CA1293676C - Detection of bilirubin and corresponding reagents - Google Patents
Detection of bilirubin and corresponding reagentsInfo
- Publication number
- CA1293676C CA1293676C CA000516268A CA516268A CA1293676C CA 1293676 C CA1293676 C CA 1293676C CA 000516268 A CA000516268 A CA 000516268A CA 516268 A CA516268 A CA 516268A CA 1293676 C CA1293676 C CA 1293676C
- Authority
- CA
- Canada
- Prior art keywords
- reagent according
- detergent
- aqueous reagent
- general formula
- aqueous
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/728—Bilirubin; including biliverdin
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
Abstract
Abstract A method for the determination of total bilirubin in a biological sample according to the diazo method as well as reagents which are suitable therefor are described.
This method comprises using in the bilirubin determination one or more detergents of the general formula I
wherein R1, R2, n, R3 and R4 have the significance mentioned in claim 1.
The method in accordance with the present invention for the determination of bilirubin guarantees a high linearity, a favourable reaction kinetics which enables the autoblanking system to be used, a simple reagent preparation, a long stability of the solution which is used and a good reproducibility.
Dodecylethyldimethylammonium bromide is a particularly preferred detergent of general formula I.
The reagents for the determination of bilirubin in accordance with the present invention can be present in aqueous solution or preferably in stabilized and lyophilized form.
This method comprises using in the bilirubin determination one or more detergents of the general formula I
wherein R1, R2, n, R3 and R4 have the significance mentioned in claim 1.
The method in accordance with the present invention for the determination of bilirubin guarantees a high linearity, a favourable reaction kinetics which enables the autoblanking system to be used, a simple reagent preparation, a long stability of the solution which is used and a good reproducibility.
Dodecylethyldimethylammonium bromide is a particularly preferred detergent of general formula I.
The reagents for the determination of bilirubin in accordance with the present invention can be present in aqueous solution or preferably in stabilized and lyophilized form.
Description
The present invention is concerned with a method for the determination of total bilirubin in a biological sample according to the diazo method as well as with reagents which are suitable therefor.
The determination of total bilirubin according to the diazo method i8 a technique which is very well known to the person skilled in the art. This method as well as the 10 reagents which are used therefor do, however, have certain disadvantages, in particular it has not been possible to date to carry out this method in automatic analyses according to the so-called autoblanking erocedure.
The method in accordance with the present invention and, re~pectively, the reagents provided therefor result in a test which is di~tinguished by the following surprising advantages:
- high linearity - favourable reaction kinectics which enable the autoblanking system to be used - simple reagent preparation - - long stability of the solution which i8 used and - good precision and good reproducibility.
As iB known, the diazo method for the determination of bilirubin comprises reacting the bilirubin which is present in the biological sample with a diazonium compound 30 to give a colouring substance, the intensity of which corresponds to the amount of bilirubin in the sample.
In the scope of the pre6ent invention it has now surprisingly been found that the above-mentioned advant-Klt/24.7.86 ~k 12~3676 ages can be achieved when one or more detergents of thegeneral formula (R2) ¦~R3 R1 - N\
wherein Rl signifies straight or branched alkyl with 10-16 C-atoms, R2 signifies oxygen or straight or branched alkyl with 1-4 C-atoms; R3, insofar as n =
0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-4 C-atoms;
R4, insofar as n = 0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R4 insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R4, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-10 C-atoms; or R2, R3 and R4 together with the nitrogen atom form a pyridinium group, whereby in this case as well as in the case where n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms a corresponding anion is present, is/are used in the bilirubin determination according to 35 the diazo method.
1~93~i76 ~ Preferred deteegents talso referred to hereinafter as accelerator reagents~ of formula I are N,N-dimethyl-decyl-amine, N,N-dimethyl-dodecylamine, N,N-dimethyl-tetradecyl-amine, N,N-dimethyl-hexadecylamine, didecyldimethyl-ammonium chloride, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, dodecylethyldimethyl-ammonium bromide, dodecylpyridinium chloride, tetradecyl-trimethylammonium bromide as well as N,N-dimethyldodecyl-amine oxide. Dodecylethyldimethylammonium bromide is an 10 especially preferred detergent of general formula I.
According to a first aspect, the present invention is concerned with an aqueous reagent for the determination of total bilirubin in a biological sample, said reagent 16 containing on the one hand a) one or more detergents of the general formula 20 (R2)n Rl I ~ 3 ~ R4 wherein Rl signifies straight or branched alkyl with 10-16 C-atoms, R2 signifies oxygen or straight or branched alkyl with 1-4 C-atoms;
R3, insofar as n = 0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -~CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-4 C-atoms: R4, insofar as 1~3676 n =0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R4 insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x i8 a whole number of 1-5, R4, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-10 C-atoms; or R2, R3 and R4 together with the nitrogen atom form a pyridinium group, whereby in this case as well as in the case where n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms a corresponding anion is present, b) an aromatic amino compound which can be converted with nitrite into a diazonium compound which is customary in the determination of bilirubin, c) a strong acid, and on the other hand d) an aqueous nitrite solution.
According to a second aspect, the present invention is 25 concerned with an aqueous reagent for the determination of total bilirubin in a biological sample, said reagent containing a) one or more detergent~ of general formula I, b) a diazonium compound which is customary in the determination of bilirubin and c) a strong acid.
In the aforementioned aqueous reagent~ the detergent 35 of general formula I is preferably present in a concentra-1~93676 tion of 0.25-lOS, especially in a concentration of 1-3%.
The aromatic amino compound is preferably present in a concentration of 0.5-40 mmol, especially preferably in a concentration of 1-30 mmol.
Amidosulphuric acid, hydrochloric acid and sulphuric acid are the preferred strong acids.
The concentration of amidosulphuric acid preferably lies in the range of 0.025-1 mol/l, especially in a range of 0.10-0.25 mol/l, and the pH of the reagent i8 preferably between 1 and 3.
