GB2414239A - Diluent for enzyme immunoassay - Google Patents

Diluent for enzyme immunoassay Download PDF

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GB2414239A
GB2414239A GB0411389A GB0411389A GB2414239A GB 2414239 A GB2414239 A GB 2414239A GB 0411389 A GB0411389 A GB 0411389A GB 0411389 A GB0411389 A GB 0411389A GB 2414239 A GB2414239 A GB 2414239A
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diluent
conjugate
polyoxyethylene
process according
enzyme
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GB0411389D0 (en
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John Martin Doyle
Cormac Gerard Kilty
Joseph Bernard Rice
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Biotrin Intellectual Properties Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus

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  • Wood Science & Technology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

A process for preparing a liquid stable, ready-to-use diluent for a protein-enzyme conjugate for use in an enzyme immunoassay for the detection of a human Parvovirus B19 antibody comprises mixing a stabilising amount of cytochrome C and a stabilising amount of bovine serum albumin, a non-ionic surfactant, a preservative and a buffer such that the diluent has a pH in the range 6.4 - 7.4 and equilibrating the diluent for a period of at least 13 days prior adding to the protein-enzyme conjugate thereto. It is found that if a "maturation period" for the diluent of at least 13 days is not allowed for equilibration, then there is an almost complete loss of the activity when the material is stored at elevated temperature.

Description

1 2414239 Process for preparing a liquid stable, ready-to-use diluent for
a protein enzyme conjugate This invention relates to diluents for proteinenzyme conjugates for use in enzyme immunoassays (EIAs) and, in particular, to a process for preparing such a diluent.
Diagnostic EIA kits are available for carrying out testing for Parvovirus B 19 IgG and IgM antibodies. These kits contain liquid stable, ready-touse conjugates, namely: streptavidin-horseradish peroxidase (S-HRP) in the Parvo B 19 IgM EIA and anti-human IgG- lo horseradish peroxidase (IgG-HRP) in the Parvo B19 IgG EIA. The conjugates are prepared by adding the S-HPR or IgG-HRP protein conjugates (bought commercially) to a medium which is the subject of Irish Patent No. S62 196 in the name of Biotrin Intellectual Properties Limited.
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 2-8OC) 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 % drop-off in activity is calculated. A low % drop off is usually indicative of long-term stability of a given component at the reference temperature.
Increased stability or, indeed, maintenance of existing stability of biological agents and reagents is an on-going aim and, indeed, requirement for those involved in the research and development of such agents and reagents, especially as the nature and source of such materials can vary with time and as a result of which manufacturers are required to modify the materials used in the kits for carrying out existing or new diagnostics assays.
During on-going process and product development for new and improved kits for parvovirus B 19 IgM and IgG antibodies an unexpected lo phenomenon was observed. As a result of this it was found necessary to develop a modified process for the manufacture of diluents for the protein-enzyme conjugates for use in such assays. When both S-HRP and IgG-HRP conjugate solutions were made up in freshly prepared conjugate diluent, the conjugates "crashed" in that there was an almost complete loss of activity in the material stored at elevated temperature.
The present invention overcomes this unexpected phenomenon.
The invention provides a process for preparing a liquid stable, ready-touse diluent for a protein-enzyme conjugate for use in an enzyme immunoassay for the detection of a human Parvovirus B 19 antibody, which process comprises mixing a stabilizing amount of cytochrome C and a stabilizing amount of bovine serum albumin, a non- ionic surfactant, a preservative and a buffer such that the diluent has a pH in the range 6.4 - 7.4 and equilibrating the diluent for a period of at least 13 days prior adding to the protein-enzyme conjugate thereto.
Thus we discovered that when the conjugate solutions used herein are prepared in a diluent that is at least 13 days old, the conjugates performed well on stress and the % drop-off in activity was acceptable, otherwise as indicated above, there is an almost complete loss of activity in the material stored at elevated temperature.
Thus, the invention is concerned with the "maturation period" of the Parvo conjugate diluent used in the preparation of conjugates in the Parvo IgG and IgM EIAs. The process of conjugate preparation requires that the diluent is matured for a period of at least 13 days for lo equilibration prior to the addition of the S-HRP or the IgG-HRP, as appropriate. If this equilibration period is omitted, the conjugate will crash on stress. Typically a maturation period of two weeks is adopted.
Preferably, the enzyme in the protein-enzyme conjugate is a peroxidase.
Further, preferably, the peroxidase is horseradish peroxidase.
According to a first embodiment of the invention, the protein- enzyme conjugate is streptavidin-horseradish peroxidase.
According to a second embodiment of the invention, the protein- enzyme conjugate is anti-human IgG - horseradish peroxidase.
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--t-octylphenols or octylphenylethylene oxide condensates, ethylene oxide condensates with fatty alcohols, polyoxyethylene nonylphenols, and mixtures of polyalkylene glycols or a mixture thereof.
Further, preferably, the protein-enzyme conjugate retains at least 60%, especially at least 70% and most especially at least 80%, reactivity following accelerated stress testing at a temperature in the range 3539 C for a period of at least 4 days relative to a positive control at a temperature in the range 2-8 C.
The invention will be further illustrated by the following
lo Examples.
In the following Examples, the following preferred conjugate diluent formulation is used: Material Quantity Trizma Base 6.05g Cytochrome C 0. 25g Tween 20 0.55g Triton X-100 5.3g BSA 1 O.Og Thiomersal 0.67g to HCI Variable Aprotinin 2mg Gentamicin 0.1 Og Deionised water up to 1L The diluent is prepared by placing 800mL of deionised water in a container. 6.05g of Trizma (Sigma Chemical Company) is added and dissolved. The solution is then titrated to 7.2+/-0.05 with HCI (BDH Ltd.) . 0.25g of cytochrome C (Merck Ltd.) is added and dissolved.
Tween 20 (Tween 20 is a trade mark) (Merck Ltd.), Triton X-100 (Triton X100 is a trade mark) (BDH Ltd.), BSA (Bovine Serum Albumin) (Sigma Chemical Company), Thiomersal (BDH Ltd.), aprotinin (Sigma Chemical Company) and gentamicin (Sigma Chemical Company) (volumes as above) are added and mixed well. The batch is then brought up to 1L with deionised water and mixed well.
The pH is then checked and adjusted, if required, to 7.2 with 5M HCl or 5M NaOH. The diluent is then filtered through a 0.22pm filter into an aluminium wrapped container.
The diluent is then stress-tested in the following manner: 0 Stress Test Method A number of preparations of Parvo conjugate diluent (A1 - A5, and PA1501) were prepared according to the formulation and methodology described above.
Diluents A1 and PA501 were prepared using raw materials of the same lot number as obtained from a given manufacturer, but PA105 was prepared 2 weeks before diluents A1 - A5.
Diluents A2 - A5 were prepared using raw materials of the same lot number as used for PA50 1 and A l with the exception of one raw material lot number change per diluent, as follows: A2 as per PA50 l with different lot of Triton X- 100 AS as per PA50 l with different lot of Thiomersal A4 as per PA50 1 with different lot of Trizma AS as per PA50 1 with different lot of Tween 20 Streptavidin-HRP was added to each of the diluents at a dilution of 1/100.
