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GB2418665A - Stabilising dilutent for HRP conjugates - Google Patents

Stabilising dilutent for HRP conjugates Download PDF

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GB2418665A
GB2418665A GB0422081A GB0422081A GB2418665A GB 2418665 A GB2418665 A GB 2418665A GB 0422081 A GB0422081 A GB 0422081A GB 0422081 A GB0422081 A GB 0422081A GB 2418665 A GB2418665 A GB 2418665A
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stabilising
diluent
added
polyoxyethylene
horseradish peroxidase
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Ian Gordon Elliott
Cormac Gerard Kilty
Joseph Bernard Rice
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Biotrin Intellectual Properties Ltd
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Biotrin Intellectual Properties Ltd
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    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

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Abstract

A stabilising diluent for a protein-horseradish peroxidase conjugate for use in an enzyme immunoassay for the detection of a human Parvovirus B19 antibody, comprises stabilising amounts of bovine serum albumin (BSA), cytochrome c and gelatin hydrolysate, an antimicrobial agent consisting of 2-methyl-4-isothiazolin-3-one and 5-bromo-5-nitro-1, 3-dioxane, a non-ionic surfactant and a biological buffer, such that the diluent has a pH in the range 7.0 to 7.5. Use of the stabilising diluent results in the protein-horseradish peroxidase conjugate retaining at least 70% reactivity following accelerated stress testing at a temperature in the range 35-39{C for a period of at least 4 days relative to a positive control at a temperature in the range 2-8{C.