When hydrochloric acid is u~ed, the concentration i5 preferably in the range of 0.01-0.5 mol/l, especially in the range of 0.05-0.2 mol/1, and the pH of the reagent is preferably 1-3.
When sulphuric acid is used, the concentration prefer-ably lies in the range of 0.01-0.5 mol/l, especially in the range of 0.05-0.25 mol/l, and the pH of the reagent is preferably between i and 3.
The aromatic amino compound, which can react with the nitrite solution to give the corre~ponding diazonium compound, is preferably 4-aminobenzenesulphonic acid, - 2,4- or 2,5-dichloroaniline, 4-nitroaniline, 2-methoxy-4--nitroaniline or 2-methoxy-5-nitroaniline.
The previou61y mentioned nitrite solution i~ prefer-ably pre6ent in a concentration of 4-100 mmol/l.
Insofar as the reagent already contain~ the formed diazonium compound, this is preferably diazotized 4-amino-benzenesulphonic acid or diazotized 2,4- or 2,5-dichloro-lZ93~76 aniline, 4-nitroaniline, 2-methoxy-4-nitroaniline or 2-methoxy-5-nitroaniline.
The concentration of diazonium compound in the aqueous solution preferably lies in the range of 0.10-5.0 g/l, especially ih a range of 0.15-3.0 g/l.
The preferred detergents of general formula I have already been mentioned earlier, but in addition to this it can also be remarked that the substituents R3 and R4 in general formula I preferably have the same significance.
~ ccording to a third aspect, the pLesent invention is concerned with a stabilized and lyophilized reagent for the determination of total bilirubin in a biological sample, said reagent containing diazotized 4-aminobenzene-sulphonic acid or diazotized 4-nitroaniline, 2-methoxy-4--nitroaniline, 2-methoxy-5-nitroaniline, 2,4-dichloro-aniline or 2,5-dichloroaniline, as well as one or more detergents of general formula I.
According to a fourth aspect, the present invention is concerned with a method for the determination of total bilirubin in a biological sample according to the diazo method at a strongly acidic pH, which method comprises using one or more detergents of general formula I.
According to a fifth aspect, the present invention is 3 concerned with the use of a detergent of general formula 1 in a method for the determination of total bilirubin in a biological sample according to the diazo method at a strongly acidic pH.
According to a sixth aspect, the present invention is concerned with a method for the determination of total bilirubin in a biological sample according to the diazo ~2936 ~
method at a strongly acidic pH according to the auto-blanking procedure, which method comprises using one or more detergents of general formula 1.
The fo}lowing Examples illustrate the invention:
ExamDle - Reaaent solutions:
The reagent solution 1 contains 20 g of dodecylethyl-dimethylammonium bromide and 5.195 g of sulehanilic acid dissolved in one litre of O.O5N hydrochloric acid. The reagent 2 i~ a 6.25 mmol aqueous sodium nitrite solution.
The reagent 1 is mixed with reagent 2 in the ratio 5 to 1.
The reagent mixture is ready-for-use after 1 minute at RT.
- Test Derformance on Cobas-Bio0 The reagent used is mixed with serum or bilirubin standard in the ritio 17.5:1 at a determined temperature (25C, 30C or 37C). The extinction is measured at 550 nm exactly after 4.5 seconds and again after 30g.5 seconds.
- Evaluation:
- by com~arsion of the extinction diffe~ence with a bilirubin standard or a control serum:
~E2P-ElP) -RB
- x bilirubin concentration (S) (E2S-ElS)-RB
P = sample S = standard or control serum.
~ 93~76 Reagent blank value tRB) is measured and deducted systematically by the apparatus.
by factor: mg/dl = 18.09 x ~E
~mol/l , 309 x ~E
Figure 1 6hows the verification of linearity with a highly concentrated bilirubin control serum t42.8 mg/dl).
ExamDle 2 - Reaaent solutions see Example 1 - Test Performance: manual Drocedure The reagent solutlons are pipetted into cuvettes tlayer thickness 1 cm) at room temperature in the following anner:
~nalvsis (A) Analvsis blank value tAB) - Reagent solution 1 - 1.0 ml - Reagent mixture 1.0 ml - Sample 0.10 ml 0.10 ml The mixture is mixed well and then left to stand at RT
for 10 minutes. In the following 60 minutes the extinc-tions of the sample against analysis blank value are read off at 546 nm or 550 nm.
The reagent blank value can be disregarded with fresh reagent mixture tl day at RT or 5 days at 4C).
i;~93~ ~6 - Evaluation by comparison o~ the extinction difference with bilirubin standard or control serum:
E(A~-E(AB) x bilirubin concentration (S) E(S)-E(SB) by factor 12.4 x ~E (mg/dl) 212 x ~E (~mol/l).
Exam~le 3 - Rea~ents - LvoDhilized dia20 reaqent In a 25 mmol aqueous amidosulphuric acid solution there are di6601ved in 6equence:
The determination of total bilirubin according to the diazo method i8 a technique which is very well known to the person skilled in the art. This method as well as the 10 reagents which are used therefor do, however, have certain disadvantages, in particular it has not been possible to date to carry out this method in automatic analyses according to the so-called autoblanking erocedure.
The method in accordance with the present invention and, re~pectively, the reagents provided therefor result in a test which is di~tinguished by the following surprising advantages:
- high linearity - favourable reaction kinectics which enable the autoblanking system to be used - simple reagent preparation - - long stability of the solution which i8 used and - good precision and good reproducibility.
As iB known, the diazo method for the determination of bilirubin comprises reacting the bilirubin which is present in the biological sample with a diazonium compound 30 to give a colouring substance, the intensity of which corresponds to the amount of bilirubin in the sample.
