CA1172561A - Igm detection method - Google Patents
Igm detection methodInfo
- Publication number
- CA1172561A CA1172561A CA000394315A CA394315A CA1172561A CA 1172561 A CA1172561 A CA 1172561A CA 000394315 A CA000394315 A CA 000394315A CA 394315 A CA394315 A CA 394315A CA 1172561 A CA1172561 A CA 1172561A
- Authority
- CA
- Canada
- Prior art keywords
- hepatitis
- igm
- solid support
- plasma
- igg
- 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5768—Hepatitis A
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Communicable Diseases (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Canada - #3836 A B S T R A C T
The invention involves an improved method for detecting IgM antibody to hepatitis A virus. It is an improvement in a three-step assay for IgM antibody to hepatitis A virus wherein plastic beads are coated with anti-human IgM
(µ chain specific) and are incubated successively with serum specimen, hepatitis A virus antigen and I125 labeled antibody to hepatitis A virus antigen; the improvement involves the pretreatment of the specimen or test sample with a dilute aqueous solution of hepatitis negative human serum, plasma, or IgG containing fraction thereof.
The invention involves an improved method for detecting IgM antibody to hepatitis A virus. It is an improvement in a three-step assay for IgM antibody to hepatitis A virus wherein plastic beads are coated with anti-human IgM
(µ chain specific) and are incubated successively with serum specimen, hepatitis A virus antigen and I125 labeled antibody to hepatitis A virus antigen; the improvement involves the pretreatment of the specimen or test sample with a dilute aqueous solution of hepatitis negative human serum, plasma, or IgG containing fraction thereof.
Description
1 ~7;Z5~L
Background Of The Invention 1. Field Of me Invention This invention is in the field of immunoassay, in general, and the immunoassay of IgM for hepatitis A virus in particular.
The invention involves a method for improving specificity of detection of IgM for hepatitis A vlrus in human serum or plasmar Description Of me Prior Art During the acute phase of hepatitis A infection, IgM-type antibody to hepatitis A virus (anti-HAV IgM) appears in the patient's serum and persists through early eonvalescence and is not deteeted in normal subjects. Thus, deteeting anti-HAV IgM provides a diagnosis for acute hepatitis A infection.
The prior art deseribes various approaches to class speeific antibody detection, for example, U.S. Patents 4,020,151;
4,048,298; and 3,904,367; Bradley et al, Journal of Clinical Micro-biology, May, 1977, pages 521-530; Lancet, Septe~ber 23, 1978, page 684; Hepatitis Scientific ~morandum, November, 1978, page 3; and Hepatitis Seientific Memorandum, February, 1979, page 2; all indieate approaehes to elass speeific an~ibody deteetion. A three-step solid phase immunoassay for IgM is kncwn. In the latter assay, a plastie bead coated with anti-human IgM (~ ehain speeifie) is ineubated sueees-sively with serum speeimen, hepatitis A virus antigen, and labeled anti-body to hepatitis A virus antigen. This invention represents an im-provement in that assay whereby the solid support eoated with ~ ehain speeifie anti-human IgM or the test sample is treated with a dilute aqueous solution of human hepatitis negative serum, plasma or IgG
eontaining fraction thereof.
Brief Deseription of the Invention This invention is an improvement in detecting the aeute phase of hepatitis A infection by meansuring IgM antibodies to hepa-titis A virus antigen. The improvement involves
Background Of The Invention 1. Field Of me Invention This invention is in the field of immunoassay, in general, and the immunoassay of IgM for hepatitis A virus in particular.
The invention involves a method for improving specificity of detection of IgM for hepatitis A vlrus in human serum or plasmar Description Of me Prior Art During the acute phase of hepatitis A infection, IgM-type antibody to hepatitis A virus (anti-HAV IgM) appears in the patient's serum and persists through early eonvalescence and is not deteeted in normal subjects. Thus, deteeting anti-HAV IgM provides a diagnosis for acute hepatitis A infection.