Description

1 24 1 8665 Stabilising diluent for a protein-horseradish peroxidase
conjugate This invention relates to diluents for protein enzyme conjugates for use in enzyme immunoassays (EIAs) and, in particular, protein horseradish peroxidase conjugates.
Diagnostic EIA kits are available for carrying out testing for Parvovirus Bl9 IgG and IgM antibodies. Parvovirus B19 IgG and IgM kits as marketed by Biotrin International Limited, Dublin Ireland, for use 0 in EIAs contain liquid stable conjugates: streptavidin-horseradish peroxidase (S-HRP) in the Parvovirus B19 IgM EIA and anti-human IgG horseradish peroxidase (IgG-HRP) in the Parvovirus B19 IgG EIA.
These conjugates are prepared by adding the S-HRP or IgG-HRP protein conjugates (bought commercially, supplier DAKO) to a diluent, which must meet certain defined criteria.
In order to ensure the requisite shelf life and stability of the conjugates, the diluents usually contain a significant concentration of stabilising proteins for example bovine serum albumin (BSA), foetal bovine serum, gelatin, etc. However, stabilising proteins provide a good growth medium for microorganisms, which can interfere with the activity of the conjugates. Hence anti-microbial agents (preservatives) are used to control the growth of microorganisms. It is conventional to use the mercury-containing antimicrobial agent thiomersal as an antimicrobial preservative in many types of solution marketed by the pharmaceutical and diagnostics industries. We have traditionally used thiomersal as a preservative, for components of our Parvovirus B19 IgG and IgM kits, including diluents for protein-HRP conjugates. Thiomersal contains mercurothiolate, which is a toxic, non-biodegradable compound, and is already banned in Japan. During the production of the diluent currently used for our HRP protein conjugates a two week maturation step is required as described in our Irish Patent No. S 83524.
The removal of this step would provide for a more convenient process.
Anti-microbial preservatives are available commercially, however as these preservatives have many applications complex biological testing l O must be performed to ensure they do not adversely affect the biological system in which they will be used. It is difficult to identify preservatives specific for the in-vitro diagnostic devices industry. Thiomersal and sodium azide are standard recommended preservatives used in this industry. We have already highlighted the problems with thiomersal set out above. Sodium abide is known to inhibit horseradish peroxidase and for this reason would not be suitable for use in a diluent for a HRP protein conjugate. Another antimicrobial agent that could potentially be used is Antibiotic Antimycotic Solution (1OOX) supplied by Sigma.
However, this could not be used in a diluent for components of a diagnostic kit, because it has a short shelf life of only seven days.
It is an object of the present invention to provide a diluent for protein-HRP conjugates which is free of a mercury containing antimicrobial preservative and which would overcome the disposal problems associated with such diluents, while at the same time maintaining acceptable stability and thus shelf life of the conjugates.
The invention provides a stabilising diluent for a protein- horseradish peroxidase conjugate for use in an enzyme immunoassay for the detection of a human Parvovirus B 19 antibody, which comprises stabilising amounts of bovine serum albumin (BSA), cytochrome c and gelatin hydrolysate, an antimicrobial agent consisting of 2-methyl-4- isothiazolin-3-one and 5-bromo-5-nitro-1, 3-dioxane, a non-ionic surfactant and a biological buffer, such that the diluent has a pH in the range 7.0 to 7.5.
0 According to a preferred embodiment of the invention, the biological buffer is tris(hydroxymethyl)aminomethane base.
In an alternative embodiment, the biological buffer is a zwittcrionic buffer.
A preferred zwitterionic buffer is morpholinepropanesulfonic acid 1 5 (MOPS).
Preferably, 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 octylphenylethylene oxide condensates, ethylene oxide condensates with fatty alcohols, polyoxyethylene nonylphenols, and mixtures of polyalkylene glycols or a mixture thereof.
Preferred non-ionic surfactants include those sold under the Trade Marks Tween 20 and Triton X- 100.
According to one aspect of the invention, the protein-horseradish peroxidase conjugate is streptavidin-horscradish peroxidase.
According to an alternative aspect of the invention, the protein horseradish peroxidase conjugate is anti-human IgG horseradish peroxidase.
In addition to ensure that the conjugate will not promote growth of microorganisms, it is important to ensure that the conjugate performance 0 remains intact over the life ofthe product. It is standard industry practice to perform accelerated stress testing on biological components to give an indication of the long-term stability thereof. This procedure usually consists of storing the material at a reference temperature (usually 2-8 C) and at an elevated temperature (usually 35-39 C) for a period of time. The activity of the material stored at the elevated temperature is then compared to the reference temperature and the percentage drop-off in activity calculated. A low percentage drop-off is indicative of long-term stability of the component at the reference temperature.
According to a preferred embodiment of the invention, the proteinhorseradish peroxidase conjugate retains at least 70% reactivity following accelerated stress testing at a temperature in the range 35- 39 C for a period of at least 4 days relative to a positive control at a temperature in the range 2-8 C. s
