CA1148859A - Simultaneous assay of two hepatitis viruses using a solid phase - Google Patents
Simultaneous assay of two hepatitis viruses using a solid phaseInfo
- Publication number
- CA1148859A CA1148859A CA000346729A CA346729A CA1148859A CA 1148859 A CA1148859 A CA 1148859A CA 000346729 A CA000346729 A CA 000346729A CA 346729 A CA346729 A CA 346729A CA 1148859 A CA1148859 A CA 1148859A
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- CA
- Canada
- Prior art keywords
- hepatitis
- sample
- solid phase
- labeled
- markers
- 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
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
Abstract
Canada #3680 A B S T R A C T
A method for simultaneously detecting in a sample at least two different markers evidencing exposure to hepatitis virus is disclosed. The method employs a solid phase reagent which is coated with at least two different, non-complimentary immunoreactants which are complimentary to the unknown markers to be detected, and a liquid reagent comprising at least two different hepatitis markers or immunoreactants, each selected to either react or compete with one of the unknown markers and each labeled with a detectably distinct tag.
A method for simultaneously detecting in a sample at least two different markers evidencing exposure to hepatitis virus is disclosed. The method employs a solid phase reagent which is coated with at least two different, non-complimentary immunoreactants which are complimentary to the unknown markers to be detected, and a liquid reagent comprising at least two different hepatitis markers or immunoreactants, each selected to either react or compete with one of the unknown markers and each labeled with a detectably distinct tag.
Description
BACKGROUND OF THE INVENTION
-This invention discloses an improvement in solid phase immunoassay methods for the detection and determina-tion of antigens and antibodies (markers) relating to hepa~
S titis.
There are at least two distinct types of viral hepatitis. Hepatitis A is believed to be caused by the hepa-titis A antigen (HAVAg) and is generally characterized by an incubation period of two to six weeks, mild prodromata and a 10 relatively mild clinical illness. The disease is generally transmitted by contaminated food or liquid, but has also been shown to be transmitted by systemic inoculation. Hepa-titis A is frequently called "infectious hepatitis", and in the United States the number of reported cases is over 50~000 15 annually.
The hepatitis B virus is believed to be the most probabl~ etiologic agent for "serum hepatitis". Hepatitis B
infection is generally transmitted by blood products or con-taminated instruments such as needles, but it may also be 20 transmitted by other means. Previously, a hepatitis B in-fection was associated with an incubation period ranging from six weeks to six months. However, incubation periods as short as two weeks have been documented. The illness may be mild or asymptomatic, but if symptomatic, manifestations may 25 be especially severe. Prodromata may include arthralgias, arthritis, rash, fever, anorexia, fatigue and pruritis with or without jaundice.
At least three distinct antigen-antibody systems can be associated with hepatitis virus B: the surface (HBsAg anti-HBs), the "core"(HBcAg: anti-HBc), and e-anti-gen(HBe~g: anti-HBe). The hepatitis B surface antigen(HBsAg) is found in the blood as 22nm spheres and as elongated tu-bules which are 22nm in diameter and variable in length and is believed to represent the protein coat of the hepatitis B
35 virus.
A 42nm particle containing DNA and a D~A polymerase is considered to represent the infectious virus (Dane parti-cle). The surface of the Dane particle is similar or identi-cal to HBsAg. In detergents, the Dane particle is degraded to a 27nm electron dense core, HBcAg. The latter is seen in nuclei of hepatocytes of patlents with serum hepatitis during the acute infection stage.
Thus, patients with viral hepatitis type B might 5 be expected to produce antibodies to the surface antigen (anti-HBs), and also to the protein core (anti-HBc). HBsAg in serum has been a consistent marker for the presence of the hepatitis B virus, and anti-HB usually appears during early viremia, often accompanying antigenemia (HB Ag), at the height 10 of liver disfunction and long before the appearance of anti-HBs.
Anti-HBc is generally associated with prolonged circulation of HBsAg suggesting that anti-HBc is produced in response to the active replication of the virus.
In 1972, the hepatitis e antigen (HBeAg) was de-15 tected and characterized. The e marker has been found in HBsAg-positive serum and is thought to occur more commonly in serum of chronic HBsAg carriers with active liver disease than in healthy carriers. In patients, during the incubation period of hepatitis B, the e antigen was shown to appear just after 20 the appearance of HBsAg and before clinically apparent liver injury. Logically, its presence in such sera would be indica-tive of a poor prognosis and on-going liver damage. Converse-ly, the presence of e antibody (anti-HBe) would be indicative of a good prognosis. These correlations are not absolute, but 25 may be useful clinical guides.
Since the serolo~ic markers for hepatitis appear in consistent, sequential order during the course of infection, acute disease and recovery, an analysis for two or more markers would be valuable to assess the time-course of the disease.
An assay for more than one marker can also provide an important double-check for HBsAg in blood doners or patients in an effort to reduce the incidence of type-B hepatitis. Such a need has been demonstrated by Goldfield et al; Am. J. Med.
