CA1080619A - Immunological reagent and method of using same - Google Patents
Immunological reagent and method of using sameInfo
- Publication number
- CA1080619A CA1080619A CA243,325A CA243325A CA1080619A CA 1080619 A CA1080619 A CA 1080619A CA 243325 A CA243325 A CA 243325A CA 1080619 A CA1080619 A CA 1080619A
- Authority
- CA
- Canada
- Prior art keywords
- polyethylene glycol
- weight
- reagent
- nonionic surfactant
- mixture
- 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
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Immunological assaying methods which require insolubilization of an antigen-antibody complex as an integral step thereof are improved by adding to the test medium in which insolubilization is desired an aqueous reagent solution containing from about 3 to 6% by weight of a mixture of polyethylene glycol and a nonionic surfactant, and which solution has a calculated HLB (Hydrophilic-Lipophilic-Balance) of about 0.7 to 1.7. This solution increases the insolubiliza-tion of the antigen-antibody complex with reduced incubation times, and produces an assay procedure of greater test range and sensitivity. A preferred embodiment involves usage in a nephelometric analysis.
Immunological assaying methods which require insolubilization of an antigen-antibody complex as an integral step thereof are improved by adding to the test medium in which insolubilization is desired an aqueous reagent solution containing from about 3 to 6% by weight of a mixture of polyethylene glycol and a nonionic surfactant, and which solution has a calculated HLB (Hydrophilic-Lipophilic-Balance) of about 0.7 to 1.7. This solution increases the insolubiliza-tion of the antigen-antibody complex with reduced incubation times, and produces an assay procedure of greater test range and sensitivity. A preferred embodiment involves usage in a nephelometric analysis.
Description
Case H-542-CIP
This invention broadly relates to an immunological reagent capable of enhancing the extent of an immunoprecipita-tion reaction. More specifically, the invention relates to an immunological reagent solution which can be utilized in a wide variety of immunological assaying methods.
The invention also relates to immunological assay-ing methods, in particular those involving a reaction of an antibody with an antigen to form an antibody-antigen complex, - the insolubilization of which is substantially increased by the utilization in the assaying method of the reagent of this invention.
There are numerous immunological assaying methods of widely varying methodology which necessitate at one stage or another in the assay, and for varying purposes, the insolu-bilization of as much of the antigen-antibody complex as possible.
For example, complex insolubilization plays an important role in such illustrative important conventional immunological assaying techniques as electrophoretic analysis, enzymatic assays, radio-immunoassays (RIA) and nephelometric assays, and much work has been directed toward developing means for increasing complex insolubilization in order to improve these assays. Normally, complex insolubilization is necessary in order to isolate the - immunological reaction product from the unreacted immunological reactants involved in a particular assay so that either the ' 25 isolated reaction product or the unreacted reactants can be separately analyzed to provide a meaningful diagnostic assay.
. ... .
. ~
~ -2-,~:
. .. - - - . .
-- , . - - . ~
.
For an immunological assaying method involving, for example, a nephelometric analysis, it is known to dilute test samples with a solution of polyethylene glycol polymer prior to both incubation of the sample and making a reading on the nephelometric instrument. While the polyethylene glycol does improve the nephelometric analysis by increasing the concentra-tion of suspended particles, thereby improving the analysis, there nevertheless remains the problem that satisfactory analysis of many biological constituents cannot be made because of inade-quate test range or sensitivity due to insufficient concentra-tion of suspended particles in the test sample. The broad ob-jective of the present invention is to solve the foregoing problem by providing an improved immunological reagent which is effective to greatly increase the insolubilization of anti-body-antigen complexes beyond that obtainable from polyethylene glycol alone, and hence increase the concentration of suspended particles in the test sample, to permit analysis of biological constituents not possible with polyethylene glycol alone, whether nephelometric or other assaying methods are used.
More specifically, we provide in accordance with the invention a reagent for clinical nephelometric analysis comprising an aqueous solution of an antiserum to the component to be assayed and a mixture of about 20% to about 40% by weight ; polyethylene glycol having a molecular weight of about 200 to about 10,000 and about 80% to about 60% by weight of a block copolymer of ethylene oxide and polyoxypropylene containing at least 50% ethylene oxide.
We also provide in accordance with the invention an immunological assaying method which involves a reaction between an antigen and an antibody to form an antigen-antibody complex, the method being characterized by carrying out said C' .
reaction in the presence of a reagent comprising an aqueous solution containing about 3 to 6% by weight of a mixture of polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than poly-ethylene glycol wherein said mixture contains about 10% to90% by weight polyethylene glycol and about 10% to 90% by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7. Significantly, the aqueous solution when not "in use", as for example in an immunolog-ical assaying method may have a-mixture concentration which is less than 3% and more than 6% and is nevertheless useful to the purpose of this invention providing the concen-tration of the mixture prior to or "in use" is adjusted to have a concentration in the range between about 3% to 6%.
Therefore, by the term "in use", it is our intention to cover a reagent the mixture concentration of which need not initially ; necessarily be in the range between about 3% to 6% so long ~ -as the concentration range, if outside the specified range of -3% to 6%~is adjusted to these limits during or prior to an assaying procedure.
Advantageously, the reagent may be introduced into the test medium for performing a nephelometric, enzymatic, electrophoretic or radioimmuno assay.
When a nephelometric assaying method, for example is conducted for analyzing biological constituents in the form of - antibody-antigen complexes, the reagent and suspended particles of the biological constituents are first incubated whereafter the nephelometric analysis is performed. According to this method, a light source is made to pass through a liquid test sample whereby ~1 .
-. - ' ~ .
the light rays are directed through the suspended particles.
As these light rays strike the particles, they are scattered or diffused at any predeter~ined angle, for example, at a right angle, from the source and are received by photocells.
This scattered light is converted to an electrical signal which is directly proportional to the amount of particulate concentration which, in turn, is thereby accurately measured on a meter face of an lnstrument.
Examples of instruments suitable for nephelometric analyses are the Laser Nephelometer manufactured by Hyland Laboratories ; the Aminco-Fluor ~ lorimeter (American Instru~
ment ~ompany); the ~inco-Bowman Spectrophotofluorometer (SPF):
and thè Autoanalyzer II with attached Fluoronephelometer (Technicon Instruments Corporation).
` By means of ~hese nephelometric principles and equip--ment, the clinical technologist can make an accurate determina-tion of small concentrations of a wide variety of specific pro-teins, for example the immunoglobulins IgG, IgA, IgM, trans-ferrin, complement C3, haptoglobin, alphal-antitrypsin, ~-lipo-protein, albumin, alpha2-macroglobulin, alphal-acid glycoprotein, and various other biological constituents such as triglyc~rides, lipoproteins, and human chorionic gonadotropins.
The most important advantages which result from use of the reagent in accordance with the invention are (a) the incubation times are significantly reduced and (b) greater con-centration of insolubilized complex particles are obtained at sh~rter incubation time with resulting improved test range and .
~ ~5-~_ .... ' !' lV~ilV~
sensitivity These advan~ages apply to any immunological assaying method which is benefited by enhanced insolubilization of the antigen-antibody complex at some point during ~he assay. The advantages also apply to immunological assay methods which rely upon an antigen-antibody complex insolubilization step at some point during the assay procedure.
As above noted, the reagent aqueous solution, in addi-tion to containing, in use, about 3% to 6% by weight of the mix-ture of polyethylene glycol and the nonionic surfactant, should also have a calculated HLB value of between about 0.7 to 1.7.
The HLB value is a well-established measure of the hydrophilic-lipophilic balance (hence "HLB"~ of a given sur-factant. The HLB system of surfactant identification was de-veloped by Atlas Chemical Industries, Inc. and is described in detail on pp. 28-36 of the Atlas publication entitled "Guide to the Use of Atlas Surfactants and Sorbitol in Cosmetic and Pharmaceutical Products" (1965). Each surfactant is - assigned an HLB number. The-lower the HLB num~er, the more lipophilic (or oil-loving) the surfactant while the higher the
This invention broadly relates to an immunological reagent capable of enhancing the extent of an immunoprecipita-tion reaction. More specifically, the invention relates to an immunological reagent solution which can be utilized in a wide variety of immunological assaying methods.
The invention also relates to immunological assay-ing methods, in particular those involving a reaction of an antibody with an antigen to form an antibody-antigen complex, - the insolubilization of which is substantially increased by the utilization in the assaying method of the reagent of this invention.
There are numerous immunological assaying methods of widely varying methodology which necessitate at one stage or another in the assay, and for varying purposes, the insolu-bilization of as much of the antigen-antibody complex as possible.
For example, complex insolubilization plays an important role in such illustrative important conventional immunological assaying techniques as electrophoretic analysis, enzymatic assays, radio-immunoassays (RIA) and nephelometric assays, and much work has been directed toward developing means for increasing complex insolubilization in order to improve these assays. Normally, complex insolubilization is necessary in order to isolate the - immunological reaction product from the unreacted immunological reactants involved in a particular assay so that either the ' 25 isolated reaction product or the unreacted reactants can be separately analyzed to provide a meaningful diagnostic assay.
. ... .
. ~
~ -2-,~:
. .. - - - . .
-- , . - - . ~
.
For an immunological assaying method involving, for example, a nephelometric analysis, it is known to dilute test samples with a solution of polyethylene glycol polymer prior to both incubation of the sample and making a reading on the nephelometric instrument. While the polyethylene glycol does improve the nephelometric analysis by increasing the concentra-tion of suspended particles, thereby improving the analysis, there nevertheless remains the problem that satisfactory analysis of many biological constituents cannot be made because of inade-quate test range or sensitivity due to insufficient concentra-tion of suspended particles in the test sample. The broad ob-jective of the present invention is to solve the foregoing problem by providing an improved immunological reagent which is effective to greatly increase the insolubilization of anti-body-antigen complexes beyond that obtainable from polyethylene glycol alone, and hence increase the concentration of suspended particles in the test sample, to permit analysis of biological constituents not possible with polyethylene glycol alone, whether nephelometric or other assaying methods are used.