In the scope of the pre6ent invention it has now surprisingly been found that the above-mentioned advant-Klt/24.7.86 ~k 12~3676 ages can be achieved when one or more detergents of thegeneral formula (R2) ¦~R3 R1 - N\
wherein Rl signifies straight or branched alkyl with 10-16 C-atoms, R2 signifies oxygen or straight or branched alkyl with 1-4 C-atoms; R3, insofar as n =
0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-4 C-atoms;
R4, insofar as n = 0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R4 insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R4, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-10 C-atoms; or R2, R3 and R4 together with the nitrogen atom form a pyridinium group, whereby in this case as well as in the case where n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms a corresponding anion is present, is/are used in the bilirubin determination according to 35 the diazo method.
1~93~i76 ~ Preferred deteegents talso referred to hereinafter as accelerator reagents~ of formula I are N,N-dimethyl-decyl-amine, N,N-dimethyl-dodecylamine, N,N-dimethyl-tetradecyl-amine, N,N-dimethyl-hexadecylamine, didecyldimethyl-ammonium chloride, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, dodecylethyldimethyl-ammonium bromide, dodecylpyridinium chloride, tetradecyl-trimethylammonium bromide as well as N,N-dimethyldodecyl-amine oxide. Dodecylethyldimethylammonium bromide is an 10 especially preferred detergent of general formula I.
According to a first aspect, the present invention is concerned with an aqueous reagent for the determination of total bilirubin in a biological sample, said reagent 16 containing on the one hand a) one or more detergents of the general formula 20 (R2)n Rl I ~ 3 ~ R4 wherein Rl signifies straight or branched alkyl with 10-16 C-atoms, R2 signifies oxygen or straight or branched alkyl with 1-4 C-atoms;
R3, insofar as n = 0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -~CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-4 C-atoms: R4, insofar as 1~3676 n =0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is a whole number of 1-5, R4 insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in which x i8 a whole number of 1-5, R4, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-10 C-atoms; or R2, R3 and R4 together with the nitrogen atom form a pyridinium group, whereby in this case as well as in the case where n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms a corresponding anion is present, b) an aromatic amino compound which can be converted with nitrite into a diazonium compound which is customary in the determination of bilirubin, c) a strong acid, and on the other hand d) an aqueous nitrite solution.
According to a second aspect, the present invention is 25 concerned with an aqueous reagent for the determination of total bilirubin in a biological sample, said reagent containing a) one or more detergent~ of general formula I, b) a diazonium compound which is customary in the determination of bilirubin and c) a strong acid.
In the aforementioned aqueous reagent~ the detergent 35 of general formula I is preferably present in a concentra-1~93676 tion of 0.25-lOS, especially in a concentration of 1-3%.
The aromatic amino compound is preferably present in a concentration of 0.5-40 mmol, especially preferably in a concentration of 1-30 mmol.
Amidosulphuric acid, hydrochloric acid and sulphuric acid are the preferred strong acids.
The concentration of amidosulphuric acid preferably lies in the range of 0.025-1 mol/l, especially in a range of 0.10-0.25 mol/l, and the pH of the reagent i8 preferably between 1 and 3.
When hydrochloric acid is u~ed, the concentration i5 preferably in the range of 0.01-0.5 mol/l, especially in the range of 0.05-0.2 mol/1, and the pH of the reagent is preferably 1-3.
When sulphuric acid is used, the concentration prefer-ably lies in the range of 0.01-0.5 mol/l, especially in the range of 0.05-0.25 mol/l, and the pH of the reagent is preferably between i and 3.
The aromatic amino compound, which can react with the nitrite solution to give the corre~ponding diazonium compound, is preferably 4-aminobenzenesulphonic acid, - 2,4- or 2,5-dichloroaniline, 4-nitroaniline, 2-methoxy-4--nitroaniline or 2-methoxy-5-nitroaniline.
The previou61y mentioned nitrite solution i~ prefer-ably pre6ent in a concentration of 4-100 mmol/l.
Insofar as the reagent already contain~ the formed diazonium compound, this is preferably diazotized 4-amino-benzenesulphonic acid or diazotized 2,4- or 2,5-dichloro-lZ93~76 aniline, 4-nitroaniline, 2-methoxy-4-nitroaniline or 2-methoxy-5-nitroaniline.
The concentration of diazonium compound in the aqueous solution preferably lies in the range of 0.10-5.0 g/l, especially ih a range of 0.15-3.0 g/l.
The preferred detergents of general formula I have already been mentioned earlier, but in addition to this it can also be remarked that the substituents R3 and R4 in general formula I preferably have the same significance.
~ ccording to a third aspect, the pLesent invention is concerned with a stabilized and lyophilized reagent for the determination of total bilirubin in a biological sample, said reagent containing diazotized 4-aminobenzene-sulphonic acid or diazotized 4-nitroaniline, 2-methoxy-4--nitroaniline, 2-methoxy-5-nitroaniline, 2,4-dichloro-aniline or 2,5-dichloroaniline, as well as one or more detergents of general formula I.
According to a fourth aspect, the present invention is concerned with a method for the determination of total bilirubin in a biological sample according to the diazo method at a strongly acidic pH, which method comprises using one or more detergents of general formula I.
According to a fifth aspect, the present invention is 3 concerned with the use of a detergent of general formula 1 in a method for the determination of total bilirubin in a biological sample according to the diazo method at a strongly acidic pH.
According to a sixth aspect, the present invention is concerned with a method for the determination of total bilirubin in a biological sample according to the diazo ~2936 ~
method at a strongly acidic pH according to the auto-blanking procedure, which method comprises using one or more detergents of general formula 1.
The fo}lowing Examples illustrate the invention:
ExamDle - Reaaent solutions:
The reagent solution 1 contains 20 g of dodecylethyl-dimethylammonium bromide and 5.195 g of sulehanilic acid dissolved in one litre of O.O5N hydrochloric acid. The reagent 2 i~ a 6.25 mmol aqueous sodium nitrite solution.
The reagent 1 is mixed with reagent 2 in the ratio 5 to 1.
The reagent mixture is ready-for-use after 1 minute at RT.
- Test Derformance on Cobas-Bio0 The reagent used is mixed with serum or bilirubin standard in the ritio 17.5:1 at a determined temperature (25C, 30C or 37C). The extinction is measured at 550 nm exactly after 4.5 seconds and again after 30g.5 seconds.
