IES62196B2 - Stabilising medium for antibody-enzyme conjugates - Google Patents

Description

Stabilising medium for antibody-enzyme conjugates This invention relates to a stabilising medium for an antibody enzyme conjugate and, in particular, to a liquid stabilising medium for an antibody-peroxidase conjugate so as to increase the shelf-life thereof.

The poor conformational stability of many proteins is a problem which affects many industrial applications of proteins and this is one of the reasons for the increasing interest shown in protein deactivation and enzyme stabilisation techniques.

The catalytic action of an enzyme is directly related to its three dimensional structure. In turn, the three dimensional structure is determined by a complex set of low energy interactions among various active groups present in the primary structure. Different inactivating interactions occur spontaneously within the protein molecule between amino acid residues, as a result, for example, of thermal agitation. Furthermore, interactions among enzyme molecules in solution can result in aggregation and subsequent precipitation thereof. External environmental modifications can also produce an increase in the rate of enzyme inactivation. For example, the structural and functional integrity of the active domain of a protein can be irreversibly disrupted by the effects of various physical, chemical and biological forces, such as, for example, heat, freezing, radiation, oxidation, reduction, solvents, metal ions, ionic strength and microbial spoilage, which variously result in enzymatic modification and/or degradation. These inactivating forces can pose a significant problem where enzyme stability is desired. It will be appreciated that enzymes which are used in the biotechnological and pharmaceutical industries must, above all, be predictably stable or otherwise serious limitations may be placed upon their use.

One area in which problems of enzymatic stability arise is that of enzyme immunoassays employing an antibody-enzyme conjugate. The stability of such conjugates (herein enzyme conjugates) is of importance in determining the shelf-life of kits containing them.

There are a number of approaches to enzyme stabilisation. However, the approach most suited to the needs of antibody-enzyme conjugates is the use of stabilising additives. It is known that the addition of certain compounds allows a very significant increase in enzyme stability to be achieved in certain instances. Thus, sugars, polyols, salts, albumins and various polymers are already used for the stabilisation of enzyme solutions.

Although there is a desire to produce enzyme conjugates in a ready to use form, the poor stability of many such conjugates in solution (despite the availabilty of a wide range of stabilising agents) means that many conjugate products have short shelf lives.

Because of this instability of enzyme conjugates in solution many such conjugates are provided in kits for enzyme immunoassays as freeze-dried powders.

There are a number of problems associated with freeze-dried enzyme conjugates which include the following: i) the expense of using a freeze dryer; ii) a freeze-drying cycle of typically three days; iii) the variability of the activity of the conjugate resulting from differences in the vacuum pressures, temperatures and times employed in individual freeze drying cycles; and iv) the reduced flexibility of the freeze dried product; the dilution of the conjugate may need to be changed, for example, and this is easier to achieve if one is dealing with a liquid conjugate.

Despite the above disadvantages it is common for conjugates to be supplied in freeze-dried form.

Accordingly, there is a need for a liquid stabilising medium for enzyme conjugates so that such conjugates can be provided in kits for enzyme immunoassays in liquid stable or ready to use form and yet have an acceptable shelf-life.

The invention provides a stabilising medium for an antibodyenzyme conjugate for use in an assay for the detection of antiParvovirus B19 antibody, comprising a stabilising amount of cytochrome c and a stabilising amount of a stabilising protein, a nonionic surfactant and a buffer, such that the medium has a pH in the range 6.5-7.2.

Preferably the enzyme of the antibody-enzyme conjugate is a peroxidase, especially a horseradish peroxidase.

Another preferred peroxidase conjugate is a biotinylated avidin (includes streptavidinj-peroxidase complex, which may be used with an antibody-biotin conjugate to amplify the enzyme assay in conventional manner. In such an enzyme assay antigen insolubilised on solid phase antibody binds to the antibody-biotin conjugate which in turn binds to the biotinylated avidin/streptavidin-peroxidase complex, whereupon the peroxidase activity is measured.

