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FluorEScEnt ana tESt SYStEm
For in vitro DIagnoStIc uSE
INTENDED USE: This is an indirect fluorescent antibody
test for the semi-quantitative detection of antinuclear
antibody in human serum. This test system is to be used
as an aid in the detection of antibodies associated with
systemic rheumatic disease.
SummarY anD EXPlanatIon oF tHE tESt
Antinuclear antibody (ANA) is a general term used to
describe autoantibodies against various cell nuclear
proteins. Early studies of these autoantibodies, using
immunofluorescent techniques, revealed a select few
nuclear protein specificities (1). Because of the high
correlation of positive ANA with systemic lupus erythe-
matosus (SLE), a negative ANA essentially ruled out the
disease (2).
Although antibodies specific to DNA continue to show a
high disease correlation with SLE (3), a number of nucle-
ar (4) and cytoplasmic (5-7) macromolecules have also
been detected and associated with other connective
tissue diseases (8-10). Some of these antibodies appear
to have diagnostic and/or prognostic significance in
progres-sive systemic sclerosis (11-12), mixed connec-
tive tissue disease (13-15), Sjögren's syndrome (16-17),
polymyositis (18), and/or rheumatoid arthritis (19). Be-
cause of these disease associations, ANA testing is now
recognized as a general screening tool for connective
tissue disease (20).
Sensitivity of the ANA test varies with the type of
substrate used, fixative procedure, and types of ANA
present in sera. Cell culture substrates generally show
greater sensitivity than tissue sections (21-24). The Im-
muno Concepts ANA Test System with mitotic* human
epithelioid cells (HEp-2), represents an advanced
immunofluorescent system for detection of ANA. HEp-2
cells with mitotic figures have been shown to have
greater sensitivity and yield sharper pattern recogni-
tion than classical mouse kidney substrate in detecting
antibodies in progressive systemic sclerosis (PSS) (25).
Mitotic figures aid in differential pattern recognition
as well as in detecting previously unreported nuclear
antigens present in higher concentrations in mitotically
active cells (26-28).
*Mitosis is a term used to describe the cell division
process. It is generally divided into six phases including
interphase, prophase, metaphase, anaphase, telo-
phase, and cytokinesis.
PrIncIPlE oF tHE tESt
The IC Fluorescent ANA Test System uses the indirect
fluorescent antibody technique first described by
Weller and Coons (29). Patient samples are incubated
with antigen substrate to allow specific binding of
autoantibodies to cell nuclei. If ANA's are present,
a stable antigen-antibody complex is formed. After
washing to remove non-specifically bound antibod-
ies, the substrate is incubated with an anti-human
antibody conjugated to fluorescein. When results are
positive, there is the formation of a stable three-part
complex consisting of fluorescent antibody bound to
human antinuclear antibody which is bound to nuclear
antigen. This complex can be visualized with the aid of
a fluorescent microscope. In positive samples, the cell
nuclei will show an apple-green fluorescence with a
staining pattern characteristic of the particular nuclear
antigen distribution within the cells. If the sample is
negative for ANA, the nucleus will not show a clearly
discernible pattern of nuclear fluorescence.
SYStEm comPonEntS
use: All components come ready to use with no
aliquoting or reconstitution required (except the PBS
buffer which must be dissolved in deionized or distilled
water before use).
Storage: All components can be stored under refrigera-
tion at 2-10°C. After reconstitution, PBS buffer should
be stored in screw cap containers under refrigeration
at 2-10°C.
Stability: All components remain stable at least 12
months from date of manufacture. Do not use any
component beyond its expiration date.
rEactIVE rEagEntS
Substrate Slides: †Catalog No. 2007 (seven-well); 2013
(thirteen-well). Seven or thirteen well ANA substrate
(matErIalS ProVIDED)
slides using HEp-2 cells (with mitotic figures) grown and
stabilized directly on the test wells. Unique moat slide
design minimizes cross contamination of wells during
testing. Slides also include additional control wells
designated positive, negative, and PBS to aid in proper
interpretation of results. The slide pouch is filled with an
inert non-toxic gas that contributes to the stability of the
cells. If the pouch does not appear to be inflated when
the slide is removed from the kit, damage to the pouch
has occured and the slide should not be used.