More specifically, we provide in accordance with the invention a reagent for clinical nephelometric analysis comprising an aqueous solution of an antiserum to the component to be assayed and a mixture of about 20% to about 40% by weight ; polyethylene glycol having a molecular weight of about 200 to about 10,000 and about 80% to about 60% by weight of a block copolymer of ethylene oxide and polyoxypropylene containing at least 50% ethylene oxide.
We also provide in accordance with the invention an immunological assaying method which involves a reaction between an antigen and an antibody to form an antigen-antibody complex, the method being characterized by carrying out said C' .
reaction in the presence of a reagent comprising an aqueous solution containing about 3 to 6% by weight of a mixture of polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than poly-ethylene glycol wherein said mixture contains about 10% to90% by weight polyethylene glycol and about 10% to 90% by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7. Significantly, the aqueous solution when not "in use", as for example in an immunolog-ical assaying method may have a-mixture concentration which is less than 3% and more than 6% and is nevertheless useful to the purpose of this invention providing the concen-tration of the mixture prior to or "in use" is adjusted to have a concentration in the range between about 3% to 6%.
Therefore, by the term "in use", it is our intention to cover a reagent the mixture concentration of which need not initially ; necessarily be in the range between about 3% to 6% so long ~ -as the concentration range, if outside the specified range of -3% to 6%~is adjusted to these limits during or prior to an assaying procedure.
Advantageously, the reagent may be introduced into the test medium for performing a nephelometric, enzymatic, electrophoretic or radioimmuno assay.
When a nephelometric assaying method, for example is conducted for analyzing biological constituents in the form of - antibody-antigen complexes, the reagent and suspended particles of the biological constituents are first incubated whereafter the nephelometric analysis is performed. According to this method, a light source is made to pass through a liquid test sample whereby ~1 .
-. - ' ~ .
the light rays are directed through the suspended particles.
As these light rays strike the particles, they are scattered or diffused at any predeter~ined angle, for example, at a right angle, from the source and are received by photocells.
This scattered light is converted to an electrical signal which is directly proportional to the amount of particulate concentration which, in turn, is thereby accurately measured on a meter face of an lnstrument.
Examples of instruments suitable for nephelometric analyses are the Laser Nephelometer manufactured by Hyland Laboratories ; the Aminco-Fluor ~ lorimeter (American Instru~
ment ~ompany); the ~inco-Bowman Spectrophotofluorometer (SPF):
and thè Autoanalyzer II with attached Fluoronephelometer (Technicon Instruments Corporation).
` By means of ~hese nephelometric principles and equip--ment, the clinical technologist can make an accurate determina-tion of small concentrations of a wide variety of specific pro-teins, for example the immunoglobulins IgG, IgA, IgM, trans-ferrin, complement C3, haptoglobin, alphal-antitrypsin, ~-lipo-protein, albumin, alpha2-macroglobulin, alphal-acid glycoprotein, and various other biological constituents such as triglyc~rides, lipoproteins, and human chorionic gonadotropins.
The most important advantages which result from use of the reagent in accordance with the invention are (a) the incubation times are significantly reduced and (b) greater con-centration of insolubilized complex particles are obtained at sh~rter incubation time with resulting improved test range and .
~ ~5-~_ .... ' !' lV~ilV~
sensitivity These advan~ages apply to any immunological assaying method which is benefited by enhanced insolubilization of the antigen-antibody complex at some point during ~he assay. The advantages also apply to immunological assay methods which rely upon an antigen-antibody complex insolubilization step at some point during the assay procedure.
As above noted, the reagent aqueous solution, in addi-tion to containing, in use, about 3% to 6% by weight of the mix-ture of polyethylene glycol and the nonionic surfactant, should also have a calculated HLB value of between about 0.7 to 1.7.
The HLB value is a well-established measure of the hydrophilic-lipophilic balance (hence "HLB"~ of a given sur-factant. The HLB system of surfactant identification was de-veloped by Atlas Chemical Industries, Inc. and is described in detail on pp. 28-36 of the Atlas publication entitled "Guide to the Use of Atlas Surfactants and Sorbitol in Cosmetic and Pharmaceutical Products" (1965). Each surfactant is - assigned an HLB number. The-lower the HLB num~er, the more lipophilic (or oil-loving) the surfactant while the higher the
2~ number, the more hydrophilic (or water-loving? the surfactant.
The method for establishing the HLB value of a~y given sur-factant is well known and is described, for example, in a publicly released Atlas printed publication entitled "The Atlas HLB
System" (Code LD~97). The HLB values of numerous surfactants are also published widely in the literature, particularly liter-ature put out by the manufacturer of the surfactant in question , .
.
.
i~.. ~ , .
.
lV~
The HLB value of a blend of surfactants, such as exist in the reagent of this invention, is a function of the concentration of each surfactant in the blend, and hence the concentration of the individual surfactants must be taken into account in computing an HLB value for the blend. The HLB value of the blend is calculated, in accordance with ac-- cepted published procedures, by multiplying the assigned HLB
value of each surfactant present in the blend by its concen-tration in the composition in question, and adding the individual calculated results. For example, if the reagent of this inven-tion contained 1% by weight polyethylene glycol (HLB = 20) and 3% by weight of a nonionic surfactant having an HLB of 30.5, the HLB value of the reagent would be computed as follows:
0.0~ X 20 = 0.2 0.03 X 30.5 = 0~915 1.115 -- HLB value of reagent To determine whether a given reagent has an HLB value which falls within the 0.7 to 1.7 range of the reagent of this in-vention a calculation as above, based on the amount of each component in the blend, can be readily made, using either pub-lished HLB values for the components in question or HLB values determined according to the Atlas method.
The present invention contemplates the use of a wide variety of nonionic surfactants in conjunction with the polyethylene glycol provided that (1) the mixture of nonionic surfactant and polyethylene glycol falls within a range of 3 to 6% by weight at the time the reagent is used in the assay, 1~8~
and (2) that the calculated HLB value of the reagent falls within a range of 0.7 to 1.7 at the same time. If at the time of use, the polyethylene glycol-nonionic surfactant mix-ture accounts for less than 3% of the reagent, or if the re-agent has an HLB less than about 0.7, it becomes difficultto insolubilize the desired amount of antigen-antibody com-plex to the extent required for a successful completion of the immunological assay. On the other hand, if the mixture accounts for more than 6% of the reagent, or if the reagent has an HLB greater than about 1.7, other proteins besides the desired antigen-antibody complex are insolubilized, thereby destroying the selectivity of the assay and rendering the re-sults meaningless.
Of course, the reagent solution could, for a variety of reasons, be prepared and marketed in such manner that it did not contain the requisite 3 to 6% mixture of polyethylene glycol and nonionic surfactant, or have the requisite HLB value of 0.7 to 1.7. Poor shelf stability could be one such reason. However, as above noted, such solutions would require adjustment, prior to or at some point during their usage in an immunological assay technique, to the 3 to 6% range and to an HLB value of about 0.7 to 1.7. For this reason, reagents which do not contain the requisite 3 to 6% of said mixture or an HLB value of about 0.7 to 1.7, but which can be readily adjusted to these values prior to or during the immunological test procedure, are within the scope of the invention, even though they may initially be out-side the specific parameters of concentration and HLB range .: ............ .
required during the assay procedure itself.
For example, the marketed reagent solution may re-quire, if the specific parameters of concentration and HLB
values fall outside the ranges of about 3% to 6% and about 0.7 to 1.7, respectively, dilution, concentration, Qr other adjustment steps prior to or at some convenient point during performance of the immunological assay method, for the purpose of bringing the solution to a mixture level of 3 to 6~/o and a cal-culated HLB value of 0.7 to 1.7. In such circumstances, the marketed solution would still fall within the scope of the present invention Thus the reagent could be marketed at a mixture con-centration higher than 6%, e.g., 8%, which would be diluted either prior to or at some suitable point during an immunological assay procedure to the 3 to 6% range. Similarly, the reagent could be marketed with a calculated HLB value outside the 0.7 to 1.7 range, but with the requirement that the reagent solution be in someway altered prior to or at some suitable point during an immunological assay procedure to bring it to a calculated HLB
range of 0.7 to 1.7.
The reagent could in certain cases be diluted ~o the proper levels with the antiserum to be used in the test, while in other cases it could be diluted with the test sample or in some cases with both the test sample and antiserum. The im-portant point is that at some point during the immunological assay procedure, usually either prior to or at the time of the antigen-antibody reaction, the requisite level of 3 to 6% of combined polyethylene glycol and nonionic surfactant, and the requisite HLB value of 0.7 to 1.7, must be provided in order for the reagent of ~he invention to function properly in the _g_ -..
assay procedure. The point in the assay procedure at which these requisite parameters must be p~ovided can vary depend-ing on the particular procedure involved. For a nephelometric analysis, for example, it is convenient to prepare and market the reagent of the invention with a concentration of poly-ethylene glycol and nonionic surfactant in excess of 6% and at an HLB value outside the 0.7 to 1.7 range. Then, ~ust prior to usage in the nephelometric analysis, the reagent is diluted with the antiserum to be used in the analysis to pro-vide a concentration of polyethylene glycol and nonionic sur-factant of between 3 to 6%,preferably about 4%, and a calcu-lated HLB value within the 0.7 to 1.7 range.
The relative proportions of polyethylene glycol and nonionic surfactant in the mixture can vary within wide limits, depending largely on the surfactant being used. Illustratively, the mixture contains from about 10 to 90% by weight polyethylene glycol and 10 to 90% nonionic surfactant. Preferably, the mix-ture contains about 15 to 85% polyethylene glycol and 15 to 85Z
nonionic surfactant.