The cytochrome c is preferably present in an amount of 0.02-2% weight by volume. Whereas the concentration of cytochrome c can be increased to above 2% weight by volume without substantially affecting stabilisation, decreasing the concentration below about 0.02% weight by volume results in a decrease in the stabilising effect of the buffer.

By cytochrome c herein is meant a cytochrome in which there are covalent linkages between the side chains of the heme moiety and the protein.

The stabilising protein is preferably a serum albumin which is present in an amount of 0.5-2% weight by volume.

An especially suitable serum albumin is bovine serum albumin (BSA). Whereas the quantity of BSA can be increased to above 2% weight by volume without substantially affecting stabilisation as in the case of the cytochrome c component, if the concentration is decreased below about 0.5% weight by volume the stabilising effect of the buffer is decreased.

The serum albumin can be supplemented by further stabilising protein, for example, foetal calf serum which is rich in BSA.

The non-ionic surfactant is preferably a non-ionic surfactant selected from polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters, polyoxyethylene alcohols, polyoxyethylene isoalcohols, polyoxyethylene ethers, polyoxyethylene esters, polyoxyethylene-p-toctylphenols or octylphenyl-ethylene oxide condensates, ethylene oxide condensates with fatty alcohols, polyoxyethylene nonylphenols, and mixtures of polyalkylene glycols or a mixture thereof.

Especially preferred non-ionic surfactants include: polyethylene sorbitan esters sold under the Trade Mark Tween, especially polyoxyethylene sorbitan monolaurate or Tween 20, but also Tween 60 and Tween 80; polyoxyethylene ethers sold under the Trade Mark Triton, such as Triton XI00, Triton XI14, Triton XI10E and Triton N101, and Brij; an octylphenyl-ethylene oxide condensate sold under the Trade Mark Nonidet P40; ethylene oxide condensates of fatty alcohols sold under the Trade Mark Lubrol, especially Lubrol PX; and a mixture of one part by weight of polyethylene glycol and four parts by weight of polypropylene glycol sold under the Trade Mark Synperonic Fl08. (Tween, Triton, Brij, Nonidet, Lubrol and Synperonic are all Trade Marks).

Suitable buffers which have a pH in the indicated pH range of 6.5-7.2 include the zwitterionic buffers described by N.E. Good and S. Izawa ((1972) Methods in Enzymol., 24, Part B, 53) including HEPES, phosphate buffered saline and Tris buffers.

The stabilising medium according to the invention can also include other additives depending on the nature of the enzyme conjugate, for example, various antimicrobial agents, preservatives and protease inhibitors, agents which stabilise protein - protein interactions, antioxidants and colouring agents which aid in identification.

A suitable antimicrobial agent, when such is present, is an antibiotic such as gentamicin.

Suitable preservatives include sodium azide (except where the enzyme is a peroxidase) and preservatives containing mercurothiolate also known as thiomersal or thiomerosal.

A suitable protease inhibitor is, for example, a trypsin inhibitor such as aprotinin.

A suitable agent which stabilises protein - protein interactions is a polyol such as glucose, glycerol, mannitol, sorbitol or sucrose or a mixture thereof. Glycerol is especially preferred. Suitably the final concentration of polyol will be in the range 5-15 v/v, more especially of the order of 10% v/v.

A suitable colouring agent is carmine dye.

While the stabilising media according to the invention contain the essential features set out above, the particular concentrations of the essential and non-essential components and the particular pH selected can vary so as to obtain an optimal stabilising effect for a particular enzyme conjugate.

The invention hereinafter will be illustrated with reference to enzyme conjugates for use in particular enzyme immunoassays and immunoblot assays, namely a number of horseradish peroxidase conjugates and strepavidin horseradish peroxidase conjugates for use in 5 the detection and qualitative or quantitative determination of IgG and IgM class antibodies to Parvo B19 virus in human serum.

The stabilising media according to the invention have been shown to provide stability and function for a number of enzyme conjugates thus ensuring kits containing such enzyme conjugates maintain an 10 acceptable shelf-life as hereinafter demonstrated.

