EP0695424A1 - A method for improving the signal to noise ratio of an immunoassay - Google Patents
A method for improving the signal to noise ratio of an immunoassayInfo
- Publication number
- EP0695424A1 EP0695424A1 EP94913403A EP94913403A EP0695424A1 EP 0695424 A1 EP0695424 A1 EP 0695424A1 EP 94913403 A EP94913403 A EP 94913403A EP 94913403 A EP94913403 A EP 94913403A EP 0695424 A1 EP0695424 A1 EP 0695424A1
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- EP
- European Patent Office
- Prior art keywords
- salt
- analyte
- sample
- immunoassay
- signal
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- 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.)
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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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Definitions
- This invention relates to a method for improving assay performance and, more particularly, to improving the signal to noise ratio of an immunoassay by adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate and perchlorate to a sample in a sufficient ' amount to improve the signal to noise ratio of the immunoassay and the salt does -not increase antigenicity of the analyte.
- at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate and perchlorate to a sample in a sufficient ' amount to improve the signal to noise ratio of the immunoassay and the salt does -not increase antigenicity of the analyte.
- Immunoassays are generally utilized for detecting and/or quantifying the amount of analyte ,in.-serum or other biological fluids and are based principally on the binding of specific binding substance, ⁇ such as an antibody, to a particular analyte which might be present in a specimen.
- the sensitivity of an assay such as an immunoassay can be defined by the ratio of the specific signal generated to the background noise of the system.
- Factors increasing assay noise include non-specific binding of reagents such as an antibody to various components of the assay and the activity of some endogeneous component of the assay matrix which reacts with the enzyme substrate to yield a reaction product interfering with the accurate detection of product formed by a labeled complex, e.g., a labeled antibody- antigen complex.
- additives are added to the assay matrix to lower assay noise by reducing non ⁇ specific binding of an enzyme-antibody conjugate or other potentially interfering component of the assay matrix.
- U.S. Patent No. 4,668,620 issued to Armenta et al . on May 26, 1987, describes a method to reduce background interference activity in enzyme immunoassays. The method involves incorporating into the sample, prior to performing the assay, an agent such as a peracid for rendering the anti-enzyme components of the sample urreactive toward the enzyme label in an amount and under conditions sufficient to substantially minimize the interference .
- This invention relates to an improved immunoassay for an analyte in a sample which comprises :• a) incubating the sample with at least one analyte specific reagent; and b) detecting the product of step (a) wherein the improvement comprises adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, and perchlorate to the sample, said salt being in an amount sufficient to improve the signal to noise ratio of the immunoassay and the salt does not increase antigenicity of the analyte.
- this invention concerns an improved immunoassay for an analyte in a sample which comprises : a) incubating the sample with a capture reagent immobilized on a solid phase; b) reacting the product of step (a) with a detector reagent; and c) detecting and/or quantitating the presence of analyte, wherein the improvement comprises adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, and perchlorate to the sample, said salt being in an amount sufficient to improve the signal to noise ratio of the immunoassay and the salt does not increase antigenicity of the analyte.
- the method of this invention improves the signal to noise ratio of an immunoassay by adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate or perchlorate to a sample wherein the salt or salts is added in an amount sufficient to improve the signal to noise ratio of the immunoassay and further wherein antigenicity of the analyte is not increased.
- antigenicity is increased by partial denaturation due to addition of certain chaotropic ions.
- the increased antigenicity results in increased interactions between the analyte (collagen) and the antibodies used.
- the salts used in the method of this invention do not increase antigenicity of the analyte, i.e., interactions between the analyte and antibodies is not increased as illustrated by the data discussed below in the examples .
- analyte specific reagent refers to a capture reagent and/or a detector reagent.
- the capture and detector reagents are members of an immune specific binding pair. Immune specific binding pairs are exemplified by antigen/antibody systems or hapten/anti-hapten systems.
- the analyte specific reagents can include both capture and detector antibodies which bind to different epitopes on the analyte or just a detector antibody. These antibodies can be polyclonal and/or monoclonal. The technology for preparing antibodies, whether polyclonal or monoclonal, is well known to those skilled in the art and, thus, no further discussion is needed.
- Immunoassays of the invention can be heterogeneous (separation) or homogeneous (non-separation) . They can be performed simultaneously or sequentially.
- blood-borne infectious agents such as Human Immunodeficiency Virus (HIV) , hepatitis, Epstein-Barr virus, cytomegalovirus, HTLV-1, HTLV-II, etc.
