Two-color Fab labeling method for antigen microarrays
Last Updated 6/25/08
Michael Kattah1, mkattah@stanford.edu
Golnaz Alemi1, galemi@stanford.edu
Donna Thibault1, thibault@stanford.edu
Imelda Balboni2, mbalboni@stanford.edu
Paul J. Utz1, pjutz@stanford.edu
1
Department of Medicine, Division of Immunology and Rheumatology; 2Department of
Pediatrics, Division of Rheumatology, Stanford University School of Medicine,
Stanford, California 94305, USA. Correspondence should be addressed to M.G.K.
(mkattah@stanford.edu) or P.J.U. pjutz@stanford.edu).
Related Journal & Article Information
Journal: Nature Methods
Article Title: A new two-color Fab labeling method for autoantigen protein
Microarrays
Abstract/Summary
We describe a simple and reproducible two-color approach for probing antigen
microarrays using fluorescently-labeled monovalent secondary Fab fragments. We preincubate these reagents with serum to allow the monovalent Fab fragments to bind and
indirectly label the serum immunoglobulins. The mixture is then used as if it were
directly labeled. Using the two-color Fab labeling method we found that we could
improve intraslide and interslide reproducibility and reliably detect changes in
autoreactivity. Overall, the novel two-color Fab-labeling method addresses some of the
difficulties that have confronted autoantigen microarrays.
Introduction
Antigen microarrays are useful tools for studying the humoral immune response
in the setting of disease. This approach involves immobilizing antigens on a slide
surface and then exposing the array to biological fluids containing immunoglobulins.
Although these arrays have proven extremely useful as research tools, they suffer from
multiple sources of variability. In order to address these issues, we have developed a
novel two-color Fab labeling method that allows two samples to be applied
simultaneously to the same array. This straightforward labeling approach improves
reproducibility and reliably detects changes in autoantibody levels.
There are two methods for detecting autoantibodies bound to autoantigen
microarrays. The single-color method is the most practical and involves probing an array
with unlabeled serum followed by detection with a secondary antibody conjugated to a
fluorophore. This approach has the advantages of simplicity and standardization with
respect to fluorophore, but it suffers from variability between array features, arrays,
samples, and laboratories. The two-color approach is an attractive alternative that can
control for some of these sources of variability. Several reports describe two-color
protein microarrays, but these techniques suffer from inherent limitations of Nhydroxysuccinimidyl (NHS)-ester chemical coupling procedures. The drawbacks of this
strategy include expense, labor, highly variable modification efficiency due to hydrolytic
side reactions, and potentially reduced binding due to modification of primary amines.
Materials
Reagents


Labeling and Probing Reagents
o 2.5% BSA PBST:
 Bovine serum albumin (BSA), Further purified Fraction V, ~99%
(agarose gel electrophoresis), lyophilized powder, Essentially γglobulin free) (Sigma, cat. no. A3059-10G). Dissolve to 25
mg/mL (2.5% w/v)
 PBS (pH 7.4) without Calcium or Magnesium (Gibco,10010)
 0.05% Tween 20 (Sigma Aldrich, cat. no. P9416-100ML)
o 5% Nonfat Dry Milk PBST
 Blotting Grade Blocker Non-Fat Dry Milk (Biorad, 170-6404)
 PBS (pH 7.4) without Calcium or Magnesium
 0.05% Tween 20 (Sigma Aldrich, cat. no. P9416-100ML)
o 96 Well Expanded Volume Polypropylene Untreated Microplate, Standard
Height, V-Bottom, 10 per Bag, Non-Sterile (Corning, Product #3343)
Fab fragments
o It is possible to purchase Cy3 and Cy5 labeled Fab fragments directly.
Note: the fluorophore-conjugated Fab fragments from commercial
sources are not guaranteed to be from the same source lot, creating the
potential for artifacts. They also tend to be weaker labeling reagents. If
this is not a concern for the user, the following reagents can be purchased:
 Cy3 Goat Anti-Human Fab fragments (Jackson ImmunoResearch
Laboratories, cat. no. 109-167-003)
 Cy5 Goat Anti-Human Fab fragments (Jackson ImmunoResearch
Laboratories, cat. no. 109-177-003)
 Cy3 Goat Anti-Mouse Fab fragments (Jackson ImmunoResearch
Laboratories, cat. no. 115-167-003)
 Cy5 Goat Anti-Mouse Fab fragments (Jackson ImmunoResearch
Laboratories, cat. no. 115-177-003).
