Note: Descriptions are shown in the official language in which they were submitted.
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.
'~
--5--
DESCRIPTIQN OP T13E PREFERRED EMBODIMENTS
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.