The combined effects of the components used give rise to buffered stabilising media which can significantly stabilise a number horseradish peroxidase conjugates. The stabilisation can be measured by subjecting the conjugates to heat stress at a temperature such as 37°C 15 and thereby predict the stability of the conjugates following long-term storage at 4°C.

Thus the stabilising media according to the invention enable one to provide liquid stable and 'ready to use' conjugates which offer a desirable alternative to freeze-dried conjugates.

It will be appreciated that the stabilising media in accordance with the invention have obvious advantages from the point of view of production apart from the desirability of providing liquid stable and ready to use conjugates for use in various assays. Liquid stable conjugates in accordance with the invention are ready to use after dilution.

Brief description of the drawings Fig. 1 is a graph of absorbance (450 nm) versus aGST concentration (ng/ml) for an anti-aGST IgG-HRP conjugate at 4°C and following stressing at 37°C for one week in Buffer A prepared in accordance with Example 1; Fig. 2 is a graph of absorbance (450 nm) versus aGST concentration (ng/ml) for an anti-aGST IgG-HRP conjugate at 4°C and following stressing at 37°C for one week in Buffer C prepared in accordance with Example 3; Fig. 3 is a graph of absorbance (450 nm) versus aGST concentration (ng/ml) for an anti-aGST IgG-HRP conjugate at 4°C and following stressing at 37°C for one week in Buffer E prepared in accordance with Example 5; Fig. 4 is a histogram which depicts the percentage stability observed when an anti-pGST IgG-HRP conjugate was stored for one week under different conditions and then used in a pGST enzyme immunoassay; Fig. 5 is a histogram which depicts the percentage stability observed when an anti-Parvo B19 IgG-HRP conjugate was stored for one week under different conditions and measured in an enzyme immunoassay for human anti-Parvo B19 IgG; and Fig. 6 is a histogram which depicts the percentage stability observed when an anti-Parvo B19 IgM-HRP conjugate was stored for one week under different conditions and measured in an enzyme immunoassay for human anti-Parvo B19IgM.

The invention will be further illustrated by the following Examples.

Example 1 Buffer A Buffer A with a pH of 6.5 was prepared from the following reagents: Reagent Quantity NaCl NaH2PO4.2H2O Na2H2PC>4.2H2O Cytochrome c Synperonic Fl08 BSA Foetal calf serum Thiomersal Gentamicin Carmine dye Concentrated HC1 (to adjust pH) Deionised water Made up to 1000 ml with deionised water. 8.000 g 0.260 g 1.425 g 0.250 g 10.000 g 10.000 g 25.000 ml 0.100 g 0.100 g 0.930 g variable variable 700 ml of deionised water was added to a glass container. To this was added the NaCl, NaH2PO4.2H2O, Na2H2PO4.2H2O and thiomersal with stirring until dissolution of the reagents occurred. The Synperonic Fl08 was then added to the solution following by stirring until the surfactant dissolved. The pH of the solution was then checked and adjusted to pH 6.5 with 5M HC1. The BSA was then added to the solution and allowed to dissolve. To this was then added the foetal calf serum with further stirring until dissolution occurred. The gentamicin, cytochrome c and carmine dye were then added with further stirring until dissolution occurred. The pH was rechecked and adjusted as necessary to pH 6.5. The final volume was adjusted to 1000 ml and the buffer was filtered through a 0.2 μκη filter ready for storage. In use nine parts of the buffer are added to one part glycerol.

Example 2 Buffer B Buffer B with a pH of 7.2 was prepared from the following reagents: Reagent Quantity Trizma base (Trizma is a Trade Mark) 9.086 g Cytochrome c 0.375 g Tween 20 (Tween is a Trade Mark) 0.825 g Triton XI00 (Triton is a Trade Mark) 7.954 g BSA 15.000 g Thiomersal 0.150 g Aprotinin 0.003 g Concentrated HC1 (to adjust pH) variable Deionised water variable Made up to 1.5 litres with deionised water. 1.3 litres of deionised water were added to a glass container to which was added the Trizma base. The solution was stirred and the pH adjusted to 7.2. The cytochrome c was added to the solution and the solution was heated to 75-78°C. in a water bath. The solution was then cooled and to this was then added the Tween 20, Triton XI00, BSA, thiomersal and aprotinin. The solution was then made up to 1.5 litres with deionised water. The pH was checked and adjusted if necessary.

