System Components - immuno concepts Colorzyme ANA Für Professionellen Gebrauch

COLORZYME® ANA TEST SYSTEM
FOR IN VITRO DIAGNOSTIC USE
SUMMARY AND EXPLANATION OF THE TEST
INTENDED USE: This is an indirect enzyme 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.
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
progressive 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
Immuno Concepts Colorzyme® ANA Test System with
mitotic* human epithelioid cells (HEp-2), represents
one of the most advanced systems for detection of
ANA. HEp-2 cells with mitotic figures have been shown
to have greater sensitivity and yield sharper pattern
recognition than classical mouse kidney substrate in
detecting antibodies in progressive systemic sclerosis
(PSS) (25). Mitotic figures aid in differential pattern rec-
ognition 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 Immuno Concepts Colorzyme® ANA Test System
is an indirect enzyme antibody technique. 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 antibodies, the substrate is incubated with an
anti-human antibody reagent conjugated to horserad-
ish peroxidase. When results are positive, there is the
formation of a stable three-part complex consisting
of enzyme antibody bound to human antinuclear
antibody which is bound to nuclear antigen. This com-
plex can be visualized by incubating the slide in color
reagent which contains an enzyme specific substrate.
The reaction between the enzyme labeled antibody
and enzyme specific substrate results in a color reaction
on the slide visible by standard light microscopy. In
positive samples, the cell nuclei will show dark blue-
purple staining with a 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 staining.
The cytoplasm may demonstrate weak staining while
the nonchromosome region of the mitotic cells may
demonstrate a darker staining.

SYSTEM COMPONENTS

Use: All components come ready to use with no
aliquoting or reconstitution required (except the PBS
buffer and color reagent 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 reagent
should be stored in a closed container under refrigera-
tion at 2-10°C. After reconstitution, Colorzyme® color
reagent should be stored in a closed container at room
temperature for up to 30 days. Depending on the rate
of use, 150 ml of Colorzyme® color reagent can be
(MATERIALS PROVIDED)
used with up to twenty slides.
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
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 demon-
strates a homogeneous staining reaction on Immuno
Concepts HEp-2 cell substrate. The chromosome region
of mitotic cells demonstrates the same homogeneous
staining reaction. This reagent contains 0.1% sodium
azide as a preservative.
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 Im-
muno Concepts HEp-2 cell substrate. The chromosome
region of mitotic cells demonstrates a negative staining
reaction. This reagent contains 0.1% sodium azide as a
preservative.
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 Immuno Concepts HEp-2 cell substrate. This
reagent contains 0.1% sodium azide as a preservative.
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 Immuno Con-
cepts HEp-2 cell substrate. The chromosome region of
mitotic cells demonstrates the same discrete speckled
staining reaction. This reagent contains 0.1% sodium
azide as a preservative.
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. This
reagent contains 0.1% sodium azide as a preservative.
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 staining of the cytoplasm with
brighter staining of the nonchromosome region of the
mitotic cell, it demonstrates no discernible pattern of
nuclear staining. This reagent contains 0.1% sodium
azide as a preservative.
Enzyme Antibody Reagent: Catalog No. 4009 (9.0 ml),
4075 (23 ml). Goat anti-human IgG (heavy and light
chains) conjugated to horseradish peroxidase (HRP).
Reagent comes ready-to-use in precision dropper
bottles with 9.0 ml for every 10 slides in complete test
kits.
Color Reagent: ††Catalog No. 4066. HRP specific en-
zyme substrate powder, containing 4 chloro 1-naphthol.
Each package contains powder to make 150 ml of
self-activating Colorzyme® color reagent.
Preparation: Dissolve contents of one pouch in 150 ml
of deionized or distilled water. Mix well until completely
dissolved. This color reagent is stable for 30 days at
room temperature in a closed container. This color
reagent may be reused for up to 30 days or until any
color change or precipitate is visible. Cloudiness or
opalescence, with no visible precipitate upon reuse
is normal. Depending on the rate of use, 150 ml of
Colorzyme® color reagent can be used with up to
twenty slides.
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