One or more different forms or types o polyethylene glycol can be used as the polyethylene glycol component of the mixture. The polyethylene glycol polymer used generally has a molecular weight of from about 200 to about 10,000, and preferably-from about 4,000 to about 6,000- A ~olecular weight range of a~ut 4,000 is especially preferred.
.
~ A -lo-.
The nonionic surfactant component of the mixture can be any nonionic surfactant which will produce in the re-agent solution a calculated HLB value of 0.7 to 1.7, and pre-ferably about 0.7 to 1.3, at a mixture concentration of 3 to 6%. Illustratively, the HLB values of the nonionic surfac-tant itself can range from about 10 to 30 more.
A particularly preferred nonionic surfactant is a block copolymer of ethylene oxide and polyoxypropylene.
This particular polymer and its preparation is described in U.S. Patent 2,674,619. These block copolymers are generally prepared by condensing ethylene oxide with polyoxypropylene polymer and can be represented by the following structural formula:
HO(CH2CH2O)a (CH3CHCH2O~b (CH2CH2O)c H-For purposes of this invention these block copolymers desirablycontain at least 50% ethylene oxide in the molecule and a poly-- 15 oxypropylene hydrophobic base molecular weight of at least 950, similarly as described in U~S. Patents 3,450,502, 3,577,522 and
The method for establishing the HLB value of a~y given sur-factant is well known and is described, for example, in a publicly released Atlas printed publication entitled "The Atlas HLB
System" (Code LD~97). The HLB values of numerous surfactants are also published widely in the literature, particularly liter-ature put out by the manufacturer of the surfactant in question , .
.
.
i~.. ~ , .
.
lV~
The HLB value of a blend of surfactants, such as exist in the reagent of this invention, is a function of the concentration of each surfactant in the blend, and hence the concentration of the individual surfactants must be taken into account in computing an HLB value for the blend. The HLB value of the blend is calculated, in accordance with ac-- cepted published procedures, by multiplying the assigned HLB
value of each surfactant present in the blend by its concen-tration in the composition in question, and adding the individual calculated results. For example, if the reagent of this inven-tion contained 1% by weight polyethylene glycol (HLB = 20) and 3% by weight of a nonionic surfactant having an HLB of 30.5, the HLB value of the reagent would be computed as follows:
0.0~ X 20 = 0.2 0.03 X 30.5 = 0~915 1.115 -- HLB value of reagent To determine whether a given reagent has an HLB value which falls within the 0.7 to 1.7 range of the reagent of this in-vention a calculation as above, based on the amount of each component in the blend, can be readily made, using either pub-lished HLB values for the components in question or HLB values determined according to the Atlas method.
The present invention contemplates the use of a wide variety of nonionic surfactants in conjunction with the polyethylene glycol provided that (1) the mixture of nonionic surfactant and polyethylene glycol falls within a range of 3 to 6% by weight at the time the reagent is used in the assay, 1~8~
and (2) that the calculated HLB value of the reagent falls within a range of 0.7 to 1.7 at the same time. If at the time of use, the polyethylene glycol-nonionic surfactant mix-ture accounts for less than 3% of the reagent, or if the re-agent has an HLB less than about 0.7, it becomes difficultto insolubilize the desired amount of antigen-antibody com-plex to the extent required for a successful completion of the immunological assay. On the other hand, if the mixture accounts for more than 6% of the reagent, or if the reagent has an HLB greater than about 1.7, other proteins besides the desired antigen-antibody complex are insolubilized, thereby destroying the selectivity of the assay and rendering the re-sults meaningless.
Of course, the reagent solution could, for a variety of reasons, be prepared and marketed in such manner that it did not contain the requisite 3 to 6% mixture of polyethylene glycol and nonionic surfactant, or have the requisite HLB value of 0.7 to 1.7. Poor shelf stability could be one such reason. However, as above noted, such solutions would require adjustment, prior to or at some point during their usage in an immunological assay technique, to the 3 to 6% range and to an HLB value of about 0.7 to 1.7. For this reason, reagents which do not contain the requisite 3 to 6% of said mixture or an HLB value of about 0.7 to 1.7, but which can be readily adjusted to these values prior to or during the immunological test procedure, are within the scope of the invention, even though they may initially be out-side the specific parameters of concentration and HLB range .: ............ .
required during the assay procedure itself.
For example, the marketed reagent solution may re-quire, if the specific parameters of concentration and HLB
values fall outside the ranges of about 3% to 6% and about 0.7 to 1.7, respectively, dilution, concentration, Qr other adjustment steps prior to or at some convenient point during performance of the immunological assay method, for the purpose of bringing the solution to a mixture level of 3 to 6~/o and a cal-culated HLB value of 0.7 to 1.7. In such circumstances, the marketed solution would still fall within the scope of the present invention Thus the reagent could be marketed at a mixture con-centration higher than 6%, e.g., 8%, which would be diluted either prior to or at some suitable point during an immunological assay procedure to the 3 to 6% range. Similarly, the reagent could be marketed with a calculated HLB value outside the 0.7 to 1.7 range, but with the requirement that the reagent solution be in someway altered prior to or at some suitable point during an immunological assay procedure to bring it to a calculated HLB
range of 0.7 to 1.7.
The reagent could in certain cases be diluted ~o the proper levels with the antiserum to be used in the test, while in other cases it could be diluted with the test sample or in some cases with both the test sample and antiserum. The im-portant point is that at some point during the immunological assay procedure, usually either prior to or at the time of the antigen-antibody reaction, the requisite level of 3 to 6% of combined polyethylene glycol and nonionic surfactant, and the requisite HLB value of 0.7 to 1.7, must be provided in order for the reagent of ~he invention to function properly in the _g_ -..
assay procedure. The point in the assay procedure at which these requisite parameters must be p~ovided can vary depend-ing on the particular procedure involved. For a nephelometric analysis, for example, it is convenient to prepare and market the reagent of the invention with a concentration of poly-ethylene glycol and nonionic surfactant in excess of 6% and at an HLB value outside the 0.7 to 1.7 range. Then, ~ust prior to usage in the nephelometric analysis, the reagent is diluted with the antiserum to be used in the analysis to pro-vide a concentration of polyethylene glycol and nonionic sur-factant of between 3 to 6%,preferably about 4%, and a calcu-lated HLB value within the 0.7 to 1.7 range.
The relative proportions of polyethylene glycol and nonionic surfactant in the mixture can vary within wide limits, depending largely on the surfactant being used. Illustratively, the mixture contains from about 10 to 90% by weight polyethylene glycol and 10 to 90% nonionic surfactant. Preferably, the mix-ture contains about 15 to 85% polyethylene glycol and 15 to 85Z
nonionic surfactant.
One or more different forms or types o polyethylene glycol can be used as the polyethylene glycol component of the mixture. The polyethylene glycol polymer used generally has a molecular weight of from about 200 to about 10,000, and preferably-from about 4,000 to about 6,000- A ~olecular weight range of a~ut 4,000 is especially preferred.
.
~ A -lo-.
The nonionic surfactant component of the mixture can be any nonionic surfactant which will produce in the re-agent solution a calculated HLB value of 0.7 to 1.7, and pre-ferably about 0.7 to 1.3, at a mixture concentration of 3 to 6%. Illustratively, the HLB values of the nonionic surfac-tant itself can range from about 10 to 30 more.
A particularly preferred nonionic surfactant is a block copolymer of ethylene oxide and polyoxypropylene.
This particular polymer and its preparation is described in U.S. Patent 2,674,619. These block copolymers are generally prepared by condensing ethylene oxide with polyoxypropylene polymer and can be represented by the following structural formula:
HO(CH2CH2O)a (CH3CHCH2O~b (CH2CH2O)c H-For purposes of this invention these block copolymers desirablycontain at least 50% ethylene oxide in the molecule and a poly-- 15 oxypropylene hydrophobic base molecular weight of at least 950, similarly as described in U~S. Patents 3,450,502, 3,577,522 and
3,590,125.
Illustrative examples of such suitable block copolymers are the F-38 and F-68 PLURONIC~ polyols sold by Wyandotte Chemicals Corporation. F-38 contains 80% of polyoxyethylene hydrophilic ; units in the molecule and the polyoxypropylene hydrophobic base has a molecular weight of 950. F-38 is a particularly preferred nonionic surfactant. F-68 also contains 80% of polyoxyethylene hydrophilic units in the molecule but the hydrophobic base has a molecular weight of 1,750. The total molecular weight of these two PLURONIC ~ polyols is 4,750 and 8,750, respectively. PLURONIC
L-125 has also been found useful. ~ further description of these .
"` . ` lV~
!
polyols is found in the bulletin of Wyandotte Chemicals Corpor-~` ation, "The Pluronic Grid" Sixth Edition.
In the case of the PLURONIC nonionic surfactants, a mixture consisting of about 20% to 40% by weight polyethylene glycol and about 80% to 6Q% block copolymer of ethylene oxide and polyoxypropylene polymer is generally preferred.