In order to determine if anti-microbial preservatives in diluents are effective against microbes, preservative efficacy challenge tests (PE l s) are performed. A PET test involves inoculating each of the diluents with test organisms, for example Staph. aureus, E. coli, Ps. aeruginosa, C. albicans, and 4. niger, at approximately I X 103 Cfu/ml (colony forming units/millilitre). The inoculated diluent is then stored in the dark at room temperature. A sample is removed at O hours, 14 days, and 28 days. These samples are tested for the inoculum and the number of colonies counted.
The invention will be further illustrated by the following
Examples.
As described in the following Examples, different formulations of diluents containing a range of anti-microbial preservatives were prepared and tested.
Example 1
(Reference) The following thiomersal-containing diluent was prepared from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Trizma base Sigma 6.05g Cytochrome c Merck 0. 25g Tween 20 Merck 0.55g lo Triton X-100 BDH 5 3g BSA Serologicals 1 O.Og Thiomersal BDH 0.67g HC1 Inhouse Variable Aprotinin Sigma 2mg Deionised water Inhouse up to 1L Gentamicin Sigma 0.1 Og The diluent was prepared as follows: 80% of the batch volume of deionised water (800ml) was added to a container. Trizma was then added and dissolved. The solution was then titrated to 7.2+/-0.05 with HCI. Cytochrome c was added and dissolved.
Tween 20, Triton X-100, BSA, thiomersal, aprotinin and gentamicin were added and mixed well.
The batch was then brought up to 1L with deionised water and mixed well.
The pH was then checked and adjusted, if required, to 7.2 with SM HCI or 5M NaOH.
The diluent was then filtered through a 0.22pm filter into an aluminium wrapped container.
Example 2
A diluent containing the antimicrobial agent 5-chloro-2-methyl-4 isothiazolin-3-one (CMIT, sold under the Trade Mark Kathon CG) was l0 prepared from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Trizma base Sigma 2.42g BSA Sigma 4g Gelatin hydrolysate Sigma 4g Aprotinin Sigma 0.4mg 5M HCI Inhouse Variable Deionised water Inhouse up to 400ml Tween 20 Merck 0.165g Triton X-100 BDH 1.59g Deionised water Inhouse up to 300ml Kathon CG Rohm and Haas 0.067ml Deionised water Inhouse up to 100ml The diluent was prepared as follows: 320ml of deionised water was added to a plastic container. The pl l was adjusted to 7.29 with 5M HCI. l he BSA was added and allowed to dissolve. The gelatin hydrolysate was added with stirring until dissolution. Aprotinin was added with stirring until mixed. The volume was adjusted to 400ml with deionised water. The pH was measured at 7.16. This solution will be referred to as solution a).
240ml of solution a) was added to a plastic container. The Tween and Triton X- l OO were added with stirring until dissolution. The volume was adjusted to 300ml with solution a).
1 OOml of this solution was added to a plastic container to which the Kathon CG was then added and stirred until dissolved.
Example 3
A stock solution, which will be referred to as stock solution X, was prepared from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Trizma base Sigma 12.1g BSA Sigma 20g Gelatin hydrolysate Sigma 20g Aprotinin Sigma 2mg SM HCI Inhouse Variable Deionised water Inhouse up to 2L The stock solution was prepared as follows: 1600ml of deionised water was added to a plastic container. Trizma base was added and stirred until dissolved. The ply of this solution was then adjusted to 7.33 with HC1. BSA was added and allowed to dissolve.
Gelatin hydrolysate was added with stirring until dissolution occurred.
Aprotinin was added with stirring until mixed. The final volume was adjusted to 2L with deionised water. The pH was measured at 7.28.
Example 4
A diluent containing a combination of 2-methyl-4-isothiazolin-3-one (MIT, sold under the Trade Mark ProClin 950) and 5-bromo-5-nitro-1, 3-dioxane (sold under the Trade Mark Bronidox L) was prepared from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Stock solution X Inhouse 100ml BronidoxL (0.02%) Sigma 0.02g ]5 ProClin 950 Supelco 50.61 (48ppm of active ingredient) Tween 20 Merck 0.0275g Triton X-100 BDH 0. 256g The diluent was prepared as follows: I OOml of stock solution X was added to a plastic container.
Bronidox L and ProClin 950 were added and stirred until dissolved, referred to herein as solution b). 40mls of this solution was added to a plastic container. The Twecn 20 and Triton X-100 were added with stirring until dissolution occurred. The solution was then adjusted to 50ml with solution b).
Example 5
A diluent containing a combination of ProClin 950 (Trade Mark) and Bronidox L (Trade Mark) was prepared from the following ingredients and in the indicated amounts.
Reagent Supplier Quantity Stock solution X Inhouse lOOml BronidoxL (0.02%) Sigma 0.02g ProClin950 Supelco 50.6,u1 (48ppm of active ingredient) Cytochrome c Sigma 0.025g Tween 20 Merck 0. 0275g Triton X-100 BDH 0.256g The diluent was prepared as follows: 100ml of stock solution X was added to a plastic container.
Bronidox L, ProClin 950 and cytochrome c were added and stirred until dissolved, referred to herein as solution c). 40ml of this solution was added to a plastic container. The Tween 20 and Triton X-100 were added with stirring until dissolution occurred. The solution was then adjusted to 50ml with solution e).
Example 6