The prior art deseribes various approaches to class speeific antibody detection, for example, U.S. Patents 4,020,151;
4,048,298; and 3,904,367; Bradley et al, Journal of Clinical Micro-biology, May, 1977, pages 521-530; Lancet, Septe~ber 23, 1978, page 684; Hepatitis Scientific ~morandum, November, 1978, page 3; and Hepatitis Seientific Memorandum, February, 1979, page 2; all indieate approaehes to elass speeific an~ibody deteetion. A three-step solid phase immunoassay for IgM is kncwn. In the latter assay, a plastie bead coated with anti-human IgM (~ ehain speeifie) is ineubated sueees-sively with serum speeimen, hepatitis A virus antigen, and labeled anti-body to hepatitis A virus antigen. This invention represents an im-provement in that assay whereby the solid support eoated with ~ ehain speeifie anti-human IgM or the test sample is treated with a dilute aqueous solution of human hepatitis negative serum, plasma or IgG
eontaining fraction thereof.
Brief Deseription of the Invention This invention is an improvement in detecting the aeute phase of hepatitis A infection by meansuring IgM antibodies to hepa-titis A virus antigen. The improvement involves
- 2 mab/~;
_ 3 _ 117~561 diluting the test sample with an aqueous solution containing hepatitis negative serum, plasma or IgG containing fraction thereof. Thus, it has been discovered that lO0 to 200 fold dilution of test sample with a diluent containing about 0.1%
hepatitis negative human serum or plasma greatly improves the specificity of a solid phase assay involving an anti-human IgM coated solid support reagent assay wherein the solid support reagent is reacted with the diluted test sample, washed, reacted with hepatitis A antigen, washed, and reacted with labeled hepatitis A antibody, washed and the amount of label on the solid support is measured. Alternatively, the solid support can be pretreated with a dilute aqueous solution of hepatitis negative human serum, plasma, or IgM containing fraction thereof. In this later embodiment, treatment with the IgG containing fraction is preferred.
Description Of The Preferred F.mbodiment For practicing a preferred embodiment of the invention the following reagents were prepared:
Anti-~ coated beads-Goat antiserum against human IgM, ~ chain specific, was obtained from Litton Bionetics. Polystyrene beads (6mm) were coated for two hours with the antiserum diluted 10 3 in O.OlM Tris buffer, pH 9.0, and were washed and dried in air. Beads were stored at 4 C until used.
Hepatitis A virus (HAV) antigen solution:
HAV was obtained from the livers of tamarin marmosets in the acute stages of hepatitis A. Liver extracts were prepared, and viral infectivity was inactivated with formalin. Extracts were diluted in O.OlM phosphate buffer, 30 pH 7.0 in normal saline (PBS) containing 5mM EDIA and 0.1%
sodium azide to give suitable cpm in the assay.
5I_anti-HAV:
IgG was isolated by DEAE cellulose chromatography from convalescent sera with high titers of anti-HAV. It was radiolabeled with l25I by the method of Greenwood. The i~dinated antibody was diluted to about 5 x 106 cpm per ml in PBS containing calf serum, normal human serum, EDTA and sodium azide.
-- ~;
- 1 ~7~561 First diluent solution;
Each specimen to be tested was first diluted 200 fold in normal saline (0.9% sodium chloride) or phosphate buffered saline (PBS 0.01 M sodium phosphate, pH 7~2 in 0.9% saline).
Second diluent solution (specimen diluent;
A second solution was used for further dilution of the specimen. An aliquot of the first dilution of the specimen in saline or PBS is placed in the reaction well ~nd is diluted 21 fold with a solution consisting of : 0.1%
normal human serum or plasma; 10% fetal calf serum; ~005 M
EDTA (ethylene diamine tetraacetate); 0.2~ Tween -20; and 0.1~ sodium azide in 0.01 PBS, pH 7.4., Assay controls:
Positive control was prepared from acute or early convalescent sera from subjects with hepatitis A. Negative control was prepared in the same manner from sera negative for anti-HAV.