Example 3 Buffer C (comparison) Buffer C with a pH of 7.2 was prepared from the following reagents: Reagent Quantity BSA 10.00 g Cytochrome c Glycerol Stock 5 x PBST 1.00 g 100.00 ml 200.00 ml The stock 5 x PBST (phosphate buffered saline/Tween 20) buffer was prepared from the following reagents: Reagents Quantity NaCl 200.00 g KC1 5.00 g Na2HPO4.12H2O 57.75 g KH2PO4 5.00 g Thiomersal 0.50 g Tween 20 13.75 g Deionised water 5,000.00 ml The NaCl, KC1, Na2HPO4.12H2O, KH2PO4 and thiomersal were added to 4.5 litres of deionised water and dissolved using a magnetic stirrer. The Tween 20 was then added to this solution and the final volume was brought to 5 litres with deionised water. The buffer so prepared can be stored at room temperature for up to two months.

To prepare Buffer C 200 ml of the stock 5 x PBST were taken and made up to 800 ml with deionised water. The BSA, cytochrome c and - ta glycerol were then added and the solution stirred until the reagents were dissolved. The pH was adjusted to 7.2 and made up to 1 litre with deionised water.

Example 4 Buffer D (comparison) Buffer D with a pH 7.2 was prepared from the following reagents: Reagent HEPES NaCl Tween 20 BSA Quantity 47.66 g 18.00 g 11.00 g .00 g The reagents were added to 900 ml of deionised water and stirred until dissolved. The pH was adjusted to 7.2 and then the solution was made up to 1 litre with deionised water.

Example 5 Buffer E (comparison) Buffer E with a pH of 7.2 was prepared from the following reagents: Reagent Quantity .00 g .00 g 200.00 ml BSA Carmine dye x PBST 200 ml of the 5 x PBST buffer prepared in the manner described in Example 3 was taken and made up to 900 ml with deionised water. The BSA and carmine were then added and the solution stirred until dissolution occurred. The pH was adjusted to 7.2 and made up to 1 litre with deionised water.

Example 6 (Comparison) The stability of the anti-aGST IgG-HRP conjugate currently provided in lyophilised form in an enzyme immunoassay kit marketed by Biotrin International Limited, Mount Merrion, County Dublin, Ireland under the trade mark Hepkit was investigated in Buffer A, a stabilising medium prepared in Example 1, under the following conditions.

Hepkit is an in vitro enzyme immunoassay which facilitates the early detection of liver damage and recovery in clinical situations such as transplant rejection. The assay specifically detects and quantifies aGST iosoenzyme in plasma and serum and other biological fluids.

The conjugate was prepared according to the procedure of Duncan R.J.S. et «/.,(1983) Anal. Biochem. 132, 68-73, with horseradish peroxidase purchased from Biozyme Laboratories Limited, Wales, United Kingdom. The same method was used in the case of the conjugates of Examples 7 and 8.

First of all, the conjugate was stored in Buffer A (nine parts Buffer A to one part glycerol) at 4°C and 37°C for a period of one week. It was found that when the conjugate was assayed for activity in accordance with the procedure of the Hepkit enzyme immunoassay, that the percentage stability retained following stressing for one week at 37°C was almost 90% of the stability observed when the conjugate was stored at 4°C. The results are depicted in Fig. 1.

The conjugate was then stored in Buffer C, a comparison buffer prepared in Example 3, using the procedure outlined for Buffer A above, and it was found that a dramatic decrease in stability was observed with the conjugate retaining only 34% of its original activity following stressing for one week at 37°C. The results are depicted in Fig. 2.

The conjugate was then stored in Buffer E, a comparison buffer prepared in Example 5, using the procedure outlined for Buffer A above, and it was found that a dramatic decrease in stability was observed with the conjugate retaining only 27% of its original activity following stressing for one week at 37°C. The results are depicted in Fig. 3.