A diluent was prepared containing ProClin 950 (Trade Mark) and gentamiein as anti-microbial preservatives from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Stock solution X Inhouse lOOml ProClin 950 Supeleo 50.61 (48ppm of active ingredient) Gentamicin Sigma 101 Tween 20 Merck 0.022g Triton X-100 BDII 0.212g The diluent was prepared as follows: 1 OOml of stock solution X was added to a plastic container.
ProClin 950 and gentamicin were added and stirred until dissolved.
40mls of this solution was added to a plastic container and the 1 ween and Triton X-100 added with stirring until dissolution occurred.
Example 7
A stock solution, which will be referred to as stock solution Y. was prepared from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Morpholinepropanesulfonic acid Sigma 4.182g (MOPS) BSA Sigma 10g Gelatin hydrolysate Sigma 10g Aprotinin Sigma Img MNaOH Inhouse Variable Deionised water Inhouse up to 1L The stock solution was prepared as follows: 800ml of deionised water was added to a plastic container. MOPS was added with stirring until dissolved. BSA was added and allowed to dissolve. Gelatin hydrolysate was added with stirring until dissolution occurred. Aprotinin was added with stirring until mixed. The pH was adjusted to 7. 25. The final volume was adjusted to lL with deionised water. l3
Example 8
A diluent was prepared using Bronidox L (Trade Mark) and ProClin 950 (Trade Mark) as anti-microbial preservatives from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Stock solution Y Inhouse lOOml BronidoxL (0.02%) Sigma 0.02g ProClin 950 Supelco 50.61 (48ppm of active ingredient) Tween 20 Merck 0.0275g Triton X-lOO BDH 0. 256g The diluent was prepared as follows: l OOml of stock solution Y was added to a plastic container.
Bronidox L and ProClin 950 were added and stirred until dissolved, referred to herein as solution d). 40ml of this solution was added to a plastic container. The Tween 20 and Triton X-lOO were added with stirring until dissolution occurred. The solution was then adjusted to 50ml with solution d).
Example 9
A diluent was prepared using Bronidox L (Trade Mark) and ProClin 950 (Trade Mark) as anti-microbial preservatives from the following s ingredients and in the indicated amounts: Reagent Supplier Quantity Stock solution Y Inhouse lOOml BronidoxL (0.02%) Sigma 0.02g lo ProClin 950 Supelco 50.61 (48ppm of active ingredient) Cytochrome c Sigma 0.025g Tween 20 Merck 0. 022g Triton X- 100 BDH 0.212g The diluent was prepared as follows: l OOml of stock solution Y was added to a plastic container.
Bronidox L, ProClin 950 and cytochrome c were added and stirred until dissolved. 40ml of this solution was added to a plastic container.
The Tween 20 and Triton X- 100 were added with stirring until dissolution occurred.
Example lO
A diluent was prepared using thiomersal and gentamicin as anti- microbial preservatives from the following ingredients and in the indicated amounts: React Supplier Quantity Trizma base Sigma 6.05g BSA Sigma I Og Gelatin hydrolysate Sigma lOg Triton X-100 BDH 5.3g Tween 20 Merck 0.55g Aprotinin Sigma 0.5ml 5M HCI Inhouse Variable Deionised water Inhouse up to lL Thiomersal Merck O.Olg Gentamicin Sigma l O The diluent was prepared as follows: 800ml of deionised water was added to a plastic container. To this was added the Trizma base with stirring until dissolution of the reagents occurred. The BSA was added and allowed to dissolve. The gelatin hydrolysate was added followed by stirring to dissolve. The Triton X- lOO, Tween 20 and aprotinin were added with further stirring until dissolution occurred. The pH was adjusted to 7.25 with 5M HCI. The fnal volume was adjusted to lOOOml with deionised water.
1 00ml of this solution was added to a glass container. Thiomersal and gentamicin were added to this diluent, followed by stirring until dissolution. The pH was measured at 7.17.
Example 11
A stock solution, which will be referred to as stock solution Z. was prepared from the following ingredients and in the indicated amounts: Reagent Supplier Quantity MOPS Sigma 4.1 85g BSA Sigma 10g Gelatin hydrolysate Sigma 10g Triton X-100 BDH 5.3g Tween 20 Merck 0.55g Aprotinin Sigma 0.5ml SM NaOH Inhouse Variable Deionised water Inhouse up to 1L The stock solution was prepared as follows: 800ml of deionised water was added to a plastic container. To this was added the MOPs with stirring until dissolution of the reagents occurred. The BSA was added and allowed to dissolve. The gelatin hydrolysate was added followed by stirring to dissolve. The Triton X 100, Tween 20 and aprotinin were added with further stirring until dissolution occurred. The pH was adjusted to 7.21 with SM HCI. The final volume was adjusted to 1000ml with deionised water.
Example 12
A diluent was prepared from stock solution Z using Bronidox L (Trade Mark) as the anti-microbial preservative in the indicated amounts as follows: Reagent Supplier Quantity Stock solution Z Inhouse lOOml Bronidox L Sigma 0.02g T he diluent was prepared as follows: I OOml of stock solution Z was added to a glass container.
Bronidox L was added to this diluent, followed by stirring until dissolution. The pI] was measured at 7.21.
Example 13
A diluent was prepared from stock solution Z using Bronidox L (Trade Mark) as the anti-microbial preservative in the indicated amounts as follows: Reagent Supplier Quantity Stock solution Z Inhouse lOOml BronidoxL (0.01%) Sigma O.Olg The diluent was prepared as follows: 1 00ml of stock solution Z was added to a glass container.
Bronidox L was added to this diluent, followed by stirring until dissolution. The pH was measured at 7. 19. s
Example 14