Homogeneous Positive control: Catalog No. 2021.
Ready-to-use dropper vial containing 1.0 ml positive
human control serum with antibody specific to DNA
and/or DNP nuclear antigens. This serum demonstrates
a homogeneous staining reaction on IC's HEp-2 cell
substrate. The chromosome region of mitotic cells dem-
onstrates the same homogeneous staining reaction.
Speckled Positive control: Catalog No. 2022. Ready-
to-use dropper vial containing 0.5 ml positive human
control serum with antibody specific to Sm and/or RNP
nuclear antigens. This serum demonstrates one of the
most common speckled staining reactions seen on IC's
HEp-2 cell substrate. The chromosome region of mitotic
cells demonstrates a negative staining reaction.
nucleolar Positive control: Catalog No. 2023. Ready-
to-use dropper vial containing 0.5 ml positive human
control serum with antibody specific to nucleolar
antigens. This serum demonstrates a nucleolar staining
reaction on IC's HEp-2 cell substrate.
centromere Positive control: Catalog No. 2025. Ready-
to-use dropper vial containing 0.5 ml positive human
control serum with antibody specific to chromosomal
centromeres (kinetochore). This serum demonstrates a
discrete speckled staining reaction on IC's HEp-2 cell
substrate. The chromosome region of mitotic cells dem-
onstrates the same discrete speckled staining reaction.
titratable control Serum: Catalog No. 2026. Ready-to-
use vial containing 1.0 ml positive human control serum
to be treated as an undiluted patient sample. See vial
label for titer value.
negative control Serum: Catalog No. 2031. Ready-
to-use dropper vial containing 1.0 ml negative human
control serum. Although the negative control serum
may demonstrate weak fluorescence of the cytoplasm
with brighter staining of the nonchromosome region of
the mitotic cell, it demonstrates no discernible pattern
of nuclear staining.
Fluorescent antibody reagent: Catalog No. 2009 (9.0
ml), 2075 (23 ml). Goat anti-human IgG (heavy and
light chains) conjugated to fluorescein isothiocyanate
(FITC). Reagent comes ready-to-use in precision drop-
per bottles with 9.0 ml for every 10 slides in complete
test kits.
†This slide is protected by one or more of the following
U.S. and foreign patents: 4387979; D-274261; D-273261;
Canadian patent 1171302 and other patents pending.
non-rEactIVE rEagEntS
PBS Buffer Powder: Catalog No. 1011. Phosphate-buff-
ered saline powder (0.01 M, pH 7.4 ± 0.2). Each pouch
contains sufficient buffer powder to make 1 liter. (One
pouch of buffer powder is supplied for every five slides
in complete test kits.)
Preparation: Dissolve one pouch of buffer powder in
1 liter of deionized or distilled water, cover, and store
refrigerated at 2-10°C for up to four weeks or until signs
of contamination or other visible changes occur.
Semi-Permanent mounting medium: Catalog No. 1111.
Ready-to-use dropper vial containing 5.0 ml glycerol-
based mounting medium.
coverslips: Catalog No. 1041. Each packet contains
ten 24x60 mm No. 1 glass coverslips.
aDDItIonal matErIalS rEQuIrED
ProVIDED)
Volumetric pipettes to deliver 20-25 µl volumes
Coplin jars or staining dishes
Squeeze bottle or Pasteur pipettes
Serological pipettes
One-liter containers (for PBS buffer)
Deionized or distilled water
Test tubes to prepare serum dilutions
Bibulous paper or paper towels
(But not
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