CA2117745A1 - Treating of lipoprotein containing samples - Google Patents
Treating of lipoprotein containing samplesInfo
- Publication number
- CA2117745A1 CA2117745A1 CA002117745A CA2117745A CA2117745A1 CA 2117745 A1 CA2117745 A1 CA 2117745A1 CA 002117745 A CA002117745 A CA 002117745A CA 2117745 A CA2117745 A CA 2117745A CA 2117745 A1 CA2117745 A1 CA 2117745A1
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- Prior art keywords
- sample
- set forth
- amino acid
- lipoprotein
- antigen
- Prior art date
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-
- 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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Endocrinology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract
ABSTRACT OF THE DISCLOSURE:
There is disclosed a sample preparatory agent for lipoprotein containing samples and for immunassaying an antigen. This agent contains a dilution buffer having a content of an amino acid, if desired, a content of a reaction accelerator, and antibodies directed against the antigen to be determined.
There is disclosed a sample preparatory agent for lipoprotein containing samples and for immunassaying an antigen. This agent contains a dilution buffer having a content of an amino acid, if desired, a content of a reaction accelerator, and antibodies directed against the antigen to be determined.
Description
. 7 ~
The invention relates to a sample preparatory agent for lipoprotein containing samples and for immunoassaying an antigen as well as a kit for the preparation of a sample preparatory agent. Likewise, the invention relates to a method of determining an antigen by an immunoassay as well as a method of reducing or eliminating turbidities in a biological liquid, e.g., in a blood or plasma sample.
A lipoprotein is a particle that has a core of ;~
hydrophobic lipids (cholesterol, cholesterol ester, phosphatides, triglycerides) surrounded by a sheath of polar lipids and apopro-teins A-1, A-2, A-4, s-48, B-100, C and E. The lipoproteins, on the one hand, solubilize highly hydrophobic lipids and, on the other hand, contain signals regulating the transfer of certain lipids on specific cells and tissues.
Lipoproteins may be subdivided into different density classes:
Chylomicrons, VLDL (very low density lipoproteins), IDL (intermediate density lipoproteins), LDL (low density lipoproteins) and HDL (high densi-ty lipoproteins).
Depending on the protein portion, lipoproteins in electrophoresis exhibit different mobilities and are classified into beta-lipoproteins, pre-beta-lipoproteins and alpha-lipoproteins. LP-X is a pathological lipoprotein consisting of phosphatides and ~.i7~ 3 cholesterol ester in a high por-tion.
Lp(a) is another lipoprotein involved in the arteriosclerotic process. The structure of Lp(a) may be derived from the structure of the LDL particle. Lp(a) is an LDL particle that is connected with the highly -glycosilated apo-lipoprotein (a) via disulfide bridges.
Lp(a) exhibits both inter- and intra-individual ~ '~
heterogenity in its lipid and protein composi-tion. It constitutes a genetically determined independent risk factor for arteriosclerosis.
The determination of lipoproteins in a biological liquid, such as a blood or plasma sample, has increased in importance in the course of preventive medical examinations for determining the risk of heart and circulatory diseases and also within the scope of therapies for controlling the lipoprotein content. In general, lipoproteins are determined by immunoassays.
To this end, antibodies against the lipopro-tein to be determined are used and the immune comple~es formed are assayed. Detection of the immune complexes formed in this case is effected by optical methods, for instance, by detecting the increase in -the turbidity of a sample.
A sample solution as clear as possible is the prerequisite for the optical tests required therefor in order to maximize the sensitivity of the assay.
The turbidity inherent in sera cons-titu-tes a problem primarily with lipemic or hyperlipoproteinemic ~ 5 7~
sera. For, such turbidity may render impossible the detection of slight amounts of lipoproteins even at a low degree of lipemia.
Thus, for instance, the detection of immuno-precipitates which are measured nephelometrically or turbidime-trically are considerably dis-turbed by the turbidity inherent in -the sample material.
The turbidimetrical determination of Lp(a), in particular, again and again encounters difficulties because of the size of the particle and its structural heterogenity, especially with samples having high inherent turbidities (chylomicrons, triglycerides) and samples that had been frozen and thawed again. With such samples, falsely high Lp(a) concentrations are fre~uently found. Similar difficulties arise with the nephelometrical determination of Lp(a).
Conse~uently, the elimination of turbidities in a serum is of extreme importance for clinical analysis, in particular, for the de-termination of lipoproteins.
A common measure for the turbidity exhibited by a solution is LSU (light scattering unit). In general, a solution is considered as -turbld if it has a measured signal of more than 70 LSU when measured in the nephelometer a-t 340 nm and a path length of 1 cm.
Measurement of an antigen-antibody reac-tion at 70 LSU
and thereabove is clearly falsified. For clinical analysis, samples should have a maximum turbidity ~ 7 7 ;~ ~j preferably ranging between 30 and 40 LSU.