A highly preferred reagent solution contains about one part by weight of polyethylene glycol having a molecular weight ~ bout 4,000 and about three parts by weight of the lC Pluronic F-3~ material. This solution can be prepared in a saline solvent (e.g., 0.9% NaCl) either at, above or below the desired level of 3 to 6% and then adjusted, if necessary, to -the desired level of 3 to 6%. Preferably, the solution is pre--- pared as an 8% solution o~ the polymer mixture in saline which is then diluted with antiserum (see Examples 1 and 5 below) or another suitable diluent prior to its usage in an imm~nological assay procedure. The diluted reagent thus con~ains about 1%
polyethylene glycol and 3% F-38. Its HLB value of l.lt5 can be calculated as follows:
1% PEG: 0.01 X 20* = 0.2 3% F-38: 0.03 X 30.5~* = ~.15 1.115 A wide variety of other nonionic surfactants can i also be used to complement the polyethylene glycol in the re-agent solution of the invention, provided they provide the desired HLB value of 0.7 to 1.7 at a mixture concentration of * the HLB of PEG from the literature i5 20 **the HLB of F-38 from the literature is 30~5 Utj~
3 to 6%. For example, the TETRONIC ~ series of nonionic sur-factants available from BASF Wyandotte Corporation and described in detail in the BASF Wyandotte Brc~chure entitled "Technical Data on Tetronic ~ Series Nonionic Surfactants" and in U.S. Patent 2,979.52~ haYe been found quite suitable, particularly those identified as TETROrlIC ~ 707,908, 1107, 1307 and 1508. The TETRONIC Q products are based on ethylene diamine and have the general formula:
H(C2H40)y(C3H60)x\ /~C3}~'6)x(C2H4)yH
H(c2H4o)y(c3H6o)x / (C3H6o)x(c2H4o)yH
The molecular weight range of the TETRONIC ~ line can vary from about 1,650 to over 26,00Q. The more preferred TETRONIC ~ sur-factants have molecular weights in the range of about 15,000 to 27,000 and HLB values of about 20 to 30.5. The proportion of polyethylene glycol to TETRONIC ~ surfactant in the mixture can vary widely, as discussed above.
Another family of useful nonionics is the PLURAFAC
line of products also put out by BASF Wyandotte, particularly- 20 those identified as PLURAFAC ~ 17R8, 25R8~ D25, A38 and A39.
The PLURAFAC ~ products are straight chain, primary aliphatic oxyalkylated alcohols described in greater detail in the BASF
Wyandotte brochure entitled "Technical Data on Typical Physical `. Properties of PLURAFAC ~ Nonionic Surfactants", The preferred PLURAFAC ~ products have HLB values of about 10 to 20 and can be used in widely varying ratios with the polyethylene glycol.
.. . . . ... .. . . . . ~, ;~08~
Other suitable nonionic surfactants include glycerol monostearate and the family of alkyl aryl sulonates. A preferred glycerol monostearate is available from Atlas Industries, Inc.
under the tradename ARLACEL ~ 165 (HLB = 11.0) while a typical useful alkyl aryl sulfonate is also available from the same source under the tradename G-3300 (HLB = 11.7).
The above listing on nonionic surfactants is illus-trative only since other such surfactants are contemplated to fall within the scope of the invention provided they provide a reagent which, when used in an immunological assay procedure, contains about 3 to 6% of a mixture of polyethylene glycol and surfactant, has a calculated HLB value based on the values of the individual components present in the solution of about 0.7 to 1.7, and produces the effect of enhancing the insolubilization of the desired antigen-antibody complex to the extent desired to improve the assay, and without insolubilizing unwanted com-ponents or in any other way detrimentally interfering with the assay procedure.
Of course, more than one nonionic surfactant can be present with the polyethylene glycol provided the requisite con-centration of the mixture and HLB value of the solution are achieved.
In selecting a nonionic surfactant for use in the invention, one should be used which also has the capability of providing a clear reagent solution at least at the time the re-agent solution is first used in the immunological assay procedure.
This is to insure that the reagent does not interfere with the test results, or does not produce non-specific precipitation of ,~
i~utiis~
the components of the biological sample or the biological reagents employed in the assay system.
The reagent system of the invention finds broad utility as an improvement in a host of conventional immunological assay methods which at one time or another, for whatever the reason, require a step to insolubilize the antigen-antibody complex formed during the assay procedure by the antigen~antibody reaction. Such assay methods are well known to those skilled in the art and need not be described in detail herein. The applicability and usefulness of the present invention in these various assay methods, and the details of how to use the reagent of the invention in such procedures would be apparent to those skilled in the art from a reading of this specification and thus these specifics bear no exhaustive repeating herein~ For example, the reagent of the invention could be used to enhance any sys-tem that depends upon the precipitation of antigen-antibody com-plexes to produce a clear supernatant fluid for fluorescence or colormetric detection. The reagent could also be used to remove interfering substances found in biological fluids or reagents (e.g., lipids, salts, and extraneous proteins) for nephelometry, enzymatic or other assay systems~ This is acco~plished by the removal of the reactants from the reaction solution for the purpose of washing and resuspending these reactants.
Specifically, the reagent of the invention may be utilized to increase the relative concentration of insoluble antigen-antibody complexes and increase a given assay system's test rsnge and sensitLvity. These principles msy be spplied ;
.
.
to quantitative light scattering from immune complexes by nephelometry, this being at present a preferred embodiment of the invention. Nephelometric analyses using the reagent of the invention are particularly useful in the analysis of various immunoglobulins. However, other test systems that are based on precipitating antibody may utilize these reagents, such as radioimmunodiffusion (RID), enzymatic analyses, and various types of electrophoretic techniques such as immuno-electrophoresis (IEP), counterelectrophoresis (CEP), and electroimmunodiffusion (EID).
The reagent of the invention may, for example, be used in enhancing immunological assays that depend upon the primary interaction of antigen-antibody coprecipitation tech-nique commonly used in radioimmunoassays (RIA). This enhanced ; 15 insolubilization characteristic of the present invention can also be utilized in the various methodologies of attachm~nt of antibodies or antigen to inert particles used as a carrier in RIA, nephelometric, and fluorometric assays in a manner well understood by those skilled in the art.
Numerous radioimmunoassay techniques have been adopted to quantitate relatively small concentrations of biological ; constituents found in body fluids~ Isotopically labeled antigen or antibody is reacted with the homologous antigen or antiserum to produce labeled immune antigen-antibody complexes. These com-plexes must then normally be rendered insoluble by an insoluble carrier, precipitating reagent or other known techniques. The free or non-reacted labeled antigen or antibody can be re ved by wa~hing techniques. The radioactive concentra~ion of the precipitated complexes is then determined by gamma or liquid io~a~is scintillation counting. An example of an instrument used for assa~s of this type is the Autogam~la~ Counter (available from Nuclear Chicago, Inc.). The reagent in accordance with this invention is useful to enhance the requisite complex insolubiliza-tion step, thus improving the analysis. Example 6 below details the use of the reagent of the invention in a radioimmunoassay procedure.
Enzymatic techniques have also been adopted to quan-titate small concentrations of biological constituents found in body fluids. Enzyme labeled antigen or antibody is reacted with the specific antibody or antigen to produce immune labeled antigen-antibody complexes. These complexes are rendered in-soluble by an insoluble carrier, precipitating reagent or other technique. The free or non-reacted enzyme labeled antibody or antigen may be removed by washing techniques. The bound or reacted enzyme can then produce a reaction with an appropriate - substrate to produce a colored or fluorescent supernant solu-tion which can be measured with a spectrophotometer or-fluoro-meter. C~xa~ les of instruments useful for this purpose are the Beckman DB ~pectrophotometer (available from Beckman ~nstruments, Inc.) and the Aminco-Bowma ~ Spectrophotofluorometer (available from the American Instrument Company). The reagent in accordance with the invention is also useful to enhance the requisite com-plex insolu~ilization step, thus improving the analysis.
The usefulness of the reagent in a nephelometric analysis has been discussed in detail above~ and is further exem~
plified in Example 5 below.
.
.
B - n .. .. , . ,~. ,.,j .. .. .. ..
It is now clear to those skilled in the art that the improved immunological reagent of the invention has wide ranging utility in the field of immunological assay procedures.
Using the improved reagent, the clinical technologist can make accurate determination of a broad range of concentrations of many specific proteins, for example, the immonoglobulins IgG, IgA, IgM, transferrin, complement C3, haptoglobin, alphal-antitrypsin, B-lipoprotein, albumin, alpha2-macroglobulin, alphal-acid glyco-protein, and various other biological constituents such as tri-iodothyonine (T3), thyroxine (T4), triglycerides, human chorionicgonadotropins, lipoproteins, and many others whose determination would benefit from the enhanced insolubilization effect produced by the present invention.
In st of the applications in which the invention finds utility, the desired test biological constituent or con-stituents are admixed with the aqueous reagent solution of the nvention, incubated for a predetermined period of time such as, for example, at room temperature (2Q-25~C) for approximately one hour, and then read on appropriate instrumentation, e..g, a nephelometer in the case of a nephelometric analysis. The results of the test samples are compared with reference samples to determine the unknown concentration.
The following examples are intended to further illus-trate the invention, although it will be understood that the in-vention is not limited to these specific examples.', . .
A reagent solution is prepared by admixing 25 parts by weight of polyethylene glycol having a molecular weight of .
c~
Illustrative examples of such suitable block copolymers are the F-38 and F-68 PLURONIC~ polyols sold by Wyandotte Chemicals Corporation. F-38 contains 80% of polyoxyethylene hydrophilic ; units in the molecule and the polyoxypropylene hydrophobic base has a molecular weight of 950. F-38 is a particularly preferred nonionic surfactant. F-68 also contains 80% of polyoxyethylene hydrophilic units in the molecule but the hydrophobic base has a molecular weight of 1,750. The total molecular weight of these two PLURONIC ~ polyols is 4,750 and 8,750, respectively. PLURONIC
L-125 has also been found useful. ~ further description of these .
"` . ` lV~
!
polyols is found in the bulletin of Wyandotte Chemicals Corpor-~` ation, "The Pluronic Grid" Sixth Edition.
In the case of the PLURONIC nonionic surfactants, a mixture consisting of about 20% to 40% by weight polyethylene glycol and about 80% to 6Q% block copolymer of ethylene oxide and polyoxypropylene polymer is generally preferred.