A diluent was prepared using Kathon CG (Trade Mark) and gentamicin as anti-microbial preservatives from the following ingredients and in the indicated amounts: Reagent Supplier Quantity Trizma base Sigma 6.05g Cytochrome c Sigma 0.25g Tween 20 Merck 0.55g Triton X-100 BDH 5.3g BSA Sigma 10g Kathon CG Rohm&Haas 0.67ml Aprotinin Sigma 2mg Gentamicin Sigma 0.1 0g SM HCI Inhouse Variable SMNaOH Inhouse Variable Deionised water Inhouse up to 1L The diluent was prepared as follows: 800ml of deionised water was added to a plastic container. To this was added the Trizma base with stirring until dissolution of the reagent occurred. The pl l was adjusted to 7.2 with SM HCI. Cytochrome c was added and allowed to dissolve. The Triton X-100 and the Tween 20 were added with stirring until dissolution occurred. The BSA was added and allowed to dissolve. The Kathon CG, aprotinin and gentamicin were added and dissolved with continuous stirring. The pH was adjusted to 7.18 with 5M NaOH. The final volume was adjusted to 1 000ml with deionised water.
Example 15
Preservative Efficacy Challenge test.
0 PETs were carried out in accordance with the procedure described in the United States Pharmacopea 27 <51> "Antimicrobial Effectiveness testing". This testing was performed to determine if diluent formulations prepared in the preceding Examples were effective in preventing the growth of microbes, which as stated above is an essential requirement of a proteinenzyme conjugate stabilising diluent.
The diluents of Examples 1, 4-6, 8 and 9 were subjected to a PET.
These diluents were selected for the PETs as they contain all of the preservatives examined and hence would allow for the determination of an effective preservative or combination of preservatives.
The following results were obtained: The diluents of Examples 1, 4, 5, 8 and 9 passed PETs. When analysed this data demonstated that ProClin 950 (at 48ppm) combined with Bronidox L (used at 0.02%) is effective in preventing microbial growth when used in this combination.
PET results for the diluent of Example 1 demonstrate that thiomersal is an effective preservative.
The diluent of Example 6 failed the PET, demonstrating that ProClin 950 (used at 48ppm) combined with gentamicin is not effective.
The PE T performed on the diluent of Example 2 could not be completed as this diluent contained microbial contamination at O hrs., clearly demonstrating that Kathon CG is not an effective preservative.
Example 16
Accelerated stability stress testing.
As already stated, S-HRP and IgG-HRP stability is essential for a diluent in accordance with the invention.
Concurrent to the PETs being performed accelerated stress stability was performed on a selection of diluents. This testing provided the data required to select diluents that provide the required stability.
Conjugates were then prepared in a selection of the example diluents as follows: IgG-HRP conjugate was prepared at a 1/12,000 dilution of IgG HRP in the diluents of Examples 4-6, 8-10 and 12-14. A sample of each of the conjugates was stored at 2-8 C and 35-39 C for a period of 7days, to be used for the accelerated stress test.
S-HRP conjugate was prepared at a 1/200 dilution of S-HRP in the following example diluents, 4-6, 8-10 and 12-14. A sample of each of the conjugates was stored at 2-8 C and 35-39 C for a period of 7days, to be used for the accelerated stress test.
The results are shown in Tables 1 and 2.
Table l
Stress testing results for IgG-HRP conjugates % drop in Preservative Diluent Positive Control O.D. values activity (Example No.) 2-8 C stored 35-39 C stored conj ugate conj ugate 4 2.744 1.748 36 B 2.643 2.250 15 B & P 6 2.558 1.931 25 P & G 8 2.607 1.614 38 B & P 9 2.819 2.202 22 B & P 2.599 1.576 39 T & G 12 2.622 1.432 45 B 13 2.635 1.547 41 B 14 2.802 2.138 24 K Abbreviations: B for Bronidox L, P for ProClin 950, G for gentamicin, T for thiomersal, K for Kathon CG.
Table 2
Stress testing results for S-HRP conjugates Diluent % drop in Preservative Positive Control O.D. values activity (Example No.) 2-8 C stored 35-39 C stored conjugate conj ugate 4 2.047 1.383 32 B 1.901 1.441 24 B & P 6 3.175 2.101 34 P & G 8 1.463 0.821 44 B & P 9 3.060 2.331 24 B & P 3.213 1.899 41 T & G 12 1.491 0.747 50 B 13 2.627 1.529 42 B 14 2.302 1.906 17 K Abbreviations: B for Bronidox L, P for ProClin 950, G for gentamicin, 1 for thiomcrsal, K for Kathon CG.
The combined data from the PETs and the accelerated stress stability was analysed and a formulation containing an effective preservative and lo providing the greatest stability for conjugates was chosen.
A target for the percentage drop off in reactivity after 7 days stress was set at < 30%, with the lowest drop off in reactivity being the most desirable.
From Tables 1 and 2 it can be concluded that the diluents of Examples 5, 9, and 14 provide the greatest stability, however Kathon CG was used as the preservative in the diluent of Example 14. Kathon CG is not an effective preservative, if reference is made to the PET results for the diluent of Example 2. This excludes the diluent of Example 14. The diluents of Example 5 and 9 both use ProClin 950 (48ppm) and Bronidox L (0.02%) which as hereinabove described has been demonstrated to be an effective combination of preservatives, having regard to the PET results of Example 15. s
As the diluent of Example 5 provides the greatest stability in Table 1 and utilises Trizma instead of MOPS, which is a more expensive raw material as used in the preparation of the diluent of Example 9, the diluent of Example 5 is the diluent of choice.