- HIV Human Immunodeficiency Virus
- An example of a commercially available immunoassay whose sensitivity and specificity can be improved using the method of the invention is the DuPont HIV-1 p24 Core Profile ELISA used in conjunction with the DuPont HIV-1 p24 Acid Disruption Difference Immune Complex Disruption Kit which is designed to dissociate antigen/antibody complexes in serum and plasma using a combination of low pH and heat.
- An important aspect of the method of the invention is the addition of at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, or perchlorate to the sample in an amount effective to improve the signal to noise ratio of an immunoassay wherein the salt does not increase antigenicity of the analyte.
- the preferred salt for practicing the invention is sodium bromide.
- magnesium bromide sodium nitrate, magnesium nitrate, and sodium perchlorate. Effective concentrations are generally between 0.05M and 0.5M.
- the DuPont HIV-1 p24 Core Profile ELISA antigen assay in conjunction with the DuPont HIV-1 p24 Acid Disruption Difference (ADD) kit described above were used to assay the samples.
- the ADD kit was used to disrupt antigen/antibody complexes in serum and plasma using a combination of low pH and heat. Samples were acidified with 1.5 M glycine reagent, pH 1.85 and were incubated at 37°C for one hour. After one hour, the samples were neutralized with 1.5 M TRIS, pH 11 and were assayed in the DuPont HIV-1 p24 Core Profile ELISA.
- the DuPont HIV-1 p24 Core Profile ELISA antigen assay utilizes an anti-HIV p24 mouse monoclonal antibody which is immobilized to microtiter plate wells.
- the immobilized monoclonal antibody then captures HIV-1 p24 antigen released upon lysis of virus in the samples.
- the captured antigen is complexed with biotinylated polyclonal antibodies to HIV-1 p24 core antigen and probed with a streptavidin-HRP (horseradish peroxidase) conjugate.
- the complex is then detected by incubation with orthophenylenediamine - HCl (OPD) which produces a yellow color that is directly proportional to the amount of HIV-1 p24 core antigen captured.
- OPD orthophenylenediamine - HCl
- the Glycine Reagent is 1.5 M glycine, pH ,1.85 % Complementary metal salts.
- the sample mixture (150 ⁇ l) was then transferred to an HIV-1 p24 Core Profile EL ⁇ SA plate and incubated for 2 hours at 37°C.
- the microtiter plate wells were then washed and 100 ⁇ l biotinylated detector antibody was added to each well and incubated for one hour at 37°C.
- the wells were then washed and streptavidin-HRP conjugate at a 1:25 dilution was added, incubated for 15 minutes at 37°C and the wells were then washed again.
- OPD substrate was added for thirty minutes at room temperature, the reaction was stopped with kit stop solution (4 N sulfuric acid) and the absorbance at 490-650 nm was determined. .
- Results are presented in Table 1 below.
- the absorbance for each of sixteen samples plus a positive control is presented using just the glycine reagent and using glycine plus sodium bromide salt.
- the results show the dramatic improvement obtained using the instant invention.
- the results also show that antigenicity of the analyte was not increased by addition of the salt in an amount sufficient to improve the signal to noise ratio of the assay. It should be noted with respect to sample (Negative 9) that the background was so low that there was very little room to improve the signal to noise ratio.
- Example 2 In this experiment, improving signal to noise was evaluated using the same DuPont HIV-1 p24 Core Profile ELISA in conjunction with the DuPont Acid Disruption Difference Kit (ADD) .
- the DuPont ELASTTM ELISA amplification system was used. The kit positive and negative controls were run in duplicate for each reagent tested. 1. Reagents Glycine Reagent Glycine HCl Distilled Water
- the mixture (150 ⁇ l) was then transferred to an HIV-1 p24 Core Profile ELISA plate and incubated for 2 hours at 37°C.
- the microtiter plate wells were then washed and 100 ⁇ l biotinylated detector antibody was added to each well and incubated for one hour at 37°C.
- the wells were then washed and streptavidin-HRP conjugate at a 1:400 dilution was added, incubated for 15 minutes at 37°C and the weTls were then washed again.
- the ELAST kit reagents were used.
- Biotinyl-tyramide reagent was prepared at 10 ⁇ l/ml of diluent, and added to the wells for 15 minutes at room temperature. The wells were washed -and streptavidin-HRP at a 1:500 dilution was added and incubated for 30 minutes at room temperature, and the wells were washed again. OPD substrate from the HIV-1 p24 Core Profile ELISA kit was added for thirty minutes at room temperature, the reaction was stopped with kit stop solution (4N sulfuric acid) and the absorbance at 490-650nm was determined.