A highly preferred reagent solution contains about one part by weight of polyethylene glycol having a molecular weight ~ bout 4,000 and about three parts by weight of the lC Pluronic F-3~ material. This solution can be prepared in a saline solvent (e.g., 0.9% NaCl) either at, above or below the desired level of 3 to 6% and then adjusted, if necessary, to -the desired level of 3 to 6%. Preferably, the solution is pre--- pared as an 8% solution o~ the polymer mixture in saline which is then diluted with antiserum (see Examples 1 and 5 below) or another suitable diluent prior to its usage in an imm~nological assay procedure. The diluted reagent thus con~ains about 1%
polyethylene glycol and 3% F-38. Its HLB value of l.lt5 can be calculated as follows:
1% PEG: 0.01 X 20* = 0.2 3% F-38: 0.03 X 30.5~* = ~.15 1.115 A wide variety of other nonionic surfactants can i also be used to complement the polyethylene glycol in the re-agent solution of the invention, provided they provide the desired HLB value of 0.7 to 1.7 at a mixture concentration of * the HLB of PEG from the literature i5 20 **the HLB of F-38 from the literature is 30~5 Utj~
3 to 6%. For example, the TETRONIC ~ series of nonionic sur-factants available from BASF Wyandotte Corporation and described in detail in the BASF Wyandotte Brc~chure entitled "Technical Data on Tetronic ~ Series Nonionic Surfactants" and in U.S. Patent 2,979.52~ haYe been found quite suitable, particularly those identified as TETROrlIC ~ 707,908, 1107, 1307 and 1508. The TETRONIC Q products are based on ethylene diamine and have the general formula:
H(C2H40)y(C3H60)x\ /~C3}~'6)x(C2H4)yH
H(c2H4o)y(c3H6o)x / (C3H6o)x(c2H4o)yH
The molecular weight range of the TETRONIC ~ line can vary from about 1,650 to over 26,00Q. The more preferred TETRONIC ~ sur-factants have molecular weights in the range of about 15,000 to 27,000 and HLB values of about 20 to 30.5. The proportion of polyethylene glycol to TETRONIC ~ surfactant in the mixture can vary widely, as discussed above.
Another family of useful nonionics is the PLURAFAC
line of products also put out by BASF Wyandotte, particularly- 20 those identified as PLURAFAC ~ 17R8, 25R8~ D25, A38 and A39.
The PLURAFAC ~ products are straight chain, primary aliphatic oxyalkylated alcohols described in greater detail in the BASF
Wyandotte brochure entitled "Technical Data on Typical Physical `. Properties of PLURAFAC ~ Nonionic Surfactants", The preferred PLURAFAC ~ products have HLB values of about 10 to 20 and can be used in widely varying ratios with the polyethylene glycol.
.. . . . ... .. . . . . ~, ;~08~
Other suitable nonionic surfactants include glycerol monostearate and the family of alkyl aryl sulonates. A preferred glycerol monostearate is available from Atlas Industries, Inc.
under the tradename ARLACEL ~ 165 (HLB = 11.0) while a typical useful alkyl aryl sulfonate is also available from the same source under the tradename G-3300 (HLB = 11.7).
The above listing on nonionic surfactants is illus-trative only since other such surfactants are contemplated to fall within the scope of the invention provided they provide a reagent which, when used in an immunological assay procedure, contains about 3 to 6% of a mixture of polyethylene glycol and surfactant, has a calculated HLB value based on the values of the individual components present in the solution of about 0.7 to 1.7, and produces the effect of enhancing the insolubilization of the desired antigen-antibody complex to the extent desired to improve the assay, and without insolubilizing unwanted com-ponents or in any other way detrimentally interfering with the assay procedure.
Of course, more than one nonionic surfactant can be present with the polyethylene glycol provided the requisite con-centration of the mixture and HLB value of the solution are achieved.
In selecting a nonionic surfactant for use in the invention, one should be used which also has the capability of providing a clear reagent solution at least at the time the re-agent solution is first used in the immunological assay procedure.
This is to insure that the reagent does not interfere with the test results, or does not produce non-specific precipitation of ,~
i~utiis~
the components of the biological sample or the biological reagents employed in the assay system.
The reagent system of the invention finds broad utility as an improvement in a host of conventional immunological assay methods which at one time or another, for whatever the reason, require a step to insolubilize the antigen-antibody complex formed during the assay procedure by the antigen~antibody reaction. Such assay methods are well known to those skilled in the art and need not be described in detail herein. The applicability and usefulness of the present invention in these various assay methods, and the details of how to use the reagent of the invention in such procedures would be apparent to those skilled in the art from a reading of this specification and thus these specifics bear no exhaustive repeating herein~ For example, the reagent of the invention could be used to enhance any sys-tem that depends upon the precipitation of antigen-antibody com-plexes to produce a clear supernatant fluid for fluorescence or colormetric detection. The reagent could also be used to remove interfering substances found in biological fluids or reagents (e.g., lipids, salts, and extraneous proteins) for nephelometry, enzymatic or other assay systems~ This is acco~plished by the removal of the reactants from the reaction solution for the purpose of washing and resuspending these reactants.
Specifically, the reagent of the invention may be utilized to increase the relative concentration of insoluble antigen-antibody complexes and increase a given assay system's test rsnge and sensitLvity. These principles msy be spplied ;
.
.
to quantitative light scattering from immune complexes by nephelometry, this being at present a preferred embodiment of the invention. Nephelometric analyses using the reagent of the invention are particularly useful in the analysis of various immunoglobulins. However, other test systems that are based on precipitating antibody may utilize these reagents, such as radioimmunodiffusion (RID), enzymatic analyses, and various types of electrophoretic techniques such as immuno-electrophoresis (IEP), counterelectrophoresis (CEP), and electroimmunodiffusion (EID).
The reagent of the invention may, for example, be used in enhancing immunological assays that depend upon the primary interaction of antigen-antibody coprecipitation tech-nique commonly used in radioimmunoassays (RIA). This enhanced ; 15 insolubilization characteristic of the present invention can also be utilized in the various methodologies of attachm~nt of antibodies or antigen to inert particles used as a carrier in RIA, nephelometric, and fluorometric assays in a manner well understood by those skilled in the art.
Numerous radioimmunoassay techniques have been adopted to quantitate relatively small concentrations of biological ; constituents found in body fluids~ Isotopically labeled antigen or antibody is reacted with the homologous antigen or antiserum to produce labeled immune antigen-antibody complexes. These com-plexes must then normally be rendered insoluble by an insoluble carrier, precipitating reagent or other known techniques. The free or non-reacted labeled antigen or antibody can be re ved by wa~hing techniques. The radioactive concentra~ion of the precipitated complexes is then determined by gamma or liquid io~a~is scintillation counting. An example of an instrument used for assa~s of this type is the Autogam~la~ Counter (available from Nuclear Chicago, Inc.). The reagent in accordance with this invention is useful to enhance the requisite complex insolubiliza-tion step, thus improving the analysis. Example 6 below details the use of the reagent of the invention in a radioimmunoassay procedure.
Enzymatic techniques have also been adopted to quan-titate small concentrations of biological constituents found in body fluids. Enzyme labeled antigen or antibody is reacted with the specific antibody or antigen to produce immune labeled antigen-antibody complexes. These complexes are rendered in-soluble by an insoluble carrier, precipitating reagent or other technique. The free or non-reacted enzyme labeled antibody or antigen may be removed by washing techniques. The bound or reacted enzyme can then produce a reaction with an appropriate - substrate to produce a colored or fluorescent supernant solu-tion which can be measured with a spectrophotometer or-fluoro-meter. C~xa~ les of instruments useful for this purpose are the Beckman DB ~pectrophotometer (available from Beckman ~nstruments, Inc.) and the Aminco-Bowma ~ Spectrophotofluorometer (available from the American Instrument Company). The reagent in accordance with the invention is also useful to enhance the requisite com-plex insolu~ilization step, thus improving the analysis.
The usefulness of the reagent in a nephelometric analysis has been discussed in detail above~ and is further exem~
plified in Example 5 below.
.
.
B - n .. .. , . ,~. ,.,j .. .. .. ..
It is now clear to those skilled in the art that the improved immunological reagent of the invention has wide ranging utility in the field of immunological assay procedures.
Using the improved reagent, the clinical technologist can make accurate determination of a broad range of concentrations of many specific proteins, for example, the immonoglobulins IgG, IgA, IgM, transferrin, complement C3, haptoglobin, alphal-antitrypsin, B-lipoprotein, albumin, alpha2-macroglobulin, alphal-acid glyco-protein, and various other biological constituents such as tri-iodothyonine (T3), thyroxine (T4), triglycerides, human chorionicgonadotropins, lipoproteins, and many others whose determination would benefit from the enhanced insolubilization effect produced by the present invention.
In st of the applications in which the invention finds utility, the desired test biological constituent or con-stituents are admixed with the aqueous reagent solution of the nvention, incubated for a predetermined period of time such as, for example, at room temperature (2Q-25~C) for approximately one hour, and then read on appropriate instrumentation, e..g, a nephelometer in the case of a nephelometric analysis. The results of the test samples are compared with reference samples to determine the unknown concentration.
The following examples are intended to further illus-trate the invention, although it will be understood that the in-vention is not limited to these specific examples.', . .
A reagent solution is prepared by admixing 25 parts by weight of polyethylene glycol having a molecular weight of .
c~
4,000 with 75 parts by weight of PLURONIC F-38, and the mixture dissolved in normal saline (0.9% NaCl) to a concent~ation of 8%
by weight of said mixture. This solution may either be diluted,such as with antiserum, to a mixture concentration of 4% (HLB ratio -1.115) as in step 3 of Example 5 below, prior to incubation, and used directly in an immunological assay procedure, or it can be kept in the 8% form until ready for use.
EX~PLE 2 Example 1 is repeated, except that polyethylene glycol having a molecular weight of 6,000 is substituted for an equivalent amount of 4,000 material.
EXA~LE 3 ~
Example 1 is repeated except that PLURONI~ F-68 is substituted for an equivalent amount of F-38.
Example 1 is repeated except that Tetronic 707, 908, 1107, 1307, 1508; Plurafac 17R8, 25R8, D25, A38 and A39; Pluronic L125, Arlacel 165; and G-3000 were substituted for the F-38 in varying amounts in a series ~f experiments to prepare a wide --~variety of reagent solutions.
