immuno concepts Colorzyme ANA Für Professionellen Gebrauch Seite 8

COLORZYME® ANA TEST PROCEDURE
RECONSTITUTE BUFFER
1.
Dissolve contents of one buffer pouch in one liter
of deionized or distilled water. The PBS buffer
may be covered and stored at 2-10°C up to four
weeks.
RECONSTITUTE COLOR REAGENT
2.
Dissolve contents of one pouch in 150 ml of deion-
ized 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 pre-
cipitate upon reuse is normal. Depending on the
rate of use, 150 ml of Colorzyme® color reagent
can be used with up to twenty slides.
DILUTE PATIENT SAMPLES
3.
Screening: Dilute patient samples to 1:40 by add-
ing 0.05 ml (50 µl) serum to 1.95 ml reconstituted
PBS.
Semi-Quantitative Titering: Make serial dilutions
of screening sample(s) (e.g. 1:80, 1:160, 1:320...1:
2560) using PBS.
PREPARE SUBSTRATE SLIDES
4.
Remove slide(s) from pouch(es) and place con-
trol sera on control wells as follows: Invert control
dropper bottle and squeeze gently until drop is
visible at the tip. Gently touch the drop to appro-
priate control well while avoiding direct contact
of dropper tip with slide surface. Place positive
control on well marked "+", negative control on
well marked "-", and one drop of PBS buffer on
well marked "PBS". Add 1 drop (20-25 µl) patient
sample to the numbered wells.
NOTE: For general screening, the homogeneous
positive control is recommended. For semi-quan-
titative titering, select the positive control illustrat-
ing the most similar pattern of staining to the
screening sample (e.g. for patient sample yielding
a speckled pattern of staining in screening, use
speckled positive control).
CAUTION: DIRECT CONTACT OF DROPPER TIP WITH
SLIDE SURFACE MAY RESULT IN DAMAGE TO THE
ANTIGEN SUBSTRATE.
INCUBATE SLIDES
5.
ature, i.e. 18-24°C)
Place slide(s) into a moist covered chamber (a
petri dish with moistened paper toweling will be
adequate). Incubate, with lid in place, for 30 min-
utes (±5 minutes) at room temperature (18-24°C).
PBS RINSE
6.
Remove slide(s) from incubator tray and rinse
briefly with PBS using a squirt bottle, Pasteur, or
serological pipette. Do not squirt buffer directly
on the wells.
NOTE: To avoid cross contamination on the slides,
direct PBS stream along midline of slide, tilting first
toward the upper row of wells followed by tilting
toward the lower row of wells.
PBS WASH
7. (10 minutes)
Wash slide(s) 10 minutes with PBS in a slide staining
dish or Coplin jar. Gentle agitation is recommend-
ed at slide immersion, midpoint, and removal.
This wash may be extended 10-30 minutes with
no variability in final test results. Discard PBS wash
solution after use. For optimal results, change PBS
at midpoint and use a magnetic stirrer.
ENZYME ANTIBODY REAGENT
8.
with 12-14 drops)
Remove one slide at a time from PBS and dip 3-5
times in deionized or distilled water. Tap slide on
its side against bibulous paper or paper toweling
to remove excess water. Immediately return slide
to the incubation chamber and cover the wells
completely using enzyme antibody reagent; be-
gin by placing a drop over each well. Repeat for
each slide. Enzyme antibody reagent has been
titered to compensate for residual deionized or
distilled water remaining on the slide after rinsing.
(PBS)
(20-25 µl/well)
(30±5 minutes at room temper-
(Cover the wells
NOTE: It is important that slide wells do not dry
during this procedure or damage to the substrate
may occur.
DO NOT BLOT OR DRY THE SLIDE IN ANY MANNER
OR ALLOW SLIDE TO SIT WITHOUT ENZYME ANTI-
BODY REAGENT FOR LONGER THAN 15 SECONDS.
INCUBATE SLIDES
9.
ature, i.e. 18-24°C)
Place lid on incubation chamber and allow
slide(s) to incubate 30 minutes (±5 minutes) at
room temperature (18-24°C).
PBS RINSE
10.
Remove slide(s) from incubator tray and rinse
briefly with PBS. Do not squirt buffer directly on
the wells.
PBS WASH
11. (10 minutes)
Wash slide(s) 10 minutes with PBS in a slide staining
dish or Coplin jar. Gentle agitation is recommend-
ed at slide immersion, midpoint, and removal.
This wash may be extended 10-30 minutes with no
variability in final test results.
COLOR REAGENT INCUBATION
12.
room temperature, i.e. 18-24°C).
Remove one slide at a time from PBS, dip 3-5
times in deionized or distilled water, and tap slide
on its side against bibulous paper or paper towel-
ing to remove excess water. Immediately place
slide(s) into a Coplin jar containing activated
color reagent and incubate for 30 minutes.
PBS RINSE
13.
Remove one slide at a time from the Coplin jar
and rinse each side of slide 4-5 seconds with PBS.
Do not squirt buffer directly on the wells. Place
each PBS rinsed slide into a Coplin jar filled with
deionized or distilled water until all slides have
been removed from the color reagent. Immedi-
ately proceed to step 14.
MOUNT COVERSLIP
14.
Remove one slide at a time from the diononized
or distilled water and tap slide on its side against
bibulous paper or paper toweling to remove
excess water.
DO NOT BLOT OR DRY THE SLIDE IN ANY MANNER
OR ALLOW TO SIT WITHOUT COVERSLIP FOR LON-
GER THAN 15 SECONDS. Add 4-5 drops of semi-
permanent mounting medium along midline of
each slide. Carefully place coverslip in position,
avoiding air pockets, by gently lowering coverslip
from one end of the slide to the other.
NOTE: Excess mounting medium on slide may
result in a lack of clear resolution of cells (blurred
image). Excess mounting medium may be
removed from slide by gently blotting coverslip
with bibulous or lens paper while avoiding any
direct movement of the coverslip. Slides may be
read immediately or stored for an extended time
at 2-10°C with no loss of reactivity.
For Technical Assistance:
1-800-251-5115 (Toll Free) or e-mail:
techservice@immunoconcepts.com
8
(30±5 minutes at room temper-
(30 minutes at