MANUALTechnology:

AlphaLISA®

AlphaLISA Human Interferon
Beta (hIFN β) Detection Kit
Part number:

AL3191HV

AL3191C

AL3191F

Assay points:

1005005,000Storage:

Store kit in the dark at 4 ºC. For reconstituted
analyte, aliquot and store at -20 ºC. Avoid
freeze-thaw cycles.

Version:

1For research only. Not for use in diagnostic procedures

Date: August 2023

ANALYTE OF INTEREST
Mature Interferon-beta (IFN-ß) contains 166 amino acids with a molecular weight of 22 kDa and is
produced in large quantities by fibroblasts in response to pathogens. IFN-ß is a cytokine involved in theregulation of unspecific humoral immune responses as well as in immune responses against viral
infections. Only small amounts of IFN-ß are produced under healthy conditions. However, levels candramatically increase during viral infection, inflamation, and autoimmunity. IFN-ß exhibits several
biological effects including antiviral and antiproliferative activities. IFN-ß has been used, with somesuccess, as a therapeutic agent in the treatment of multiple sclerosis, as well as in chronic active
hepatitis B where it appears to be most promising if the disease has not lasted longer than 5 years.

DESCRIPTION OF THE ALPHALISA ASSAY
AlphaLISA technology allows the detection of molecules of interest in cell culture medium and serum ina highly sensitive, quantitative, reproducible, and user-friendly mode. In this AlphaLISA assay, abiotinylated anti-hIFN ß antibody binds to the streptavidin coated AlphaLISA Donor beads, while an antihIFN ß antibody is conjugated to AlphaLISA Acceptor beads. In the presence of hIFN ß, the beads comeinto proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules thattriggers a cascade of energy transfer within the Acceptor beads, resulting in emission with λmax at 615nm.

PRECAUTIONS••The Alpha Donor beads are light-sensitive. All the other assay reagents can be used under normal
light conditions. All Alpha assays using the Donor beads should be performed under subdued
laboratory lighting (< 100 lux). Green filters (LEE 090 filters) can be applied to light fixtures.
Take precautionary measures to avoid contamination of the reagent solutions.

Manual AL3191 V11•The biotinylated anti- hIFN ß antibody contains sodium azide. Contact with skin or inhalation should
be avoided.

KIT CONTENT: REAGENTS AND MATERIALSKit components
AlphaLISA Anti-hIFN ß
Acceptor beads stored inPBS, 0.05% Kathon
CG/ICP II, pH 7.2
Streptavidin (SA)-coatedDonor beads stored in 25
mM HEPES, 100 mM NaCl,
0.05% Kathon CG/ICP II,
pH 7.4
Biotinylated Anti-hIFN ßAntibody stored in PBS,
0.1% Tween-20, 0.05%NaN3, pH 7.4
Lyophilized RecombinantAnti-hIFN ß *AlphaLISA Immunoassay
Assay Buffer (10X)

AL3191HV***

AL3191C****

AL3191F****

25 µL @ 5 mg/mL(1 brown tube, white cap)

50 µL @ 5 mg/mL(1 brown tube, white cap)

500 µL @ 5 mg/mL(1 brown tube, white cap)

75 µL @ 5 mg/mL(1 brown tube, black cap)

150 µL @ 5 mg/mL(1 brown tube, black cap)

1.5 mL @ 5 mg/mL(1 brown tube, black caps)

50 µL @ 500 nM(1 tube, black cap)

100 µL @ 500 nM(1 tube, black cap)

1 mL @ 500 nM(1 tube, black cap)

0.1 µg(1 tube, clear cap)

0.1 µg(1 tube, clear cap)

0.1 µg(1 tube, clear cap)