- The immunological reagent solutions prepared in ; Examples 1-4 are used in separate nephelometric assays o~
immunoglobulins (IgA, IgG, IgM), complement-C3 and transferrin, with each assay being conducted as follows:
1. Reference controls ~ 2, #3, #4, ~5, ~6 ~of known assay) for each said biological constituent are diluted 1:100 in saline.
2. The unknowns are then similarly diluted 1:100 i 30 in saline.
3. Prediluted antiserums to IgG, IgM, IgA, C3 and transferrin are each diluted 1:2 with the 8% mix-... . . . . .. . . __ _ .. ... . .. , _.. . . _ . . _. ... . . . _ _~ __ . .. .. _ _ .. _ ~ ;' ' - , ture of the immonological reagent from Examples 1, 2, 3, or 4 (as the case may be) and mixed by inversion to produce a 4% concen-tration of the polyethylene glycol and nonionic surfactant in the solution and a calculated HLB
of between 0.7 and 1.7 in all cases.
4. The antiserum is filtered through a 0.45 Millipore filter.
by weight of said mixture. This solution may either be diluted,such as with antiserum, to a mixture concentration of 4% (HLB ratio -1.115) as in step 3 of Example 5 below, prior to incubation, and used directly in an immunological assay procedure, or it can be kept in the 8% form until ready for use.
EX~PLE 2 Example 1 is repeated, except that polyethylene glycol having a molecular weight of 6,000 is substituted for an equivalent amount of 4,000 material.
EXA~LE 3 ~
Example 1 is repeated except that PLURONI~ F-68 is substituted for an equivalent amount of F-38.
Example 1 is repeated except that Tetronic 707, 908, 1107, 1307, 1508; Plurafac 17R8, 25R8, D25, A38 and A39; Pluronic L125, Arlacel 165; and G-3000 were substituted for the F-38 in varying amounts in a series ~f experiments to prepare a wide --~variety of reagent solutions.
- The immunological reagent solutions prepared in ; Examples 1-4 are used in separate nephelometric assays o~
immunoglobulins (IgA, IgG, IgM), complement-C3 and transferrin, with each assay being conducted as follows:
1. Reference controls ~ 2, #3, #4, ~5, ~6 ~of known assay) for each said biological constituent are diluted 1:100 in saline.
2. The unknowns are then similarly diluted 1:100 i 30 in saline.
3. Prediluted antiserums to IgG, IgM, IgA, C3 and transferrin are each diluted 1:2 with the 8% mix-... . . . . .. . . __ _ .. ... . .. , _.. . . _ . . _. ... . . . _ _~ __ . .. .. _ _ .. _ ~ ;' ' - , ture of the immonological reagent from Examples 1, 2, 3, or 4 (as the case may be) and mixed by inversion to produce a 4% concen-tration of the polyethylene glycol and nonionic surfactant in the solution and a calculated HLB
of between 0.7 and 1.7 in all cases.
4. The antiserum is filtered through a 0.45 Millipore filter.
5. A series of test tubes (10 mm x 75 mm disposable culture tubes) appropriately labeled blank, ref-erence controls #1, #2, #3, #4, #5, #6, and un-known, are prepared.
6. To each tube, 1 ml of the diluted mixture of antiserum and reagent prepared in step 3 is added.
7. To the appropriate tube 25~1 of reference and unknown dilutions are added for IgG, IgA, and transferrin (100 ~1 for Ig~ and C3).
8. The appropriate blank tube correspondingly re-ceived 25 or 100 ~1 of saline.
9. The tubes are mixed by inversion and incubated for 1 hour at room temperature (20-25C).
10. Sample blanks are prepared by using 1 ml of the .~ filtered reagents from Examples 1-4, (as the case may be) prepared as in step 3 except that the 8% so-' lution was diluted with saline instead of antiserum.
The blanks are placed in identical labeled tubes as before steps (5 and 6).
.
... . . .
The blanks are placed in identical labeled tubes as before steps (5 and 6).
.
... . . .
11. The same reference and sample volumes are added to each tube as before (step 7)
12. All tubes are read in the Laser Nephelometer PDQTM (Hyland Laboratories) for relative percent light scatter.
13. The blanks are read and subtracted electronically from the reaction values by the instrument.
14. The reference results are plotted on linear graph paper as relative percent light scatter versus concentration of references.
15. The unknown values are determined by reading from the reference curve.
The immunological reagents of the invention used in this sample produced substantially greater precipitation of the antigen-; 15 antibody complexes, more linearity over a wider range, and greater sensitivity than was obtained from a reagent containing - polyethylene glycol alone.
I This example describes the use of the immonological reagent of the invention in a radioimmunoassay procedure for the determination of human thyroid stimulating hormone (HTSH).
A sample of the immunological reagent of the inven-tion was prepared by mixing 8.4 ml of a 5% solution of polyethy-lene glycol in 0.9 saline solution with 20 ml of 6% Pluronic F-38 nonionic surfactant. The volume of the mixture was then adjusted to 30 ml with 0.9 saline solution to give a final reagent con-centration of 1.4% polyethylene glycol and 4.2% F-38.
The radioimmunoassay was performed as follows:
0.050 ml of Rabbit anti-HTSH absorbed with human chorionic gonadotropin (HCG) was mixed with 0.200 ml of HTSH standards .
. .
of varying strength. 0.050 ml of a phosphare buffer, pH 7.4, was then added to the mixture and the mixture incubated for 2 hours at 37C. At this point, 0.100 ml of HTSH tagged with I125 was added and the mixture further incubated for 3 hours at 37C. 1.0 ml of the reagent of the invention prepared above was then added and the mixtu~e incubated for one hour at room temperature. Due to dilution of the reagent upon addition to the mixture, the concentration of polyethylene glycol and F-38 was reduced from 1.4 and 4.2% to 1 and 3% by weight respectively.
The mixture was centrifuged at 1000 x 9 for 10 minutes. If a wash of the centrifuged solids is required, the wash solution must contain the same concentration as the reagent of the inven-tion at the time the reagent was first used in the assay, i.e., 1% polyethylene glycol and 3% F-38. The supernatant liquid was then decanted and the precipitate counted. This procedure was repeated for a variety of differing HTSH standards and a stan-dard curve was obtained by plotting the counts in the various precipitates versus the concentration of corresponding HTSH
standard.
~nce the standard curve had been obtained, the assay was then performed on unknown test samples, using the procedure described above, except that the HTSH standard was replaced by the test sample. The HTSH level in the test sample could then be readily determined from the location of the precipitate co~nt ` 25 on the standard curve.
; The use of the reagent sol-ution of the invention en-hanced the extent of precipitate which was formed beyond that obtainable from a reagent using polyethylene glycol alone, and resulted in an improved radioimmunoassay.
- - . . . - - . ~
Suitable nephelometric assay results can also be achieved without the presence of the polyethylene glycol, for example, with an aqeous solution containing about 4% by weight of the block copolymer of ethylene oxide and polyoxypropylene polymer. However, the mixture of block copolymer and polyethylene glycol described above is preferred for optimum results.
; -23-. ~
' '' ', . ' '' '
The immunological reagents of the invention used in this sample produced substantially greater precipitation of the antigen-; 15 antibody complexes, more linearity over a wider range, and greater sensitivity than was obtained from a reagent containing - polyethylene glycol alone.
I This example describes the use of the immonological reagent of the invention in a radioimmunoassay procedure for the determination of human thyroid stimulating hormone (HTSH).
A sample of the immunological reagent of the inven-tion was prepared by mixing 8.4 ml of a 5% solution of polyethy-lene glycol in 0.9 saline solution with 20 ml of 6% Pluronic F-38 nonionic surfactant. The volume of the mixture was then adjusted to 30 ml with 0.9 saline solution to give a final reagent con-centration of 1.4% polyethylene glycol and 4.2% F-38.
The radioimmunoassay was performed as follows:
0.050 ml of Rabbit anti-HTSH absorbed with human chorionic gonadotropin (HCG) was mixed with 0.200 ml of HTSH standards .
. .
of varying strength. 0.050 ml of a phosphare buffer, pH 7.4, was then added to the mixture and the mixture incubated for 2 hours at 37C. At this point, 0.100 ml of HTSH tagged with I125 was added and the mixture further incubated for 3 hours at 37C. 1.0 ml of the reagent of the invention prepared above was then added and the mixtu~e incubated for one hour at room temperature. Due to dilution of the reagent upon addition to the mixture, the concentration of polyethylene glycol and F-38 was reduced from 1.4 and 4.2% to 1 and 3% by weight respectively.
The mixture was centrifuged at 1000 x 9 for 10 minutes. If a wash of the centrifuged solids is required, the wash solution must contain the same concentration as the reagent of the inven-tion at the time the reagent was first used in the assay, i.e., 1% polyethylene glycol and 3% F-38. The supernatant liquid was then decanted and the precipitate counted. This procedure was repeated for a variety of differing HTSH standards and a stan-dard curve was obtained by plotting the counts in the various precipitates versus the concentration of corresponding HTSH
standard.
~nce the standard curve had been obtained, the assay was then performed on unknown test samples, using the procedure described above, except that the HTSH standard was replaced by the test sample. The HTSH level in the test sample could then be readily determined from the location of the precipitate co~nt ` 25 on the standard curve.
; The use of the reagent sol-ution of the invention en-hanced the extent of precipitate which was formed beyond that obtainable from a reagent using polyethylene glycol alone, and resulted in an improved radioimmunoassay.
- - . . . - - . ~
Suitable nephelometric assay results can also be achieved without the presence of the polyethylene glycol, for example, with an aqeous solution containing about 4% by weight of the block copolymer of ethylene oxide and polyoxypropylene polymer. However, the mixture of block copolymer and polyethylene glycol described above is preferred for optimum results.