To eliminate disturbing turbidities in llpemic samples, treatment with lipases or high speed centrifugation is, for instance, recommended in Clinica Chimica Acta (135 (1983) 203-208). However, it is decisively advised against using de-tergents, since ;
these do reduce the turbidity inherent in a sample, yet also result in a strong reduction of the immunochemical response. Polyethylene glycol is proposed as a reaction accelerator for the immunochemical reaction.
In the lyophilization of control sera -turbidities occur, in particular, with lipemic samples on account of the procedures of lyophilization and subsequent reconstitution of the originally clear sera. As a~ -precautionary measure in the prevention of -turbidities, EP-0 141 922 proposes the addition of a detergent and of proline prior to lyophilization of the control serum. ~he sera are mixed with proline and sodium-desoxycholate and are lyophilized. After reconstitution of the lyophilisate it could be seen that the clear sera had not become turbid. Since it is always departed from clear sera when stabilizing control sera, clarification of the solution, i.e., reduction of its turbidi-ty by detergen-t/proline could not be effected.
EP-0 058 959 also mentions various additions for the preparation of control sera that are to remain clear after lyophilization and reconstitution. Organic : '' ~ ~ ~ 7 ~
non-sugar-like substances, such as methanol, alanine, triethylene glycol, valine, acetate, lactate or sodium-2-hydroxymethylbutyrate, for instance, are suggested as agents for the prevention of turbidities caused by changes in the state of aggregation. But not even EP-0 058 959 discloses a way of treating a sample -to be assayed in a manner that undisturbed determination of the lipoprotein content is ~easible.
The invention has as its object to provide a technology by which, on the one hand, possibly present turbidies inherent in biological liquids can be reduced or eliminated and, on the other hand, analysis of an antigen in a sample as unfalsified as possible is feasible.
In accordance with the invention, this object is achieved by an agent whi.ch, for sample treatment, contains an amino acid reducing or eliminating turbid.ity in a sample while laaving unaffected the immunochemical determination of an antigen, preferably of a lipoprotein, by aid of an antibody contained in the agent and directed against the antigen to be determined. The agent according to -the invention advantageously contains a reaction accelerator, for instance, polyethylene glycol. The amino acid is present in a buffered medium, preferably at a concentration of from 0.05 to 3 M, most preferred from 0.1 to 0.5 M. I-t has been shown that proline, above ~.
- 5 - .
~ -~ i 7 7 ~
all, is excellently suited to eliminate the turbidity in a lipemic or turbid sample irrespec-tive of the pretreatment of the sample.
A preferred embodiment of -the agent contains antibodies directed against a lipoprotein and is used for determining this lipoprotein by an immunoassay.
For instance, the agent of the invention may be used for assaying Lp(a), the agent in such a case containing Lp(a) antibodies, preferably an Lp(a) antiserum. The agent of the invention can be prepared in a simple manner by modification of commercially available reagents or reagent components.
The agent according to the invention enables the undisturbed determination preferably of lipoproteins in a sample irrespective of whether the sample has been previously deepfrozen, stored in the cooled state or freshly drawn. As such a sample, a biological liquid, such as a blood, serum or plasma sample, which is optically turhid due to its lipoprotein content may be particularly applied.
Surprisingly, the agent according to the invention substantially increases the sensitivity of the determination of, for instance, Lp(a), wherein it can simultaneously be proved by a reference method that the results obtained are of high relevance. The addition of proline to a sample preparatory agent clearly lowers the blank value of a serum sample and increases the 7 7 ~
nephelometrically determined difference in absorbance from the time of the addition of antibody.
The advantageous effect of amino acids, in particular, of proline, in the agent according to the invention is the more surprising as amino acids generally have been proposed in connection with detergents only for preventing -the formation of turbidity in a lyophilized control serum, yet such admixtures (detergents) to a sample are considered as disturbing in an immunoassay. Moreover, such samples are not lyophilized, whence there has not been any need to prevent the formation of turbidities during lyophilization and reconstitution by such admixtures.
It is advantageous also with the agent according to the invention that the sample is not admixed with a tenside in order to render feasible -the undisturbed immunochemical determina~ion of an antigen. The agent according to the invention or the kit for preparing such agent as well as the components o such a kit advantageously are substantially f ee of tensides.
Solutions are regarded as "substantially" free of tensides if they have a tenside conten-t of less than about 0.01 %.
An imunoassay in a solution having a tenside content of more than 0.05 % is clearly falsified by the tenside such that the measured results obtained are no longer relevant.
. "' .
~ ~ . 1 7 J j It has been shown that it is advantageous to provide the agent according to the invention as a kit of at least two components. Thus, the amino acid and, if desired, a reaction accelerator may be made available in the first component in the presence of buffer substances, the second component containing antibodies directed against the antigen to be assayed.
According to a further aspect, the invention relates to a method of determining an antigen by an immunoassay, wherein an amino acid is added to a sample proposed for determining by an immunoassay, antibodies agains-t the an-tigen to be assayed are introduced into the sample subsequently or simultaneously and the ;~
amount of antibody-antigen complex formed is determined.