Sci., 270: 335-342 (1975), who in careful follow-ups of recip-35 ients of blood negative for HBsAg, found evidence of exposure to the hepatitis antigen in 7 of 465 patients. Clearly, a method of detecting more than one marker to the hepatitis B
virus should decrease or prevent the occurrence of false nega-tives. Similarly, in many instances a ~ ~.fl~;~S9 positive response to two tests minimizes the possibility of a false positive occurring in one test.
While the reagents and methods described and claim-ed herein are similar to known commercial products and proced-5 ures for detecting the various markers to viral hepatitis,heretofore there has been no disclosure or suggestion describ-ing any techniques for detecting said markers simultaneously.
SU~MARY OF THE INVENTION
Accordingly, this specification describes in de-10 tail reagents and a method useful for detecting simultaneously in a sample at least two different markers evidencing exposure to hepatitis virus.
The method comprises contacting ~he sample with a solid support which has been coated with at least two differ-15 ent, non-complimentary immunoreactants which are complimentary to the unknown markers; incubating the sample with the coated solid support for a period up to 24 hours;
separating the coated solid support from the sample;
washing the coated solid support;
contacting the washed solid support with a liquid reagent comprising at least two different hepatitis markers or immunoreactants, each selected to either react or compete with one of the unknown hepatitis markers and each labeled with detectably distinct tags;
separating the solid phase from the liquid reagent;
and determining the presence of labeled markers on the solid support by detecting each distinct tag.
DESCRIPTION OF THE PREFERRED EMBODI~NTS
The following examples will demonstrate the prepa-30 ration and use of representative reagents within the scope of the claimed invention. The ~irst example describes the method of preparing a solid support coated with two different non-complimentary immunoreactants which are complimentary to markers evidencing exposure to hepatitis.
More specifically, a solid support (bead) has been coated with two different, noncomplimentary immunoreactants in such a manner that both retain their reactivity and are able to combine with complimentary markers in an unknown 8~5~
ds ~
specimen.
In the first example a polystyrene bead has been coated with an antibody to HBsAg and with the core antigen, HBcAg. The antibody retains its avidity and will react with 5 any HBsAg in the unknown specimen and the affixed core anti-gen will retain its antigenicity and react with any antibody to the core (anti-HBc) in the unknown specimen. The crux of this invention is that both immunoreactions will occur simultaneously with a single solid phase reagent.
EXAMPLE I
PREPAR~TION OF COATED SUPPORT
A formulation of HBcAg was made by exposing Dane particles to a solution of 2% 2-mercaptoethanol (2-ME) and 5~ Tween~80 in Tris-EDTA-saline (TSE) buffers at 37C for 2 15 hours. That solution was diluted 10-fold in 5% 2~-TSE and allowed to stand overnight before diluting to final concen-tration of 1:8000 in TSE. Separately, polystyrene beads (1/4") were coated with a 1:2000 dilution of anti-HBs serum ~guinea pig) in phosphate buffered saline (PBS) by soaking 20 for two hours. The beads were removed, washed and dried.
The core preparation was then poured over the coated beads and the core antigen was allowed to adhere to the bead for 2 days at room temperature. The beads were removed from the buffer solukion, washed, coated with a 10~ sucrose solution 25 to stabilize the adherents and air dried.
Although polystyrene beads are preferred because they are easily coated and manipulated, any solid support of either macro or micro dimensions, fashioned from a variety of plastics, metal and glass could just as easily be coated and 30 used to demonstrate the claimed invention.
While it is possible that the beads may be coated first with core antigen and subsequently coated with anti-HBS, it has been observed that coating the heads first withthe guinea pig anti-HBsserum significantly augments the ad-35 herence of core antigen.
It is also important to note that other combina-tions of non-complimentary immunoreactants relating to hepa-titis virus may also be employed. The immunoreactants must, of course, be non-complimentary because a complimentary at-traction would diminish the antigenicity and avidity of the reagent.
The following example will demonstrate the prepara-5 tion of a liquid phase rea~ent containing an immunoreactant,anti-HBs , tagged with a radio label (125I) and a marker, anti-HBC , tagged with a reactive enzyme label (horseradish peroxidase).
While the terms "marker" and "immunoreactant"
10 might be used interchangably, it is preferable if the term "marker" is used to designate the antigen or antibody, and their equivalents, to be detected in the unknown sample. The term "immunoreactant" is used to define either the antigen or antibody complimentary to the markers to be detected.
Accordingly, in the following examples, the anti-gen and antibody employed on the solid support are always complementary to one of the unknown markers, and, therefore, are referred to as immunoreactants.
In the liquid phase reagent, the labeled surface ~0 antibody is complimentary to the unknown marker (HBsAg) and is, therefore, referred to as the labeled immunoreactant. The labeled core antibody, however, is identical to the marker to be detected and is, therefore, referred tc as a labeled marker.