Test Procedure:
The test procedure can be summarized as follows:
1. Bead.anti ~ + IgM ~ Bead.anti ~-IgM
2~ Bead.anti ~-IgM + HAV ~ Bead.anti ~-IgM-HAV
_ 3 _ 117~561 diluting the test sample with an aqueous solution containing hepatitis negative serum, plasma or IgG containing fraction thereof. Thus, it has been discovered that lO0 to 200 fold dilution of test sample with a diluent containing about 0.1%
hepatitis negative human serum or plasma greatly improves the specificity of a solid phase assay involving an anti-human IgM coated solid support reagent assay wherein the solid support reagent is reacted with the diluted test sample, washed, reacted with hepatitis A antigen, washed, and reacted with labeled hepatitis A antibody, washed and the amount of label on the solid support is measured. Alternatively, the solid support can be pretreated with a dilute aqueous solution of hepatitis negative human serum, plasma, or IgM containing fraction thereof. In this later embodiment, treatment with the IgG containing fraction is preferred.
Description Of The Preferred F.mbodiment For practicing a preferred embodiment of the invention the following reagents were prepared:
Anti-~ coated beads-Goat antiserum against human IgM, ~ chain specific, was obtained from Litton Bionetics. Polystyrene beads (6mm) were coated for two hours with the antiserum diluted 10 3 in O.OlM Tris buffer, pH 9.0, and were washed and dried in air. Beads were stored at 4 C until used.
Hepatitis A virus (HAV) antigen solution:
HAV was obtained from the livers of tamarin marmosets in the acute stages of hepatitis A. Liver extracts were prepared, and viral infectivity was inactivated with formalin. Extracts were diluted in O.OlM phosphate buffer, 30 pH 7.0 in normal saline (PBS) containing 5mM EDIA and 0.1%
sodium azide to give suitable cpm in the assay.
5I_anti-HAV:
IgG was isolated by DEAE cellulose chromatography from convalescent sera with high titers of anti-HAV. It was radiolabeled with l25I by the method of Greenwood. The i~dinated antibody was diluted to about 5 x 106 cpm per ml in PBS containing calf serum, normal human serum, EDTA and sodium azide.
-- ~;
- 1 ~7~561 First diluent solution;
Each specimen to be tested was first diluted 200 fold in normal saline (0.9% sodium chloride) or phosphate buffered saline (PBS 0.01 M sodium phosphate, pH 7~2 in 0.9% saline).
Second diluent solution (specimen diluent;
A second solution was used for further dilution of the specimen. An aliquot of the first dilution of the specimen in saline or PBS is placed in the reaction well ~nd is diluted 21 fold with a solution consisting of : 0.1%
normal human serum or plasma; 10% fetal calf serum; ~005 M
EDTA (ethylene diamine tetraacetate); 0.2~ Tween -20; and 0.1~ sodium azide in 0.01 PBS, pH 7.4., Assay controls:
Positive control was prepared from acute or early convalescent sera from subjects with hepatitis A. Negative control was prepared in the same manner from sera negative for anti-HAV.
Test Procedure:
The test procedure can be summarized as follows:
1. Bead.anti ~ + IgM ~ Bead.anti ~-IgM
2~ Bead.anti ~-IgM + HAV ~ Bead.anti ~-IgM-HAV
3. Bead~anti ~-IgM-HAV + I anti-HAV
Bead.anti ~-IgM-H,AV- I-anti-HAV
Tests including positive and negative control are conducted according to the following protocol:
1. Add 10 ~Q of each specimen to 2 ml normal saline.
2. Pipette 10 ~Q of each control or diluted specimen into one of the well of a reaction tray.
3. Add 200 ~Q specimen diluent to each well, tap tray to mix.
Bead.anti ~-IgM-H,AV- I-anti-HAV
Tests including positive and negative control are conducted according to the following protocol:
1. Add 10 ~Q of each specimen to 2 ml normal saline.
2. Pipette 10 ~Q of each control or diluted specimen into one of the well of a reaction tray.
3. Add 200 ~Q specimen diluent to each well, tap tray to mix.
4. Add one bead coated with antibody to human IgM to each well.
5. Cover tray and tap to mix. Incubate at room te~perature for two hours.
6. Aspirate liquid, then wash each bead twice with 5 ml of water.
* trade mark - 5 - 117ZS~l
* trade mark - 5 - 117ZS~l
7. Add 200 ~Q HAV solution to each well~
Cover and incubate at room temperature for 18 to 22 hours.
Cover and incubate at room temperature for 18 to 22 hours.
8. Repeat step 6.
9~ Add 200 ~Q anti-HAV 5I to each well~
Cover and incubate at 45~ C for four hours.
Cover and incubate at 45~ C for four hours.
