CA1300057C - Lactoferrin-containing incubation medium for solid-phase immunometric methods and its use - Google Patents
Lactoferrin-containing incubation medium for solid-phase immunometric methods and its useInfo
- Publication number
- CA1300057C CA1300057C CA000551697A CA551697A CA1300057C CA 1300057 C CA1300057 C CA 1300057C CA 000551697 A CA000551697 A CA 000551697A CA 551697 A CA551697 A CA 551697A CA 1300057 C CA1300057 C CA 1300057C
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- Prior art keywords
- lactoferrin
- solid
- incubation medium
- phase
- phase immunometric
- Prior art date
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- Expired - Lifetime
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Abstract
Abstract of the disclosure Incubation media intended for solid-phase immunometric assays and containing lactoferrin are described. Compared with the media used hitherto, these incubation media have the advantage that they prevent, especially in the case where heated sample material is used in the assays, false results on determination of antigens or antibodies.
Description
~3(~00S~
~EHRING~ERKE Aktiengesellschaft 86/~ 038~- Ma 583 Dr. Ha/3n/Li.
A lactoferrin-containing incubation medium for solid-phase immunometric methods and its use The invention relates to a lactoferrin-containing incuba-tion ~edium for solid-phase immunometric assays and to the use of th~s medium.
For solid-phase immunometric assays, for example ELISA, the composition of the required incubation media t"buffer solutions", "incubation milieus") must be such that non-specific binding of concomitant substances in the sample to the solid phase is prevented. Additives known for this purpose are proteins such as albumin~ casein, mix~
tures of proteins such as animal sera, hydrolyzed gelatin or its derivatives, as ~ell as surfactants.
Incubation media provided with such additives are unable to prevent measurements being false, ~hich particularly occurs ~hen samples have been heated. Heating is expe-dient to reduce the infectiosity of the samples, ~hich may derive from, for example, HIV (human immunodeficiency virus~.
Hence the object was to find an incubation medium ~ith which there is no occurrence, ~hen it is used, of false values caused by heating of the sample.
:
It has been found, surprisingly, that an incubation medium ~hich contain~ lactoferrin achieves this object~
The invention relates to an incubation medium for solid-phase immunometric assays containing lactoferrin~
The invention also relates to the use of lactoferrin in an incubation medium ~hich is used for a solid-phase immunometrlc assay.
~30~
An incubation medium wi~hin the meaning of the invention is the liquid phase,in ~hich the immunochemical reaction takes place,of a solid-phase immunometric assay.
Lactof~rrin is added in a concentration o~ from O.OS to 20 g/l. A concentration of 0.15-0.25, in particular 0.2, g/l is preferred.
The lactoferrin can be added to incubation media or buffer solutions kno~n per se for solid-phase immunometric assays. One of these is, for example, the solution des-cribed in Çerman Patent A-27 448 36 on page ~4, ~hich, apart from buffer substance, contains 5 g/l bovine serum albumin, 5 g/l polyoxyethylene (20) sorbitan monolaurate ~R)Tween 20) in phosphate-buffered physiological saline solution (P9S). ~Another example which may be mentioned is the solution described in German Patent A-31 15 115 on page 10, a 0.2 mol/l NaH2P04/Na2HP04 buffer, pH 6.5, which contains 2 g/l bovine serum albu~in and 2~0 ml/l normal goat serum. A preferred buffer solution is used in examPle 1.
A med;um of this type can advantageously contain a prote;n kno~n per se, preferably casein, and/or a hydrolysis pro-duct of gelatin, or hydrolyzed and chemically treated gelatin.
The incubation medium of this invention can also advantageously 25 include a surfactant and/or a neutral salt.
The lactoferrin to be used according to the invention can be obtiined from the 0ilk of a mammal by removing ehe cream from the milk, adjusting the milk, fro0 ~hich the cream has been removed, to a p~ of 3.5-4.5, removing the protein precipitate ~hich has formed, and removing and isolating the lactoferrin from the supernatant by chroma-tography on an ion exchanger.