2 mL, 1 small bottle

10 mL, 1 medium bottle

100 mL, 1 large bottle* Reconstitute lyophilized analyte in 100 µL Milli-Q® grade H2O. The reconstituted analyte should be usedwithin 60 minutes or aliquoted into screw-capped 0.5 mL polypropylene vials and stored at -20 °C for
future experiments. The aliquoted analyte at -20°C is stable up to 4 weeks. Avoid freeze-thaw cycles. Onevial contains an amount of analyte sufficient for performing 10 standard curves. Additional vials can beordered separately (cat # AL3191S).
** Extra buffer can be ordered separately (cat # AL000C: 10 mL, cat # AL000F: 100 mL).
*** The number of assay points is based on an assay volume of 100 µL in 96-well plates.
**** The number of assay points is based on an assay volume of 50 µL in 384-well assay plates using thekit components at the recommended concentrations.
Sodium azide should not be added to the stock reagents. High concentrations of sodium azide (> 0.001%
final in the assay) might decrease the AlphaLISA signal. Note that sodium azide from the biotinylated antihIFN ß antibody stock solution will not interfere with the AlphaLISA signal (0.0001% final in the assay).

Additional reagents and materials:
The following materials are recommended but not provided in the kit:
Item
Light gray AlphaPlate™384
TopSeal™-A Plus Adhesive
Sealing Film
EnVision®-Alpha Reader
Manual AL3191 V1

Suggested source
Revvity Inc.
Revvity Inc.
Revvity Inc.

2

RECOMMENDATONSIMPORTANT: PLEASE READ THE RECOMMENDATIONS BELOW BEFORE USE•

The volume indicated on each tube is guaranteed for single pipetting. Multiple pipetting of thereagents may reduce the theoretical amount left in the tube. To minimize loss when pipetting beads,
it is preferable not to pre-wet the tip.

••

Centrifuge all tubes (including lyophilized analyte) before use to improve recovery of content (2000g,10-15 sec).
Re-suspend the Donor and Acceptor beads by vortexing before use.

•Use Milli-Q® grade H2O to dilute buffers and to reconstitute the lyophilized analyte.

•When diluting the standard or samples, change tips between each standard or sample dilution.
When loading reagents in the assay microplate, change tips between each standard or sample
addition and after each set of reagents.

•When reagents are added to the microplate, make sure the liquids are at the bottom of the well.

•Small volumes may be prone to evaporation. It is recommended to cover microplates with TopSealA Adhesive Sealing Film to reduce evaporation during incubation. Microplates can be read with theTopSeal-A Film in place.

•The AlphaLISA signal is detected with an EnVision Multimode Plate Reader equipped with the Alpha
option using the AlphaScreen standard settings (e.g. Total Measurement Time: 550 ms, Laser 680 nm
Excitation Time: 180 ms, Mirror: D640as, Emission Filter: M570w, Center Wavelength 570 nm,Bandwidth 100 nm, Transmittance 75%).

•AlphaLISA signal will vary with temperature and incubation time. For consistent results, identical
incubation times and temperature should be used for each plate. It is recommended to avoid
multiple reads of the same well of the assay plate.

•The standard curves shown in this manual are provided for information only. A standard curve mustbe generated for each experiment.

ASSAY PROCEDURE•The protocol described below is an example for generating one standard curve in a 50 µL final assay
volume (48 wells, triplicate determinations). The protocols also include testing samples in 452 wells.
If different amounts of samples are tested, the volumes of all reagents must be adjusted
accordingly, as shown in the table below. These calculations do not include excess reagent toaccount for losses during transfer of solutions or dead volumes.

•The standard dilution protocol is provided for information only. As needed, the number of replicatesor the range of concentrations covered can be modified.

•Use of four background points in triplicate (12 wells) is recommended when LDL/LLOQ is calculated.
One background point in triplicate (3 wells) can be used when LDL/LLOQ is not calculated.