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Abstract
A method for improving immunoassay performance by adding at least one salt having a cation selected from sodium or magnesium and an anion selected from bromide, nitrate or perchlorate to a sample in an amount sufficient to improve the signal to noise ratio and the salt does not increase antigenicity of the analyte.
Description
Title A Method For Improving the Signal to Noise Ratio Of An
Immunoassay
Field of the Invention This invention relates to a method for improving assay performance and, more particularly, to improving the signal to noise ratio of an immunoassay by adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate and perchlorate to a sample in a sufficient' amount to improve the signal to noise ratio of the immunoassay and the salt does -not increase antigenicity of the analyte.
Background of the Invention Assays and, in particular, immunoassays are widely used as diagnostic tools in bacterial, viral and parasitic diseases as well as 'infectious diseases such as AIDS which constitute major health problems around the world.
Immunoassays are generally utilized for detecting and/or quantifying the amount of analyte ,in.-serum or other biological fluids and are based principally on the binding of specific binding substance, ■ such as an antibody, to a particular analyte which might be present in a specimen.
The sensitivity of an assay such as an immunoassay can be defined by the ratio of the specific signal generated to the background noise of the system. Factors increasing assay noise include non-specific binding of reagents such as an antibody to various components of the assay and the activity of some endogeneous component of the assay matrix which reacts
with the enzyme substrate to yield a reaction product interfering with the accurate detection of product formed by a labeled complex, e.g., a labeled antibody- antigen complex. Typically, additives are added to the assay matrix to lower assay noise by reducing non¬ specific binding of an enzyme-antibody conjugate or other potentially interfering component of the assay matrix.
Efforts to increase sensitivity and specificity of assay systems to improve detection and/or quantification of analyte have been undertaken as illustrated by the following:
PCT International Publication No. W092/16846 published October 1, 1992 describes a collagen immunoassay in which the sample is pretreated with chaotropic ions. Such ions are described on pages 5 and 6 as being monovalent anions with a protein denaturing action and large ionic radius represented by thiocyanic acid ions, iodine ions, perchloric acid ions, bromine ions, nitric acid ions, chlorine ions, salicylic acid ions, etc. In addition, it is indicated on page 27 that the invention described therein is not limited to a collagen assay but might be useful to determine substances whose antigenicity is accelerated by partial denaturation of the antigen using an immune reaction. Chaotropic ions as described in W092/16846 above are generally not useful in assays where antigenicity is destroyed by partial denaturation.
U.S. Patent No. 4,810,630, issued to Craig et al. on March 7, 1989, describes improving the signal to noise ratio of enzyme immunoassays employing peroxidase conjugates by including polyoxyehylene ether detergent in the assay buffer.
U.S. Patent No. 4,668,620, issued to Armenta et al . on May 26, 1987, describes a method to reduce background
interference activity in enzyme immunoassays. The method involves incorporating into the sample, prior to performing the assay, an agent such as a peracid for rendering the anti-enzyme components of the sample urreactive toward the enzyme label in an amount and under conditions sufficient to substantially minimize the interference .
U.S. Patent No. 4,758,508 issued to Schnabel et al . on July 19 1988 describes the use of certain salts to accelerate ester-cleaving enzymes.
European Patent Application Publication No. 261,493 published on March 30, 1988 describes a method for determining IgM and IgA antibodies by removing IgG antibody and rheumatoid factor by precipitating them with a zinc ion and separating from the remaining liquid.
U.S. Patent No. 4,180,556, issued to Kim et al. on December 25, 1979, describes a method for measuring carcinoembryonic antigen (CEA) by pretreating with perchloric acid to dissociate 'CEA from binding proteins and precipitating out potassium perchlorate.
Summary of the Invention
This invention relates to an improved immunoassay for an analyte in a sample which comprises :• a) incubating the sample with at least one analyte specific reagent; and b) detecting the product of step (a) wherein the improvement comprises adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, and perchlorate to the sample, said salt being in an amount sufficient to improve the signal to noise ratio of the immunoassay and the salt does not increase antigenicity of the analyte.