Lactoferrin is isolated from milk, preferably from cow's .
13~0 [3S7 milk. In a preferred process for obtaining a lactoferrin suitable for the purpose according to the ;nvention, the fat is removed from the milk by, for exampLe, centrifuging it. The upper of the t~o phases which form is removed and discarded~ The lower phase ;s adjusted to pH 3.5-4.5 with an ac;d. This results in a precip;tate wh;ch is sedjmented, for example by centrifugation, and is dis-carded. The supernatant is adjusted to 3 conductivity of 6-7 mS/cm by dilution with ~ater. Then a cation exchanger, for example CM-cellulose or SP-RSephadex, ~hich has been swollen and equilibrated with a buffer of oH 4-5.5 and a conductivity of, preferably, 6-7 mS/cm is introduced.
The suspension is filtered, and the residue ;s washed several t;mes with the same buffer, and is then suspended therein and packed into a column. It is eluted with a gradient of, preferably, 0-1 mol/l NaCl at pH 5. The fraction which is eluted in the range 500-650 mmol/l NaCl as a homogeneous peak measured at 280 nm contains the lactoferrin which is particularly suitable for the incu-bation medium. On SDS-PAGE electrophoresis, this lacto-ferrin appears as a band with a molecular ~eight of 80,000 ~ 3,000.
On chromatography on an anion exchanger, for example on DEAE- or QAE-RSephadex, or on DEAE-cellulose, the super-natant from the prec;p;tation at pH 3.5-4.5 which has been descr;bed above ;s adjusted to pH 6.5-7.5~ and a conduc-tiv;ty of 6-8 mStcm, and ;s appl;ed to 3 column packed with the ;on exchanger which has previously been equ;l;-brated ~ith a buffer of pH 6.5-7.5 and a conduct;v;ty of 6-8 mS/cm. The lactoferrin then passes through the column or ;s eluted as the f;rst Peak on elution ~;th an NaCl grad;ent.
The lactoferrin must be present on incubation of the sam-ple or of the analyte w;th the reactants wh;ch are bound to the sol;d phase. It can be added to the solut;on which contains the analyte or to the solution or the buffer ~3~057 ~ith ~hich the analyte is diluted. However, it can also be contacted in dissolved form with the reactant which is bound to the solid phase and/or dried onto the solid phase before this is contacted with the analyte.
s Suitable reactants which are bound to solid phases are the components of a bioaffinity binding system wh;ch are able to bind an analyte of this typeO In ~he case of an i~munDlogical binding system, these reactants are either antigens or antibodies. Solid-phase immunometric assays can be both competitive and two- side immunometric (sand-wich) assays or even indirect assays. In the latter case the analyte is an antibody, and the solid-phase reactant is the corresponding antigen, and the amount of antibody which is bound to the solid phase is determined by a second, labeled antibody.
The assay system can be a single-step or multistep assay.
The lactoferrin prevents the b;nding of those constituents of the sampLe which do not belong to the particular bio-aftinity binding system. It thus averts false results in the detection and determination of the analyte.
The examples which follow illustrate the invention and demonstrate the appropriateness of lactoferrin as a com-ponent of the incubation medium.
The examples are intended to show that Lactoferr;n can be used in a variety of incubation media, introduced in various amounts.
Example 1 .
1. Isolation of lactoferrin Z0 l of cow's milk were centrifuged at 3400 x 9 for 30 m;n, and the milk fat ~as removed in the ~3~0~ii7 supernatant. Small amounts of insoluble constituents ~ere removed as sediment and likewise discarded.
The pH of the milk from which the fat had been removed was adjusted to pH 4.0 by addition of hydrochLoric acid. The protein precipitate ~hich ~as produced by this ~as removed by centrifugation at 3400 x 9 The supernatant, which had, after adj~ustment to pH 5.Q
with sodium hydroxide solution, a conductivity of 12 mS/cm at 20C, was diluted to 7 mS/c~ with water and then mixed with 200 ml of the swollen ion exchanger carboxymethylcellulose CM 32 supplied by What~ann, and the mixture was stirred at room temperature for 4 h.