EXAMPLE II
TAGGED ANTIBODY REAGENT
The iodination (125I) of antibody to HBsAg (anti-HBs) entailed adding approximately 0.1 ml of a 0.5M phosphate buffer at pH7.5, a small volume of 5-6 mci of Nal25I, and 100 mg. of purified anti-HBs to a one dram vial, adjusting the pH
to 7.5-8.0 and mixing all ingredients. To this mixture was added 50 microliters of a freshly prepared solution of chloramine T (35mg in 10 ml. 0.05M phosphate buffer, pH 7.5).
After additional mixing, the reaction was allowed to proceed at room temperature for 60 seconds. Fifty microliters of a freshly prepared solution of sodium metasulfite (35 mg in 10 ml of 0.05M phosphate buffer at pH 7.5) were added to the reaction to reduce the chloramine T and thereby stop the re-action.
-- 6 ~
The reaction efficiency was checked by placing 5 microliters of the reaction mlxture on a strip of Whatman No. 1 paper strip and chromatographing the mixture in 70%
methanol. The iodinated protein remained at the origin while 5 the free 125I migrated with the solvent. The ~ of 125I in protein is~ccns de~ed t~ be th~ %l o~ re~c~i~o~ efic~ncy.
The crude iodinated antibody was purified using a Sephadex~G-50 gel column with 0.1 molar Tris buffer con-taining 0.9% sodium chloride at pH 7.8. The column was pre-10 treated with a small volume of a 30~ aqueous solution ofbovine serum albumin followed by an equal additional volume of 0.1 molar Tris buffer solution containing 0.9% sodium chloride at pH 7.8. The iodinated antibody was added to the top of the column and washed through using the saline 15 buffered Tris solution. The labeled antibody was the first eluate collected from the column.
The conjugation of antibody to hepatitis B core to horseradish peroxidase (HRP) entailed activating peroxi-dase by using sodium meta periodate. The excess periodate 20 and by-products were seParated ~rom the active peroxidase by ~ e~
gel filtration (4~u~x~ G-25) column. The activated peroxi-dase was then reacted with the antibody (anti-HBc). The re-action occurred spontaneously. Sodium borohydride was then added to stabilize the bond formed between peroxidase and 25 the antibody, and acetone was added to destroy the remaining sodium borohydride.
~hen employed according to the teaching of this invention, the two tagged antibodies are diluted into a diluent containing:
~0 50% fetal calf serum 15~ normal human serum .005 M EDT~
0.1% Tween-20 0.01% ~ in PBS
It should be apparent that a variety of detect-able tags may be employed. The only requirement, naturally, is that they be detectably distinct. Any of a variety of isotopes could be utilized just as easily as 125I. It is not necessary that one marker or immunoreactant be radio-5~
labeled and t~e other enzymatically or florescently labeled since different isotopes are, themselves, detectably distinct.
Similarly, all labeled components of the reagent might just as easily employ different enzymes requiring different sub-strates yielding detectably distinct reaction products.
It should be noted that any of the antibodies and antigens of hepatitis A and B could be labeled to perform according to the disclosed invention. The only immunochemical requirements are that the labeled components not be compli-lO mentary with each other or to the markers to be determined.
EXAMPLE III
SIMULT~EOUS DETECTION OF HB Ag AND ~TI-HBC
-- s Serum samples containing the "unknown" hepatitis markers were added to the individual wells of a reaction tray and a polystyrene bead coated in accordance with Example I was added to each specimen sample and allowed to incubate for 2 hours at 45C. The beads were removed from the reaction well, washed with water to remove any excess reagent, and added to 0.2 ml of the liquid phase reagent prepared in accordance 20 with Example II. The solid and liquid phases were incubated for a period of one hour at 45C. The solid phase was then separated from the antibody reagent and washed four times with water to remove excess reagent. The washed beads were placed in test tubes containing o-phenylenediamine(30 mg in 10 ml of 0.1 M citrate buffer, pH 5.5) and allowed to incubate for 30 minutes. Following that period the enzyme reaction was stopped by the addition of 1 ml of 1 M HCl and the tubes were visually examined for the presence or absence of color resulting from the reaction of the enzyme tag and the substrate. The ab-sence or low incidence of color indicated that anti-HBc was present in the unknown sample and competed with the labeled marker for reaction sites on the coated support. Next, radio-activity on the beads was counted in a gamma counter and the CPM's were recorded. Radioactivity on the bead indicated that there was HBsAg in the unknown sample which adhered to the affixed antibody and provided a binding site for the radio-labeled antibody.
The invention may now be practiced in the various B~9 ways suggested by the foregoing elaboration and all such ways are now defined by the terms of the following claims.
-This invention discloses an improvement in solid phase immunoassay methods for the detection and determina-tion of antigens and antibodies (markers) relating to hepa~
S titis.
There are at least two distinct types of viral hepatitis. Hepatitis A is believed to be caused by the hepa-titis A antigen (HAVAg) and is generally characterized by an incubation period of two to six weeks, mild prodromata and a 10 relatively mild clinical illness. The disease is generally transmitted by contaminated food or liquid, but has also been shown to be transmitted by systemic inoculation. Hepa-titis A is frequently called "infectious hepatitis", and in the United States the number of reported cases is over 50~000 15 annually.