Manual AL3191 V1

3

Volumes for 2-Step High Concentration Protocol
Format# of datapoints

AL3191HVAlphaLISA
Acceptor Beads+ biotinylated
Ab MixSADonor
beads

Sample100100 µL

10 µL

10 µL

80 µL

White OptiPlate-96White ½ AreaPlate-96

250

100 µL

10 µL

10 µL

80 µL

White OptiPlate-96

500

50 µL

5 µL

5 µL

40 µL

1 25020 µL

2 µL

2 µL

16 µL

2 500

10 µL

1 µL

1 µL

8 µL

5 000

50 µL

5 µL

5 µL

40 µL

12 500

20 µL

2 µL

2 µL

16 µL

25 000

10 µL

1 µL

1 µL

8 µL

AL3191C

AL3191F

Plate recommendation

Final½ Area AlphaPlate-96
White OptiPlate-384
Light gray AlphaPlate™-384
Light gray AlphaPlate-384
ProxiPlate™-384 Plus
White OptiPlate-384
Light gray AlphaPlate-1536½ Area AlphaPlate-96
White OptiPlate-384
Light gray AlphaPlate-384
Light gray AlphaPlate-384
ProxiPlate-384 Plus
White OptiPlate-384
Light gray AlphaPlate-1536

The 2-Step High Concentration protocol described below is for 500 assay points including one
standard curve (48 wells) and samples (452 wells). If different amounts of samples are tested, thevolumes of all reagents must be adjusted accordingly.
1)

Preparation of 1X AlphaLISA Immunoassay Buffer:
Add 2.5 mL of 10X AlphaLISA Immunoassay Buffer to 22.5 mL Milli-Q® grade H2O.

2)

Preparation of hIFN ß analyte standard dilutions:a. Reconstitute lyophilized hIFN ß (0.1µg) in 100 μL Milli-Q® grade H2O. The remaining
reconstituted analyte should be aliquoted immediately and stored at -20 ºC for future assays(see page 2 for more details).
b. A standard curve must be generated for each experiment. The standard curve should be
performed in a similar matrix diluent as the samples (e.g. cell culture media for cellsupernatant samples, FBS for serum samples). Use of the 1X AlphaLISA Immunoassay Bufferis recommended as a diluent to confirm assay performance.
c. Prepare standard dilutions as follows (change tip between each standard dilution).

Manual AL3191 V14

*[hIFN ß] in standard curve

TubeVol. of hIFN ß (µL)

Vol. of diluent (µL)*

A

10 µL ofreconstituted hIFNß90

B

60 µL of tube A

140

C60 µL of tube B120

D

60 µL of tube C140

E

60 µL of tube D120

F

60 µL of tube E140

G

60 µL of tube F120

H

60 µL of tube G140

I60 µL of tube H120

J

60 µL of tube I140K60 µL of tube J120L

60 µL of tube K140

M ** (background)

010000N ** (background)

010000O ** (background)

010000P ** (background)

010000(g/mL in 5 µL)(pg/mL in 5 µL)

1.00E-07

100 000

3.00E-08
1.00E-08
3.00E-09
1.00E-09
3.00E-10
1.00E-10
3.00E-11
1.00E-11
3.00E-12
1.00E-12
3.00E-1330 000
10 000
3 000
1 000
300
10030
10
310.3

Dilute standards in appropriate diluent (e.g. 1X AlphaLISA Immunoassay buffer Buffer).
At low concentrations of analyte, a significant amount of analyte can bind to the vial. Therefore,load the analyte standard dilutions in the assay microplate within 60 minutes of preparation.

**Four background points in triplicate (12 wells) are used when LDL is calculated. If LDL does not need
to be calculated, one background point in triplicate can be used (3 wells).

3) Preparation of 10X MIX AlphaLISA Anti hIFN ß Acceptor beads (100 µg/mL) + Biotinylated Anti hIFN ßAntibody (20 nM):a. Prepare just before use.
Add 5 µL of 5 mg/mL AlphaLISA Anti hIFN ß Acceptor Bead and 10 µL of 500 nM
Biotinylated Anti hIFN ß Antibody to 235 µL of 1X AlphaLISA Immunoassay Buffer.
4)

Preparation of 1.25X Streptavidin (SA) Donor beads (37.5 µg/mL):

Manual AL3191 V1

5

a.

Prepare just before use.

b. Keep the beads under subdued laboratory lighting.
c.

Add 15 µL of 5 mg/mL SA-Donor beads to 1985 µL of 1X AlphaLISA Immunoassay Buffer.