Buffer A was selected following earlier experimental work using a range of buffers containing a number of the components present in Buffer A but not all of the essential components hereinabove specified. None of these buffers was successful in stabilising the conjugate to any great extent. Accordingly, it appeared that the particular combination of components present in Buffer A was necessary for successful stabilisation.

Example 7 (Comparison) The stability of the anti-pGST IgG-HRP conjugate currently provided in lyophilised form in an enzyme immunoassay kit marketed by Biotrin International Limited under the trade mark Mukit was investigated in Buffer A prepared in Example 1.

Mukit is an immunoassay which provides for the qualitative estimation of human pGST. The study of the presence of pGST is of importance when investigating the metabolism of potential toxins and carcinogens and in identifying risk factors associated with the development of cancers and other adverse reactions due to environmental exposure to toxins.

As in the case of Example 6, a similar successful stabilisation was observed when the conjugate was stressed in Buffer A at 37°C for one week at a number of different concentrations and in different containers. Stabilities between 77% and 100% were observed as shown in Fig. 4. Even after a total of fifteen days at 37°C, all conjugates retained approximately 70% stability, including a 1 x 'ready to use' conjugate. The same vial of 1 x conjugate also showed 100% stability real time after five months at 4°C. This represented a very considerable improvement on previous freeze-dried conjugates. For example, when the conjugate was freeze-dried in Buffer D prepared in Example 4, followed by stressing at 37°C for four days, the stability attained was only 30% of that observed when the conjugate was stored at 4°C.

The histogram of Fig. 4 depicts the percentage (%) stability observed when the conjugate was (1) stored at 4°C for one week in Buffer A; (2) stressed at 37°C for one week as a 1 x 'ready to use' conjugate in Buffer A; (3) stressed at 37°C for one week as a 5 x concentrate in Buffer A; (4) stressed at 37°C for one week as a 10 x concentrate in Buffer A; and (5) stressed at 37°C for four days after freeze-drying in Buffer D.

Example 8 (Comparison) The stability of the anti-aGST IgG-HRP conjugate currently provided in lyophilised form in an enzyme immunoassay kit marketed by Biotrin International Limited under the trade mark Nephkit was investigated in Buffer A prepared in Example 1. The Nephkit assay provides a method for the quantitative determination of ocGST in urine and can be indicative of proximal tubule damage in the kidney. 100% stability of a 10 x concentrate was obtained when the conjugate was stored for twenty four hours at room temperature in Buffer A.

Example 9 The stability of the HRP-labelled rabbit anti-human IgG conjugate currently used in lyophilised form in an enzyme immunoassay kit for the qualitative detection of anti-Parvo virus B19 IgG in human serum and marketed by Biotrin International Limited was investigated in Buffer B prepared in Example 2. Whole kit stabilities were performed. The conjugate was obtained from Dako A/S, Glastrop, Denmark.

Parvo virus has been identified as the causative agent of the common rash illness erythema infectiosum (Anderson, N.J., et al. (1984) J.Hyg., 93, 85-93). This is mainly a disease of school children which occurs during localised outbreaks and epidemics during the winter and spring months. Parvo B19 infection in adults, especially women, may cause acute arthritis which can persist for some time (Reid, D.M. et al., (1985) Lancet 1, 422-425). Parvo B19 replicates in human red cell precursors and infection can lead to life threatening anaemia in immunocompromised patients and individuals with underlying haemolytic disorders. In addition, Parvo B19 can complicate pregnancy resulting in spontaneous abortion and hydrops fetalis.

After stress at 37°C for one week there was an approximately 20% drop off with the positive control. Therefore, this represents an 80% stability of the whole kit. The use of Buffer B, a stabilising medium in accordance with the invention, enabled a significant improvement to be achieved with the stability of this kit. The results are depicted in Fig. 5.

The histogram corresponding to Fig. 5 depicts the percentage (%) stability observed when the IgG-HRP conjugate was: (1) stored at 4°C for one week in Buffer B; (2) stressed at 37°C for one week in Buffer B; and (3) stressed at 37°C for one week in Buffer A. Accordingly, it will be appreciated that Buffer B was the stabilising buffer of choice used for this particular assay, rather than Buffer A which was found to be the buffer with the maximum stabilising effects in the case of the various enzyme immunoassays for glutathione S-transferases described in Examples 6-8.