The hepatitis B virus is believed to be the most probabl~ etiologic agent for "serum hepatitis". Hepatitis B
infection is generally transmitted by blood products or con-taminated instruments such as needles, but it may also be 20 transmitted by other means. Previously, a hepatitis B in-fection was associated with an incubation period ranging from six weeks to six months. However, incubation periods as short as two weeks have been documented. The illness may be mild or asymptomatic, but if symptomatic, manifestations may 25 be especially severe. Prodromata may include arthralgias, arthritis, rash, fever, anorexia, fatigue and pruritis with or without jaundice.
At least three distinct antigen-antibody systems can be associated with hepatitis virus B: the surface (HBsAg anti-HBs), the "core"(HBcAg: anti-HBc), and e-anti-gen(HBe~g: anti-HBe). The hepatitis B surface antigen(HBsAg) is found in the blood as 22nm spheres and as elongated tu-bules which are 22nm in diameter and variable in length and is believed to represent the protein coat of the hepatitis B
35 virus.
A 42nm particle containing DNA and a D~A polymerase is considered to represent the infectious virus (Dane parti-cle). The surface of the Dane particle is similar or identi-cal to HBsAg. In detergents, the Dane particle is degraded to a 27nm electron dense core, HBcAg. The latter is seen in nuclei of hepatocytes of patlents with serum hepatitis during the acute infection stage.
Thus, patients with viral hepatitis type B might 5 be expected to produce antibodies to the surface antigen (anti-HBs), and also to the protein core (anti-HBc). HBsAg in serum has been a consistent marker for the presence of the hepatitis B virus, and anti-HB usually appears during early viremia, often accompanying antigenemia (HB Ag), at the height 10 of liver disfunction and long before the appearance of anti-HBs.
Anti-HBc is generally associated with prolonged circulation of HBsAg suggesting that anti-HBc is produced in response to the active replication of the virus.
In 1972, the hepatitis e antigen (HBeAg) was de-15 tected and characterized. The e marker has been found in HBsAg-positive serum and is thought to occur more commonly in serum of chronic HBsAg carriers with active liver disease than in healthy carriers. In patients, during the incubation period of hepatitis B, the e antigen was shown to appear just after 20 the appearance of HBsAg and before clinically apparent liver injury. Logically, its presence in such sera would be indica-tive of a poor prognosis and on-going liver damage. Converse-ly, the presence of e antibody (anti-HBe) would be indicative of a good prognosis. These correlations are not absolute, but 25 may be useful clinical guides.
Since the serolo~ic markers for hepatitis appear in consistent, sequential order during the course of infection, acute disease and recovery, an analysis for two or more markers would be valuable to assess the time-course of the disease.
An assay for more than one marker can also provide an important double-check for HBsAg in blood doners or patients in an effort to reduce the incidence of type-B hepatitis. Such a need has been demonstrated by Goldfield et al; Am. J. Med.
Sci., 270: 335-342 (1975), who in careful follow-ups of recip-35 ients of blood negative for HBsAg, found evidence of exposure to the hepatitis antigen in 7 of 465 patients. Clearly, a method of detecting more than one marker to the hepatitis B
virus should decrease or prevent the occurrence of false nega-tives. Similarly, in many instances a ~ ~.fl~;~S9 positive response to two tests minimizes the possibility of a false positive occurring in one test.
While the reagents and methods described and claim-ed herein are similar to known commercial products and proced-5 ures for detecting the various markers to viral hepatitis,heretofore there has been no disclosure or suggestion describ-ing any techniques for detecting said markers simultaneously.
SU~MARY OF THE INVENTION
Accordingly, this specification describes in de-10 tail reagents and a method useful for detecting simultaneously in a sample at least two different markers evidencing exposure to hepatitis virus.
The method comprises contacting ~he sample with a solid support which has been coated with at least two differ-15 ent, non-complimentary immunoreactants which are complimentary to the unknown markers; incubating the sample with the coated solid support for a period up to 24 hours;
separating the coated solid support from the sample;
washing the coated solid support;
contacting the washed solid support with a liquid reagent comprising at least two different hepatitis markers or immunoreactants, each selected to either react or compete with one of the unknown hepatitis markers and each labeled with detectably distinct tags;
separating the solid phase from the liquid reagent;
and determining the presence of labeled markers on the solid support by detecting each distinct tag.
DESCRIPTION OF THE PREFERRED EMBODI~NTS
The following examples will demonstrate the prepa-30 ration and use of representative reagents within the scope of the claimed invention. The ~irst example describes the method of preparing a solid support coated with two different non-complimentary immunoreactants which are complimentary to markers evidencing exposure to hepatitis.
More specifically, a solid support (bead) has been coated with two different, noncomplimentary immunoreactants in such a manner that both retain their reactivity and are able to combine with complimentary markers in an unknown 8~5~
ds ~
specimen.