Anti-human IgG-HRP was also prepared in each of the diluents at a dilution of 1/4,500 A sample of each conjugate in each diluent was stored at 2-8 C and at 35-39 C, respectively.
The percentage drop in reactivity in each diluent was measured lo after 4 days storage.
Example 1
TABLE 1
S-HRP conjugates Conjugate Positive Control O.D. value % drop in Age of diluent prior I.D. reactivity to conjugate addition 2-8 C stored 35-39 C stored Al 1.810 0.841 54 1 day A2 1.730 0.620 65 1 day AS 1.535 0.234 85 1 day A4 1.690 0.568 66 1 day A5 1.573 1.332 15 1 day PAI501 2.127 1.722 > 2 months s The percentage drop in reactivity was measured after 4 days storage.
TABLE 2
Anti-human IgG-HRP conjugates Conjugate Positive Control O.D. value % drop in Age of diluent I.D. reactivity prior to 2-8 C stored 35-39 C stored conjugate addition Al 2.087 0.875 58 1 day
_
A2 2.133 0.909 57 1 day AS 2.091 0.832 60 1 day A4 1.973 1.061 46 1 day A5 1.997 0.952 52 1 day PA1501 1.447 0.959 34 2 months The percentage drop in reactivity was measured after 6 days storage.
The S-HPR in diluent AS appears to retain a significant level of reactivity after the stress test. A similar observation is not seen with the IgG-HRP. However diluents Al to A4 all show a significant % drop-off with both conjugates. It is notable that diluent PA1501 appears to confer lo a greater degree of stability on both S-HRP and IgG-HRP conjugate than the other diluents. It is significant that diluent PA1501 while prepared from the same raw materials as Al is approximately 2 months older.
It appears therefore that no particular raw material is responsible for the significant loss of activity of the conjugate on stress. /
Example 2
Additional conjugate preparations in diluents Al, A3 and PA105 were made 13 days after the diluent has been prepared. S-HRP was used at a dilution of l/100 as used previously.
The conjugates were then subjected to the 2-8 C and 35-39 C stress test.
TABLE 3
S-HRP conjugates Conjugate Positive Control O.D. value % drop in Age of diluent prior I.D. reactivity to conjugate addition 2-8 C stored 35-39 C stored A1 2.008 1.852 8 13 days A3 2.104 1.861 12 13 days PA1501 1.973 1.716 _ >2 months lo The percentage drop in reactivity was measured after 5 days storage.
These results show a signification retention of activity in diluents A l and A3 after stress. This compares to drop-off values of 54 and 85%, respectively as compared to Table 1. Therefore, there appears to be a significant improvement in the stability achieved when the diluents were stored for a period of time.
It appears therefore that the age of the diluent prior to addition of the conjugate is critical to the performance of the conjugate on stress.
Example 3
2 additional Parvo conjugate diluents (TC2A and TC2B) were s prepared as described above with the exception that one (TC2B) was prepared without thiomersal.
S-HRP and IgG-HRP conjugates were prepared in both diluents at 1/100 and 1/4,500 dilutions, respectively. The conjugate solutions were prepared after the following time intervals: lo 1 hour post diluent preparation 1 day post diluent preparation 7 days post diluent preparation 14 days post diluent preparation Each of the conjugates was then subjected to a 7 day stress test.
TABLE 4
Results of 7 day stress for S-HRP conjugate in diluents with and without thiomersal Time of S- Diluent O.D. for Positive Control % drop off HRP addition 2-8 C stored 35-39 C stored in reactivity after diluent preparation 1 hour TC2A 1.163 0.011 99 TC2B 1.675 1.140 31 1 day TC2A 1.475 1.102 24 TC2B 1.767 1.322 25 7 days TC2A 1.572 1.080 31 TC2B 1.693 1.444 14 14 days TC2A 1.712 1.231 28 TC2B 1.755 1.461 17 It appears from these results that addition of the S-HRP conjugate protein within 1 hour after diluent preparation causes an almost complete loss of activity when the conjugate is stressed. While some activity is lost in the other diluents, a significant level is retained.
The presence of thiomersal appears to have an effect on the lo conjugate prepared within 1 hour, suggesting that this component may be responsible for the crashing on stress. The thiomersal effect is less marked in the other diluents.
TABLE 5
Results of 7 day stress for Anti-human IgG-HRP conjugate
_
Time of IgG- Diluent O.D. for Positive Control % drop off HRP addition 28 C stored 35-39 C stored in reactivity after diluent preparation 1 hour TC2A 1.575 1.378 26 TC2B 1.842 1.415 23
_
1 day TC2A 1.676 0.952 43 TC2B 1.862 1.504 19 7 days TC2A 1.821 1.257 31 TC2B 1.838 1.441 21 14 days TC2A 1.844 1.410 24 TC2B 1.877 1.557 17 It appears from these results that the age of the diluent is not as critical a factor in the stability of the IgG-HRP conjugate on stress.
As with S-HRP, the presence/absence of thiomersal does appear to have some effect on the level of drop-off after stress, particularly as the diluents age.
Thiomersal is a critical component in the diluent as it acts as a lo preservative and it must therefore be retained in the diluent.
Example 4
A range of Parvo conjugate diluents which previously gave rise to unacceptable stress results were re-assessed by preparing fresh S-HRP conjugates at various times after diluent manufacture (i.e. the diluents had been stored for a number of weeks prior to addition of the S-HRP).
Each of these diluents was at least 2 weeks old when re-tested.
The drop-off in activity was calculated and compared to the drop- off obtained when conjugates were freshly prepared in each diluent.
The following diluents were re-tested: lo Al, A2, As, A4: B2, B3, PA1675, Trial BSA-1 and Trial BSA-2 Note: All of these diluents were made up to the same formulation and has been stored for different periods of time prior to addition of the S-HRP protein conjugate.
The results are shown in Table 6.
TABLE 6
Diluent lot Time of S-HRP addition post Stress result Number of No. diluent manufacture drop in days on stress _ reactivity A1 _ 1 day 54% 4 Al 13 days 8% 5 A2 1 1 day 1 65% 14 A2 1 8 weeks 1 27% 16 AS 1 1 day 1 85% 14 A3 13 days 12% 5 A4 1 1 day 1 66% 14 A4 1 8 weeks 1 33% 16 B2 | O days (Same day addition) | 58% |2 B2 1 7 weeks 1 32% 16 B3 | O days (same day addition) | 75% |_ 4 B3 1 7weeks 1 29% 16 PA1675 1 2 days 1 98% 16 PA1675 9 days 43% 6 PAl675 14 days 12% 5
_
Trial BSA-1 O days (same day addition) 55% 5 Trial BSA-1 19 days _ 12% 6 TrialBSA-2 | O days (same day addition) | 69% | 5 TnalBSA-2 1 l9 days 1 32% 1 6 As can be seen from these results, the diluents all showed a lower % drop-off of activity when they had been stored for a period of time.
The minimum period appeared to be 13 days.