Another aspect of the invention relates to a method of reducing or eliminating turbidities in a turbid biological liquid, which method is characterized in that an amino acid is added to said -turbid biological liquid. Thus, a method of treating a sample liquid is provided at the same time.
With the methods according to the invention, proline preferably is used as the amino acid.
The methods according -to the invention preferably are employed with blood or plasma samples as well as with turbid lipoprotein containing samples of an antigen provided for determining by an immunoassay.
~ ~ '3" ~ 7 ~, ,r,3 Another aspect of the invention is related to the use of an amino acid, in particular, proline for reducing or eliminating turbidities in a biological liquid, preferably in a turbid lipoprotein containing sample of an antigen provided for determining by an immunoassay, as well as the use of an amino acid, in particular, proline, for treating lipoprotein containing samples, preferably in a sample provided for determining an antigen by an immunoassay.
The invention will be explained in even more detail ~
by the following examples. The examples illustrate the -determination of Lp(a) in serum samples by aid of appropriately modified commercially available individual reagents of Immuno AG according to a valid working prescription (anti-human Lp(a) of sheep, ~-reference standard Lp(a) human, norm control Lp(a) human). The difference in absorbance is detected by aid of a photometer or an automatic analyzer, in the examples indicated by aid of a COBAMSIRA device (Hoffmann-La Roche).
Exam~e 1: Absorbance course of calibration curve The absorbance was measured, and a calibration curve was established, of a series of Lp(a) solutions having different concentrations of Lp(a). To one Lp(a) containing sample dilution buffer containing 1 x PBS (1 x P8S: 10 mM Na/K phosphate, 0.8 % NaCl, 0.2 % HCl, pH
7.2) and 4 % PEG with or without addition of O.1 mol/l .,'. : :.
~ 7 7 .~ ~
proline and, after 5 min, an Lp( a) antibody solution were added. In Fig. lA the absorbance course of the calibration curve with -the addition of 0.1 mol/l proline in dilution medium and in Fig. lB the same course without addition are represented.
As is apparent, the calibrator having -the highest Lp(a) concentration, i.e., 108 mg/dl, has an absorbance of 0.09 RATES as compared to 0.125 RATES of the same calibrator in dilution medium with 0.1 mol/l proline. ~-By the addition of proline, the reaction signal is, :
thus, intensified. RATES in this case correspond to the measured signal of the antigen-antibody reaction after subtraction of the measured signal that occurred upon admixture of dilution buffer as compared -to the original solution.
Furthermore, it is apparent from the course of the calibration curve that the calibration curve, without addition in the dilution medium, flattens towards -~
higher aoncentrations (Fig. lB), whereas the curve shown in Fig. lA has a linear course.
E~am~le 2: Lp(a) determination with pre-incubation A serum sample having a high inherent -turbidity is incubated for 5 min with dilution buffer containing 0.1 mol/l proline. After this, Lp(a) an-tibodies are added.
The absorbance is measured at a wave length of 340 nm and plotted against -the -time unit (Fig. 2A). The same assay was carried out also without (Fig. 2B) addition i 7 7 !1 ;3 of proline in dilu-tion buffer. From this, it is apparent that the absorbance corresponding to the blank value of the serum sample could be lowered from 0.36 (without addition) to 0.24 (with proline).
The absorbance difference from the time of addition of the antibodies in the reaction course of the mixture with proline (Fig. 2A) is greater by 200 ~ than in the mixture without proline (Fig. 2s). Hence an Lp(a) concentration of 103.3 mg/dl in the mixture with proline, yet, at the same time, a falsely low value of 19.2 mg/dl in the mixture without proline are calcula-ted. In general, an Lp(a) concentration of below 30 is considered as normal, values thereabove being regarded as pathological. In the instant case, the sample, thus, would have been regarded as "normal"
(19.2 mg/dl) on grounds of conventional measuring methods, although the correct concen-tra-tion (determined by the reerence method) clearly constitutes a pathological value.
Example 3: Lp(a) determination with pre-incubation Analogous to Example 2, the Lp(a) content o a sample was turbidimetrically determined by the addition of Lp(a) antibodies. The same reaction course as in :, , .
Figs. 2A and 2B is represented in Figs. 3A and 3B with ~;
the calibrator of the highest Lp( a) concentra-tion (=
108 mg/dl). In this example, the reaction signal (without addition, Example 3B) could be intensified by ,~
~ ~ i ' 7 ~ ~3 ~ .
50 ~ by the addition of 0.1 mol/l proline.
Example 4: Correlation between fresh and thawed samples As mentioned above, discrepancies occurred in the turbidime-trical determination of Lp(a) with sera that had been frozen and rethawed as compared to freshly tested samples because of the turbidity of the thawed ~;~
sample material.
The improvement obtained by the admixture of proline to the agent is demonstrated by the following example:
A panel of 25 serum samples was aliquoted, one portion having been assayed on the same day the blood had been drawn, the other portion having been frozen.
The latter aliquot was thawed on the next day and assayed afterwards.