In the first example a polystyrene bead has been coated with an antibody to HBsAg and with the core antigen, HBcAg. The antibody retains its avidity and will react with 5 any HBsAg in the unknown specimen and the affixed core anti-gen will retain its antigenicity and react with any antibody to the core (anti-HBc) in the unknown specimen. The crux of this invention is that both immunoreactions will occur simultaneously with a single solid phase reagent.
EXAMPLE I
PREPAR~TION OF COATED SUPPORT
A formulation of HBcAg was made by exposing Dane particles to a solution of 2% 2-mercaptoethanol (2-ME) and 5~ Tween~80 in Tris-EDTA-saline (TSE) buffers at 37C for 2 15 hours. That solution was diluted 10-fold in 5% 2~-TSE and allowed to stand overnight before diluting to final concen-tration of 1:8000 in TSE. Separately, polystyrene beads (1/4") were coated with a 1:2000 dilution of anti-HBs serum ~guinea pig) in phosphate buffered saline (PBS) by soaking 20 for two hours. The beads were removed, washed and dried.
The core preparation was then poured over the coated beads and the core antigen was allowed to adhere to the bead for 2 days at room temperature. The beads were removed from the buffer solukion, washed, coated with a 10~ sucrose solution 25 to stabilize the adherents and air dried.
Although polystyrene beads are preferred because they are easily coated and manipulated, any solid support of either macro or micro dimensions, fashioned from a variety of plastics, metal and glass could just as easily be coated and 30 used to demonstrate the claimed invention.
While it is possible that the beads may be coated first with core antigen and subsequently coated with anti-HBS, it has been observed that coating the heads first withthe guinea pig anti-HBsserum significantly augments the ad-35 herence of core antigen.
It is also important to note that other combina-tions of non-complimentary immunoreactants relating to hepa-titis virus may also be employed. The immunoreactants must, of course, be non-complimentary because a complimentary at-traction would diminish the antigenicity and avidity of the reagent.
The following example will demonstrate the prepara-5 tion of a liquid phase rea~ent containing an immunoreactant,anti-HBs , tagged with a radio label (125I) and a marker, anti-HBC , tagged with a reactive enzyme label (horseradish peroxidase).
While the terms "marker" and "immunoreactant"
10 might be used interchangably, it is preferable if the term "marker" is used to designate the antigen or antibody, and their equivalents, to be detected in the unknown sample. The term "immunoreactant" is used to define either the antigen or antibody complimentary to the markers to be detected.
Accordingly, in the following examples, the anti-gen and antibody employed on the solid support are always complementary to one of the unknown markers, and, therefore, are referred to as immunoreactants.
In the liquid phase reagent, the labeled surface ~0 antibody is complimentary to the unknown marker (HBsAg) and is, therefore, referred to as the labeled immunoreactant. The labeled core antibody, however, is identical to the marker to be detected and is, therefore, referred tc as a labeled marker.
EXAMPLE II
TAGGED ANTIBODY REAGENT
The iodination (125I) of antibody to HBsAg (anti-HBs) entailed adding approximately 0.1 ml of a 0.5M phosphate buffer at pH7.5, a small volume of 5-6 mci of Nal25I, and 100 mg. of purified anti-HBs to a one dram vial, adjusting the pH
to 7.5-8.0 and mixing all ingredients. To this mixture was added 50 microliters of a freshly prepared solution of chloramine T (35mg in 10 ml. 0.05M phosphate buffer, pH 7.5).
After additional mixing, the reaction was allowed to proceed at room temperature for 60 seconds. Fifty microliters of a freshly prepared solution of sodium metasulfite (35 mg in 10 ml of 0.05M phosphate buffer at pH 7.5) were added to the reaction to reduce the chloramine T and thereby stop the re-action.
-- 6 ~
The reaction efficiency was checked by placing 5 microliters of the reaction mlxture on a strip of Whatman No. 1 paper strip and chromatographing the mixture in 70%
methanol. The iodinated protein remained at the origin while 5 the free 125I migrated with the solvent. The ~ of 125I in protein is~ccns de~ed t~ be th~ %l o~ re~c~i~o~ efic~ncy.
The crude iodinated antibody was purified using a Sephadex~G-50 gel column with 0.1 molar Tris buffer con-taining 0.9% sodium chloride at pH 7.8. The column was pre-10 treated with a small volume of a 30~ aqueous solution ofbovine serum albumin followed by an equal additional volume of 0.1 molar Tris buffer solution containing 0.9% sodium chloride at pH 7.8. The iodinated antibody was added to the top of the column and washed through using the saline 15 buffered Tris solution. The labeled antibody was the first eluate collected from the column.
The conjugation of antibody to hepatitis B core to horseradish peroxidase (HRP) entailed activating peroxi-dase by using sodium meta periodate. The excess periodate 20 and by-products were seParated ~rom the active peroxidase by ~ e~
gel filtration (4~u~x~ G-25) column. The activated peroxi-dase was then reacted with the antibody (anti-HBc). The re-action occurred spontaneously. Sodium borohydride was then added to stabilize the bond formed between peroxidase and 25 the antibody, and acetone was added to destroy the remaining sodium borohydride.