In another embodiment this invention concerns an improved immunoassay for an analyte in a sample which comprises : a) incubating the sample with a capture reagent immobilized on a solid phase; b) reacting the product of step (a) with a detector reagent; and c) detecting and/or quantitating the presence of analyte, wherein the improvement comprises adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, and perchlorate to the sample, said salt being in an amount sufficient to improve the signal to noise ratio of the immunoassay and the salt does not increase antigenicity of the analyte.
Detailed Descrip ion of the Invention The method of this invention improves the signal to noise ratio of an immunoassay by adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate or perchlorate to a sample wherein the salt or salts is added in an amount sufficient to improve the signal to noise ratio of the immunoassay and further wherein antigenicity of the analyte is not increased.
In some instances such as that described above in PCT International Publication No. 092/16846 antigenicity is increased by partial denaturation due to addition of certain chaotropic ions. The increased antigenicity results in increased interactions between the analyte (collagen) and the antibodies used.
In contrast, the salts used in the method of this invention do not increase antigenicity of the analyte, i.e., interactions between the analyte and antibodies is not increased as illustrated by the data discussed below in the examples .
The sensitivity and specificity of any conventional immunoassay can be improved using the method of the invention.
The term "analyte specific reagent" as used herein refers to a capture reagent and/or a detector reagent. The capture and detector reagents are members of an immune specific binding pair. Immune specific binding pairs are exemplified by antigen/antibody systems or hapten/anti-hapten systems. As those skilled in the art will appreciate, the analyte specific reagents can include both capture and detector antibodies which bind to different epitopes on the analyte or just a detector antibody. These antibodies can be polyclonal and/or monoclonal. The technology for preparing antibodies, whether polyclonal or monoclonal, is well known to those skilled in the art and, thus, no further discussion is needed.
In addition, the method of this invention is not limited to a particular assay format. Immunoassays of the invention can be heterogeneous (separation) or homogeneous (non-separation) . They can be performed simultaneously or sequentially.
Examples of analytes which can be evaluated using the method of the invention include viral proteins, bacterial proteins, hormones, drugs, etc. In particular there can be mentioned blood-borne infectious agents such as Human Immunodeficiency Virus (HIV) , hepatitis, Epstein-Barr virus, cytomegalovirus, HTLV-1, HTLV-II, etc.
An example of a commercially available immunoassay whose sensitivity and specificity can be improved using the method of the invention is the DuPont HIV-1 p24 Core Profile ELISA used in conjunction with the DuPont HIV-1 p24 Acid Disruption Difference Immune Complex Disruption Kit which is designed to dissociate antigen/antibody complexes in serum and plasma using a combination of low pH and heat. This assay is discussed in greater detail in the examples below. An important aspect of the method of the invention is the addition of at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, or perchlorate to the sample in an amount effective to improve the signal to noise ratio of an immunoassay wherein the salt does not increase antigenicity of the analyte.
The preferred salt for practicing the invention is sodium bromide. There can also be mentioned magnesium bromide, sodium nitrate, magnesium nitrate, and sodium perchlorate. Effective concentrations are generally between 0.05M and 0.5M.
Selection of the optimum or preferred concentrations can be done in many ways. , These ways are obvious to those skilled in the art of developing immunoassays and are ways which can be used to optimize other components of the immunoassay.
Exs l l The DuPont HIV-1 p24 Core Profile ELISA antigen assay in conjunction with the DuPont HIV-1 p24 Acid Disruption Difference (ADD) kit described above were used to assay the samples. The ADD kit was used to disrupt antigen/antibody complexes in serum and plasma using a combination of low pH and heat. Samples were
acidified with 1.5 M glycine reagent, pH 1.85 and were incubated at 37°C for one hour. After one hour, the samples were neutralized with 1.5 M TRIS, pH 11 and were assayed in the DuPont HIV-1 p24 Core Profile ELISA. The DuPont HIV-1 p24 Core Profile ELISA antigen assay utilizes an anti-HIV p24 mouse monoclonal antibody which is immobilized to microtiter plate wells. The immobilized monoclonal antibody then captures HIV-1 p24 antigen released upon lysis of virus in the samples. The captured antigen is complexed with biotinylated polyclonal antibodies to HIV-1 p24 core antigen and probed with a streptavidin-HRP (horseradish peroxidase) conjugate. The complex is then detected by incubation with orthophenylenediamine - HCl (OPD) which produces a yellow color that is directly proportional to the amount of HIV-1 p24 core antigen captured. The absorbance of each well is determined using a micropϊate reader and calibrated against the absorbance of an HIV-1 p2 core antigen standard curve.