5) In a light gray AlphaPlate (384 wells):

Add 5 µL of each analyte standard dilution or 5 µL of samples

Add 5 µL of 10X MIX of Anti-hIFN ß Acceptor Beads 10 µg/mL final) and Biotinylated
hIFN ß Antibody (2 nM final)

Incubate 3 hours at 23 °C

Add 40 µL of 1.25X SA-Donor beads (30 µg/mL final)

Incubate 30 minutes at 23˚C in the dark

Read using EnVision-Alpha Reader (615 nm)

DATA ANALYSIS•Calculate the average count value for the background wells.

•Generate a standard curve by plotting the AlphaLISA counts versus the concentration of analyte. Alog scale can be used for either or both axes. No additional data transformation is required.

•Analyze data according to a nonlinear regression using the 4-parameter logistic equation (sigmoidal
dose-response curve with variable slope) and a 1/Y2 data weighting (the values at maximal
concentrations of analyte after the hook point should be removed for correct analysis).

•The LDL is calculated by interpolating the average background counts (12 wells without analyte) + 3
x standard deviation value (average background counts + [3xSD]) on the standard curve.

•The LLOQ as measured here is calculated by interpolating the average background counts (12 wellswithout analyte) + 10 x standard deviation value (average background counts + [10xSD]) on thestandard curve. Alternatively, the true LLOQ can be determined by spiking known concentrations ofanalyte in the matrix and measuring the percent recovery, and then determining the minimal amountManual AL3191 V1

6

of spiked analyte that can be quantified within a given limit (usually +/- 20% or 30% of the realconcentration).
•Read from the standard curve the concentration of analyte contained in the samples.

•If samples have been diluted, the concentration read from the standard curve must be multiplied by
the dilution factor.

ASSAY PERFORMANCE CHARACTERISTICS
AlphaLISA assay performance described below was determined by using a 2-step high concentration
protocol using 1X AlphaLISA Immunoassay Buffer as an assay buffer. The analytes (standards) wereprepared in different matrix diluents depending on sample type. All other components were prepared in1X AlphaLISA Immunoassay Buffer.

Standard curve:

AlphaLISA Signal (cps)A typical sensitivity curve is shown below, using the 2-step high concentration protocol described on
page 4, using 1X AlphaLISA Immunoassay Buffer to dilute the standard.

1,000,000
100,000
10,0001,000-∞ -12 -11 -10 -9 -8
Log [hIFNβ](g/ml)-7-6

Assay sensitivity:
The LDL was calculated by interpolating the average background counts (12 wells without analyte) + 3 x
standard deviation value (average background counts + [3xSD]) on the standard curve. The values
correspond to the lowest concentration of analyte that can be detected in a volume of 5 µL of sample
using the recommended assay conditions.
Analyte diluent
Immunoassay Buffer
RPMI + 10% FBS
DMEM + 10% FBS

Manual AL3191 V1

LDL (pg/mL)
1.05
1.68
0.48

LLOQ (pg/mL)
3.85
9.47
3.39

7

Assay precision:
The following assay precision data were calculated from three independent assays using two different
kit lots. In each lot, the analytes were prepared in RPMI + 10% FBS, DMEM + 10% FBS and serum. Allother components were prepared in AlphaLISA Immunoassay Buffer. Each assay consisted of one
standard curve comprising 12 data points (each in triplicate) and 12 background wells (no analytes). Theassays were performed in a 384-well plate format.

Intra-assay precision:
The intra-assay precision was determined using a total of 24 independent determinations. Shown as
CV% of measured concentration.
hIFN ß
CONCENTRATION
Sample at 397 pg/mL

Immunoassay buffer

RPMI + 10% FBS

DMEM + 10% FBS

4.0%

6.8%

4.9%

Inter-assay precision:
The inter-assay precision was determined using a total of 6 independent determinations with 6measurements for 100 ng/mL sample. Shown as CV% as measured concentration.

hIFN ßSample at 100 pg/mLImmunoassay buffer
2.5%

RPMI + 10% FBS
5.9%

DMEM + 10% FBS
3.5%

Spike and recovery:
Three known concentrations of analyte were spiked into RPMI + 10% FBS, DMEM + 10% FBS and humanserum. All samples, including non-spiked diluents, were measured in the assay. Note that the analytes
for the respective standard curves were prepared in RPMI + 10% FBS, DMEM + 10% FBS and humanserum. All other assay components were diluted in AlphaLISA Immunoassay Buffer.