; -23-. ~
' '' ', . ' '' '
Claims (42)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A reagent for clinical nephelometric analysis comprising an aqueous solution of an antiserum to the component to be assayed and a mixture of about 20% to about 40% by weight polyethylene glycol having a molecular weight of about 200 to about 10,000 and about 80% to about 60% by weight of a block copolymer of ethylene oxide and polyoxy-propylene containing at least 50% ethylene oxide.
2. The reagent of claim 1 in which the concentration of said mixture is from about 3% to about 6% by weight.
3. The reagent of claim 2 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
4. The reagent according to any one of claim 1, 2 or 3 in which the concentration of said mixture is about 4%
by weight, and wherein said mixture contains about 25% by weight polyethylene glycol and about 75% by weight of said block copolymer.
by weight, and wherein said mixture contains about 25% by weight polyethylene glycol and about 75% by weight of said block copolymer.
5. In an immunological assaying method which involves a reaction between an antigen and an antibody to form an antigen-antibody complex, the improvement which comprises carrying out said reaction in the presence of a reagent comprising an aqueous solution containing about 3 to 6% by weight of a mixture of polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than polyethylene glycol wherein said mixture contains about 10% to 90% by weight polyethylene glycol and about 10% to 90%
by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7.
by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7.
6. The method of claim 5 wherein said reagent is utilized in a nephelometric analysis.
7. The method of claim 5 wherein said reagent is utilized in a radioimmunoassay.
8. The method of any one of claims 5 to 7 wherein said reagent is utilized in an enzymatic assay.
9. The method of claim 5 wherein said reagent is utilized in an electrophoretic assay.
10. The method according to any one of claims 5 to wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
11. A nephelometric immunoassay method comprising incubating a test sample, prior to conducting light scattering measurements, in a reagent comprising an aqueous solution con-taining about 3 to 6% by weight of a mixture of polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than polyethylene glycol wherein said mixture contains about 10% to 90% by weight polyethylene glycol and about 10% to 90% by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7.
12. The method of claim 11 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
13. The method of claim 11 or 12 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000 and said mixture contains about 15% to 85% polyethylene glycol and about 15% to 85% nonionic surfactant.
14. A nephelometric immunoassay method, comprising incubating a test sample, prior to conducting light scattering measurements, in a reagent comprising an aqueous solution containing an antiserum to said constituent and about 3 to 6% by weight of a mixture of polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than polyethylene glycol wherein said mixture contains about 10% to 90% by weight polyethylene glycol and about 10% to 90% by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7.
15. The method of claim 14 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
16. A nephelometric analysis of an immunoglobulin which involves a reaction between said immunoglobulin with its antibody, the improvement comprising incubating said immunoglobulin selected from the group consisting of IgG, IgA, IgM, transferrin, complement C3, haptoglobin, alpha1-antitrypsin, B-lipoprotein, albumin, alpha2-macroglobulin and alpha1-acid glyco-protein, prior to conducting light scattering measurements, in a reagent comprising an aqueous solution containing about 3 to 6% by weight of a mixture of about 20% to about 40% by weight polyethylene glycol having a molecular weight of about 200 to about 10,000 and about 80 to about 60% by weight of a block copolymer of ethylene oxide and polyoxypropylene, said solution having a calculated HLB value of from 0.7 to 1.7.
17. The method of claim 16 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
18. In an immunological assaying method which involves a reaction between an antigen and an antibody to form an antigen-antibody complex, the improvement which comprises carrying out said reaction in the presence of a reagent comprising an aqueous solution containing about 3 to 6% by weight of a mixture of polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant selected from the group consisting of :
a) a block copolymer of ethylene oxide and polyoxypropylene, b) straight chain primary aliphatic oxyalkylated alcohols, and c) glycol monostearate, wherein said mixture contains about 10% to 90% by weight poly-ethylene glycol and about 10% to 90% by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7.
a) a block copolymer of ethylene oxide and polyoxypropylene, b) straight chain primary aliphatic oxyalkylated alcohols, and c) glycol monostearate, wherein said mixture contains about 10% to 90% by weight poly-ethylene glycol and about 10% to 90% by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7.
19. The method of claim 18 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
20. A reagent for use in an immunological assay comprising an aqueous solution containing a mixture of (1) polyethylene glycol having a molecular weight of about 4,000 to about 6,000, (2) a block copolymer of ethylene oxide and polyoxypropylene and (3) an antiserum to the component to be assayed, wherein the combined amount of polyethylene glycol and block copolymer present in the solution is about 3 to 6% by weight and comprises about 20% to about 40% by weight polyethylene glycol and about 80% to about 60% by weight block copolymer, and wherein the calculated HLB value of the solution is about 0.7 to 1.7.
21. The reagent of claim 20 wherein the antiserum is an antiserum to a member selected from the group consisting of IgG, IgM, IgA, complement C3 and transferrin.
22. A reagent for use in an immunoassay comprising an aqueous solution containing about 3 to 6% by weight of a mixture of polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than polyethylene glycol, said mixture containing about lO
to 90% by weight polyethylene glycol and about 10 to 90% by weight nonionic surfactant, and wherein said solution has a calculated HLB value of about 0.7 to 1.7 provided, however, that when the nonionic surfactant is a block copolymer of ethylene oxide and polyoxypropylene the block copolymer contains at least 50% ethylene oxide.
to 90% by weight polyethylene glycol and about 10 to 90% by weight nonionic surfactant, and wherein said solution has a calculated HLB value of about 0.7 to 1.7 provided, however, that when the nonionic surfactant is a block copolymer of ethylene oxide and polyoxypropylene the block copolymer contains at least 50% ethylene oxide.
23. The reagent of claim 22 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
24. A nephelometric immunoassay method comprising diluting an aqueous solution containing polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than polyethylene glycol to produce a diluted solution containing about 3 to 6% by weight of a mixture of polyethylene glycol and said nonionic surfactant having a calculated HLB value of about 0.7 to 1.7, said mixture containing about 10-90% by weight polyethylene glycol and about 10-90% by weight nonionic surfactant, and contacting a test sample with said diluted solution either prior to or at the time of said immunoreaction and prior to conducting the light scattering measurements.
25. The method of claim 24 wherein the test sample is selected from the group consisting of IgG, IgA, IgM, transferrin, complement C3, haptoglobin, alphal-antitrypsin, albumin, alpha2-macroglobulin, alpha1-acid glycoprotein, iodo-thyonine (T3), thyroxine (T4), triglycerides, human chorionic ganodotropins and lipoproteins.
26. The immunoassay of claim 24 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
27. The immunoassay of claim 24 wherein the polyethylene glycol has a mQlecular weight of about 4,000 to 6,000 and said mixture contains about 15% to 85% polyethylene glycol and about 15% to 85% nonionic surfactan.
28. The immunoassay of claim 27 wherein the nonionic surfactant is a block copolymer of ethylene oxide and poly-oxypropylene.
29. A nephelometric immunoassay method comprising diluting with an antiserum for a test sample an aqueous solution containing polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than polyethylene glycol to produce a diluted solution con-taining about 3 to 6% by weight of a mixture of polyethylene glycol and said nonionic surfactant wherein said mixture contains about 10% to 90% by weight polyethylene glycol and about 10% to 90% by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7, and contacting said test sample with said diluted solution prior to conducting the light scattering measurements.
30. The immunoassay of claim 29 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
31. The immunoassay of claim 29 wherein the antiserum is a prediluted antiserum.
32. The immunoassay of claim 31 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
33. The method of claim 29 wherein the test sample is selected from the group consisting of IgG, IgA, IgM. transferrin, complement C3, haptoglobin, alpha1-antitrypsin, albumin, alpha2-macroglobulin, alpha1-acid glycoprotein, T-3, T-4, triglycerides, human chorionic ganadotropins and lipoproteins.
34. In an immunoassay which produces an antigen-antibody complex, the improvement comprising diluting with an antiserum for said antigen or antibody an aqueous solution containing polyethylene glycol having a molecular weight of about 200 to about 10,000 and a nonionic surfactant other than poly-ethylene glycol to produce a diluted solution containing about 3% to 6% by weight of a mixture of polyethylene glycol and said nonionic surfactant wherein said mixture contains about 10% to 90% by weight polyethylene glycol and about 10% to 90%
by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7, and producing the antigen-antibody complex in the presence of said diluted solution.
by weight nonionic surfactant and said solution has a calculated HLB value of about 0.7 to 1.7, and producing the antigen-antibody complex in the presence of said diluted solution.
35. The method of claim 34 wherein the biological component is selected from the group consisting of IgG, IgA, IgM, transferrin, complement C3, haptoglobin, alpha1-antitrypsin, albumin, alpha2-macroglobulin, alpha1-acid glycoprotein, T-3, T-4, triglycerides, human chorionic ganadotropins and lipoproteins.
36. The method of claim 34 wherein the nonionic surfactant is a block copolymer of ethylene oxide and poly-oxypropylene, the calculated HLB value of the diluted solution is about 0.7 to 1.3, and the biological component is selected from the group consisting of IgG, IgA, IgM, component C3, transferrin T3 and T4.
37. The immunoassay according to any one of claims 34 to 36 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
38. The immunoassay of claim 34 wherein the antiserum is a prediluted antiserum.
39. The immunoassay of claim 38 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
40. In an immunological assaying method which involves a reaction between an antigen and an antibody to form an antigen-antibody complex, the improvement which comprises carrying out said reaction in the presence of a reagent comprising an aqueous solution containing about 3% to 6% of a mixture of polyethylene glycol having a molecule weight of about 200 to about 10,000 and an alkyl aryl sulfonate wherein said mixture contains about 10% to 90% by weight polyethylene glycol and about 10% to 90% by weight alkyl aryl sulfonate and said solution has a calculated HLB value of about 0.7 to 1.7.