- Evaluation:
- by com~arsion of the extinction diffe~ence with a bilirubin standard or a control serum:
~E2P-ElP) -RB
- x bilirubin concentration (S) (E2S-ElS)-RB
P = sample S = standard or control serum.
~ 93~76 Reagent blank value tRB) is measured and deducted systematically by the apparatus.
by factor: mg/dl = 18.09 x ~E
~mol/l , 309 x ~E
Figure 1 6hows the verification of linearity with a highly concentrated bilirubin control serum t42.8 mg/dl).
ExamDle 2 - Reaaent solutions see Example 1 - Test Performance: manual Drocedure The reagent solutlons are pipetted into cuvettes tlayer thickness 1 cm) at room temperature in the following anner:
~nalvsis (A) Analvsis blank value tAB) - Reagent solution 1 - 1.0 ml - Reagent mixture 1.0 ml - Sample 0.10 ml 0.10 ml The mixture is mixed well and then left to stand at RT
for 10 minutes. In the following 60 minutes the extinc-tions of the sample against analysis blank value are read off at 546 nm or 550 nm.
The reagent blank value can be disregarded with fresh reagent mixture tl day at RT or 5 days at 4C).
i;~93~ ~6 - Evaluation by comparison o~ the extinction difference with bilirubin standard or control serum:
E(A~-E(AB) x bilirubin concentration (S) E(S)-E(SB) by factor 12.4 x ~E (mg/dl) 212 x ~E (~mol/l).
Exam~le 3 - Rea~ents - LvoDhilized dia20 reaqent In a 25 mmol aqueous amidosulphuric acid solution there are di6601ved in 6equence:
4 g of D-mannitol 1 g of Dextran 32 and 2.4 g of diazotized sulphanilic acid tetrafluoro-borate salt.
The solution is made up tD 1 litre and divided into 3 ml portions for the lyophilization. When the temperature in the lyophilizator rearheç -40C, the vacuum i6 adju6ted (0.1 mm Hg). The plate6 are then heated to 10C under a - vacuum for about 16 hour~. Finally, the lyophilizate i6 dried at 20C for 2 hours under a vacuum.
- Accelecator rea~ent To a 0.25M aqueous amidosulphuric acid 601ution there are added in 6equence:
* trade mark ~' . ..~.
75 ml of dodecyldimethylamine oxide (30% aqueous solution) 1 g of methylparaben and 25 g of D-mannitol.
The solution is made up to 1 litre.
This solution can be used as the liquid reagent or, a~
above, can be lyophilized after division into 6 ml portions.
- Rea~ents used The lyophilized diazo reagent i8 dissolved in 3 ml of deionized water. Insofar as the accelerator reagent is lyophilized, it i6 dissolved in 6 ml of deionized water.
- Test Performance on Cobas BioO
The accelerator reagent solution is mixed with a 6erum sample (P) or a bilirubin standard (S) in the ratio 12.5:1 at a determined temperature (25C, 30C or 37C). ~fter an incubation period of 10 seconds the extinction is measured at 550 nm (. analysis blank value). 40 microlitres of diazo reagent solution are then added to the cuvette and the extinction is read off after 1 second and again after 301 seconds.
- Evaluation:
By comparison of the extinction difference with a - bilirubin standard or with a control serum:
(E2P-ElP)-~B-RB
_ __ x bilirubin concentration (S) (E2S-ElS)-SB-RB
36~6 P = sample S = standard AB - analysis blank value, SB = standard blank value RB - reagent blank value measured and deducted system-atically by the apparatu6 by factor: 16.10 x ~E for mgJdl 275 x ~E for ~mol/l - Fiaure 2: Kinetics of the reaction with a dilution series of control serum mixture (40.4 mg bilirubin/dl).
ExamDle 4 - Reaaents - lyophilized diazo reagent see Example 3 - accelerator reagent - Reaaents used: see Example 3 - te t Derformance: manual procedure 25. The reagent solutions are pipetted into cuvettes (layer thickness 1 cm) at room temperature in the following manner:
- sample or standard: 0.10 ml -.accelerator reagent solution 1.00 ml mixed well and the extinction (El) is read off at 546 nm or 550 nm against water.
- Diazo reagent solution 0.20 ml ..
is added, mixed well and after 6 minutes the extinction (E2) i8 read off against water.
- Evaluation - by comparison of the extinction difference with a bilirubin standard or with a control serum:
E2P-ElP
x bilirubin concentration (S) E2 S-El S
- by factor: 16.3 x ~E (mg/dl) 279 x ~E (~mol/l) - Fiaure 3 Accuracy with control sera (concentration in mg/dl).
ExamDle 5 - Reaaents - lvo~hilized diazo reaaent see Example 3 - accelerator reaaent - Reaaents used - Insofar as the accelerator reagent is lyophilized, it is dissolved in 6 ml of deionized water.
-- The diazo reagent is dissolved in 18 ml of acceler-ator reagent solution.
- Test Performance on Cobas BioO
The reagent used is mixed with sérum or bilirubin -3t~7f~
standard in the ratio 17.5:1 at a determined temperature (25C, 30C or 37C). The extinction i8 measured exactly after 4.5 seconds at 550 nm and aqain after 304.5 6econds.
- Evaluation:
- comparison of the extinction difference as in Example 3 - with factor , 16.80 x ~E (mg/dl) , 287 x AE (~mol/l) - Fiaure 4:
Xinetics of the reaction with the dilution series of a control serum mixture (40.4 mg/dl).
Exam~le 6 - Reaa~ents see Example 5 - Test ~erformance: (manual procedure) The solutions given below are pipetted into cuvettes (layer thickness 1 cm) at room temperature as follows:
AnalYsis (A) Analvsi6 blank value (AB) - Accelerator reagent golution - 1.0 ml - Reagent solution used 1.0 ml - Serum or standard 0.10 ml 0.10 ml The mixture is mixed well and after 6 minutes the extinction is read off against the extinction (AB) at 546 nm or 500 nm.
36'76 .
A reagent blank value is measured for each series (reagent solution used).
- Evaluation - by extinction difference with a standard EtA~ E~(AB R x bilirubin concentration (S) E(S)-E(SB)-RB
with factor: 13.20 x ~E (mg/dl) 226 x ~E (~mol/l) ExamDle 7 - LYoDhilized monoreaaent To a 0.333M aqueous amidosulphuric acid solution there are added and admixed in sequence 60 g of dodecylethy}dimethylammonium bromide 3 g of Dextran 32 and 1 g of diazotized sulphanilic acid tetrafluoroborate salt.
The solution is made up to 1 litre and divided into 2 ml portions for the lyophilization. The lyophilization i8 carried out as described in Example 3.
- Reaaent used:
The lyophilizate is disgolved iA 6 ml of deionized water. This reagent is especially favourable for automatic analyses.
. "
`-' lZ93676 - Test Performance on Cobas Bio as described in Example 1.
- Evaluation:
- by comparison with standard or control serum; see Example 1 - by factor 16.95 x aE (mg/dl) 290 x ~E (~mol/l).
- Fiaure 5 Recovery study with control serum mixtures (23-366 ~mol/l).
Example 8 Lyophilized monoreagent with diazotized 2,4-dichloroaniline 20 - LYohilized monoreaaent To a 0.33M aqueous amidosulphuric acid solution there are added and admixed in sequence:
30 g of dodecylethyldimethylammonium bromide 6 g of Dextran 32 and 1.4 g of diazotized 2,4-dichloroaniline stabilized with naphthalenedisulphonic acid sodium ~alt.
The solution is made up to 1 litre and divided into 2 ml 30 portions for the lyophilization (see Example 3).
- Reaaent used The lyophilizate i8 di6601ved in 6 ml of deionized 3~5 water-3t~76 - Test Procedure on Cobas-Bio as described in Example 1 - Evaluation-- by comparison with standard or control serum as described in Example 1 - with factor: 19.80 x ~E (mg/dl) - Fiaure 6:
Linearity verification with a highly concentrated bilirubin control serum (37.7 mg/dl).
ExamPle 9 Lyophilized monoreagent with diazotized 2,5-dichloroaniline - LvoPhilized monoreaaent To a 0.333M aqueous amidosulphuric acid solution there are added and admixed in sequence:
30 g of dodecylethyldimethylammonium bromide 6 g of Dextran 32 and 1.4 g of diazotized 2,5-dichloroaniline stabilized with naphthalenedisulphonic acid sodium salt.
The solution is made up to 1 litre and divided into 2 ml portions for the lyophilization (see Example 3).
- Reaaent used The lyophilizate i8 dissolved in 6 ml of deionized water.
, .
- Test Procedure on Cobas-Bio~
.
The reagent used is mixed with serum or bilirubin standard in the ratio 17.5:1 at 25C. The extinction is measured at 550 nm exactly after 4.5 seconds and again after 304.5 seconds.
- Evaluation - by comparison of the extinction difference with a bilirubin standard or a control serum - by factor: mg/dl = 21.11 x aE
~mol/l = 361 x aE.
ExamPle 10 Lyophilized monoreagent with 4-nitrobenzenediazonium tetrafluoroborate salt - LYoPhilized monoreaaent In a 0.333M aqueous amidosulphuric acid solution there are added and admixed in sequence:
54 ml of dodecyldimethylamine 6 g of Dextran 32 and 0.6 g of 4-nitrobenzenediazonium tetrafluoroborate salt.
The solution is made up to 1 litre and divided into 2 ml portions for the lyophilization (see Example 3).
- Reaaent uged The lyophilizate is dissolved in 6 ml of deionized water.
1~93676 - Test ~rocedure on Cobas-Bio as described in Example 9.
- Evaluation - by comparison of the extinction difference with a bilirubin standard or a control serum - by factor: mg/dl = 18.44 x ~E
~mol/l = 315.1 x ~E.
The solution is made up tD 1 litre and divided into 3 ml portions for the lyophilization. When the temperature in the lyophilizator rearheç -40C, the vacuum i6 adju6ted (0.1 mm Hg). The plate6 are then heated to 10C under a - vacuum for about 16 hour~. Finally, the lyophilizate i6 dried at 20C for 2 hours under a vacuum.
- Accelecator rea~ent To a 0.25M aqueous amidosulphuric acid 601ution there are added in 6equence:
* trade mark ~' . ..~.
75 ml of dodecyldimethylamine oxide (30% aqueous solution) 1 g of methylparaben and 25 g of D-mannitol.
The solution is made up to 1 litre.
This solution can be used as the liquid reagent or, a~
above, can be lyophilized after division into 6 ml portions.
- Rea~ents used The lyophilized diazo reagent i8 dissolved in 3 ml of deionized water. Insofar as the accelerator reagent is lyophilized, it i6 dissolved in 6 ml of deionized water.
- Test Performance on Cobas BioO
The accelerator reagent solution is mixed with a 6erum sample (P) or a bilirubin standard (S) in the ratio 12.5:1 at a determined temperature (25C, 30C or 37C). ~fter an incubation period of 10 seconds the extinction is measured at 550 nm (. analysis blank value). 40 microlitres of diazo reagent solution are then added to the cuvette and the extinction is read off after 1 second and again after 301 seconds.
- Evaluation:
By comparison of the extinction difference with a - bilirubin standard or with a control serum:
(E2P-ElP)-~B-RB
_ __ x bilirubin concentration (S) (E2S-ElS)-SB-RB
36~6 P = sample S = standard AB - analysis blank value, SB = standard blank value RB - reagent blank value measured and deducted system-atically by the apparatu6 by factor: 16.10 x ~E for mgJdl 275 x ~E for ~mol/l - Fiaure 2: Kinetics of the reaction with a dilution series of control serum mixture (40.4 mg bilirubin/dl).
ExamDle 4 - Reaaents - lyophilized diazo reagent see Example 3 - accelerator reagent - Reaaents used: see Example 3 - te t Derformance: manual procedure 25. The reagent solutions are pipetted into cuvettes (layer thickness 1 cm) at room temperature in the following manner:
- sample or standard: 0.10 ml -.accelerator reagent solution 1.00 ml mixed well and the extinction (El) is read off at 546 nm or 550 nm against water.
- Diazo reagent solution 0.20 ml ..
is added, mixed well and after 6 minutes the extinction (E2) i8 read off against water.
- Evaluation - by comparison of the extinction difference with a bilirubin standard or with a control serum:
E2P-ElP
x bilirubin concentration (S) E2 S-El S
- by factor: 16.3 x ~E (mg/dl) 279 x ~E (~mol/l) - Fiaure 3 Accuracy with control sera (concentration in mg/dl).
ExamDle 5 - Reaaents - lvo~hilized diazo reaaent see Example 3 - accelerator reaaent - Reaaents used - Insofar as the accelerator reagent is lyophilized, it is dissolved in 6 ml of deionized water.
-- The diazo reagent is dissolved in 18 ml of acceler-ator reagent solution.
- Test Performance on Cobas BioO
The reagent used is mixed with sérum or bilirubin -3t~7f~
standard in the ratio 17.5:1 at a determined temperature (25C, 30C or 37C). The extinction i8 measured exactly after 4.5 seconds at 550 nm and aqain after 304.5 6econds.
- Evaluation:
- comparison of the extinction difference as in Example 3 - with factor , 16.80 x ~E (mg/dl) , 287 x AE (~mol/l) - Fiaure 4:
Xinetics of the reaction with the dilution series of a control serum mixture (40.4 mg/dl).
Exam~le 6 - Reaa~ents see Example 5 - Test ~erformance: (manual procedure) The solutions given below are pipetted into cuvettes (layer thickness 1 cm) at room temperature as follows:
AnalYsis (A) Analvsi6 blank value (AB) - Accelerator reagent golution - 1.0 ml - Reagent solution used 1.0 ml - Serum or standard 0.10 ml 0.10 ml The mixture is mixed well and after 6 minutes the extinction is read off against the extinction (AB) at 546 nm or 500 nm.
36'76 .
A reagent blank value is measured for each series (reagent solution used).
- Evaluation - by extinction difference with a standard EtA~ E~(AB R x bilirubin concentration (S) E(S)-E(SB)-RB
with factor: 13.20 x ~E (mg/dl) 226 x ~E (~mol/l) ExamDle 7 - LYoDhilized monoreaaent To a 0.333M aqueous amidosulphuric acid solution there are added and admixed in sequence 60 g of dodecylethy}dimethylammonium bromide 3 g of Dextran 32 and 1 g of diazotized sulphanilic acid tetrafluoroborate salt.
The solution is made up to 1 litre and divided into 2 ml portions for the lyophilization. The lyophilization i8 carried out as described in Example 3.
- Reaaent used:
The lyophilizate is disgolved iA 6 ml of deionized water. This reagent is especially favourable for automatic analyses.
. "
`-' lZ93676 - Test Performance on Cobas Bio as described in Example 1.
- Evaluation:
- by comparison with standard or control serum; see Example 1 - by factor 16.95 x aE (mg/dl) 290 x ~E (~mol/l).
- Fiaure 5 Recovery study with control serum mixtures (23-366 ~mol/l).
Example 8 Lyophilized monoreagent with diazotized 2,4-dichloroaniline 20 - LYohilized monoreaaent To a 0.33M aqueous amidosulphuric acid solution there are added and admixed in sequence:
30 g of dodecylethyldimethylammonium bromide 6 g of Dextran 32 and 1.4 g of diazotized 2,4-dichloroaniline stabilized with naphthalenedisulphonic acid sodium ~alt.
The solution is made up to 1 litre and divided into 2 ml 30 portions for the lyophilization (see Example 3).
- Reaaent used The lyophilizate i8 di6601ved in 6 ml of deionized 3~5 water-3t~76 - Test Procedure on Cobas-Bio as described in Example 1 - Evaluation-- by comparison with standard or control serum as described in Example 1 - with factor: 19.80 x ~E (mg/dl) - Fiaure 6:
Linearity verification with a highly concentrated bilirubin control serum (37.7 mg/dl).
ExamPle 9 Lyophilized monoreagent with diazotized 2,5-dichloroaniline - LvoPhilized monoreaaent To a 0.333M aqueous amidosulphuric acid solution there are added and admixed in sequence:
30 g of dodecylethyldimethylammonium bromide 6 g of Dextran 32 and 1.4 g of diazotized 2,5-dichloroaniline stabilized with naphthalenedisulphonic acid sodium salt.
The solution is made up to 1 litre and divided into 2 ml portions for the lyophilization (see Example 3).
- Reaaent used The lyophilizate i8 dissolved in 6 ml of deionized water.
, .
- Test Procedure on Cobas-Bio~
.
The reagent used is mixed with serum or bilirubin standard in the ratio 17.5:1 at 25C. The extinction is measured at 550 nm exactly after 4.5 seconds and again after 304.5 seconds.
- Evaluation - by comparison of the extinction difference with a bilirubin standard or a control serum - by factor: mg/dl = 21.11 x aE
~mol/l = 361 x aE.
ExamPle 10 Lyophilized monoreagent with 4-nitrobenzenediazonium tetrafluoroborate salt - LYoPhilized monoreaaent In a 0.333M aqueous amidosulphuric acid solution there are added and admixed in sequence:
54 ml of dodecyldimethylamine 6 g of Dextran 32 and 0.6 g of 4-nitrobenzenediazonium tetrafluoroborate salt.
The solution is made up to 1 litre and divided into 2 ml portions for the lyophilization (see Example 3).
- Reaaent uged The lyophilizate is dissolved in 6 ml of deionized water.
1~93676 - Test ~rocedure on Cobas-Bio as described in Example 9.
- Evaluation - by comparison of the extinction difference with a bilirubin standard or a control serum - by factor: mg/dl = 18.44 x ~E
~mol/l = 315.1 x ~E.
Claims (42)
1. An aqueous reagent for the determination of total bilirubin in a biological sample, said reagent containing on the one hand a) one or more detergents of the general formula I
wherein R1 signifies straight or branched alkyl with 10-16 C-atoms, R2 signifies oxygen or straight or branched alkyl with 1-4 C-atoms;
R3, insofar as n = 0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-4 C-atoms: R4, insofar as n =0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH
x is a whole number of 1-5, R4 insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH in which x is a whole number of 1-5, R4, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-10 C-atoms; or R2, R3 and R4 together with the nitrogen atom form a pyridinium group, whereby in this case as well as in the case where n signifies 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms a corresponding anion is present, b) an aromatic amino compound which can be converted with nitrite into a diazonium compound which is customary in the determination of bilirubin, c) a strong acid, and on the other hand d) an aqueous nitrite solution.
wherein R1 signifies straight or branched alkyl with 10-16 C-atoms, R2 signifies oxygen or straight or branched alkyl with 1-4 C-atoms;
R3, insofar as n = 0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH in which x is a whole number of 1-5, R3, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-4 C-atoms: R4, insofar as n =0, signifies hydrogen or straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH
x is a whole number of 1-5, R4 insofar as n = 1 and R2 signifies oxygen, signifies straight or branched alkyl with 1-4 C-atoms or -(CH2-CH2-O)xH in which x is a whole number of 1-5, R4, insofar as n = 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms, signifies straight or branched alkyl with 1-10 C-atoms; or R2, R3 and R4 together with the nitrogen atom form a pyridinium group, whereby in this case as well as in the case where n signifies 1 and R2 signifies straight or branched alkyl with 1-4 C-atoms a corresponding anion is present, b) an aromatic amino compound which can be converted with nitrite into a diazonium compound which is customary in the determination of bilirubin, c) a strong acid, and on the other hand d) an aqueous nitrite solution.
2. An aqueous reagent for the determination of total bilirubin in a biological sample, said reagent containing a) one or more detergents of general formula I, b) a diazonium compound which is customary in the determination of bilirubin and c) a strong acid.
3. An aqueous reagent according to claim 1 or 2, wherein the detergent of general formula I is present in a concentration of 0.25-10%.
4. An aqueous reagent according to claim 1 or 2 wherein the detergent of general formula I is present in a concentration of 1-3%.
5. An aqueous reagent according to claim 1, wherein the aromatic amino compound is present in a concentration of 0.5-40 mmol.
6. An aqueous reagent according to claim 5, wherein the aromatic amino compound is present in a concentration of 1-30 mmol.
7. An aqueous reagent according to claim 1 or 2, wherein the strong acid is amidosulphuric acid.
8. An aqueous reagent according to claim 1 or 2, wherein the strong acid is amidosulphuric acid which is present in a concentration of 0.025-1 mol/l and the pH of the reagent is 1-3.
9. An aqueous reagent according to claim 1 or 2, wherein the strong acid is amidosulphuric acid which is present in a concentration of 0.10-0.25 mol/l and the pH of the reagent is 1-3.
10. An aqueous reagent according to claim 1 or 2, wherein the strong acid is hydrochloric acid.
11. An aqueous reagent according to claim 1 or 2, wherein the strong acid is hydrochloric acid which is present in a concentration of 0.01-0.5 mol/l and the pH of the reagent is 1-3.
12. An aqueous reagent according to claim 1 or 2, wherein the strong acid is hydrochloric acid which is present in a concentration of 0.05-0.2 mol/l and the pH of the reagent is 1-3.
13. An aqueous reagent according to claim 1 or 2, wherein the strong acid is sulphuric acid.
14. An aqueous reagent according to claim 1 or 2, wherein the strong acid is sulphuric acid which is present in a concentration of 0.01-0.5 mol/l and the pH of the reagent is 1-3.
15. An aqueous reagent according to claim 1 or 2, wherein the strong acid is sulphuric acid which is present in a concentration of 0.05-0.25 mol/l and the pH of the reagent is 1-3.
16. An aqueous reagent according to claim 2, wherein the diazonium compound is present in a concentration of 0.10-5.0 g/l.
17. An aqueous reagent according to claim 16, wherein the diazonium compound is present in a concentration of 0.15-3.0 g/l.
18. An aqueous reagent according to claim 1, wherein the nitrite solution is present in a concentration of 4-100 mmol/l.
19. An aqueous reagent according to claim 1 or 2, wherein the substituents R3 and R4 in general formula I have the same significance.
20. An aqueous reagent according to claim 1, wherein the aromatic amino compound is 4-aminobenzenesulphonic acid, 2,4- or 2,5-dichloroaniline, 4-nitroaniline, 2-methoxy-4-nitroaniline or 2-methoxy-5-nitroaniline.
21. An aqueous reagent according to claim 2, wherein the diazonium compound is diazotized 4-aminobenzene-sulphonic acid or diazotized 2,4- or 2,5-dichloroaniline, 4-nitroaniline, 2-methoxy-4-nitroaniline or 2-methoxy-5-nitroaniline.
22. An aqueous reagent according to claim 1 or 2, wherein the detergent of general formula 1 is N,N-dimethyl-decylamine.
23. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is N,N-dimethyl-dodecylamine.
24. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is N,N-dimethyl-tetradecylamine.
25. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is N,N-dimethyl-hexadecylamine.
26. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is didecyldi-methylammonium chloride.
27. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is dodecyltri-methylammonium chloride.
28. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is dodecyltri-methylammonium bromide.
29. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is dodecylethyl-dimethylammonium bromide.
30. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is dodecyl-pyridinium chloride.
31. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is tetradecyltri-methylammonium bromide.