The resul-ts of the assays without proline are represented in Fi~. 4A. The correlation between the freshly tested and the thawed samples was R = 0.908 and the linear equation was y = 1.01 + 0.88x. In a parallel assay, the thawed samples with proline were determined.
Comparison to the freshly -tested samples is illustrated ~ -in Fig. 4B. The correlation was improved to R = 0.972, also the linear equation with y= 3.52 -~ l.O9x showed a marked improvemen-t in the consistency with the ideal values having the gradient 1.
Example 5: Lp(a) determination by electroimmuno-~' ' 7 -~
diffusion Electroimmunodiffusion serves as a reference method of immunologic Lp(a) determination.
The frozen and thawed samples used in Example 4 were tested in a parallel assay also in electro-immunodiffusion. (Method according to Laurell, Anal.
Biochem. 15 (1966), 45).
In Fig. 5A, the resul-ts of the turbidimetric determination without proline are compared to those determined by elec-troimmunodiffusion. The correlation was R = 0.918 and the linear equation for the elec-troimmunodiffusion curve was y = 1.95 + 0.54x. (y =
1.01 + 0.88x for turbidimetric determina-tion: Fig. 4A).
Thus, there is a distinct difference between the two methods in respect of the results obtained. `
By adding proline, not only the correlation coefficient could be raised to R = 0.989, but also the linear equation y = 3.34 + 0.92x implied an almost perfect consistency of the methods (Fig. 5B); y = 3.52 ~ l.O9x for the turbidimitrical determination in the presence of proline, Fig. 4B).
~"'~ `'~'"
- 13 - ~
The invention relates to a sample preparatory agent for lipoprotein containing samples and for immunoassaying an antigen as well as a kit for the preparation of a sample preparatory agent. Likewise, the invention relates to a method of determining an antigen by an immunoassay as well as a method of reducing or eliminating turbidities in a biological liquid, e.g., in a blood or plasma sample.
A lipoprotein is a particle that has a core of ;~
hydrophobic lipids (cholesterol, cholesterol ester, phosphatides, triglycerides) surrounded by a sheath of polar lipids and apopro-teins A-1, A-2, A-4, s-48, B-100, C and E. The lipoproteins, on the one hand, solubilize highly hydrophobic lipids and, on the other hand, contain signals regulating the transfer of certain lipids on specific cells and tissues.
Lipoproteins may be subdivided into different density classes:
Chylomicrons, VLDL (very low density lipoproteins), IDL (intermediate density lipoproteins), LDL (low density lipoproteins) and HDL (high densi-ty lipoproteins).
Depending on the protein portion, lipoproteins in electrophoresis exhibit different mobilities and are classified into beta-lipoproteins, pre-beta-lipoproteins and alpha-lipoproteins. LP-X is a pathological lipoprotein consisting of phosphatides and ~.i7~ 3 cholesterol ester in a high por-tion.
Lp(a) is another lipoprotein involved in the arteriosclerotic process. The structure of Lp(a) may be derived from the structure of the LDL particle. Lp(a) is an LDL particle that is connected with the highly -glycosilated apo-lipoprotein (a) via disulfide bridges.
Lp(a) exhibits both inter- and intra-individual ~ '~
heterogenity in its lipid and protein composi-tion. It constitutes a genetically determined independent risk factor for arteriosclerosis.
The determination of lipoproteins in a biological liquid, such as a blood or plasma sample, has increased in importance in the course of preventive medical examinations for determining the risk of heart and circulatory diseases and also within the scope of therapies for controlling the lipoprotein content. In general, lipoproteins are determined by immunoassays.
To this end, antibodies against the lipopro-tein to be determined are used and the immune comple~es formed are assayed. Detection of the immune complexes formed in this case is effected by optical methods, for instance, by detecting the increase in -the turbidity of a sample.
A sample solution as clear as possible is the prerequisite for the optical tests required therefor in order to maximize the sensitivity of the assay.
The turbidity inherent in sera cons-titu-tes a problem primarily with lipemic or hyperlipoproteinemic ~ 5 7~
sera. For, such turbidity may render impossible the detection of slight amounts of lipoproteins even at a low degree of lipemia.
Thus, for instance, the detection of immuno-precipitates which are measured nephelometrically or turbidime-trically are considerably dis-turbed by the turbidity inherent in -the sample material.
The turbidimetrical determination of Lp(a), in particular, again and again encounters difficulties because of the size of the particle and its structural heterogenity, especially with samples having high inherent turbidities (chylomicrons, triglycerides) and samples that had been frozen and thawed again. With such samples, falsely high Lp(a) concentrations are fre~uently found. Similar difficulties arise with the nephelometrical determination of Lp(a).
Conse~uently, the elimination of turbidities in a serum is of extreme importance for clinical analysis, in particular, for the de-termination of lipoproteins.
A common measure for the turbidity exhibited by a solution is LSU (light scattering unit). In general, a solution is considered as -turbld if it has a measured signal of more than 70 LSU when measured in the nephelometer a-t 340 nm and a path length of 1 cm.
