-terminal residues is determined by the dansyl effect technique described by Woods and Wang (1967). Peak 5 in Fig. 1 or fraction 80 in Fig. 4 indicated that the amino-acid was isoleucine. The third protein peak in Fig. 1 or fraction 30 in Fig. 4 had leucine as the N-terminal amino acid. The fourth protein peak in Fig. 1 or fraction 45 in Fig. 4 had glutamate as the N-terminal residue.

Example 5 The separation of spray-died Carica papaya extract as carried out by Buttle & Barrett (1984, their Fig. 1) using FPLC associated with a mono S column was repeated and the same fractions were obtained. The papain peak obtained from the mono S column was rerun on a mono 0 column and gave a single peak in the area of peak 3 shown in Fig. 1 of the drawings. Without wishing to be bound to the theory it is our understanding the majority of papain does not appear when spray-dried latex is separated on the mono S column system using acetate buffer and this is possibly because the papain either binds to the column and is not eluted under the conditions em ployed, or because the papain is present in the main peaks.In support of the second theory that papain appears associated with other peak it was found that the main peaks from mono S column separation were heterogeneous on SDS polyacrylamide gels. In a further comparison between the acidic separation on mono S columns and the alkaline separation on mono Q columns it was found that the small peak eluted just before papaya proteinase on mono S column had a relative molecular mass of 28K, however there was also some of this material in the major peaks of the mono S column separation. The main peaks from the mono S column separation correspond to fractions 30, 45 and 62-64 shown in Fig. 3.

All previous work based on the ion exchange FPLC separations (e.g. Zucker et al., 1984 and Buttle and Barrett, 1984) have used acidic buffer systems. Alternative buffer systems have not been used with ion exchange FPLC separations, because it was believed on theoretical grounds that alkaline buffer systems would lead to high pl proteases not being bound to the column and eluting before the eluting salt gradient begins. Subsequently using the alkaline buffer system it would be expected that the chymopapains would elute early in the gradient followed by papain. In the alkaline buffer system papain is positively charged, the chymopapains are either positively charged or just at their isoelectric point and the high pl proteases are negatively charged.

Protein was determined by the method of Bradford (1976) and protease activity was assayed by the release of blue dye from hide powder azure with incubations at 15"C for 1 min at pH 6.0.

Protein from fraction 45 and 62 was centrifuged in a Beckman model E centrifuge and the molecular weight calculated according to the formula:

where R=gas constant (8.314x 107) T=absolute temperature =the angular velocity v=the partial specific volume of the solute p=the density of the solution o=the effective reduced molecular weight v was calculated as 0728 using a Pharr density meter and the concentration was determined by a synthetic boundary centrifugation.

The molecular weight of the protein was 23K and was the average of three different concentrations of protein.

Example 6 Fig. 6 shows the data from a digestion of ss-casein after 1 min of digestion by the protease fractions from Example 3. Separation was towards the anode, the gel was 15% pH 8.3. Total protein was 100dum/track and current was 3mA for 2 hours and staining was Coomassie Blue R250. The digestion by the separate protein fractions is similar and there appear to be 10 separate fragments produced by each of the four proteases.

The results shown in the Figures identify papain and papaya proteinase which have been characterised both by isoelectric points and N-terminal analysis. In addition there are three other identifiable protease molecules which are separable by acid gel electrophoresis and also by denaturing gel electrophoresis into high and low molecular mass forms. The data displayed in Fig. 5 shows the higher molecular mass protein (28K) has a pl of 11 whereas the other, lower molecular mass forms 24K to 25K have pls of 10.3 to 10.7 and amino acids of N-terminal glutamic acids.

These lower molecular mass forms are members of the chymopapain family and have characteristics suggesting they are of the chymopapain A series. The separation displayed in Fig. 4 is evidence that they are two closely related enzymes.

Having isolated and characterised the five protease enzymes from the spray-dried latex, samples of the protease enzymes are used in the production of antibodies specific for the enzymes.

Monoclonal antibody production Protease enzymes isolated by the polyacrylamide gel electrophoresis step are injected into a mouse which is then left for an incubation period. Spleen cells from the mouse are hybridised with myeloma cells producing hybridoma cells which are subjected to selection for the specific anti-protease enzyme monoclonal antibody producing cell lines which are of interest.

Example 7 Fractions from the mono Q column containing the protease enzyme of interest are collected and freeze dried before redissolving in phosphate buffered saline (PBS). The protein concentrations are measured and aliquots of the fractions separated using polyacryamide gel electrophoresis (as described above). Other aliquots of the protease enzyme showing no contamination are used for mouse immunization.

Immunization A BALB/c mouse is injected subcutaneously with 50ug of the protease enzyme. The enzyme is in 200 jell PBS, emulsified with Freunds complete adjuvant. 49 days later the animal is injected with another 50,us of the enzyme in 200,u1 PBS. Cell fusion is carried on the 52nd day.

Fusion Spleen cells 1.1 X 108 are fused with mouse myeloma cells X63Ag8653 1.19x 107 using freshly prepared 50% polyethylene glycol (supplied by Sigma Ltd) in RPMI medium (Rosweel Park Memorial Institute).

The fusion cells are resuspended in 10 mls of hypoxanthine-aminopterin-thymidine medium+insulin (HIAT, Feit et. al. 1984) RPMI 1640 with 20% foetal calk serum (FCS) and placed into 96 well microtitre plates using 50,u1/well, after seeding each well with a macrophage feeder layer. The macrophage feeder cells are obtained from BALB/c mice by peritoneal lavage with lOmls DMEM medium (Dulbeccos modified Eagles medium)+ 10 units Heparin/ml.

Preliminary screening of culture supernatants.