The ;on exchanger had previously been equilibrated ~ith 50 mmol/l sodium acetate buffer, pH 5Ø The ion exchanger was then ren~oved by filtration on a suction funnel and suspended ;n the sodium acetate buffer and transferred into a chromatography column. The column was then washed with 400 ml o~ the acetate buffer pre-viously described. The proteins bound to the cation exchanger were eluted with a solution o~ 1 mol/l sodium chloride.
The eluate was adjusted to pH 5.0 ~ith 1-normal sodium hydroxide solution and was converted by dialysis into 50 mmol/l sodium acetate, pH 5Ø The prote;n m;x-ture was then rechromatographed on 200 ml of carboxy-methylcellulose CM 32. The prote;ns ~ere eluted w;th a linear gradient from 50 mmol/l sodium acetate, pH 5, to 50 mmol/l sodium acetate, pH 5, and l mol/l sodium chloride.
The 4th peak of the elution d;agram, whose solut;on had a conduct;vity of 18-20 mS/cm, was collec~ed as one fraction, concentrated by ultraf;ltration and sub-jected to gel f;ltration on RSepharose AcA 34 wh;ch had been egu;librated ~ith PBS containing O.S g/l sod;um azide. The main component from the gel filtration, ~3~
which amounted to 1.2 9 of protein, contained the lactoferrin.
~EHRING~ERKE Aktiengesellschaft 86/~ 038~- Ma 583 Dr. Ha/3n/Li.
A lactoferrin-containing incubation medium for solid-phase immunometric methods and its use The invention relates to a lactoferrin-containing incuba-tion ~edium for solid-phase immunometric assays and to the use of th~s medium.
For solid-phase immunometric assays, for example ELISA, the composition of the required incubation media t"buffer solutions", "incubation milieus") must be such that non-specific binding of concomitant substances in the sample to the solid phase is prevented. Additives known for this purpose are proteins such as albumin~ casein, mix~
tures of proteins such as animal sera, hydrolyzed gelatin or its derivatives, as ~ell as surfactants.
Incubation media provided with such additives are unable to prevent measurements being false, ~hich particularly occurs ~hen samples have been heated. Heating is expe-dient to reduce the infectiosity of the samples, ~hich may derive from, for example, HIV (human immunodeficiency virus~.
Hence the object was to find an incubation medium ~ith which there is no occurrence, ~hen it is used, of false values caused by heating of the sample.
:
It has been found, surprisingly, that an incubation medium ~hich contain~ lactoferrin achieves this object~
The invention relates to an incubation medium for solid-phase immunometric assays containing lactoferrin~
The invention also relates to the use of lactoferrin in an incubation medium ~hich is used for a solid-phase immunometrlc assay.
~30~
An incubation medium wi~hin the meaning of the invention is the liquid phase,in ~hich the immunochemical reaction takes place,of a solid-phase immunometric assay.
Lactof~rrin is added in a concentration o~ from O.OS to 20 g/l. A concentration of 0.15-0.25, in particular 0.2, g/l is preferred.
The lactoferrin can be added to incubation media or buffer solutions kno~n per se for solid-phase immunometric assays. One of these is, for example, the solution des-cribed in Çerman Patent A-27 448 36 on page ~4, ~hich, apart from buffer substance, contains 5 g/l bovine serum albumin, 5 g/l polyoxyethylene (20) sorbitan monolaurate ~R)Tween 20) in phosphate-buffered physiological saline solution (P9S). ~Another example which may be mentioned is the solution described in German Patent A-31 15 115 on page 10, a 0.2 mol/l NaH2P04/Na2HP04 buffer, pH 6.5, which contains 2 g/l bovine serum albu~in and 2~0 ml/l normal goat serum. A preferred buffer solution is used in examPle 1.
A med;um of this type can advantageously contain a prote;n kno~n per se, preferably casein, and/or a hydrolysis pro-duct of gelatin, or hydrolyzed and chemically treated gelatin.
The incubation medium of this invention can also advantageously 25 include a surfactant and/or a neutral salt.
The lactoferrin to be used according to the invention can be obtiined from the 0ilk of a mammal by removing ehe cream from the milk, adjusting the milk, fro0 ~hich the cream has been removed, to a p~ of 3.5-4.5, removing the protein precipitate ~hich has formed, and removing and isolating the lactoferrin from the supernatant by chroma-tography on an ion exchanger.
Lactoferrin is isolated from milk, preferably from cow's .
13~0 [3S7 milk. In a preferred process for obtaining a lactoferrin suitable for the purpose according to the ;nvention, the fat is removed from the milk by, for exampLe, centrifuging it. The upper of the t~o phases which form is removed and discarded~ The lower phase ;s adjusted to pH 3.5-4.5 with an ac;d. This results in a precip;tate wh;ch is sedjmented, for example by centrifugation, and is dis-carded. The supernatant is adjusted to 3 conductivity of 6-7 mS/cm by dilution with ~ater. Then a cation exchanger, for example CM-cellulose or SP-RSephadex, ~hich has been swollen and equilibrated with a buffer of oH 4-5.5 and a conductivity of, preferably, 6-7 mS/cm is introduced.
The suspension is filtered, and the residue ;s washed several t;mes with the same buffer, and is then suspended therein and packed into a column. It is eluted with a gradient of, preferably, 0-1 mol/l NaCl at pH 5. The fraction which is eluted in the range 500-650 mmol/l NaCl as a homogeneous peak measured at 280 nm contains the lactoferrin which is particularly suitable for the incu-bation medium. On SDS-PAGE electrophoresis, this lacto-ferrin appears as a band with a molecular ~eight of 80,000 ~ 3,000.
On chromatography on an anion exchanger, for example on DEAE- or QAE-RSephadex, or on DEAE-cellulose, the super-natant from the prec;p;tation at pH 3.5-4.5 which has been descr;bed above ;s adjusted to pH 6.5-7.5~ and a conduc-tiv;ty of 6-8 mStcm, and ;s appl;ed to 3 column packed with the ;on exchanger which has previously been equ;l;-brated ~ith a buffer of pH 6.5-7.5 and a conduct;v;ty of 6-8 mS/cm. The lactoferrin then passes through the column or ;s eluted as the f;rst Peak on elution ~;th an NaCl grad;ent.
The lactoferrin must be present on incubation of the sam-ple or of the analyte w;th the reactants wh;ch are bound to the sol;d phase. It can be added to the solut;on which contains the analyte or to the solution or the buffer ~3~057 ~ith ~hich the analyte is diluted. However, it can also be contacted in dissolved form with the reactant which is bound to the solid phase and/or dried onto the solid phase before this is contacted with the analyte.
s Suitable reactants which are bound to solid phases are the components of a bioaffinity binding system wh;ch are able to bind an analyte of this typeO In ~he case of an i~munDlogical binding system, these reactants are either antigens or antibodies. Solid-phase immunometric assays can be both competitive and two- side immunometric (sand-wich) assays or even indirect assays. In the latter case the analyte is an antibody, and the solid-phase reactant is the corresponding antigen, and the amount of antibody which is bound to the solid phase is determined by a second, labeled antibody.
The assay system can be a single-step or multistep assay.
The lactoferrin prevents the b;nding of those constituents of the sampLe which do not belong to the particular bio-aftinity binding system. It thus averts false results in the detection and determination of the analyte.
The examples which follow illustrate the invention and demonstrate the appropriateness of lactoferrin as a com-ponent of the incubation medium.
The examples are intended to show that Lactoferr;n can be used in a variety of incubation media, introduced in various amounts.
Example 1 .
1. Isolation of lactoferrin Z0 l of cow's milk were centrifuged at 3400 x 9 for 30 m;n, and the milk fat ~as removed in the ~3~0~ii7 supernatant. Small amounts of insoluble constituents ~ere removed as sediment and likewise discarded.