Measurement of an antigen-antibody reac-tion at 70 LSU
and thereabove is clearly falsified. For clinical analysis, samples should have a maximum turbidity ~ 7 7 ;~ ~j preferably ranging between 30 and 40 LSU.
To eliminate disturbing turbidities in llpemic samples, treatment with lipases or high speed centrifugation is, for instance, recommended in Clinica Chimica Acta (135 (1983) 203-208). However, it is decisively advised against using de-tergents, since ;
these do reduce the turbidity inherent in a sample, yet also result in a strong reduction of the immunochemical response. Polyethylene glycol is proposed as a reaction accelerator for the immunochemical reaction.
In the lyophilization of control sera -turbidities occur, in particular, with lipemic samples on account of the procedures of lyophilization and subsequent reconstitution of the originally clear sera. As a~ -precautionary measure in the prevention of -turbidities, EP-0 141 922 proposes the addition of a detergent and of proline prior to lyophilization of the control serum. ~he sera are mixed with proline and sodium-desoxycholate and are lyophilized. After reconstitution of the lyophilisate it could be seen that the clear sera had not become turbid. Since it is always departed from clear sera when stabilizing control sera, clarification of the solution, i.e., reduction of its turbidi-ty by detergen-t/proline could not be effected.
EP-0 058 959 also mentions various additions for the preparation of control sera that are to remain clear after lyophilization and reconstitution. Organic : '' ~ ~ ~ 7 ~
non-sugar-like substances, such as methanol, alanine, triethylene glycol, valine, acetate, lactate or sodium-2-hydroxymethylbutyrate, for instance, are suggested as agents for the prevention of turbidities caused by changes in the state of aggregation. But not even EP-0 058 959 discloses a way of treating a sample -to be assayed in a manner that undisturbed determination of the lipoprotein content is ~easible.
The invention has as its object to provide a technology by which, on the one hand, possibly present turbidies inherent in biological liquids can be reduced or eliminated and, on the other hand, analysis of an antigen in a sample as unfalsified as possible is feasible.
In accordance with the invention, this object is achieved by an agent whi.ch, for sample treatment, contains an amino acid reducing or eliminating turbid.ity in a sample while laaving unaffected the immunochemical determination of an antigen, preferably of a lipoprotein, by aid of an antibody contained in the agent and directed against the antigen to be determined. The agent according to -the invention advantageously contains a reaction accelerator, for instance, polyethylene glycol. The amino acid is present in a buffered medium, preferably at a concentration of from 0.05 to 3 M, most preferred from 0.1 to 0.5 M. I-t has been shown that proline, above ~.
- 5 - .
~ -~ i 7 7 ~
all, is excellently suited to eliminate the turbidity in a lipemic or turbid sample irrespec-tive of the pretreatment of the sample.
A preferred embodiment of -the agent contains antibodies directed against a lipoprotein and is used for determining this lipoprotein by an immunoassay.
For instance, the agent of the invention may be used for assaying Lp(a), the agent in such a case containing Lp(a) antibodies, preferably an Lp(a) antiserum. The agent of the invention can be prepared in a simple manner by modification of commercially available reagents or reagent components.
The agent according to the invention enables the undisturbed determination preferably of lipoproteins in a sample irrespective of whether the sample has been previously deepfrozen, stored in the cooled state or freshly drawn. As such a sample, a biological liquid, such as a blood, serum or plasma sample, which is optically turhid due to its lipoprotein content may be particularly applied.
Surprisingly, the agent according to the invention substantially increases the sensitivity of the determination of, for instance, Lp(a), wherein it can simultaneously be proved by a reference method that the results obtained are of high relevance. The addition of proline to a sample preparatory agent clearly lowers the blank value of a serum sample and increases the 7 7 ~
nephelometrically determined difference in absorbance from the time of the addition of antibody.
The advantageous effect of amino acids, in particular, of proline, in the agent according to the invention is the more surprising as amino acids generally have been proposed in connection with detergents only for preventing -the formation of turbidity in a lyophilized control serum, yet such admixtures (detergents) to a sample are considered as disturbing in an immunoassay. Moreover, such samples are not lyophilized, whence there has not been any need to prevent the formation of turbidities during lyophilization and reconstitution by such admixtures.
It is advantageous also with the agent according to the invention that the sample is not admixed with a tenside in order to render feasible -the undisturbed immunochemical determina~ion of an antigen. The agent according to the invention or the kit for preparing such agent as well as the components o such a kit advantageously are substantially f ee of tensides.
Solutions are regarded as "substantially" free of tensides if they have a tenside conten-t of less than about 0.01 %.
An imunoassay in a solution having a tenside content of more than 0.05 % is clearly falsified by the tenside such that the measured results obtained are no longer relevant.
. "' .
~ ~ . 1 7 J j It has been shown that it is advantageous to provide the agent according to the invention as a kit of at least two components. Thus, the amino acid and, if desired, a reaction accelerator may be made available in the first component in the presence of buffer substances, the second component containing antibodies directed against the antigen to be assayed.