SpikedhIFN ß (pg/mL)
30 000
3 000
300% Recovery
RPMI + 10% FBS
91%
88%
85%

DMEM + 10% FBS
88%
96%
108%

Specificity:
Cross-reactivity of the AlphaLISA human IFN ß Detection Kit was tested using recombinant IFN ß frommouse and rat as analytes in an assay using the protocol as described above. Human isoforms were alsotested (IFNγ, IFNα 2 and IL6). The cross reactivities were established using the hIFN ß related proteins
below at a test concentration from 300 000 to 1pg/mL. Percentage recovery was computed bycomparing the measured Interpolated concentration versus the theoretical one.
Manual AL3191 V18

Proteins
Mouse IFN ßRat IFN ß
Human IFNγ
Human IFNα 2b
Human IL6

Cross reactivity (%)
0.00
0.00
0.00
0.00
0.00

Calibration:
Target NIBSC/WHO International Standard (code 01/420) was tested using the AlphaLISA hIFN ßdetection kit: 1 unit of Standard NIBSC 01/420 corresponds to 42 pg/mL of AlphaLISA hIFN ß
respectively.

Cell experiments:
Cell culture conditions
PBMC cells were collected following a Ficoll gradient treatment of whole blood.
Cells were prepared at 30 millions cells In 1mL.
Pharmacological rreatment
A cGAMP treatment was done on cells In order to induce cytokine release. A -ènM of cGAMP was addedon top of the cells during 16 hours.
Supernatant are then collected and diluted In a 2-fold serial dilution and tested In HP hIFN ß kit.

PBMC treated with cGAMP
sample dosage[hIFNb] pg/ml

2 000
1 500
1 000500t
ne1/21/
41/80

sample dilution

Manual AL3191 V19Human serum experiments:Dilution linearityNormal human serum and hIFN ß spiked (7 600 pg/mL) normal human serum samples were diluted with
100% FBS and the assay was performed along with a standard curve prepared in 100% FBS.
Concentrations of hIFN ß in diluted samples were determined by interpolating from the standard curve.
The other components (anti-hIFN ß Acceptor beads, biotinylated anti-hIFN ß antibody, and SA-Donorbeads) of the assays were prepared in 1X AlphaLISA ImmunoAssay Buffer.
Basal levels of hIFN ß was not detected in human serum from presumably health subjects. Excellent
dilution linearity (R2 >0.99, slope=0.98) and dilution recovery was achieved in the hIFN ß spiked humanserum samples (in the range of 2- down to 16-fold dilution. The results are shown in the table below.

Serum dilution
factor (x)
neat
2
4
8
16

Expected hIFN ß(pg/mL)
7633
3817
1908
954
477

Observed hIFN ß(pg/mL)
7633
4118
1829
972
348

Dilution Recovery
(%)
108
96
10273

Spike recovery
Three known amounts of human IFN ß were spiked into normal human serum (30 000, 3 000 and 300
pg/mL). The samples were assayed along with the standard prepared in 100% FBS. Other components(anti- hIFN ß Acceptor beads, biotinylated anti- hIFN ß antibody, and SA-Donor beads) of the assays wereprepared in AlphaLISA ImunoAssay Buffer. The spike recoveries of human IFN ß were determined. The
results shown in the table below indicate that excellent recoveries were achieved for all three spikes
tested.
hIFN ßSpike (pg/mL)
30 000
3 000
300

Diluent: 100% FBS
Spiked sample (normal human serum)
Concentration (pg/mL)
Recovery (%)
29 45398%
3 386
113%
309
103%

TROUBLESHOOTING
To find detailed recommendations for common situations you might encounter with your AlphaLISA
assay kit, please visit our website at www.revvity.com.

Manual AL3191 V110

Manual AL3191 V111