10. Repeat step 6.
11. Transfer beadstocounting tubes and count in a suitable counter for one minute.
12. Determine the average cpm of the positive control and of the negative control~
Calculate the positive-negative cut off by adding one-tenth of the positive control mean to the negative control mean; e~g., x PC + x NC = cutoff.
A specimen is judged positive if the cpm values are e~ual to or greater than the cutoff.
Calculate the positive-negative cut off by adding one-tenth of the positive control mean to the negative control mean; e~g., x PC + x NC = cutoff.
A specimen is judged positive if the cpm values are e~ual to or greater than the cutoff.
13. As a check on test performance, determine the positive to negative (P:N) ratio using net cpm values for each control.
A P:N value must be ~ 5.0 to ensure a valid run~ . .
Effect of specimen dilution and of the presence of normal human plasma (NHP) on Hepatitis A IgM Specificity is shown in Table I.
, .
.
1~7~Stii~
TABLE I
No NHP in Diluent 0.1% NHP in Diluent Specimen Tested Dilution 1:50 1:200 1:50 1:200 * S: CO +/o S_ +/oS: CO +/o S: ~70 High IgM anti-HAV 6.32 + 5.88 + 6.61 + 6.44 +
Weak IgM anti-HAV 5.17 + 2~69 + 4.80 + 2.33 +
Patient I IgG anti-HAV 1.10 + 1.03 + 0.85 o 0.54 o Patient II IgG anti-HAV 1.10 + 0.84 o 0.96 o 0.63 o *S:CO is the sample to cut off ratio It can be seen from patients I and II that strong or elevated IgG anti-lIAV levels can give a false positive but the false positive is eliminated by dilution with a diluent con-taining 0.1% normal human plasma. Thus, diluting a sample 50-500 fold with normal saline in the presence of about a 10-25 times dilution with a .1% hepatitis negative human serum or plasma solution increases the sensitivity of the TgM/IgG
ratio of the reagent-human anti-IgM coated solid support.
Those skilled in the immunoassay arts will recognize that the antihuman IgM coated solid support may be reacted with the aqueous solution of hepatitis negative serum, plasma, or IgG
fraction thereof before the assay proceduxe is begun or, as above, during the assay procedure. Generally, only very small amounts of the hepatitis negative serum or plasma are required to increase the IgM/IgG sensitivity ratio of the reagent.
_ . .
A P:N value must be ~ 5.0 to ensure a valid run~ . .
Effect of specimen dilution and of the presence of normal human plasma (NHP) on Hepatitis A IgM Specificity is shown in Table I.
, .
.
1~7~Stii~
TABLE I
No NHP in Diluent 0.1% NHP in Diluent Specimen Tested Dilution 1:50 1:200 1:50 1:200 * S: CO +/o S_ +/oS: CO +/o S: ~70 High IgM anti-HAV 6.32 + 5.88 + 6.61 + 6.44 +
Weak IgM anti-HAV 5.17 + 2~69 + 4.80 + 2.33 +
Patient I IgG anti-HAV 1.10 + 1.03 + 0.85 o 0.54 o Patient II IgG anti-HAV 1.10 + 0.84 o 0.96 o 0.63 o *S:CO is the sample to cut off ratio It can be seen from patients I and II that strong or elevated IgG anti-lIAV levels can give a false positive but the false positive is eliminated by dilution with a diluent con-taining 0.1% normal human plasma. Thus, diluting a sample 50-500 fold with normal saline in the presence of about a 10-25 times dilution with a .1% hepatitis negative human serum or plasma solution increases the sensitivity of the TgM/IgG
ratio of the reagent-human anti-IgM coated solid support.
Those skilled in the immunoassay arts will recognize that the antihuman IgM coated solid support may be reacted with the aqueous solution of hepatitis negative serum, plasma, or IgG
fraction thereof before the assay proceduxe is begun or, as above, during the assay procedure. Generally, only very small amounts of the hepatitis negative serum or plasma are required to increase the IgM/IgG sensitivity ratio of the reagent.
_ . .