According to a further aspect, the invention relates to a method of determining an antigen by an immunoassay, wherein an amino acid is added to a sample proposed for determining by an immunoassay, antibodies agains-t the an-tigen to be assayed are introduced into the sample subsequently or simultaneously and the ;~
amount of antibody-antigen complex formed is determined.
Another aspect of the invention relates to a method of reducing or eliminating turbidities in a turbid biological liquid, which method is characterized in that an amino acid is added to said -turbid biological liquid. Thus, a method of treating a sample liquid is provided at the same time.
With the methods according to the invention, proline preferably is used as the amino acid.
The methods according -to the invention preferably are employed with blood or plasma samples as well as with turbid lipoprotein containing samples of an antigen provided for determining by an immunoassay.
~ ~ '3" ~ 7 ~, ,r,3 Another aspect of the invention is related to the use of an amino acid, in particular, proline for reducing or eliminating turbidities in a biological liquid, preferably in a turbid lipoprotein containing sample of an antigen provided for determining by an immunoassay, as well as the use of an amino acid, in particular, proline, for treating lipoprotein containing samples, preferably in a sample provided for determining an antigen by an immunoassay.
The invention will be explained in even more detail ~
by the following examples. The examples illustrate the -determination of Lp(a) in serum samples by aid of appropriately modified commercially available individual reagents of Immuno AG according to a valid working prescription (anti-human Lp(a) of sheep, ~-reference standard Lp(a) human, norm control Lp(a) human). The difference in absorbance is detected by aid of a photometer or an automatic analyzer, in the examples indicated by aid of a COBAMSIRA device (Hoffmann-La Roche).
Exam~e 1: Absorbance course of calibration curve The absorbance was measured, and a calibration curve was established, of a series of Lp(a) solutions having different concentrations of Lp(a). To one Lp(a) containing sample dilution buffer containing 1 x PBS (1 x P8S: 10 mM Na/K phosphate, 0.8 % NaCl, 0.2 % HCl, pH
7.2) and 4 % PEG with or without addition of O.1 mol/l .,'. : :.
~ 7 7 .~ ~
proline and, after 5 min, an Lp( a) antibody solution were added. In Fig. lA the absorbance course of the calibration curve with -the addition of 0.1 mol/l proline in dilution medium and in Fig. lB the same course without addition are represented.
As is apparent, the calibrator having -the highest Lp(a) concentration, i.e., 108 mg/dl, has an absorbance of 0.09 RATES as compared to 0.125 RATES of the same calibrator in dilution medium with 0.1 mol/l proline. ~-By the addition of proline, the reaction signal is, :
thus, intensified. RATES in this case correspond to the measured signal of the antigen-antibody reaction after subtraction of the measured signal that occurred upon admixture of dilution buffer as compared -to the original solution.
Furthermore, it is apparent from the course of the calibration curve that the calibration curve, without addition in the dilution medium, flattens towards -~
higher aoncentrations (Fig. lB), whereas the curve shown in Fig. lA has a linear course.
E~am~le 2: Lp(a) determination with pre-incubation A serum sample having a high inherent -turbidity is incubated for 5 min with dilution buffer containing 0.1 mol/l proline. After this, Lp(a) an-tibodies are added.
The absorbance is measured at a wave length of 340 nm and plotted against -the -time unit (Fig. 2A). The same assay was carried out also without (Fig. 2B) addition i 7 7 !1 ;3 of proline in dilu-tion buffer. From this, it is apparent that the absorbance corresponding to the blank value of the serum sample could be lowered from 0.36 (without addition) to 0.24 (with proline).
The absorbance difference from the time of addition of the antibodies in the reaction course of the mixture with proline (Fig. 2A) is greater by 200 ~ than in the mixture without proline (Fig. 2s). Hence an Lp(a) concentration of 103.3 mg/dl in the mixture with proline, yet, at the same time, a falsely low value of 19.2 mg/dl in the mixture without proline are calcula-ted. In general, an Lp(a) concentration of below 30 is considered as normal, values thereabove being regarded as pathological. In the instant case, the sample, thus, would have been regarded as "normal"
(19.2 mg/dl) on grounds of conventional measuring methods, although the correct concen-tra-tion (determined by the reerence method) clearly constitutes a pathological value.
Example 3: Lp(a) determination with pre-incubation Analogous to Example 2, the Lp(a) content o a sample was turbidimetrically determined by the addition of Lp(a) antibodies. The same reaction course as in :, , .
Figs. 2A and 2B is represented in Figs. 3A and 3B with ~;
the calibrator of the highest Lp( a) concentra-tion (=
108 mg/dl). In this example, the reaction signal (without addition, Example 3B) could be intensified by ,~
~ ~ i ' 7 ~ ~3 ~ .
50 ~ by the addition of 0.1 mol/l proline.
Example 4: Correlation between fresh and thawed samples As mentioned above, discrepancies occurred in the turbidime-trical determination of Lp(a) with sera that had been frozen and rethawed as compared to freshly tested samples because of the turbidity of the thawed ~;~
sample material.