1. Reagents Glycine Reagent
This is used to acidify sample to about pH 2.0. The Glycine Reagent is 1.5 M glycine, pH ,1.85 % Comp
Glycine 11.3%
HCl 9.6%
Distilled Water 79.1%
Glycine plus NaBr
Glycine Reagent 30%
5M NaBr in water 15%
Distilled Water 55%
Tris Reagent
This is 1.5 M Tris, pH 11 which is used to neutralize samples after disruption.
% Comp Tris Base 17.0%
Distilled Water 83.0%
2. Sample Preparation
Sixteen HIV-negative serum/plasma samples and the ADD kit positive control were evaluated.
The following were added to the wells of a blank microtiter plate: a) 20 μl of 5% Triton X-100, b) 90 μl of sample or positive control, c) 90 μl of glycine reagent or glycine reagent plus sodium bromide salt .
These were incubated for one hour at 37°C. After one hour, 90 μl of 1.5M Tris pH 11 was added to each well and incubated for ten minutes at room temperature.
3. Assay
The sample mixture (150 μl) was then transferred to an HIV-1 p24 Core Profile ELΪSA plate and incubated for 2 hours at 37°C. The microtiter plate wells were then washed and 100 μl biotinylated detector antibody was added to each well and incubated for one hour at 37°C. The wells were then washed and streptavidin-HRP conjugate at a 1:25 dilution was added, incubated for 15 minutes at 37°C and the wells were then washed again. OPD substrate was added for thirty minutes at room temperature, the reaction was stopped with kit stop solution (4 N sulfuric acid) and the absorbance at 490-650 nm was determined.
. Results
Results are presented in Table 1 below. The absorbance for each of sixteen samples plus a positive control is presented using just the glycine reagent and using glycine plus sodium bromide salt. The results show the dramatic improvement obtained using the instant invention. The results also show that antigenicity of the analyte was not increased by addition of the salt in an amount sufficient to improve the signal to noise ratio of the assay. It should be noted with respect to sample (Negative 9) that the background was so low that there was very little room to improve the signal to noise ratio.
Table 1 Absorbance 490 - 650nm
1Signal To Noise = Positive Control/Negative Control
Example 2 In this experiment, improving signal to noise was evaluated using the same DuPont HIV-1 p24 Core Profile ELISA in conjunction with the DuPont Acid Disruption Difference Kit (ADD) . In addition, the DuPont ELAST™ ELISA amplification system was used. The kit positive and negative controls were run in duplicate for each reagent tested.
1. Reagents Glycine Reagent Glycine HCl Distilled Water
Glycine plus NaBr Glycine Reagent 5M NaBr in Water Distilled Water
Glycine plus MσBr Glycine Reagent 2.5M gBr2 in Water Distilled Water
Glycine plus Na Qi Glycine Reagent 2.5M NaNθ3 in Water Distilled Water
Glycine plus Mg(NOj?) - Glycine Reagent 2.5M Mg(NO'3>2 n water Distilled Water
Glycine plus NaCLOj, Glycine Reagent 2.5M NaCL04 in Water Distilled Water
2. Sample Preparation
The following were added to the wells of a blank microtiter plate: a) 20 μl of 5% Triton X-100 b) 90 μl of positive or negative control, c) 90 μl of glycine reagent or glycine reagent plus salt . These were incubated for one hour at 37°C. After one hour, 90 μl of 1.5M Tris pH 11 was added to each well and incubated for ten minutes at room temperature.
3. Assays
The mixture (150 μl) was then transferred to an HIV-1 p24 Core Profile ELISA plate and incubated for 2 hours at 37°C. The microtiter plate wells were then washed and 100 μl biotinylated detector antibody was added to each well and incubated for one hour at 37°C. The wells were then washed and streptavidin-HRP conjugate at a 1:400 dilution was added, incubated for 15 minutes at 37°C and the weTls were then washed again. At this point in the assay, the ELAST kit reagents were used. Biotinyl-tyramide reagent was prepared at 10 μl/ml of diluent, and added to the wells for 15 minutes at room temperature. The wells were washed -and streptavidin-HRP at a 1:500 dilution was added and incubated for 30 minutes at room temperature, and the wells were washed again. OPD substrate from the HIV-1 p24 Core Profile ELISA kit was added for thirty minutes at room temperature, the reaction was stopped with kit stop solution (4N sulfuric acid) and the absorbance at 490-650nm was determined.