32. An aqueous reagent according to claim 1 or 2, wherein the detergent of formula I is N,N-dimethyl-dodecylamine oxide.
33. A stabilized and lyophilized reagent for the determination of total bilirubin in a biological sample, said reagent containing diazotized 4-aminobenzenesulphonic acid or diazotized 4-nitroaniline, 2-methoxy-4-nitro-aniline, 2-methoxy-5-nitroaniline, 2,4-dichloroaniline or 2,5-dichloroaniline, as well as one or more detergents of general formula I in accordance with claim 1.
34. A reagent according to claim 33, wherein the detergent of general formula I is N,N-dimethyldodecylamine oxide.
35. A reagent according to claim 33, wherein the detergent of general formula I is N,N-dimethyldodecylamine.
36. A reagent according to claim 33, wherein the detergent of general formula I is dodecylethyldimethyl-ammonium bromide.
37. A reagent according to claim 33, wherein the detergent of general formula I is dodecyltrimethylammonium bromide.
38. A reagent according to claim 33, wherein the detergent of general formula I is tetradecyltrimethyl-ammonium bromide.
39. A reagent according to claim 33, wherein the detergent of general formula I is dodecylpyridinium chloride.
40. A method for the determination of total bilirubin in a biological sample according to the diazo method at a strongly acidic pH, which method comprises using one or more detergents in accordance with general formula I in accordance with claim 1.
41. A method for the determination of total bilirubin in a biological sample according to the diazo method at a strongly acidic pH according to the auto-blanking procedure, which method comprises using one or more detergents of general formula I in accordance with claim 1.
42. The use of a detergent of general formula I in accordance with claim 1 in a method for the determination of total bilirubin in a biological sample according to the diazo method at a strongly acidic pH.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH4172/85 | 1985-09-26 | ||
| CH417285 | 1985-09-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1293676C true CA1293676C (en) | 1991-12-31 |
Family
ID=4271143
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000516268A Expired - Fee Related CA1293676C (en) | 1985-09-26 | 1986-08-19 | Detection of bilirubin and corresponding reagents |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0217197B1 (en) |
| JP (1) | JPS6275349A (en) |
| AT (1) | ATE68602T1 (en) |
| BR (1) | BR8604639A (en) |
| CA (1) | CA1293676C (en) |
| DE (1) | DE3682014D1 (en) |
| DK (1) | DK165765C (en) |
| ES (1) | ES2001988A6 (en) |
| IL (1) | IL80094A0 (en) |
| NO (1) | NO167482C (en) |
| NZ (1) | NZ217624A (en) |
| ZA (1) | ZA867154B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1233755B (en) * | 1989-09-21 | 1992-04-14 | Diesse Diagnostica | REACTIVE FOR THE RESEARCH AND DETERMINATION OF BILIRUBIN IN URINE. |
| DE69534877T2 (en) * | 1994-12-02 | 2006-10-05 | Nitto Boseki Co., Ltd. | METHOD FOR DETERMINING BILIRUBINE |
| DE102005015213A1 (en) | 2005-04-02 | 2006-10-05 | Pierburg Gmbh | Wet running pump for use as cooling water pump in motor vehicles has stators that are connected to printed circuit board (PCB) that separates wet and dry areas |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4078892A (en) * | 1975-06-30 | 1978-03-14 | Becton, Dickinson And Company | Novel means and method for diagnostic quantitation of serum or plasma bilirubin |
| US4119401A (en) * | 1977-06-07 | 1978-10-10 | Technicon Instruments Corporation | Total bilirubin assay |
| US4246133A (en) * | 1979-06-22 | 1981-01-20 | Sherwood Medical Industries Inc. | Stabilized diazotized sulfanilic acid solutions |
| US4404286A (en) * | 1982-02-22 | 1983-09-13 | The Dow Chemical Company | Bilirubin assay |
| EP0103628A4 (en) * | 1982-03-11 | 1984-09-19 | Arthur Babson | Stabilization of diazonium salt solutions. |
-
1986
- 1986-08-19 CA CA000516268A patent/CA1293676C/en not_active Expired - Fee Related
- 1986-09-12 DE DE8686112620T patent/DE3682014D1/en not_active Expired - Lifetime
- 1986-09-12 EP EP86112620A patent/EP0217197B1/en not_active Expired - Lifetime
- 1986-09-12 AT AT86112620T patent/ATE68602T1/en not_active IP Right Cessation
- 1986-09-18 NZ NZ217624A patent/NZ217624A/en unknown
- 1986-09-19 IL IL80094A patent/IL80094A0/en not_active IP Right Cessation
- 1986-09-19 ZA ZA867154A patent/ZA867154B/en unknown
- 1986-09-25 DK DK458986A patent/DK165765C/en not_active IP Right Cessation
- 1986-09-25 BR BR8604639A patent/BR8604639A/en unknown
- 1986-09-25 ES ES8602146A patent/ES2001988A6/en not_active Expired
- 1986-09-25 NO NO863827A patent/NO167482C/en unknown
- 1986-09-25 JP JP61225087A patent/JPS6275349A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| ZA867154B (en) | 1987-05-27 |
| ATE68602T1 (en) | 1991-11-15 |
| BR8604639A (en) | 1987-06-09 |
| DE3682014D1 (en) | 1991-11-21 |
| JPS6275349A (en) | 1987-04-07 |
| NZ217624A (en) | 1989-10-27 |
| IL80094A0 (en) | 1986-12-31 |
| NO167482B (en) | 1991-07-29 |
| DK458986A (en) | 1987-03-27 |
| EP0217197A3 (en) | 1987-11-25 |
| EP0217197A2 (en) | 1987-04-08 |
| NO167482C (en) | 1991-11-06 |
| EP0217197B1 (en) | 1991-10-16 |
| NO863827L (en) | 1987-03-27 |
| NO863827D0 (en) | 1986-09-25 |
| DK165765C (en) | 1993-06-07 |
| DK458986D0 (en) | 1986-09-25 |
| ES2001988A6 (en) | 1988-07-01 |
| DK165765B (en) | 1993-01-11 |
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