Claims (3)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. In a method for improving the IgM/IgG
sensitivity ratio of IgM antibody to hepatitis A virus antigen of the type wherein an antihuman IgM coated solid support is reacted with test sample, washed, reacted with hepatitis A antigen, washed, reacted with labeled anti-body to hepatitis A antigen, washed, and the amount of labeled antibody bound to the solid support is measured, the improvement comprising diluting the test sample with an aqueous solution of hepatitis negative human serum, plasma, or IgG containing fraction thereof.
sensitivity ratio of IgM antibody to hepatitis A virus antigen of the type wherein an antihuman IgM coated solid support is reacted with test sample, washed, reacted with hepatitis A antigen, washed, reacted with labeled anti-body to hepatitis A antigen, washed, and the amount of labeled antibody bound to the solid support is measured, the improvement comprising diluting the test sample with an aqueous solution of hepatitis negative human serum, plasma, or IgG containing fraction thereof.
2. A method for improving the IgM/IgG
sensitivity ratio of a reagent for detecting IgM for hepa-titis A virus in a test sample wherein the reagent is an antihuman IgM coated solid support comprising:
contacting the antihuman IgM coated solid support with an aqueous solution of hepatitis-negative serum, plasma, or IgG containing fraction thereof.
sensitivity ratio of a reagent for detecting IgM for hepa-titis A virus in a test sample wherein the reagent is an antihuman IgM coated solid support comprising:
contacting the antihuman IgM coated solid support with an aqueous solution of hepatitis-negative serum, plasma, or IgG containing fraction thereof.
3. An immunoassay reagent comprising anti-human IgM coated solid support said reagent further treated with a dilute aqueous solution of hepatitis nega-tive human serum, plasma, or IgG containing fraction thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US22925081A | 1981-01-28 | 1981-01-28 | |
| US229,250 | 1981-01-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1172561A true CA1172561A (en) | 1984-08-14 |
Family
ID=22860424
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000394315A Expired CA1172561A (en) | 1981-01-28 | 1982-01-18 | Igm detection method |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS57144462A (en) |
| BE (1) | BE891901A (en) |
| CA (1) | CA1172561A (en) |
| DE (1) | DE3202559A1 (en) |
| FR (1) | FR2498760B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9523685B2 (en) | 2007-06-08 | 2016-12-20 | Bio-Rad Innovations | Multiplex method for detecting an infection |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1240937A (en) * | 1982-07-26 | 1988-08-23 | Gordon R. Dreesman | Monoclonal igm antibodies and method of preparation |
| JPH05126829A (en) * | 1991-04-03 | 1993-05-21 | Syntex Usa Inc | Immunoassay of immunogloblin |
| DE19548375A1 (en) * | 1995-12-27 | 1997-07-03 | Behringwerke Ag | Immunological method of determination |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1059901A (en) * | 1974-06-17 | 1979-08-07 | Chung-Mei Ling | Direct radioimmunoassay for antigens and their antibodies |
| US3992517A (en) * | 1975-02-19 | 1976-11-16 | Pfizer Inc. | Detection of hepatitis B surface antigen by latex agglutination |
| US4273756A (en) * | 1978-07-28 | 1981-06-16 | Abbott Laboratories | Immunoassay for class specific antibodies |
| US4464474A (en) * | 1980-07-09 | 1984-08-07 | Connaught Laboratories Limited | Non-A, non-B hepatitis assay and vaccine |
-
1982
- 1982-01-18 CA CA000394315A patent/CA1172561A/en not_active Expired
- 1982-01-26 BE BE0/207141A patent/BE891901A/en not_active IP Right Cessation
- 1982-01-27 FR FR8201260A patent/FR2498760B1/en not_active Expired
- 1982-01-27 DE DE19823202559 patent/DE3202559A1/en not_active Ceased
- 1982-01-28 JP JP57011053A patent/JPS57144462A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9523685B2 (en) | 2007-06-08 | 2016-12-20 | Bio-Rad Innovations | Multiplex method for detecting an infection |
Also Published As
| Publication number | Publication date |
|---|---|
| DE3202559A1 (en) | 1982-08-05 |
| JPS57144462A (en) | 1982-09-07 |
| FR2498760A1 (en) | 1982-07-30 |
| JPH0368346B2 (en) | 1991-10-28 |
| FR2498760B1 (en) | 1986-04-04 |
| BE891901A (en) | 1982-07-26 |
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