4. Results
Results for each reagent are presented in Table 2 below. These results show that the presence of at least
one salt of a bromide, nitrate or perchlorate improved the signal to noise ratio and did not increase antigenicity of the analyte.
Reagent
Glycine (Gly) Gly plus NaBr Gly plus gBr2 Gly plus Na θ3 Gly plus Mg (Nθ3) 2 Gly plus NaC104
1Signal to Noise = Positive Control/Negative Control
Claims
1. In an immunoassay for an analyte in a sample which comprises:
a) incubating the sample with at least one analyte specific reagent; and
b) detecting the product of step (a)
wherein the improvement comprises adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, and perchlorate to the sample, said salt being in an amount sufficient to improve the signal to noise ratio of the immunoassay and said salt does not increase antigenicity of the analyte.
2. An assay according to claim 1 -wherein the salt is sodium bromide.
3. An assay according td claim 1 wherein the analyte is HIV.
4. In an immunoassay for an analyte in a sample which comprises:
a) incubating the sample with a capture reagent immobilized on a solid phase;
b) reacting the product of step (a) with a detector reagent; and
c) detecting and/or quantitating the presence of analyte,
wherein the improvement comprises adding at least one salt having a cation selected from the group consisting of sodium and magnesium and an anion selected from the group consisting of bromide, nitrate, and perchlorate to the sample, said salt being in an amount sufficient to improve the signal to noise ratio of the immunoassay and said salt does not increase antigenicity of the analyte.
5. An assay according to claim 4 wherein the salt is sodium bromide.
6. An assay according to claim 4 wherein the analyte is HIV.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US4974693A | 1993-04-19 | 1993-04-19 | |
US49746 | 1993-04-19 | ||
PCT/US1994/003963 WO1994024558A1 (en) | 1993-04-19 | 1994-04-12 | A method for improving the signal to noise ratio of an immunoassay |
Publications (1)
Publication Number | Publication Date |
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EP0695424A1 true EP0695424A1 (en) | 1996-02-07 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP94913403A Withdrawn EP0695424A1 (en) | 1993-04-19 | 1994-04-12 | A method for improving the signal to noise ratio of an immunoassay |
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EP (1) | EP0695424A1 (en) |
JP (1) | JPH08508101A (en) |
AU (1) | AU6558094A (en) |
WO (1) | WO1994024558A1 (en) |
Families Citing this family (3)
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US5962340A (en) * | 1994-11-02 | 1999-10-05 | Wako Pure Chemical Industries Ltd. | Homogeneous immunoassay method utilizing 5-300 mM magnesium |
CA2316130A1 (en) * | 1999-08-19 | 2001-02-19 | Masanori Fukui | Method for detection or determination of hcv core antigens and reagent for detection or determination for use therein |
CN114137222A (en) * | 2021-11-22 | 2022-03-04 | 江西乐成生物医疗有限公司 | Kit for detecting tissue metal protease inhibitor-1 in body fluid sample |
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DE3413118A1 (en) * | 1984-04-06 | 1985-10-24 | Miles Laboratories, Inc., Elkhart, Ind. | ANALYSIS METHOD AND MEANS FOR DETECTING ESTEROLYTIC AND / OR PROTEOLYTIC ENZYMS |
IL80129A0 (en) * | 1986-09-23 | 1986-12-31 | Savyon Diagnostics Ltd | Method for the determination of igm and iga immunoglobulins and reagents therefor |
DE3901458A1 (en) * | 1989-01-19 | 1990-07-26 | Behringwerke Ag | USE OF TWO OR THREE-VALUE CATIONS IN IMMUNCHEMICAL TESTS |
DE4035174A1 (en) * | 1990-11-06 | 1992-05-07 | Biotest Ag | METHOD FOR DETERMINING PROTEINS IN BODY LIQUIDS AND MEANS FOR IMPLEMENTING THE METHOD |
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1994
- 1994-04-12 JP JP6523375A patent/JPH08508101A/en active Pending
- 1994-04-12 EP EP94913403A patent/EP0695424A1/en not_active Withdrawn
- 1994-04-12 WO PCT/US1994/003963 patent/WO1994024558A1/en not_active Application Discontinuation
- 1994-04-12 AU AU65580/94A patent/AU6558094A/en not_active Abandoned
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AU6558094A (en) | 1994-11-08 |
WO1994024558A1 (en) | 1994-10-27 |
JPH08508101A (en) | 1996-08-27 |
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