96 well polyvinyl chloride) microtitre plates are coated with a 1ug/ml solution of the enzyme in coating buffer at pH 9.6 which is prepared according to Kilshaw and comprises sodium carbonate 0.1 M, sodium bicarbonate 0.1 M and sodium azide 0.001 M. The coating step is carried out either overnight in "the cold room" or for 1 hour at 20"C and the wells are subsequently washed six times with PBS containing 0.015% Thiomersal.

100,u1 of medium from the feeder plates is incubated in the washed wells for 1 hour at room temperature, the wells are then washed again as described above and a 100,ul aliquot of a 1/5000 diluted peroxidase conjugated rabbit immunoglobulin anti-mouse serum (Dacopatts a/s Denmark) is added to each well.

The diluent used is normal rabbit serum. The plate is left to incubate for 1 hour at 20"C and all the wells are washed 6 times (in the same way as before) after which 100ul of ophenylenediamine substrate solution is added to each well. The reactants in the solutions are:- citric acid, sodium phosphate, o-phenylenediamine, hydrogen peroxide. The plate is then left to incubate at 20"C until colour develops in the wells. When colour has developed the reaction is stopped by the addition of 2581 of 12.5% sulphuric acid to each well.

Cloning.

In this particular example eleven cultures were cloned by the technique of limiting dilution and sub-clones of five of the eleven cultures were injected into pristane-primed mice in order to generate ascites fluid.

2F6/FB Line 5 1H1/G4 Line 8 3E9/C11 Line 3 1F6/E4 Line 7 3A11/H5 Line 1 The above mentioned five sub-clones were tested according to the procedure of Kilshaw and were each found to produce only one subclass of IgG.

Ascites fluid from these mice was tapped and preliminary screening done using the microtitre plates.

Two cell lines prepared by the techniques given above and yielding papaya proteinases A and B, respectively, were deposited on 7th August 1986 at the European Collection of Animal Cell Lines, Porton Down, Wiltshire, U.K. under the accession numbers 86080702 and 86080701, respectively.

Claims (28)

1. An antibody capable of specifically binding to a protease enzyme of the papain family.

2. An antibody specific for papaya proteinase A.

3. An antibody specific for papaya proteinase B.

4. An antibody specific for a protease enzyme having a relative molecular mass of approximately 28K and an isoelectric point of 11.

5. An antibody specific for a protease enzyme of the chymopapain group having a relative molecular mass of 24 to 25K, an isoelectric point of 10.0 to 10.7 and an N-terminal of glutamic acid.

6 A monoclonal antibody according to Claim 1, 2, 3, 4 or 5.

7. A monoclonal antibody according to Claim 6 produced by an immune-derived cell line.

8. Cell line 86080702 or 86080701 as deposited on 7th August 1986 at the European Collection of Animal Cell Lines, Porton Down, U.K.

9. A substantially biologically pure protease enzyme of the papain family having a relative molecular mass of approximately 28K and an isoelectric point of 11.

10. A substantially biologically pure protease enzyme of the chymopapain group having a relative molecular mass of 24 to 25K, an isoelectric point of 10.0 to 10.7 and an Nterminal of glutamic acid.

11. Substantially biologically pure papaya proteinase A.

12. Substantially biologically pure papaya proteinase B.

13. A process for isolating protease enzymes of the papain family from Carica papaya using Fast Protein Liquid Chromatography characterised by using separation on an amion exchange column.

14. A process according to Claim 13, characterised by using an alkaline buffer system to elute the enzymes from the column.

15. A process according to Claim 14 wherein the alkaline buffer system is prepared by mixing 1,3-diamino- propane and distilled water to produce a 20 mM concentration of the said 1,3-diamino-propane and by adjusting the buffer pH to 10.8.

16. A process according to any one of Claims 13 to 15, wherein the enzyme is that defined in Claim 9, 10, 11 or 12.

17. A method of producing monoclonal antibodies with binding specificity for a specific enzyme of the papain family comprising cultivating a hybridoma cell formed by fusing a cell producing antibody with binding specificity for said specific enzyme and a myeloma cell and recovering the monoclonal antibody produced.

18. A method of producing monoclonal antibodies with binding specificity for a specific enzyme of the papain family, comprising immunising a BALB/c mouse with said specific enzyme, forming hybridoma cells from antibody producing cells from said BALB/c mouse and mouse myeloma cells X63Ag8653, cloning said hybridoma cells and selecting clones which produce antibodies that demonstrate specificity for the said specific enzyme.

19. A method according to Claim 17 or 18, wherein the specific enzyme is that defined in Claim 9, 10, 11 or 12.

20. A method of detecting a specific enzyme of the papain family present in a sample, comprising treating the sample with antibodies having binding affinity for the enzyme so as to bind therewith, and detecting the presence of bound antibodies.

21. A method according to Claim 20, wherein said antibodies are as claimed in any one of Claims 1 to 7.

22. A method according to Claim 20 or 21, including determining the quantity of bound antibodies in said treated sample as a measure of the quantity of said enzyme in the sample.

23. A method according to Claim 20, 21 or 22, wherein the specific enzyme is that defined in Claim 9, 10, 11 or 12.

24. An enzyme according to any one of Claims 9 to 12 bound to a support and suitable for use in an assay technique for the said enzyme.

25. An antibody according to any one of Claims 1 to 7 bound to a support and suitable for use in an assay technique for the enzyme for which the antibody is specific.

26. A diagnostic kit comprising an enzyme according to any one of Claims 9 to 12 or an antibody according to any one of Claims 1 to 7.

27. The use of an enzyme according to any one of Claims 9 to 12 in medicine.

28. The use of an enzyme according to any one of Claims 9 to 12 in the manufacture of a medicament for the degradation of proteinaceous material in vivo.