Example 10 Buffer B as prepared in Example 2 was used to stabilise the streptavidin-HRP conjugate (obtained from Dako A/S) currently provided in lyophilised form in an enzyme immunoassay kit marketed by Biotrin International Limited for the qualitative detection of antiParvo virus B19 IgM in human serum. The presence of anti-Parvo virus B19 IgM is indicative of a current Parvo virus infection. The stability of the conjugate in Buffer B under various conditions was investigated.

After stress at 37°C for one week there was only a 12% drop off in the positive control value relative to the value obtained after storage for one week at 4°C. The results are shown in Table 1.

Table 1 Mean value of positive control cv* Max. value Min. value % stability 4°C 0.932 12.2% 1.024 0.480 37°C 0.818 3.9% 0.890 0.765 88 * Coefficient of variability.

The stability obtained of almost 90% represented a significant improvement over other buffers which have been used to stabilise the conjugate as depicted in Fig. 6. For example, when Buffer A was used with this conjugate, only a 60% stability was observed.

The histogram corresponding to Fig. 6 depicts the percentage (%) stability observed when the streptavidin-HRP conjugate was: (1) stored at 4°C for one week in Buffer B; (2) stressed at 37°C for one week in Buffer B; (3) stressed at 37°C for one week in Buffer B minus cytochrome c; and (4) stressed at 37°C for one week in Buffer B minus BSA.

Example 11 Stability studies were carried out on conjugates (obtained from Dako A/S) for two immunoblot kits for the detection of Parvo B19 IgG and Parvo B19 IgM. Buffer A, prepared in Example 1, was found to be the most suitable stabilising buffer for each of the conjugates. When the 10 conjugates were stored in Buffer A as 50 x concentrates and subjected to stress at 37°C for one week, both the IgG and IgM-HRP conjugate gave stable results. Stabilisation was defined to have occurred if weak positive control values were observed after stress.

Examples 6-11 above show that it is possible to provide stable liquid enzyme conjugates for various immunoassays, where freeze-dried or lyophilised conjugates have previously been used and, thereby, to extend the storage time at 4°C.

Claims (5)

1. Claims:-

1. A stabilising medium for an antibody-enzyme conjugate for use in an assay for the detection of anti-Parvovirus B19 antibody, comprising a stabilising amount of cytochrome c and a stabilising amount of a stabilising protein, a non-ionic surfactant and a buffer, such that the medium has a pH in the range 6.5-7.2.

2. A stabilising medium according to Claim 1, wherein the enzyme of the antibody-enzyme conjugate is a peroxidase, in particular horseradish peroxidase.

3. A stabilising medium according to Claim 1 or 2, wherein the non-ionic surfactant is selected from polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters, polyoxyethylene alcohols, polyoxyethylene isoalcohols, polyoxyethylene ethers, polyoxyethylene esters, polyoxyethylene-p-t-octylphenols or octylphenyl-ethylene oxide condensates, ethylene oxide condensates with fatty alcohols, polyoxyethylene nonylphenols, and mixtures of polyalkylene glycols or a mixture thereof.

4. A stabilising medium according to any preceding claim which has the following composition: Trizma (tris(hydroxymethyl) aminomethane) base 9.086 g Cytochrome c 0.375 g Tween 20 (polyoxyethylene sorbitan monolaurate) 0.825 g Triton XI00 (octylphenol ethoxylate) 7.954 g Bovine serum albumin 15.000 g Thiomersal 0.150 g Aprotinin 0.003 g Concentrated HC1 (to adjust pH) variable Deionised water variable Made up to 1.5 litres with deionised water, wherein the cytochrome c in the Trizma base is heated to 75-78°C 5 and cooled prior to the addition of the remaining components.

5. A stabilising medium according to any preceding claim, which retains at least 80% stability after aging at 37°C for one week relative to a positive control at 4°C.