~hen employed according to the teaching of this invention, the two tagged antibodies are diluted into a diluent containing:
~0 50% fetal calf serum 15~ normal human serum .005 M EDT~
0.1% Tween-20 0.01% ~ in PBS
It should be apparent that a variety of detect-able tags may be employed. The only requirement, naturally, is that they be detectably distinct. Any of a variety of isotopes could be utilized just as easily as 125I. It is not necessary that one marker or immunoreactant be radio-5~
labeled and t~e other enzymatically or florescently labeled since different isotopes are, themselves, detectably distinct.
Similarly, all labeled components of the reagent might just as easily employ different enzymes requiring different sub-strates yielding detectably distinct reaction products.
It should be noted that any of the antibodies and antigens of hepatitis A and B could be labeled to perform according to the disclosed invention. The only immunochemical requirements are that the labeled components not be compli-lO mentary with each other or to the markers to be determined.
EXAMPLE III
SIMULT~EOUS DETECTION OF HB Ag AND ~TI-HBC
-- s Serum samples containing the "unknown" hepatitis markers were added to the individual wells of a reaction tray and a polystyrene bead coated in accordance with Example I was added to each specimen sample and allowed to incubate for 2 hours at 45C. The beads were removed from the reaction well, washed with water to remove any excess reagent, and added to 0.2 ml of the liquid phase reagent prepared in accordance 20 with Example II. The solid and liquid phases were incubated for a period of one hour at 45C. The solid phase was then separated from the antibody reagent and washed four times with water to remove excess reagent. The washed beads were placed in test tubes containing o-phenylenediamine(30 mg in 10 ml of 0.1 M citrate buffer, pH 5.5) and allowed to incubate for 30 minutes. Following that period the enzyme reaction was stopped by the addition of 1 ml of 1 M HCl and the tubes were visually examined for the presence or absence of color resulting from the reaction of the enzyme tag and the substrate. The ab-sence or low incidence of color indicated that anti-HBc was present in the unknown sample and competed with the labeled marker for reaction sites on the coated support. Next, radio-activity on the beads was counted in a gamma counter and the CPM's were recorded. Radioactivity on the bead indicated that there was HBsAg in the unknown sample which adhered to the affixed antibody and provided a binding site for the radio-labeled antibody.
The invention may now be practiced in the various B~9 ways suggested by the foregoing elaboration and all such ways are now defined by the terms of the following claims.
Claims (10)
1. A method of simultaneously detecting in a sample at least two different markers evidencing exposure to hepatitis virus, said method comprising:
a) contacting the sample with a solid phase coated with at least two different, noncomplimentary immunoreac-tants complimentary to the unknown markers;
b) incubating the sample with the coated solid phase for a period of 1-24 hours;
c) separating the coated solid phase from the sample;
d) washing the coated solid phase to remove unbound sample;
e) contacting the washed solid support with a liquid re-agent comprising at least two different hepatitis markers or immunoreactants, each selected to either react or compete with one of the unknown hepatitis markers, and each labeled with detectably distinct tags;
f) separating the solid phase from the labeled reagent;
g) and determining the presence of labeled markers or immunoreactants on the solid support by detecting the distinct tags.
a) contacting the sample with a solid phase coated with at least two different, noncomplimentary immunoreac-tants complimentary to the unknown markers;
b) incubating the sample with the coated solid phase for a period of 1-24 hours;
c) separating the coated solid phase from the sample;
d) washing the coated solid phase to remove unbound sample;
e) contacting the washed solid support with a liquid re-agent comprising at least two different hepatitis markers or immunoreactants, each selected to either react or compete with one of the unknown hepatitis markers, and each labeled with detectably distinct tags;
f) separating the solid phase from the labeled reagent;
g) and determining the presence of labeled markers or immunoreactants on the solid support by detecting the distinct tags.
2. A method of simultaneously detecting in a sample hepatitis B surface antigen and antibody to hepatitis B core antigen, said method comprising:
a) contacting the sample with a solid phase coated with both hepatitis B core antigen and an antibody to hepa-titis B surface antigen;
b) incubating the sample with the coated solid phase for a period of 1-24 hours;
c) separating the coated solid phase from the sample;
d) washing the coated solid phase to remove unbound sample;
e) contacting the washed solid support with a liquid re-agent comprising antibodies to hepatitis B surface antigen and hepatitis s core antigen each labeled with detectably distinct tags.
f) separating the solid phase from the labeled antibody reagent; and g) determining the presence of labeled antibodies on the solid support by detecting the distinct tags.
a) contacting the sample with a solid phase coated with both hepatitis B core antigen and an antibody to hepa-titis B surface antigen;
b) incubating the sample with the coated solid phase for a period of 1-24 hours;
c) separating the coated solid phase from the sample;
d) washing the coated solid phase to remove unbound sample;
e) contacting the washed solid support with a liquid re-agent comprising antibodies to hepatitis B surface antigen and hepatitis s core antigen each labeled with detectably distinct tags.
f) separating the solid phase from the labeled antibody reagent; and g) determining the presence of labeled antibodies on the solid support by detecting the distinct tags.