o For conjugating Fab fragments to fluorophore
 Unconjugated Goat Anti-Human Fab fragments (Jackson
ImmunoResearch Laboratories, cat. no. 109-007-003) and
Unconjugated Goat Anti-Mouse Fab fragments (Jackson
ImmunoResearch Laboratories, cat. no. 115-007-003)







Cy3 NHS ester (GE Healthcare, cat. no. PA13101) and Cy5 NHS
ester (GE Healthcare, cat. no. PA15101)
0.5ml Zeba Desalt Spin Columns (Pierce, cat. no. 89882) or 2ml
Zeba Desalt Spin Columns (Pierce, cat. no. 89889)
Vivaspin 6 3,000 MWCO PES spin concentration columns
(Vivascience, cat. no. VS0691)
Dimethyl Sulfoxide (DMSO), anhydrous 99.9+% (Sigma Aldrich,
cat. no. 276855-100ML). Store under vacuum or with dessicant as
the reagent is hygroscopic.
10X Modification Buffer pH 8.0:
 2M Sodium Phosphate Monobasic (Fisher Scientific S3691): Dissolve 27.6 g NaH2PO4×H2O in 100 ml ddH2O
 2M Sodium Phosphate Dibasic anhydrous (Fisher Scientific
S374-1): Dissolve 28.4 g Na2HPO4 in 100ml ddH2O
 3M NaCl: Dissolve 175g of Sodium Chloride (Sigma
Aldrich S-9888) in 1 liter of ddH2O
 Mix 5.3ml of 2M Sodium Phosphate Monobasic with 94.7
ml of 2M Sodium Phosphate Dibasic and dilute with 100ml
of 3M NaCl. Filter and store for up to 3 months.
10X PBS pH 7.4 without Calcium and Magnesium (Gibco, 70011)
100X Sodium azide: dissolve sodium azide (Sigma Aldrich, cat.
no. S8032-100G) in at ddH2Oat a final concentration of 50mg/ml
(5% w/v)
Equipment
 Polarseal Foil adhesive tape for Multiwell Plates (E & K scientific, cat. no.
T592100)
 FAST slide incubation chambers and slide holders (Whatman)
 slide racks
 glass washing chambers
 microarray scanner
 arrays
 software for analyzing array data (Genepix)
Timing
Minimum total time: 3 hrs
 1 hour to block slides and pre-incubate serum sample with Fab fragments
 1.5 hours for incubation on arrays
 35 min for washing drying slides.
Procedure
1. Prepare or purchase cyanine dye-labeled Fab fragments
Conjugation to Cyanine or other dyes is relatively straightforward and can be performed
in bulk from the same lot of Fabs to generate reagents that have a long shelf-life.
a. Concentrate unconjugated Fab fragments to 10mg/ml (high concentration
is critical for predictably modifying protein with NHS-esters) using spin
column concentrators according to manufacturer’s protocol. Quantify
protein concentration by any method. Absorbance at 280nm is sufficient
for IgG quantification using an extinction coefficient of 13.7 at 280 nm for
a 1% (10 mg/ml) IgG solution. Add 10X Modification Buffer.
b. Aliquot fragments into two tubes. Calculate the moles of Fab fragment.
Multiply by 4 to get the amount of dye to add (since the goal is to get
about 2 dye molecules/Fab fragment, and the reaction is usually about
50% efficient when the protein is at 10mg/ml). Calculate the mass of Cy3
or Cy5 to add.
c. Resuspend 1mg of Cy3 and Cy5 using 100ul DMSO. Use immediately.
d. Allow reaction to proceed at room temperature for 1.5 hours.
e. Pass over three consecutive desalting spin columns. Three columns are
necessary to remove all the unbound cyanine dye. As a control, we
perform a mock conjugation with no protein to look at how the desalting
removes the unbound dye. Dialysis is too time-consuming and less
efficient than desalting spin-columns for small molecule removal.
f. Quantify Cy3 and Cy5 conjugates by absorbance. Correct for dye
absorbance at 280nm. Resuspend reagent at a final concentration of 510mg/ml in PBS 0.05% Sodium azide and store at 4oC for several months.
The Fab fragments can be stored for over a year at -20oC in 50% glycerol.
2. Block arrays
a. Number slides and load arrays in a slide rack and block with 5% milk
PBST at room temperature (21-23oC). Blocking with milk is critical for
nitrocellulose-coated surfaces, such as FAST slides from Schleicher &
Schull, or for any other high protein binding membrane.
b. Immediately prior to probing, rinse slides three times in PBST (see
below).