The pH of the milk from which the fat had been removed was adjusted to pH 4.0 by addition of hydrochLoric acid. The protein precipitate ~hich ~as produced by this ~as removed by centrifugation at 3400 x 9 The supernatant, which had, after adj~ustment to pH 5.Q
with sodium hydroxide solution, a conductivity of 12 mS/cm at 20C, was diluted to 7 mS/c~ with water and then mixed with 200 ml of the swollen ion exchanger carboxymethylcellulose CM 32 supplied by What~ann, and the mixture was stirred at room temperature for 4 h.
The ;on exchanger had previously been equilibrated ~ith 50 mmol/l sodium acetate buffer, pH 5Ø The ion exchanger was then ren~oved by filtration on a suction funnel and suspended ;n the sodium acetate buffer and transferred into a chromatography column. The column was then washed with 400 ml o~ the acetate buffer pre-viously described. The proteins bound to the cation exchanger were eluted with a solution o~ 1 mol/l sodium chloride.
The eluate was adjusted to pH 5.0 ~ith 1-normal sodium hydroxide solution and was converted by dialysis into 50 mmol/l sodium acetate, pH 5Ø The prote;n m;x-ture was then rechromatographed on 200 ml of carboxy-methylcellulose CM 32. The prote;ns ~ere eluted w;th a linear gradient from 50 mmol/l sodium acetate, pH 5, to 50 mmol/l sodium acetate, pH 5, and l mol/l sodium chloride.
The 4th peak of the elution d;agram, whose solut;on had a conduct;vity of 18-20 mS/cm, was collec~ed as one fraction, concentrated by ultraf;ltration and sub-jected to gel f;ltration on RSepharose AcA 34 wh;ch had been egu;librated ~ith PBS containing O.S g/l sod;um azide. The main component from the gel filtration, ~3~
which amounted to 1.2 9 of protein, contained the lactoferrin.
2. Comparison of sample dilution buffer containing lacto-ferrin with sample dilution buffer without addition The EnzygnostR anti HSV ELISA kit of ~ehringwerke was used for the solid-phase immunometric method. The procedure described in the pack insert was followed for carrying out the assay, and it is described briefly below.
The samples ~ere diluted in the ratio 1:25, instead of 1:44 as recommended, with sample dilution butfer (137 mmol/l NaCl, 7.247 mmol/l Na2HP04.2H20 and 2.7~0 mmol/l KH2P04 with the addition of 2.7 mmol/l KCl, 0.4 mmol/l MgCl2, 3 mmol/l NaN3, 10 mltl fetal calf serum and 40 ml/l (~)Tween 20) without or with the addition of 0.2 g/l lactoferrin, and were incubated with the antigens immobilized in the wells of a micro-assay plate, the unbound ant;bodies were vashed out, an enzyme was bound, using a conjugate solution, to the ant;body/antigen complex ~hich had formed, the excess conjugate solution was washed out, a chromogenic substrate solution was used to develop a color via ~he enzyme, and then the reaction was stopped ~ith a stop solution. The samples which had thus been prepared and had developed a color depending on the content of the virus-specific antibodies were then evaLuated by determination of their extinction tE) at 405 nm by photometry.
One portion of each of 9 subjects' sera was heated at 56C for 30 min. The other portion remained un-treated.
It was found that, in particular, in the case of heated samples ;n dilution buffer without lactoferrin the extinctions measured in the control wells were very 130~ 5~
high, but the extinctions in the antigen-coated wells ~ere also raised. This resulted ;n 5 false-negative results. The control wells are treated ("coated") with the preparation of non-virus-infected cells ~hich are used for obtaining the viruses. When ~he 9 heated samples were assayed using the diLution buffer con-taining ~actoferrin, no false-negative value was found.
The unhea~ed samples ~ere tested using the dilution buffer containing no lactoferrin. The results served to check the results with the heated sera.