3. The method according to claim 2 wherein the antibody to the surface antigen is radiolabeled and the antibody to the core antigen is labeled with an enzyme.
4. The method according to claim 3 wherein the radiolabel is 125I and the enzyme is horseradish peroxidase.
5. A reagent useful for simultaneously detecting in a sample at least two different markers evidencing exposure to hepatitis virus said reagent comprising:
at least two different hepatitis markers or immunoreactants each selected to either react or compete with an unknown hepatitis marker and each labeled with detectably distinct tags.
at least two different hepatitis markers or immunoreactants each selected to either react or compete with an unknown hepatitis marker and each labeled with detectably distinct tags.
6. A reagent according to claim 5 wherein the reagent is further defined as comprising antibodies to hepatitis B
surface antigen and hepatitis B core antigen.
surface antigen and hepatitis B core antigen.
7. A reagent according to claim 6 wherein the antibody to hepatitis B surface antigen is radiolabeled.
8. A reagent according to claim 7 wherein the radiolabel is 125I.
9. A reagent according to claim 6 wherein the antibody to hepatitis B core antigen is labeled with an enzyme.
10. A reagent according to claim 9 wherein the enzyme label is horseradish peroxidase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US4831979A | 1979-06-14 | 1979-06-14 | |
| US48,319 | 1979-06-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1148859A true CA1148859A (en) | 1983-06-28 |
Family
ID=21953924
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000346729A Expired CA1148859A (en) | 1979-06-14 | 1980-02-29 | Simultaneous assay of two hepatitis viruses using a solid phase |
Country Status (12)
| Country | Link |
|---|---|
| JP (2) | JPS562558A (en) |
| AT (1) | AT368813B (en) |
| AU (1) | AU530580B2 (en) |
| BE (1) | BE883824A (en) |
| CA (1) | CA1148859A (en) |
| CH (1) | CH654113A5 (en) |
| DE (1) | DE3021891C2 (en) |
| FR (1) | FR2459482A1 (en) |
| GB (1) | GB2051357B (en) |
| IT (1) | IT1140846B (en) |
| NL (1) | NL8001661A (en) |
| ZA (1) | ZA801318B (en) |
Families Citing this family (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0058428B1 (en) * | 1981-02-18 | 1985-10-09 | Eisai Co., Ltd. | An enzyme immuno-assay for simultaneously measuring a plurality of samples and test vessel for carrying out this method |
| GB2165046B (en) * | 1982-02-10 | 1986-10-15 | Baker Terence S | Ligand molecule |
| IE54109B1 (en) * | 1982-02-10 | 1989-06-21 | Boots Celltech Diagnostics | Assay |
| JPS58187861A (en) * | 1982-04-26 | 1983-11-02 | Otsuka Pharmaceut Co Ltd | Assay of antibody related to adult type t leukemia |
| GB2125547B (en) * | 1982-07-31 | 1986-04-23 | Mochida Pharm Co Ltd | Simultaneous immunoassay of two or more substances |
| JPS5984162A (en) * | 1982-11-05 | 1984-05-15 | Toshiba Corp | Immunological analysis method |
| EP0131974A1 (en) * | 1983-06-08 | 1985-01-23 | Akzo N.V. | Determination of delta-antigens in body fluids |
| EP0139316B1 (en) * | 1983-09-14 | 1989-04-05 | Akzo N.V. | Method for the immunochemical determination of hepatitis b core antigens |
| US4701421A (en) * | 1984-08-27 | 1987-10-20 | Akzo, N.V. | Determination of protecting anti-HBV immunoglobulins |
| GB8607101D0 (en) * | 1986-03-21 | 1986-04-30 | Serono Diagnostics Ltd | Immunoassay |
| AU1334588A (en) * | 1987-03-23 | 1988-09-22 | Ortho Pharmaceutical Corporation | Assay for detecting hepatitis antigens and aids antibodies |
| CA1335880C (en) * | 1988-07-14 | 1995-06-13 | Thomas P. O'connor | Detection of an antibody and antigen in an immunoassay |
| AU4346989A (en) * | 1988-10-14 | 1990-05-01 | Cecil Czerkinsky | A method for simultaneous detection of different types of antibodies and/or antigens produced by individual cells |
| DE3919810A1 (en) * | 1989-06-16 | 1990-12-20 | Biochrom Beteiligungs Gmbh & C | SOLID-PHASE IMMUNOASSAY AND TEST EQUIPMENT FOR DETERMINING ANTIGENS AND ANTIBODIES |