3. Prepare Fab-labeled sample
a. Add Fab fragments to sample and incubate 30 minutes at roomtemperature
i. This step is flexible but here are a few guidelines (Table 1). An
Fab:IgG ratio of 2:1 or 3:1 is a good place to start. The example
below is for a murine serum with 5mg/ml IgG, labeling at a molar
ratio of 2:1, in a final volume of 500ul and a dilution of 1:250.
ii. The order of addition is important.
1. Dilute each serum sample. Example:
8ul PBST (add first)
+2ul serum (10ug IgG murine samples)
10ul diluted serum (pipet up and down)
2. Prepare cocktail of diluted Fab fragments
Example:
a. (N+1) x
8.7ul PBST
(N+1) x
+1.3ul Fab fragments
(N+1) x
10ul (pipet up and down)
Where N=number of samples.
Add 10ul of diluted Fab fragments to each sample, pipet
up and down.
3. Prepare reference. Reference can be prepared in bulk
or separately, but bulk preparation improves interslide
comparison.
b. Incubate serum samples with Fab fragments for 30min at room
temperature, then move samples to 4oC.
Table 1. Typical values for Human and Mouse Serum.
Species
Mouse
Human
Serum
2ul
1ul
Typical IgG
~10ug
~10ug
Fab
6.7ug
6.7ug
Molar Ratio
2
2
Labeling Volume
20ul
20ul
Probing Volume
500ul
500ul
c. Dilute unlabeled Fab fragments to a final concentration of 65ug/ml in
2.5% BSA PBST.
N+1 x 475ul 2.5% BSA PBST
N+1 x
25ul of 1.3 mg/ml unlabeled GAM Fab fragments
N+1 x 500ul
d. Add 480ul to each sample, pipet up and down and incubate at room
temperature for 10 min at 4oC.
e. Place 96-well plate/tubes on ice or at 4oC immediately. Move to
probing arrays immediately. This step is important since the Fab
fragments bind to the antibodies with finite half-lives.
4. Probe and scan arrays
a. Rinse pre-blocked slides 3 times, and apply the sample to the array.
Incubate 1.5 hours at 4oC.
b. When incubation on the array is over, place each slide in its own 50ml
conical tube filled with PBST at 4oC. Once all the slides are in 50ml
tubes, the slides can then be individually moved to racks for further
washing. Rinsing each slide in this manner is critical due to the large
amount of labeled sample on each slide. Placing slides directly in a
washing rack will cause cross-contamination.
c. Wash slides in the slide racks in glass chambers one time for four minutes
in 4oC PBST, then three times for four minutes in PBST. Wash once in
PBS for four minutes. Dunk in 0.2x PBS for 1 second and centrifuge
immediately at 600xg for 7 minutes at room temperature. It is important
to perform this step at room temperature to avoid condensation on the
slide.
d. Store dry slides under vacuum until scanning.
5. Analyzing two-color data
a. To set the two photomultiplier tube voltages it is helpful to have both IgG
and capture IgG on the array. For example, when looking at murine
samples, print Goat anti-mouse IgG as well as mouse IgG. Use these
ratios to calibrate the detectors. The ratio should be set close to 1.0 during
scanning and then adjusted mathematically by dividing ratios for each
antigen by the ratio at this feature. Essentially this normalizes the data
with respect to label and IgG levels.
b. If the IgG levels are known to be different, for example if the IgG of one
sample is twice as high as another, then one can adjust the data so that the
ratio at the IgG spot is normalized to 2.0 or 0.5. Measuring IgG can be
performed with commercially available ELISA kits.
Troubleshooting
1. Systematic dye bias
a. Some antigens demonstrate binding to Cy3 or Cy5 preferentially, creating
a bias so that even in a self-self experiment the ratio is significantly
different from 1.0. There are several ways to control this.
i. Perform a dye-swap and average the ratios. An antigen that is
consistently Cy5 positive will drop out.
ii. Use the same reference for all the samples. The reference can be
labeled with Cy3 and then all of the samples can be labeled with
Cy5. One then compares all of the ratios.
iii. Perform a self-self experiment and determine which antigens show
bias, then normalize the ratios to this observed ratio.
2. Intensity of signal
a. Fab labeling is generally weaker than labeling with a secondary antibody.
It is possible to try increasing the Fab:IgG ratio to increase the signal.
Increasing beyond 6:1 however is not recommended.
3. Cross-labeling
a. To avoid cross-labeling it is important to use the Fab-labeled samples
within a few minutes after quenching with unlabeled Fab fragments. The
labeling is more stable at 4oC than room-temperature, so the samples
should be stored in the fridge or on ice while not in use.
b. Incubation of the samples on the array can be performed as long as
overnight at 4oC but should not be allowed to go beyond 2 hours at roomtemperature.
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