Exam The effect of adding lactoferrin was tested in a single-step sandwich assay. ~hen, in an assay of this type for H3sAg, 7.5 g/l lactoferrin were added, all of 14 unheated critical samples were correctly found to be ne~ative, whereas all had been false-positive determinations with-out addit;on of lactoferrin.
~0 Example 3 The effect of addition of lactoferrin was tested in a three-step sandwich assay for detecting influenza A anti-gen. Lactoferrin was used in the second and third stepsof this sol;d-phase immunochemical method.
When, in these two steps, first an antibody/biotin con-jugate diluted for use and then a streptavidin/POD con-jugate d;luted for use and conta;ning lactoferrin (~ore~han 0005 g/l) were incubated, it was poscible to reduce the non-specific reactions, in terms of background and influenza B foreign ant;gen, by more than 80~. An ELISA
result ~easured at 450 nm is given here as representative:
Non-specific binding ~ithout lactoferrin = 2.892 U.
Non-specific binding with lactoferrin = O.Z31 U.
The samples ~ere diluted in the ratio 1:25, instead of 1:44 as recommended, with sample dilution butfer (137 mmol/l NaCl, 7.247 mmol/l Na2HP04.2H20 and 2.7~0 mmol/l KH2P04 with the addition of 2.7 mmol/l KCl, 0.4 mmol/l MgCl2, 3 mmol/l NaN3, 10 mltl fetal calf serum and 40 ml/l (~)Tween 20) without or with the addition of 0.2 g/l lactoferrin, and were incubated with the antigens immobilized in the wells of a micro-assay plate, the unbound ant;bodies were vashed out, an enzyme was bound, using a conjugate solution, to the ant;body/antigen complex ~hich had formed, the excess conjugate solution was washed out, a chromogenic substrate solution was used to develop a color via ~he enzyme, and then the reaction was stopped ~ith a stop solution. The samples which had thus been prepared and had developed a color depending on the content of the virus-specific antibodies were then evaLuated by determination of their extinction tE) at 405 nm by photometry.
One portion of each of 9 subjects' sera was heated at 56C for 30 min. The other portion remained un-treated.
It was found that, in particular, in the case of heated samples ;n dilution buffer without lactoferrin the extinctions measured in the control wells were very 130~ 5~
high, but the extinctions in the antigen-coated wells ~ere also raised. This resulted ;n 5 false-negative results. The control wells are treated ("coated") with the preparation of non-virus-infected cells ~hich are used for obtaining the viruses. When ~he 9 heated samples were assayed using the diLution buffer con-taining ~actoferrin, no false-negative value was found.
The unhea~ed samples ~ere tested using the dilution buffer containing no lactoferrin. The results served to check the results with the heated sera.
Exam The effect of adding lactoferrin was tested in a single-step sandwich assay. ~hen, in an assay of this type for H3sAg, 7.5 g/l lactoferrin were added, all of 14 unheated critical samples were correctly found to be ne~ative, whereas all had been false-positive determinations with-out addit;on of lactoferrin.
~0 Example 3 The effect of addition of lactoferrin was tested in a three-step sandwich assay for detecting influenza A anti-gen. Lactoferrin was used in the second and third stepsof this sol;d-phase immunochemical method.
When, in these two steps, first an antibody/biotin con-jugate diluted for use and then a streptavidin/POD con-jugate d;luted for use and conta;ning lactoferrin (~ore~han 0005 g/l) were incubated, it was poscible to reduce the non-specific reactions, in terms of background and influenza B foreign ant;gen, by more than 80~. An ELISA
result ~easured at 450 nm is given here as representative:
Non-specific binding ~ithout lactoferrin = 2.892 U.
Non-specific binding with lactoferrin = O.Z31 U.
Claims (10)
1. An incubation medium for solid-phase immunometric assays containing lactoferrin.
2. An incubation medium as claimed in claim 1, wherein the lactoferrin is present in the concentration range 0.05-20 g/l.