41. The method of claim 40 wherein the immunological assaying method is a nephelometric analysis.
42. The method of claim 40 or 41 wherein the polyethylene glycol has a molecular weight of about 4,000 to 6,000.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US54506675A | 1975-01-29 | 1975-01-29 | |
| US55590875A | 1975-03-06 | 1975-03-06 | |
| US05/615,024 US4148869A (en) | 1975-03-06 | 1975-09-19 | Immunological reagent and method of using same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1080619A true CA1080619A (en) | 1980-07-01 |
Family
ID=27415448
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA243,325A Expired CA1080619A (en) | 1975-01-29 | 1976-01-12 | Immunological reagent and method of using same |
Country Status (15)
| Country | Link |
|---|---|
| JP (1) | JPS604938B2 (en) |
| BE (1) | BE837675A (en) |
| BR (1) | BR7600322A (en) |
| CA (1) | CA1080619A (en) |
| CH (1) | CH613047A5 (en) |
| DE (2) | DE2602068C2 (en) |
| DK (1) | DK150809C (en) |
| ES (1) | ES444543A1 (en) |
| FR (1) | FR2299645A1 (en) |
| GB (1) | GB1540097A (en) |
| IL (1) | IL48804A (en) |
| IT (1) | IT1062433B (en) |
| NL (1) | NL183852C (en) |
| NO (1) | NO149864C (en) |
| SE (1) | SE441392B (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2901327B1 (en) * | 1979-01-15 | 1980-07-24 | Boehringer Mannheim Gmbh | Method for determining an opsonizing surface-binding alpha-glycoprotein |
| FI792576A (en) * | 1979-08-20 | 1981-02-21 | Orion Yhtymae Oy | METHOD OF ENZYME IMMUNOLOGICAL ASSESSMENT |
| JPS5943362A (en) * | 1982-09-06 | 1984-03-10 | Kainosu:Kk | Immunological measuring reagent |
| JPH0672881B2 (en) * | 1982-10-13 | 1994-09-14 | バイオウィッテッカー・インコーポレーテッド | Fluorescence analysis of allergic reaction |
| GB8317855D0 (en) * | 1983-06-30 | 1983-08-03 | Iq Bio Ltd | Biochemical detection method |
| DE3327642A1 (en) * | 1983-07-30 | 1985-02-14 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETERMINING A PARTNER OF AN IMMUNE REACTION AND REAGENT FOR CARRYING OUT THIS METHOD |
| DE3532626A1 (en) * | 1985-09-12 | 1987-03-19 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING A PARTNER OF AN IMMUNE REACTION |
| JPS62159047A (en) * | 1985-12-31 | 1987-07-15 | Chemo Sero Therapeut Res Inst | Reagent for quantitative determination of plasma protein |
| US4810638A (en) * | 1986-07-24 | 1989-03-07 | Miles Inc. | Enzyme-labeled antibody reagent with polyalkyleneglycol linking group |
| JP2684069B2 (en) * | 1988-10-13 | 1997-12-03 | 昇一 首藤 | Method for measuring immunologically active substance and reagent therefor |
| US5403716A (en) * | 1991-01-10 | 1995-04-04 | Teijin Limited | Method for measurement of tissue factor in high sensitivity and measurement kit therefor |
| AU6525494A (en) * | 1993-03-26 | 1994-10-24 | Curative Technologies, Inc. | Method and assay involving improved specific binding reactivity of a polypeptide |
| WO2004090613A1 (en) * | 2003-04-03 | 2004-10-21 | Seed Co., Ltd. | Ophthalmic lenses capable of sustained drug release and preservative solutions therefor |
| JP4915638B2 (en) * | 2005-08-26 | 2012-04-11 | パナソニック株式会社 | Electrodeless discharge lamp device and lighting fixture equipped with the electrodeless discharge lamp device |
| JP6242068B2 (en) * | 2013-04-10 | 2017-12-06 | 積水メディカル株式会社 | Latex immunoagglutination measurement reagent |
| TWI842764B (en) * | 2018-11-09 | 2024-05-21 | 日商積水醫療股份有限公司 | Methods for suppressing abnormal detections in immunoassays conducted with an automatic analyzer, detection methods, immunoassay reagents, and immunoassay reagent kits |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1598151A1 (en) * | 1966-11-19 | 1970-05-27 | Kleine Dr Rer Nat Norbert | Agglutination enhancers |
| DE2361169C3 (en) * | 1973-12-07 | 1978-09-14 | Boehringer Mannheim Gmbh, 6800 Mannheim | Process for the activation of cholesterol oxidase freed from detergent traces |
| US3947250A (en) * | 1974-06-21 | 1976-03-30 | Baxter Laboratories, Inc. | Method of immunodiffusion |
-
1976
- 1976-01-07 IL IL48804A patent/IL48804A/en unknown
- 1976-01-12 SE SE7600232A patent/SE441392B/en not_active IP Right Cessation
- 1976-01-12 CA CA243,325A patent/CA1080619A/en not_active Expired
- 1976-01-15 FR FR7600986A patent/FR2299645A1/en active Granted
- 1976-01-16 DK DK016976A patent/DK150809C/en not_active IP Right Cessation
- 1976-01-16 GB GB1784/76A patent/GB1540097A/en not_active Expired
- 1976-01-16 IT IT19348/76A patent/IT1062433B/en active
- 1976-01-19 BR BR7600322A patent/BR7600322A/en unknown
- 1976-01-19 BE BE163598A patent/BE837675A/en not_active IP Right Cessation
- 1976-01-21 DE DE2602068A patent/DE2602068C2/en not_active Expired
- 1976-01-21 DE DE2661010A patent/DE2661010C2/en not_active Expired
- 1976-01-22 NO NO760203A patent/NO149864C/en unknown
- 1976-01-22 ES ES444543A patent/ES444543A1/en not_active Expired
- 1976-01-23 JP JP51007067A patent/JPS604938B2/en not_active Expired
- 1976-01-23 NL NLAANVRAGE7600697,A patent/NL183852C/en not_active IP Right Cessation
- 1976-01-27 CH CH102276A patent/CH613047A5/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| DE2602068C2 (en) | 1986-09-11 |
| BR7600322A (en) | 1976-08-31 |
| ES444543A1 (en) | 1977-06-01 |
| FR2299645A1 (en) | 1976-08-27 |
| DE2661010C2 (en) | 1987-03-19 |
| IT1062433B (en) | 1984-10-10 |
| BE837675A (en) | 1976-07-19 |
| IL48804A0 (en) | 1976-03-31 |
| FR2299645B1 (en) | 1980-11-28 |
| SE7600232L (en) | 1976-07-30 |
| JPS51101121A (en) | 1976-09-07 |
| CH613047A5 (en) | 1979-08-31 |
| NL183852B (en) | 1988-09-01 |
| SE441392B (en) | 1985-09-30 |
| NO760203L (en) | 1976-07-30 |
| IL48804A (en) | 1979-05-31 |
| DK16976A (en) | 1976-07-30 |
| NL183852C (en) | 1989-02-01 |
| DK150809C (en) | 1988-05-02 |
| GB1540097A (en) | 1979-02-07 |
| DE2602068A1 (en) | 1976-08-05 |
| NL7600697A (en) | 1976-08-02 |
| NO149864B (en) | 1984-03-26 |
| DK150809B (en) | 1987-06-22 |
| JPS604938B2 (en) | 1985-02-07 |
| NO149864C (en) | 1984-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4148869A (en) | Immunological reagent and method of using same | |
| CA1080619A (en) | Immunological reagent and method of using same | |
| Killingsworth et al. | Manual nephelometric methods for immunochemical determination of immunoglobulins IgG, IgA, and IgM in human serum | |
| Tiffany et al. | Specific protein analysis by light-scatter measurement with a miniature centrifugal fast analyzer | |
| EP0968422B1 (en) | Improving performance of binding assays by use of more than one label | |
| US5721105A (en) | Method for the immunological determination of proteins and kit for carrying out the method | |
| CA2422856C (en) | Methods and kits for decreasing interferences of assay samples containing plasma or serum in specific binding assays by using a large polycation | |
| Nishikawa et al. | Competitive nephelometric immunoassay of theophylline in plasma | |
| Parry et al. | Detection of rubella antibody using an optical immunosensor | |
| Blanchard et al. | Two immunofluorescent methods compared with a radial immunodiffusion method for measurement of serum immunoglobulins. | |
| CN101446586A (en) | Immunological assay reagents and assay method | |
| De Jonge et al. | Time-resolved immunofluorometric assay (TR-IFMA) for the detection of the schistosome circulating anodic antigen | |
| CN116008524A (en) | Protein-free universal protection liquid, preparation method thereof and application thereof in mycotoxin quantitative detection kit | |
| Malkus et al. | An automated turbidimetric rate method for immunoglobulin assays | |
| EP0439611B1 (en) | Method for assaying immunologically active substance and reagent therefor | |
| Faulkner et al. | Measurement of IgA levels in human cord serum by a new radioimmunoassay | |
| EP2207036B1 (en) | Methods for determining the negative control value for multi-analyte assays | |
| Finley et al. | Immunochemical determination of human immunoglobulins: use of kinetic turbidimetry and a 36-place centrifugal analyzer. | |
| Wang et al. | A simplified solid-phase immunofluorescence assay for measurement of serum immunoglobulins | |
| Killingsworth et al. | Automated immunochemical method for determination of urinary protein of plasma origin | |
| JPS6113135A (en) | Method of measuring other party of reaction and device for said method | |
| NO149865B (en) | IMMUNOLOGICAL MEASUREMENT PROCEDURES. | |
| EP0643832A1 (en) | Immunoassay method | |
| JPH08297123A (en) | Immunological measuring method for protein and kit for excecuting method thereof | |
| Matsuzawa et al. | Quantitation of DNP, digoxin, HCG and anti-DNP antibody by electronic data processing of latex antiglobulin reaction results |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |