CA1206092A - Glucose oxidase immunohistochemical detection of antinuclear antibodies - Google Patents
Glucose oxidase immunohistochemical detection of antinuclear antibodiesInfo
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- CA1206092A CA1206092A CA000410409A CA410409A CA1206092A CA 1206092 A CA1206092 A CA 1206092A CA 000410409 A CA000410409 A CA 000410409A CA 410409 A CA410409 A CA 410409A CA 1206092 A CA1206092 A CA 1206092A
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- glucose
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
- G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
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Abstract
GLUCOSE OXIDASE IMMUNOHISTOCHEMICAL
DETECTION OF ANTINUCLEAR ANTIBODIES
ABSTRACT
For use in the immunohistochemical detec-tion of antigens and related antibodies in a test serum, an antigen source fixed on a support and stabilized by dehydration treatment with an organic solvent mixture, such as acetone methanol, or mixtures thereof. The support is stored under nitrogen in a sealed polyethylene bag. Further, an immunoenzymatic method for the detec-tion of biological components for a test sample, the method comprising the steps of: contacting the stabilized antigen source with the test sample, contacting the antigen source with an antibody conjugated with glucose oxidase and reactive with the component; incubating the antigen souce with a solution containing glucose and a chromagenic mixture; and analyzing the antigen source for the presence of color, preferably with a light microscope.
DETECTION OF ANTINUCLEAR ANTIBODIES
ABSTRACT
For use in the immunohistochemical detec-tion of antigens and related antibodies in a test serum, an antigen source fixed on a support and stabilized by dehydration treatment with an organic solvent mixture, such as acetone methanol, or mixtures thereof. The support is stored under nitrogen in a sealed polyethylene bag. Further, an immunoenzymatic method for the detec-tion of biological components for a test sample, the method comprising the steps of: contacting the stabilized antigen source with the test sample, contacting the antigen source with an antibody conjugated with glucose oxidase and reactive with the component; incubating the antigen souce with a solution containing glucose and a chromagenic mixture; and analyzing the antigen source for the presence of color, preferably with a light microscope.
Description
GLUCOSE OXIDASE IMMUNOHISTOCHEMICAL
DETECTION OF ANTINUCLEAR ANTIBODIES
BAC~GROUND O~ THE INVENTION
This invention relates generally to immuno-enzymatic processes for use in medical di.agnoses 9 and more particularl~, to an immunohistochemical process for the detection of antinuclear antibodies in test serum~
In recent years, an increasing amount of research has focused on improving clinical laboratory tests, A primary thrust of this research has been to replace the traditional radiotracer labels with safer and more stable labels.
~ 15 The res~arch eff~rts have been quite successful : for a number of clinical assaysO For example, in tests for antinucle~r antibodies ~ANA), fl~orescent markers have found wide accept~nce. While fluvrescent markers give quick results~ they have not proven entirely satis-factory for a number of assays bec~use once placed in a detectable state they have a very limited shelf life, fading after a few days or more. Moreover, fluorescent assay~ frequently eachibit high background due ~o auto-flu~rescence caused by non-speci~ic binding ~o various proteins7 and are impr~ctical for ~ome laboratories beca~se they re~uire thè use of relatively expensive fluorescent microscopes.
~ .
Anoth~r procedure for replacing radiotracer based ~ssays, utiliæing enzyme markers in immuno~ssay and immunohistochemical tests, has been practiced by a number of clinical laboratories in recent years. One of -5 ~he mos~ commonly used enzymes is horseradish peroxidase, which has been succ~ssfully used as a marker in immuno~
histochemical procedures for the detection of a number of ti~sue antigens and related antibodies~ ~ue to the relatively small ~ize and high cellular penetration of this enzyme, it has ~und particular utility in the field of electron microscopy. For the light microscope, horseradish peroxidase has nst prov~n entirely satis-factory, hcwever, because of a high tendency to produce non-specific background ~tain, eYen in spite of attempts ~o ~uench the presence o~ endogenou~ peroxidase~like actiYity. Another objectionable feature of the peroxi-dase label is that it generally requires the use of a highly carcinogenic material, di~minobenzidine, for superior s~ain developmen~O A.lthough ~ubstitutes for this material have been develo]ped, their contrast and intensity are typically not entirely satisf2ctory.
From the foregoing, it will be appre~iated that ~here exi~ts a definite need for a simple, inexpensive~
~a~e and reliable antinucle~r antibody detection method ~hat can produce a rela~ively permanent record o~ the assay substantially in the ab~enc@ of non~specific backgro~nd~ The presen~ invention fulfills ~his need.
S~MMARY O~ THE INVENTIQN
The present inventlon provides an immunohisto-chemical method which ~ubstantially reduces the amo~nt ofbackground stain caused by endo~enous materials~ while producing a relatively permanent record of the assay.
~IL2()~
Moreover, 'che reagent~ of the present inventi~n are relatively inexpensive to manufac~ure, are seable for long period~ c)f time even when stored at room tempera-ture, and have minimal carcinogenic characteristics.
In accordance with the inveneion, an immuno-enzy~atic process for the detection of a c~mponerlt of a test sample is provided, said proeess comprisin~ s~eps o~:
contacting the test ~ample with a c~llular ar~igen source f ixed onto a suppc~rt and stabili ed by d~hydration;
contacting the antigen source with an antibody conjugaeed with glucose cxidase and reac~cive with the component;
incubating 'che antigen ~our~e with the olution containin9 ~lucose and a chromoclenic mixture; and analyzing the antigen source for the presence o~ color from the incubation~
The chromogenic mixt:ureg preferably con~l:ain t-nitrohlue tetrazolium chloricle and l-methoxyphenazine me~ho-sulfate, which permit ~nalysis with a light microscope.
When the com~nent of the test sample is an antinuclear antibody, the antigen source fixed ~n a slass slide is preferably E~ep-2 cell~ or rae kidney tissue, and the conjugated antibody is goat immunoglobulin reactive with ihuman immun~globulirls.
I~ore particularly" an immunohistochemical method for detecting antinuclear antibodies in a te~t serum, substantially in the absence of backgrourlld stain ing, is provided. This me~hod co~pri~es s~eps of:
providing a cellular nuclear antigen source ixed onto a slide and dehydrated with acetone at about 4 C.
~'s:,, ~L2C~ 2 contacting the nuclear antigen so~rce with the test ~erum;
contacting the nuclear antigen source with goa~
immunoglobulin conjug~ed with glucose oxidalse and reactive with the antinuclear antibodies;
in~ubatin~ the nu~lear antigen source with a solution containing beta-D-glucose~ t-nitrohlue tetra-201lum, and l-methoxyphena2ine methosulfate; and analyzing the nuclear antigen source under 1~ a light microscope for the presence of a formazan stain~
Another aspect of the invention is a m~ethod of stabilizing an immunochemical substrate fixed on a solid support ~ the method comprisirlg the steps of:
providing a ~ubstrate f ixed on the support;
contacting the sub~;trate wi'ch ~n organic solvent reagent to 6~bstantially dehydrate the substrate;
placing the support into a protective sheath;
backfilling the sheath with a gas; and seal ing the support in the sheath .
The suppor~ is pre$erably a glass slide, and the s~bstrate Hep-2 cells or rat kidney ti~sue. The organic solvent reagent can be composed of ace~one, me~chanol ~ or mixtures ~hereof, and the in~eracting step performed between about 4 and 8 C. The ~heath can be 25 ~ omposed of a polyes~er film, and can cont~ a des-icant. The preferred gas is ni'crogen gas,, Other aspects and advantages of the presen inven~ion will become apparen~ from the following de-scription of the preferred em~odiment, which discloses, by way of ex~mple, the principles of the invention.
'~
Exemplary ~tarting materials useful ln prac-~icing the present inVentiQn are slides fixed with human epethelial ~lls (Hep-2) or wi~h rat kidney tissue S suitable for use in antinuclear antibody assays. Such lides can be purchased commercially from a number of sources, including An~ibodies Incorporated, Davis, Californi~, and Behring, La Jslla, California, or, alternatively, can be manufactured according ~o con-1~ vention~l techniques. Generally, these slicles should bestored at -2~ C. to prevent degrad~tion.
:rn accordance with the present invention, the 51 ides are immersed for about 10 minutles in a bath containing histological grade 2~cetone~ meth~nol, or a 1~ combination of these organic solven~s. The ba~h is maintained a,t a temperature preferably ranging from about 4C to about 8~C. After removing the slides from the bath, they are allowed to air dry ~or about 2 ~o 3 minutes~ Although the dehydration caused by treatment with the organi~ solvent mixl~ure has significantly stabiliz~d the substrate fixed on the slides9 the slides are preferably ~tored at about 5C during con~inued processingO
After dehydration~ the slides are placed into a protective sheath 9 prefer~bly ~ade from a polyethylene ~ilm~ sueh as MYLAR bags with an aluminum outer l~yer.
A small pack~t containing a standard dessicant is placed in the bag, and ~he bag is partially sealed. Ni~rogen gas is then introduced through the unseal*d portion of the bag unti1 the bag bulges 51 ightly. The bag is then totally sealed to prevent the nitrogen gas from esc3ping~
,, * trade mark.
Goat immunoglc~bulin con jug~ted with the glucose o~idase enæyme can be prepared a; follows.
Gluc0s2 oxidase, which is available from a var:iety of s~ommercial sources~ ineluding Sigm~, St. Louis~ ~Sissouri, 5 and ~ehringer Manneheirn, Indianapolis, Indiana, is di~solved in ~istilled water at a concentration of abou~ 12 mg/ml . To 'chis solution is added about 0. 2 ml of ~0 o l M ~odium periodate. The mix~ure i~ stirred for about 20 minutes and then dialyzed, preferably against 1 10 mM soàium acetate bufferr pH 4.4, for about 16 hours.
After dialysis, the glucose oxidase solution is mixed wlth 1 ml of 0.01 M sodium bicarbonate buffer, pH 9.5, containing about B mg of the IgG fraction of goat anti-human immunQglobulins ~IgG, IgA, and ~gM)I After stir-ring for abou~ ~wo hours~ 0.1 mls of an aqueous solutioncontaininy about 4 mg/ml sodium borohydrate water solu-tion is ~ddedr When ~hat reaction h~s completed (about 2 hours at 4C), the entire mixture can be chro~atographed on a gel filtration column~ such as a Sephaeryl* S-300, available from Pharmacia, Piscal:away, New Jersey, and the fractions containing the enzyme-IgG conjugate pooled.
This pool can then be titrated to determine working dilutions.
In accordance with another aspec~ of the invention, a method or detecting antinuclear antibodies util iæing these reagent~ is performed as follows O
small amount t~:E test serum, or other te~t solution, is contact2d with l:he nuclear antigen f ixed source on the slide. After about ~hir~y minutes at room temperature, 30 ~he serum is wa~hed from the slide with phosplla~e buf-fered saline (PESS); ~nd ~hen soaked in a cold PBS solu-kion for abou~ 10 minlates . The &l ides are removed rom ~he PBS and excess moisture blotted wi~h absorbent pape r .
* trade mark.
A ~mall amoun~ ~f the enzyme-IgG con~ugate, preferably about 50 ~1~ is eontacted with the antigen ~ource for ~bout 30 minutes at room temperature.
The slid~ is again washed with PB~, so~ked for 10 S minutes in a cold PBS ~olution, removed, and excess moisture ~lotted with absorbent paper.
At this time, the color reagent may be pre-pared. It oonsists of a~out 2 volumes beta-D-gluoose, lS
mg/ml in 0.1 M sodium phosphate buffer, pH 6~9~ one 10 volume t-nitroblue tetrazolium (t-NBT), 2 mg/ml in the same buffer (if necessary, this may he filtered through Whatman No. 1 filter paper just prior to use)l and 1 volume of l-methoxyphenazine methosulfate (m-PMS), 0.8 mg/ml in the same buffer. Although the preferred chromo-genic reag~ants are t-N~T and m-PMS, vther materials can also be utilized~ To en~ure mutarotation of the glucose molecules ~o their beta form, the mixture may be prepared about 1 hour before the st~ining reaction.
A small volume, prefer~,bly about 100 ~1, Qf the ~0 color reagen~ are contacted w:ith the antigen source.
The slides are placed in a ~lide chamber and incubated for about 30 minutes At 55 C~ to permit forma~ion of ~he formazan st~in. The e~cess co~or reagent is then w~shed ~rom the slide wi~h PBS, soaked for about 15 minutes in ~old PBS ~nd the excess moisture again removed with the absorbent paper.
The slide i then prepared for light miero~cope viewin~ according to standard techniquesa Briefly, a glycer~1-gelatin mounting medium is placed in a 55C.
incubator for about 5 to 10 minutes for liqu~fication~
and a 13rge drop placed over the antigen source 9 The antigen source is then covered with a cover slip, taking care to avoid form~tion of air bubbles.
~2~
When the slides are examined under a light microscope, the existence of antinuclear antibodies in the test serum will cause slightly enlarged, homo-geneous dark nuclei to appear, substantially without dark background staining~
From the foregoing descriptionr it should be apparent that the present invention provides an ef-fective, inexpensive, highly visible, and permanent record of an antinuclear antibody assay. Further, the nuclear an~igen source fixed onto the slide is extremely stable and does not require storage at low temperatures.
While a particular form of the invention has been described in detail with reference to its presently preferred embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, it is not intended that the invention be limi~ed except by the appended claims.
DETECTION OF ANTINUCLEAR ANTIBODIES
BAC~GROUND O~ THE INVENTION
This invention relates generally to immuno-enzymatic processes for use in medical di.agnoses 9 and more particularl~, to an immunohistochemical process for the detection of antinuclear antibodies in test serum~
In recent years, an increasing amount of research has focused on improving clinical laboratory tests, A primary thrust of this research has been to replace the traditional radiotracer labels with safer and more stable labels.
~ 15 The res~arch eff~rts have been quite successful : for a number of clinical assaysO For example, in tests for antinucle~r antibodies ~ANA), fl~orescent markers have found wide accept~nce. While fluvrescent markers give quick results~ they have not proven entirely satis-factory for a number of assays bec~use once placed in a detectable state they have a very limited shelf life, fading after a few days or more. Moreover, fluorescent assay~ frequently eachibit high background due ~o auto-flu~rescence caused by non-speci~ic binding ~o various proteins7 and are impr~ctical for ~ome laboratories beca~se they re~uire thè use of relatively expensive fluorescent microscopes.
~ .
Anoth~r procedure for replacing radiotracer based ~ssays, utiliæing enzyme markers in immuno~ssay and immunohistochemical tests, has been practiced by a number of clinical laboratories in recent years. One of -5 ~he mos~ commonly used enzymes is horseradish peroxidase, which has been succ~ssfully used as a marker in immuno~
histochemical procedures for the detection of a number of ti~sue antigens and related antibodies~ ~ue to the relatively small ~ize and high cellular penetration of this enzyme, it has ~und particular utility in the field of electron microscopy. For the light microscope, horseradish peroxidase has nst prov~n entirely satis-factory, hcwever, because of a high tendency to produce non-specific background ~tain, eYen in spite of attempts ~o ~uench the presence o~ endogenou~ peroxidase~like actiYity. Another objectionable feature of the peroxi-dase label is that it generally requires the use of a highly carcinogenic material, di~minobenzidine, for superior s~ain developmen~O A.lthough ~ubstitutes for this material have been develo]ped, their contrast and intensity are typically not entirely satisf2ctory.
From the foregoing, it will be appre~iated that ~here exi~ts a definite need for a simple, inexpensive~
~a~e and reliable antinucle~r antibody detection method ~hat can produce a rela~ively permanent record o~ the assay substantially in the ab~enc@ of non~specific backgro~nd~ The presen~ invention fulfills ~his need.
S~MMARY O~ THE INVENTIQN
The present inventlon provides an immunohisto-chemical method which ~ubstantially reduces the amo~nt ofbackground stain caused by endo~enous materials~ while producing a relatively permanent record of the assay.
~IL2()~
Moreover, 'che reagent~ of the present inventi~n are relatively inexpensive to manufac~ure, are seable for long period~ c)f time even when stored at room tempera-ture, and have minimal carcinogenic characteristics.
In accordance with the inveneion, an immuno-enzy~atic process for the detection of a c~mponerlt of a test sample is provided, said proeess comprisin~ s~eps o~:
contacting the test ~ample with a c~llular ar~igen source f ixed onto a suppc~rt and stabili ed by d~hydration;
contacting the antigen source with an antibody conjugaeed with glucose cxidase and reac~cive with the component;
incubating 'che antigen ~our~e with the olution containin9 ~lucose and a chromoclenic mixture; and analyzing the antigen source for the presence o~ color from the incubation~
The chromogenic mixt:ureg preferably con~l:ain t-nitrohlue tetrazolium chloricle and l-methoxyphenazine me~ho-sulfate, which permit ~nalysis with a light microscope.
When the com~nent of the test sample is an antinuclear antibody, the antigen source fixed ~n a slass slide is preferably E~ep-2 cell~ or rae kidney tissue, and the conjugated antibody is goat immunoglobulin reactive with ihuman immun~globulirls.
I~ore particularly" an immunohistochemical method for detecting antinuclear antibodies in a te~t serum, substantially in the absence of backgrourlld stain ing, is provided. This me~hod co~pri~es s~eps of:
providing a cellular nuclear antigen source ixed onto a slide and dehydrated with acetone at about 4 C.
~'s:,, ~L2C~ 2 contacting the nuclear antigen so~rce with the test ~erum;
contacting the nuclear antigen source with goa~
immunoglobulin conjug~ed with glucose oxidalse and reactive with the antinuclear antibodies;
in~ubatin~ the nu~lear antigen source with a solution containing beta-D-glucose~ t-nitrohlue tetra-201lum, and l-methoxyphena2ine methosulfate; and analyzing the nuclear antigen source under 1~ a light microscope for the presence of a formazan stain~
Another aspect of the invention is a m~ethod of stabilizing an immunochemical substrate fixed on a solid support ~ the method comprisirlg the steps of:
providing a ~ubstrate f ixed on the support;
contacting the sub~;trate wi'ch ~n organic solvent reagent to 6~bstantially dehydrate the substrate;
placing the support into a protective sheath;
backfilling the sheath with a gas; and seal ing the support in the sheath .
The suppor~ is pre$erably a glass slide, and the s~bstrate Hep-2 cells or rat kidney ti~sue. The organic solvent reagent can be composed of ace~one, me~chanol ~ or mixtures ~hereof, and the in~eracting step performed between about 4 and 8 C. The ~heath can be 25 ~ omposed of a polyes~er film, and can cont~ a des-icant. The preferred gas is ni'crogen gas,, Other aspects and advantages of the presen inven~ion will become apparen~ from the following de-scription of the preferred em~odiment, which discloses, by way of ex~mple, the principles of the invention.
'~
Exemplary ~tarting materials useful ln prac-~icing the present inVentiQn are slides fixed with human epethelial ~lls (Hep-2) or wi~h rat kidney tissue S suitable for use in antinuclear antibody assays. Such lides can be purchased commercially from a number of sources, including An~ibodies Incorporated, Davis, Californi~, and Behring, La Jslla, California, or, alternatively, can be manufactured according ~o con-1~ vention~l techniques. Generally, these slicles should bestored at -2~ C. to prevent degrad~tion.
:rn accordance with the present invention, the 51 ides are immersed for about 10 minutles in a bath containing histological grade 2~cetone~ meth~nol, or a 1~ combination of these organic solven~s. The ba~h is maintained a,t a temperature preferably ranging from about 4C to about 8~C. After removing the slides from the bath, they are allowed to air dry ~or about 2 ~o 3 minutes~ Although the dehydration caused by treatment with the organi~ solvent mixl~ure has significantly stabiliz~d the substrate fixed on the slides9 the slides are preferably ~tored at about 5C during con~inued processingO
After dehydration~ the slides are placed into a protective sheath 9 prefer~bly ~ade from a polyethylene ~ilm~ sueh as MYLAR bags with an aluminum outer l~yer.
A small pack~t containing a standard dessicant is placed in the bag, and ~he bag is partially sealed. Ni~rogen gas is then introduced through the unseal*d portion of the bag unti1 the bag bulges 51 ightly. The bag is then totally sealed to prevent the nitrogen gas from esc3ping~
,, * trade mark.
Goat immunoglc~bulin con jug~ted with the glucose o~idase enæyme can be prepared a; follows.
Gluc0s2 oxidase, which is available from a var:iety of s~ommercial sources~ ineluding Sigm~, St. Louis~ ~Sissouri, 5 and ~ehringer Manneheirn, Indianapolis, Indiana, is di~solved in ~istilled water at a concentration of abou~ 12 mg/ml . To 'chis solution is added about 0. 2 ml of ~0 o l M ~odium periodate. The mix~ure i~ stirred for about 20 minutes and then dialyzed, preferably against 1 10 mM soàium acetate bufferr pH 4.4, for about 16 hours.
After dialysis, the glucose oxidase solution is mixed wlth 1 ml of 0.01 M sodium bicarbonate buffer, pH 9.5, containing about B mg of the IgG fraction of goat anti-human immunQglobulins ~IgG, IgA, and ~gM)I After stir-ring for abou~ ~wo hours~ 0.1 mls of an aqueous solutioncontaininy about 4 mg/ml sodium borohydrate water solu-tion is ~ddedr When ~hat reaction h~s completed (about 2 hours at 4C), the entire mixture can be chro~atographed on a gel filtration column~ such as a Sephaeryl* S-300, available from Pharmacia, Piscal:away, New Jersey, and the fractions containing the enzyme-IgG conjugate pooled.
This pool can then be titrated to determine working dilutions.
In accordance with another aspec~ of the invention, a method or detecting antinuclear antibodies util iæing these reagent~ is performed as follows O
small amount t~:E test serum, or other te~t solution, is contact2d with l:he nuclear antigen f ixed source on the slide. After about ~hir~y minutes at room temperature, 30 ~he serum is wa~hed from the slide with phosplla~e buf-fered saline (PESS); ~nd ~hen soaked in a cold PBS solu-kion for abou~ 10 minlates . The &l ides are removed rom ~he PBS and excess moisture blotted wi~h absorbent pape r .
* trade mark.
A ~mall amoun~ ~f the enzyme-IgG con~ugate, preferably about 50 ~1~ is eontacted with the antigen ~ource for ~bout 30 minutes at room temperature.
The slid~ is again washed with PB~, so~ked for 10 S minutes in a cold PBS ~olution, removed, and excess moisture ~lotted with absorbent paper.
At this time, the color reagent may be pre-pared. It oonsists of a~out 2 volumes beta-D-gluoose, lS
mg/ml in 0.1 M sodium phosphate buffer, pH 6~9~ one 10 volume t-nitroblue tetrazolium (t-NBT), 2 mg/ml in the same buffer (if necessary, this may he filtered through Whatman No. 1 filter paper just prior to use)l and 1 volume of l-methoxyphenazine methosulfate (m-PMS), 0.8 mg/ml in the same buffer. Although the preferred chromo-genic reag~ants are t-N~T and m-PMS, vther materials can also be utilized~ To en~ure mutarotation of the glucose molecules ~o their beta form, the mixture may be prepared about 1 hour before the st~ining reaction.
A small volume, prefer~,bly about 100 ~1, Qf the ~0 color reagen~ are contacted w:ith the antigen source.
The slides are placed in a ~lide chamber and incubated for about 30 minutes At 55 C~ to permit forma~ion of ~he formazan st~in. The e~cess co~or reagent is then w~shed ~rom the slide wi~h PBS, soaked for about 15 minutes in ~old PBS ~nd the excess moisture again removed with the absorbent paper.
The slide i then prepared for light miero~cope viewin~ according to standard techniquesa Briefly, a glycer~1-gelatin mounting medium is placed in a 55C.
incubator for about 5 to 10 minutes for liqu~fication~
and a 13rge drop placed over the antigen source 9 The antigen source is then covered with a cover slip, taking care to avoid form~tion of air bubbles.
~2~
When the slides are examined under a light microscope, the existence of antinuclear antibodies in the test serum will cause slightly enlarged, homo-geneous dark nuclei to appear, substantially without dark background staining~
From the foregoing descriptionr it should be apparent that the present invention provides an ef-fective, inexpensive, highly visible, and permanent record of an antinuclear antibody assay. Further, the nuclear an~igen source fixed onto the slide is extremely stable and does not require storage at low temperatures.
While a particular form of the invention has been described in detail with reference to its presently preferred embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, it is not intended that the invention be limi~ed except by the appended claims.
Claims (8)
1. An immunoenzymatic process for the de-tection of a component of a test sample, said process comprising the steps of:
contacting said test sample with a cellular antigen source fixed onto a support and stabilized by dehy-dration;
contacting the antigen source with an anti-body conjugated with glucose oxidase and reactive with said component;
incubating the antigen source with a so-lution containing glucose and a chromogenic mixture;
and analyzing the antigen source for the presence of color from the incubation.
contacting said test sample with a cellular antigen source fixed onto a support and stabilized by dehy-dration;
contacting the antigen source with an anti-body conjugated with glucose oxidase and reactive with said component;
incubating the antigen source with a so-lution containing glucose and a chromogenic mixture;
and analyzing the antigen source for the presence of color from the incubation.
2. The method of Claim 1, wherein the chrom-ogenic mixture consists essentially of t-nitroblue tetrazolium chloride and 1-methoxyphenazine methosulfate.
3. The method of Claim 1, wherein said an-tigen source is Hep-2 cell or rat kidney tissue.
4. The method of Claim 1, wherein said sup-port is a glass slide.
5. The method of Claim 1, wherein said con-jugated antibody is goat immunoglobulin reactive with human immunoglobulins.
6. The method of Claim 1, wherein said com-ponent is an antinuclear antibody.
7. The method of Claim 1, wherein said an-alysis is accomplished with a light microscope.
8. An immunohistochemical method for de-tecting antinuclear antibodies in a test serum sub-stantially in the absence of background staining, said method comprising the steps of:
providing a cellular nuclear antigen source fixed onto a slide and dehydrated with acetone at about 4°
C.;
contacting the nuclear antigen source with said test serum;
contacting the nuclear antigen source with goat immunogloblulin conjugated with glucose oxidase and reactive with said antinuclear antibodies;
incubating said nuclear antigen source with a solution containing beta-D-glucose, t-nitroblue tetrazolium, and 1-methoxyphenazine methosulfate;
and analyzing said nuclear antigen source under a light microscope for the presence of a formazan stain.
providing a cellular nuclear antigen source fixed onto a slide and dehydrated with acetone at about 4°
C.;
contacting the nuclear antigen source with said test serum;
contacting the nuclear antigen source with goat immunogloblulin conjugated with glucose oxidase and reactive with said antinuclear antibodies;
incubating said nuclear antigen source with a solution containing beta-D-glucose, t-nitroblue tetrazolium, and 1-methoxyphenazine methosulfate;
and analyzing said nuclear antigen source under a light microscope for the presence of a formazan stain.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US29811281A | 1981-08-31 | 1981-08-31 | |
| US298,112 | 1981-08-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1206092A true CA1206092A (en) | 1986-06-17 |
Family
ID=23149085
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000410409A Expired CA1206092A (en) | 1981-08-31 | 1982-08-30 | Glucose oxidase immunohistochemical detection of antinuclear antibodies |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0091911A4 (en) |
| CA (1) | CA1206092A (en) |
| ES (2) | ES8400773A1 (en) |
| IT (1) | IT1153188B (en) |
| WO (1) | WO1983000877A1 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4487830A (en) * | 1982-05-14 | 1984-12-11 | American Hoechst Corporation | Enzyme/immunofluorescent assay for autoantibodies |
| US4588682A (en) * | 1982-12-13 | 1986-05-13 | Integrated Genetics, Inc. | Binding nucleic acid to a support |
| CA1279818C (en) * | 1985-03-29 | 1991-02-05 | Cenfold Holdings S.A. | Diagnostic testing for micro-organisms |
| JPS6232363A (en) * | 1985-06-21 | 1987-02-12 | モダン・ダイアグノステイツクス・インコ−ポレ−テツド | Method and device for detecting antinuclear antibody |
| EP0241025A1 (en) * | 1986-04-08 | 1987-10-14 | Whittaker Bioproducts, Inc. | Method and substrate for immunofluorescent microscopy |
| CA1313818C (en) * | 1987-06-03 | 1993-02-23 | Ross Leon Coppel | Nuclear antigen la |
| EP0335293A3 (en) * | 1988-03-30 | 1990-09-12 | Hoechst Japan Limited | A method of preparing an analytical element for the determination of anti-nuclear antibodies |
| WO2005079814A1 (en) * | 2004-02-20 | 2005-09-01 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A pharmaceutical composition used for treating recurrent spontaneous abortion and method thereof |
| WO2005080980A1 (en) * | 2004-02-20 | 2005-09-01 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A method for diagnosing immunity recurrent spontaneous abortion and method for treating and monitoring |
| US7763418B2 (en) * | 2005-07-05 | 2010-07-27 | Cytoskeleton, Inc. | Detection of Rho proteins |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3146163A (en) * | 1962-01-23 | 1964-08-25 | John H Brewer | Apparatus for separating certain components from blood |
| NL154600B (en) * | 1971-02-10 | 1977-09-15 | Organon Nv | METHOD FOR THE DETERMINATION AND DETERMINATION OF SPECIFIC BINDING PROTEINS AND THEIR CORRESPONDING BINDABLE SUBSTANCES. |
| US3997657A (en) * | 1973-10-15 | 1976-12-14 | American Cyanamid Company | Dry slide reagent employed in immunofluorescent test for detection of human antinuclear factor |
| US3876504A (en) * | 1974-06-10 | 1975-04-08 | Early Warning Co | Procedure for determination of antigens and antibodies and articles for use therewith |
| US3979509A (en) * | 1974-09-03 | 1976-09-07 | General Electric Company | Opaque layer method for detecting biological particles |
| DE2836362A1 (en) * | 1978-08-19 | 1980-03-06 | Behringwerke Ag | DIAGNOSTIC MEANS |
-
1982
- 1982-08-12 EP EP19820902930 patent/EP0091911A4/en not_active Withdrawn
- 1982-08-12 WO PCT/US1982/001107 patent/WO1983000877A1/en not_active Application Discontinuation
- 1982-08-30 ES ES515350A patent/ES8400773A1/en not_active Expired
- 1982-08-30 CA CA000410409A patent/CA1206092A/en not_active Expired
- 1982-08-31 IT IT23075/82A patent/IT1153188B/en active
-
1983
- 1983-07-16 ES ES524183A patent/ES524183A0/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| EP0091911A1 (en) | 1983-10-26 |
| IT8223075A0 (en) | 1982-08-31 |
| EP0091911A4 (en) | 1984-04-06 |
| ES515350A0 (en) | 1983-11-01 |
| ES8400773A1 (en) | 1983-11-01 |
| WO1983000877A1 (en) | 1983-03-17 |
| ES8500999A1 (en) | 1984-11-01 |
| ES524183A0 (en) | 1984-11-01 |
| IT1153188B (en) | 1987-01-14 |
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