3. An incubation medium as claimed in claim 1, which contains lactoferrin which can be obtained from milk of a mammal by a) removing the cream from the milk, b) adjusting the milk, from which the cream has been removed, to a pH of 3.5-4.5, c) removing the protein precipitate which has been produced in (b), and d) removing and isolating the lactoferrin from the supernatant produced in (c) by chromatography on an ion exchanger.
4. An incubation medium for solid-phase immunometric assays containing a buffer salt, lactoferrin and, which may also contain one or more of, a protein, a surfactant and, a neutral salt.
5. An incubation medium for solid-phase immunometric assays containing a buffer salt, lactoferrin, bovine serum albumin, and a surfactant and which may also contain a neutral salt.
6. An incubation medium for solid-phase immunometric assays containing a buffer salt, lactoferrin, a gelatin hydrolysis product or hydrolyzed and chemically treated, or chemically treated and hydrolyzed, gelatin, and a surfactant and, which may also contain a neutral salt.
7. The use of lactoferrin in a solid-phase immunometric assay.
8. The use of an incubation medium as claimed in claim 1 in solid-Phase immunometric assays for the detection and determination of an antibody in a biological fluid which has been heated.
9. The use of an incubation medium as claimed in claim 1 in solid-phase immunometric assays for the detection and determination of an antigen in a biological fluid which has been heated.
10. The use of an incubation medium as claimed in claim 1 in solid-phase immunometric assays for the detection and determination of an antibody or an antigen in a biological fluid which contains aggregated constitu-ents in dissolved form.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19863638767 DE3638767A1 (en) | 1986-11-13 | 1986-11-13 | INCUBATION MEDIUM CONTAINING LACTOTRINE FOR SOLID-PHASE IMMUNOMETRIC METHOD AND ITS USE |
| DEP3638767.3 | 1986-11-13 | ||
| EP87105621.4 | 1987-04-15 | ||
| EP87105621A EP0270729B1 (en) | 1986-11-13 | 1987-04-15 | Incubation medium for solid fase immunometric methods and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1300057C true CA1300057C (en) | 1992-05-05 |
Family
ID=25849347
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000551697A Expired - Lifetime CA1300057C (en) | 1986-11-13 | 1987-11-12 | Lactoferrin-containing incubation medium for solid-phase immunometric methods and its use |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP2714572B2 (en) |
| AU (1) | AU616869B2 (en) |
| CA (1) | CA1300057C (en) |
| MX (1) | MX172176B (en) |
| NO (1) | NO169980C (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5642142A (en) * | 1979-08-31 | 1981-04-20 | Amano Pharmaceut Co Ltd | Removing method for nonspecific reaction inhibiting action in immunity measuring method |
| EP0210684A1 (en) * | 1985-07-31 | 1987-02-04 | MALLINCKRODT, INC.(a Missouri corporation) | Composition comprising a radioactive metal compound for radioassaying |
| JPS6234059A (en) * | 1985-08-07 | 1987-02-14 | Sankyo Co Ltd | Stabilizer for solid phase reaction reagent |
| JPS62159047A (en) * | 1985-12-31 | 1987-07-15 | Chemo Sero Therapeut Res Inst | Reagent for quantitative determination of plasma protein |
-
1987
- 1987-11-12 AU AU81135/87A patent/AU616869B2/en not_active Ceased
- 1987-11-12 NO NO874721A patent/NO169980C/en not_active IP Right Cessation
- 1987-11-12 MX MX928187A patent/MX172176B/en unknown
- 1987-11-12 JP JP62284411A patent/JP2714572B2/en not_active Expired - Fee Related
- 1987-11-12 CA CA000551697A patent/CA1300057C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| NO874721L (en) | 1988-05-16 |
| JP2714572B2 (en) | 1998-02-16 |
| AU616869B2 (en) | 1991-11-14 |
| MX172176B (en) | 1993-12-07 |
| NO169980B (en) | 1992-05-18 |
| JPS63135865A (en) | 1988-06-08 |
| AU8113587A (en) | 1988-05-19 |
| NO169980C (en) | 1992-08-26 |
| NO874721D0 (en) | 1987-11-12 |
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