The improvement obtained by the admixture of proline to the agent is demonstrated by the following example:
A panel of 25 serum samples was aliquoted, one portion having been assayed on the same day the blood had been drawn, the other portion having been frozen.
The latter aliquot was thawed on the next day and assayed afterwards.
The resul-ts of the assays without proline are represented in Fi~. 4A. The correlation between the freshly tested and the thawed samples was R = 0.908 and the linear equation was y = 1.01 + 0.88x. In a parallel assay, the thawed samples with proline were determined.
Comparison to the freshly -tested samples is illustrated ~ -in Fig. 4B. The correlation was improved to R = 0.972, also the linear equation with y= 3.52 -~ l.O9x showed a marked improvemen-t in the consistency with the ideal values having the gradient 1.
Example 5: Lp(a) determination by electroimmuno-~' ' 7 -~
diffusion Electroimmunodiffusion serves as a reference method of immunologic Lp(a) determination.
The frozen and thawed samples used in Example 4 were tested in a parallel assay also in electro-immunodiffusion. (Method according to Laurell, Anal.
Biochem. 15 (1966), 45).
In Fig. 5A, the resul-ts of the turbidimetric determination without proline are compared to those determined by elec-troimmunodiffusion. The correlation was R = 0.918 and the linear equation for the elec-troimmunodiffusion curve was y = 1.95 + 0.54x. (y =
1.01 + 0.88x for turbidimetric determina-tion: Fig. 4A).
Thus, there is a distinct difference between the two methods in respect of the results obtained. `
By adding proline, not only the correlation coefficient could be raised to R = 0.989, but also the linear equation y = 3.34 + 0.92x implied an almost perfect consistency of the methods (Fig. 5B); y = 3.52 ~ l.O9x for the turbidimitrical determination in the presence of proline, Fig. 4B).
~"'~ `'~'"
- 13 - ~
Claims (40)
1. A sample preparatory agent intended for treating a lipoprotein containing sample and for determining an antigen by an immunoassay, which sample preparatory agent comprises a dilution buffer having an amino acid content and antibodies directed against said antigen to be determined.
2. A sample preparatory agent as set forth in claim 1, wherein said dilution buffer further comprises a reaction accelerator.
3. A sample preparatory agent intended for treating a lipoprotein containing sample and for determining a lipoprotein by an immunoassay, which sample preparatory agent comprises a dilution buffer having an amino acid content and antibodies directed against said lipoprotein to be determined.
4. A sample preparatory agent as set forth in claim 3, wherein said dilution buffer further comprises a reaction accelerator.
5. A sample preparatory agent intended for treating an Lp(a) containing sample and for determining Lp(a) by an immunoassay, which sample preparatory agent comprises a dilution buffer having an amino acid content and antibodies directed against Lp(a).
6. A sample preparatory agent as set forth in claim 5, wherein said dilution buffer further comprises a reaction accelerator.
7. A sample preparatory agent as set forth in claim 1, 3 or 5, wherein said amino acid is present at a concentration of from 0.05 to 3 M.
8. A sample preparatory agent as set forth in claim 7, wherein said concentration ranges from 0.1 to 0.5 M.
9. A sample preparatory agent as set forth in claim 1, 3 or 5, wherein said amino acid is proline.
10. A sample preparatory agent as set forth in claim 2, 4 or 6, wherein said reaction accelerator is polyethylene glycol.
11. A kit destined for preparing a sample preparatory agent intended for treating a lipoprotein containing sample and for determining an antigen by an immunoassay, which kit comprises a first sample preparatory agent component having a content of buffer substances and of an amino acid, and a second sample preparatory agent component containing antibodies directed against said antigen to be determined by an immunoassay.
12. A kit as set forth in claim 11, wherein said first sample preparatory agent component further has a content of a reaction accelerator.
13. A kit as set forth in claim 11, wherein said amino acid is contained at a concentration of from 0.05 to 3 M in said first sample preparatory agent component.
14. A kit as set forth in claim 13, wherein said concentration ranges from 0.1 to 0.5 M.
15. A kit as set forth in claim 11, wherein said amino acid is proline.
16. A kit as set forth in claim 12, wherein said first sample preparatory agent component comprises polyethylene glycol as said reaction accelerator.
17. A kit as set forth in claim 11, wherein said second sample preparatory agent component comprises antibodies directed against a lipoprotein.
18. A kit as set forth in claim 11, wherein said second sample preparatory agent component comprises antibodies directed against Lp(a).
19. A component comprised by a kit destined for treating a lipoprotein containing sample and for determining an antigen by an immunoassay, which component comprises buffer sustances and an amino acid.