Claims (9)

  1. Claims: 1. A stabilising diluent for a protein-horseradish peroxidase
    conjugate for use in an enzyme immunoassay for the detection of a human Parvovirus B19 antibody, comprises stabilising amounts of s bovine serum albumin (BSA), cytochrome c and gelatin hydrolysate, an antimicrobial agent consisting of 2-methyl-4-isothiazolin-3-one and 5- bromo-5-nitro-1, 3-dioxane, a non-ionic surfactant and a biological buffer, such that the diluent has a pH in the range 7.0 to 7.5.
  2. 2. A stabilising diluent according to Claim 1, wherein the biological buffer is tris(hydroxymethyl)aminomethane base.
  3. 3. A stabilising diluent according to Claim 1, wherein the biological buffer is a zwitterionic buffer.
  4. 4. A stabilising diluent according to Claim 3, wherein the zwitterionie buffer is morpholinepropanesulfonie acid (MOPS).
  5. 5. A stabilising diluent according to any preceding claim, wherein the non-ionic surfactant is selected from polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters, polyoxyethylene alcohols, polyoxyethylene isoaleohols, polyoxyethylene ethers, polyoxyethylene esters, polyoxyethylene-p-t-oetylphenols or octylphenyl-ethylene oxide condensates, ethylene oxide condensates with fatty alcohols, polyoxyethylene nonylphenols, and mixtures of polyalkylene glyeols or a mixture thereof.
  6. 6. A stabilising diluent according to any preceding claim, wherein the protein-horseradish peroxidase conjugate is streptavidin- horseradish peroxidase.
  7. 7. A stabilising diluent according to any one of Claims 1-5, wherein the protein-horseradish peroxidase conjugate is anti-human IgG horseradish peroxidase.
  8. 8. A stabilising diluent according to any preceding claim, wherein the protein-horseradish peroxidase conjugate retains at least 70% reactivity following accelerated stress testing at a temperature in the range 35-39 C for a period of at least 4 days relative to a positive control at a temperature in the range 2-8 C.
  9. 9. A stabilising diluent according to Claim 1, substantially as hereinbefore described and exemplified.
GB0422081A 2004-10-04 2004-10-04 Stabilising diluent for a protein-horseradish peroxidase conjugate Expired - Lifetime GB2418665B8 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2466379A (en) * 2008-12-19 2010-06-23 Chisso Corp Protein stabilizers
CN105974121A (en) * 2016-04-18 2016-09-28 武汉云克隆科技股份有限公司 Biological product stabilizer containing fish gelatin