Claims (8)

  1. Claims: 1. A process for preparing a liquid stable, ready-to-use diluent
    for a protein-enzyme conjugate for use in an enzyme immunoassay for the detection of a human Parvovirus B 19 antibody, which process comprises mixing a stabilising amount of cytochrome C and a stabilising amount of bovine serum albumin, a non-ionic surfactant, a preservative and a buffer such that the diluent has a pH in the range 6.4 - 7.4 and equilibrating the diluent for a period of at least 13 days prior adding to the proteinenzyme conjugate "hereto.
    lo
  2. 2. A process according to Claim 1, wherein the enzyme in the proteinenzyme conjugate is a peroxidase.
  3. 3. A process according to Claim 2, wherein the peroxidase is horseradish peroxidase.
  4. 4. A process according to any preceding claim, wherein the protein-enzyme conjugate is streptavidin-horseradish peroxidase.
  5. 5. A process according to any one of Claims 1-3, wherein the proteinenzyme conjugate is anti-human IgG - horseradish peroxidase.
  6. 6. A process according to any preceding claim, 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--t-octylphenols or octylphenyl-ethylene oxide condensates, ethylene oxide condensates with fatty alcohols, polyoxyethylene nonylphenols, and mixtures of polyalkylene glycols or a mixture thereof.
  7. 7. A process according to any preceding claim, wherein the protein-enzyme conjugate retains at least 60% 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.
  8. 8. A process according to Claim 1 for preparing a liquid stable ready-touse diluent for a protein-enzyme conjugate for use in an lo enzyme immunoassay for the detection of a human Parvovirus B19 antigen or antibody, substantially as hereinbefore described and exemplified.
GB0411389A 2004-05-21 2004-05-21 Process for preparing a liquid stable, ready-to-use diluent for a protein-enzyme conjugate Expired - Lifetime GB2414239B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0456309A1 (en) * 1990-05-11 1991-11-13 Johnson & Johnson Clinical Diagnostics, Inc. Use of heme-containing proteins as stabilizers for enzyme-labeled immunoreactants
US5268299A (en) * 1988-04-14 1993-12-07 Eastman Kodak Company Diluent composition useful in the detection of antibodies in assays
WO1994010294A1 (en) * 1992-10-23 1994-05-11 New York University Human monoclonal antibodies to human parvovirus and methods of making and using thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5268299A (en) * 1988-04-14 1993-12-07 Eastman Kodak Company Diluent composition useful in the detection of antibodies in assays
EP0456309A1 (en) * 1990-05-11 1991-11-13 Johnson & Johnson Clinical Diagnostics, Inc. Use of heme-containing proteins as stabilizers for enzyme-labeled immunoreactants
WO1994010294A1 (en) * 1992-10-23 1994-05-11 New York University Human monoclonal antibodies to human parvovirus and methods of making and using thereof

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