20. A component as set forth in claim 19, wherein said component further comprises a reaction accelerator.
21. A component as set forth in claim 19, wherein said amino acid is contained at a concentration of from 0.05 to 3 M.
22. A component as set forth in claim 21, wherein said concentration ranges from 0.1 to 0.5 M.
23. A component as set forth in claim 19, wherein said amino acid is proline.
24. A component as set forth in claim 20, wherein said reaction accelerator is polyethylene glycol.
25. A method of determining an antigen by an immunoassay, which method comprises providing a sample to be determined by an immunoassay, admixing an amino acid to said sample, introducing antibodies directed against said antigen to be determined by an immunoassay into said sample so as to form an antibody-antigen complex, and determining the amount of said antibody-antigen complex.
26. A method as set forth in claim 25, wherein said antibodies are introduced subsequent to said amino acid.
27. A method as set forth in claim 25, wherein said antibodies are introduced simultaneously with said amino acid.
28. A method as set forth in claim 25, wherein said amino acid is proline.
29. A method as set forth in claim 25, wherein said sample is selected from the group consisting of a blood sample, a serum sample and a plasma sample.
30. A method as set forth in claim 29, wherein said sample is optically turbid due to its content of lipoprotein.
31. A method of reducing or eliminating turbidities in a turbid biological sample, which method comprises adding an amino acid to said turbid biological sample.
32. A method as set forth in claim 31, wherein said amino acid is proline.
33. A method as set forth in claim 31, wherein said sample is selected from the group consisting of a blood sample, a serum sample and a plasma sample.
34. A method as set forth in claim 33, wherein said sample is optically turbid due to its content of lipoprotein.
35. A method of treating a lipoprotein containing sample, which method comprises the step of adding an amino acid to said lipoprotein containing sample.
36. A method as set forth in claim 35, wherein said amino acid is proline.
37. A method as set forth in claim 35, wherein said sample is provided for determining an antigen by an immunoassay.
38. A method of reducing or eliminating turbidities in a biological liquid containing a lipoprotein, which method comprises the step of adding an amino acid to said lipoprotein containing biological liquid.
39. A method as set forth in claim 38, wherein said amino acid is proline.
40. A method as set forth in claim 38, wherein said biological liquid is provided for determining an antigen by an immunoassay.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ATA2043/93 | 1993-10-11 | ||
| AT0204393A AT400199B (en) | 1993-10-11 | 1993-10-11 | TREATMENT OF LIPOPROTE CONTAINING SAMPLES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2117745A1 true CA2117745A1 (en) | 1995-04-12 |
Family
ID=3526548
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002117745A Abandoned CA2117745A1 (en) | 1993-10-11 | 1994-10-07 | Treating of lipoprotein containing samples |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0647850B1 (en) |
| JP (1) | JPH07181182A (en) |
| AT (2) | AT400199B (en) |
| CA (1) | CA2117745A1 (en) |
| DE (1) | DE59409638D1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008079290A2 (en) * | 2006-12-21 | 2008-07-03 | Amgen Inc | Stable buffered formulations containing polypeptides |
| EP3330703A3 (en) * | 2007-06-08 | 2018-08-08 | Quest Diagnostics Investments Incorporated | Lipoprotein analysis by differential charged-particle mobility |
| JP5093087B2 (en) * | 2008-12-18 | 2012-12-05 | 東ソー株式会社 | Method for enhancing immune response with polyethylene glycol and urea |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3107060A1 (en) * | 1981-02-25 | 1982-09-09 | Boehringer Mannheim Gmbh, 6800 Mannheim | CONTROL OR CALIBRATION SERUM AND METHOD FOR THE PRODUCTION THEREOF |
| DE3329952A1 (en) * | 1983-08-19 | 1985-02-28 | Behringwerke Ag, 3550 Marburg | METHOD FOR REDUCING TURBIDITY IN CONTROL SERIES |
| DE3330770A1 (en) * | 1983-08-26 | 1985-03-14 | Behringwerke Ag, 3550 Marburg | METHOD FOR PASTEURIZING HUMAN PLASMA |
| JPS62261962A (en) * | 1986-05-08 | 1987-11-14 | Chemo Sero Therapeut Res Inst | Liquid control serum for lipid component |
-
1993
- 1993-10-11 AT AT0204393A patent/AT400199B/en not_active IP Right Cessation
-
1994
- 1994-10-05 DE DE59409638T patent/DE59409638D1/en not_active Expired - Fee Related
- 1994-10-05 AT AT94890160T patent/ATE198797T1/en not_active IP Right Cessation
- 1994-10-05 EP EP94890160A patent/EP0647850B1/en not_active Expired - Lifetime
- 1994-10-07 CA CA002117745A patent/CA2117745A1/en not_active Abandoned
- 1994-10-11 JP JP6245147A patent/JPH07181182A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07181182A (en) | 1995-07-21 |
| ATA204393A (en) | 1995-02-15 |
| ATE198797T1 (en) | 2001-02-15 |
| DE59409638D1 (en) | 2001-02-22 |
| EP0647850B1 (en) | 2001-01-17 |
| AT400199B (en) | 1995-10-25 |
| EP0647850A3 (en) | 1996-07-31 |
| EP0647850A2 (en) | 1995-04-12 |
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