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117007791B (en) * 2023-07-18 2024-08-09 广州市进德生物科技有限公司 Horseradish peroxidase conjugate dilution preservation solution

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Publication number Priority date Publication date Assignee Title
US5460944A (en) * 1991-10-28 1995-10-24 Boehringer Mannheim Gmbh Storable protein solution
EP0997527A1 (en) * 1998-09-22 2000-05-03 Ortho-Clinical Diagnostics Stabilisation of peroxidases
GB2353797A (en) * 1999-09-01 2001-03-07 Ortho Clinical Diagnostics Stabilisation of peroxidases
US6383766B1 (en) * 1998-10-02 2002-05-07 Ortho-Clinical Diagnostics, Inc. Reduced cortisol conjugates

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5460944A (en) * 1991-10-28 1995-10-24 Boehringer Mannheim Gmbh Storable protein solution
EP0997527A1 (en) * 1998-09-22 2000-05-03 Ortho-Clinical Diagnostics Stabilisation of peroxidases
US6383766B1 (en) * 1998-10-02 2002-05-07 Ortho-Clinical Diagnostics, Inc. Reduced cortisol conjugates
GB2353797A (en) * 1999-09-01 2001-03-07 Ortho Clinical Diagnostics Stabilisation of peroxidases

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2466379A (en) * 2008-12-19 2010-06-23 Chisso Corp Protein stabilizers
GB2466379B (en) * 2008-12-19 2013-07-31 Jnc Corp Protein stabilizer
CN105974121A (en) * 2016-04-18 2016-09-28 武汉云克隆科技股份有限公司 Biological product stabilizer containing fish gelatin
CN105974121B (en) * 2016-04-18 2018-02-06 武汉云克隆科技股份有限公司 A kind of biological products stabilizer containing isinglass

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