| AT394114B (en) * | 1989-07-13 | 1992-02-10 | Immuno Ag | METHOD FOR DETERMINING ANTIGENS AND / OR ANTIBODIES IN HUMAN BODY LIQUIDS, AND SET FOR CARRYING OUT THE METHOD |
| EP0473065A3 (en) * | 1990-08-29 | 1992-08-26 | Abbott Laboratories | Simultaneous assay for detecting two or more analytes |
| DE4236189A1 (en) * | 1992-10-27 | 1994-04-28 | Boehringer Mannheim Gmbh | Method for the simultaneous determination of antigens and antibodies |
| EP1020727B1 (en) * | 1998-07-30 | 2008-10-29 | Advanced Life Science Institute, Inc. | Method for assaying hepatitis c virus |
| EP1672366A3 (en) * | 1999-06-11 | 2007-11-28 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Methods and compositions for opsonophagocytic assays |
| US7101683B2 (en) * | 2001-06-26 | 2006-09-05 | Abbott Laboratories | Methods for the simultaneous detection of HCV antigens and HCV antibodies |
| WO2003002749A2 (en) * | 2001-06-26 | 2003-01-09 | Abbott Laboratories | Methods for the simultaneous detection of hcv antigens and hcv antibodies |
| US7332269B2 (en) * | 2001-11-11 | 2008-02-19 | Ortho-Clinical Diagnostics, Inc. | HCV core protein sequences |
| CN109828110A (en) | 2013-04-26 | 2019-05-31 | 生物辐射实验室股份有限公司 | Multiple hepatitis B test |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3867517A (en) * | 1971-12-21 | 1975-02-18 | Abbott Lab | Direct radioimmunoassay for antigens and their antibodies |
| GB1506017A (en) * | 1974-03-04 | 1978-04-05 | Int Diagnostic Tech | Fluorometric system and method for the detection of biologically derived substances |
| SE7610683L (en) * | 1975-09-29 | 1977-06-10 | Cordis Corp | METHOD OF DETERMINING THE NERVARON OF AN ANTIGEN ASSOCIATE WITH HEPATITIS |
| AT343822B (en) * | 1976-08-20 | 1978-06-26 | Immuno Ag | RADIOIMMUNOLOGICAL METHOD AND EQUIPMENT FOR DETERMINING ANTIGENES |
| DE2803154C2 (en) * | 1977-01-27 | 1986-07-24 | Becton, Dickinson and Co., Paramus, N.J. | Simultaneous radio testing of folate and vitamin B 12 |
| CA1100037A (en) * | 1977-03-11 | 1981-04-28 | Chung-Mei Ling | Hb.sub.c ag coated on solid phase |
| US4102996A (en) * | 1977-04-20 | 1978-07-25 | Merck & Co., Inc. | Method of preparing hepatitis B core antigen |
| US4189464A (en) * | 1978-05-05 | 1980-02-19 | Institute For Cancer Research | Hepatitis B testing reagent and method |
| IL55816A (en) * | 1978-10-30 | 1982-04-30 | Ames Yissum Ltd | Method for simultaneous immunoassay of several different antibodies and a kit therefor |
-
1980
- 1980-02-29 CA CA000346729A patent/CA1148859A/en not_active Expired
- 1980-03-06 AU AU56226/80A patent/AU530580B2/en not_active Ceased
- 1980-03-06 ZA ZA00801318A patent/ZA801318B/en unknown
- 1980-03-11 GB GB8008095A patent/GB2051357B/en not_active Expired
- 1980-03-20 NL NL8001661A patent/NL8001661A/en not_active Application Discontinuation
- 1980-05-07 IT IT21874/80A patent/IT1140846B/en active
- 1980-06-06 JP JP7568180A patent/JPS562558A/en active Granted
- 1980-06-11 DE DE3021891A patent/DE3021891C2/en not_active Expired
- 1980-06-12 AT AT0309980A patent/AT368813B/en not_active IP Right Cessation
- 1980-06-13 FR FR8013217A patent/FR2459482A1/en active Granted
- 1980-06-13 CH CH4579/80A patent/CH654113A5/en not_active IP Right Cessation
- 1980-06-13 BE BE0/201040A patent/BE883824A/en not_active IP Right Cessation
-
1990
- 1990-01-16 JP JP2004676A patent/JPH02276969A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CH654113A5 (en) | 1986-01-31 |
| GB2051357B (en) | 1983-05-18 |
| DE3021891C2 (en) | 1983-08-04 |
| AU530580B2 (en) | 1983-07-21 |
| GB2051357A (en) | 1981-01-14 |
| FR2459482B1 (en) | 1984-10-26 |
| FR2459482A1 (en) | 1981-01-09 |
| ZA801318B (en) | 1981-02-25 |
| DE3021891A1 (en) | 1980-12-18 |
| BE883824A (en) | 1980-12-15 |
| IT1140846B (en) | 1986-10-10 |
| JPH0220945B2 (en) | 1990-05-11 |
| NL8001661A (en) | 1980-12-16 |
| AU5622680A (en) | 1980-12-18 |
| ATA309980A (en) | 1982-03-15 |
| JPS562558A (en) | 1981-01-12 |
| IT8021874A0 (en) | 1980-05-07 |
| AT368813B (en) | 1982-11-10 |
| JPH02276969A (en) | 1990-11-13 |
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