Green DJ1, Morrison C2, Rudd EA2, Smejkal G2, Skea W2, Laugharn JA2

Covaris, Inc. • 14 Gill Street, Unit H, Woburn, Massachusetts 01801-1721 USA Tel: +1 781-932-3959 • Fax: +1 781-932-8705 • Email: info@covarisinc.com

Web: www.covarisinc.com

Adaptive Focused Acoustics improves the performance of microplate ELISA’sOVERVIEWObjective—To develop a new technology platform (“dry AFA”) that enables the

improved performance (faster antigen/antibody binding, lower NSB, lower LOD’s, higher Z’)

of off-the-shelf microplate ELISA’s by Adaptive Focused Acoustics (AFA) to be achieved in a

HTS compatible format to benefit HST applications.

MetHODS—AFA was applied in a prototype “dry AFA” format instrument to microplate

ELISA’s during the first step of the assay in which antigen is captured by immobilized capture

antibody. Conventional passive binding during the first assay step was performed in control

plates. All other assay steps were performed in both AFA and control assays per the protocol

stipulated by the kit manufacturer. Temperatures of samples during antigen/antibody

binding were measured with a thermocouple Dose response curves for levels of acoustic

energy and frequency of application to samples were determined. Optimal AFA instrument

settings were used for comparison of assays performed with an AFA-mediated antigen/

antibody binding step to assays performed per manufacturers’ kit protocols.

ReSULtS—AFA-mediated ELISA’s (in which AFA was applied during the initial antigen/

antibody binding step) exhibited superior performance to assays performed with

conventional passive binding protocols.

cONcLUSiON—the “dry AFA” format instrumentation has the potential to bring

significant improvements to processing time, precision, and sensitivity of ELISA’s used in

applications such as HTS and in vitro diagnostics.

INTRODUCTIONCovaris L-Series Adaptive Focused Acoustics (AFA) instruments - one of which (the LE220) was a winner of a New Product Award at the SLAS 2011 Annual Meeting - are now in widespread use in laboratories worldwide for applications such as precise shearing of DNA prior to sequencing, chromatin shearing, and compound management. See Figure1.

Recently, Covaris has demonstrated that the L-Series instruments used in the above applications can also be used at low acoustic power to provide significant improvements in the performance of microplate ELISA’s compared to assays performed with conventional passive binding. Improvements demonstrated to date include –

• Faster antigen/binding rates • Better assay precision • Lower NSB

• Lower LOD’s • Higher Z’ factors • Efficient washing

Figure 1— Adaptive Focused Acoustics™ (AFA)

AFA TECHNOLOGYAdaptive Focused Acoustics™ (AFA) is Covaris’ patented acoustic technology empowering Focused-ultrasonicators to mechanically process samples. AFA employs highly controlled bursts of focused high-frequency acoustic energy to efficiently and reproducibly process samples in a temperature controlled, non-contact, and closed environment. The very high frequency ultrasound utilized in AFA results in a wavelength of only a few millimeters, enabling the acoustic energy to be focused into a discrete zone within a sample vessel. This focused and efficient delivery method requires a minimal amount of energy avoiding the adverse effects of excess energy such as damaging heat, experimental variability, and sample over-processing typical of ordinary sonicators.

Sample processing with AFA ultrasonic energy is accomplished by controlling the creation and collapse of millions of cavitation bubbles within the closed sample vessel. Acoustic energy passing through an aqueous medium causes localized pressure

fluctuations which forms small cavities (or bubbles) in the regions of relative low pressure. The cavitation bubbles oscillate or grow to a critical size and then collapse. The oscillation and collapse of the cavitation bubbles generates acoustic microstreaming, which creates hydrodynamic shear stress in the sample. AFA™ Focused-ultrasonicators provide exquisite control of the acoustic bursts delivered to a sample. The tuning of peak incident power, duration, and duty factor, controls the microstreaming, and in turn the generation of shearing forces.

AFA can be precisely tuned to process samples in a variety of applications, from low-power gentle mixing of solutions and protein extraction, to higher-power applications such as DNA fragmentation, liposome formation, and the creation of nanosuspensions.

Focused-ultrasonic Transducer

Sample Vessel

Focal Zone

LE220’s unique linear transducer for High Throughput applications

Figures 2-4 The commercially available L-Series AFA instruments used to obtain the data shown in Figures 2-4 use open water baths to (a) couple ultrasonic energy from the transducer into test samples, and (b) to maintain sample temperature control, however, plate bottoms become wetted during the AFA procedures. Although they can be readily blotted to remove water, wet microplates are sub-optimal for both integration into automated HTS platforms and for clinical in vitro diagnostic laboratories.

The objective of the work presented here was to develop a new “dry format” technology platform for applying AFA to microplate ELISA’s in a way that ensures that-

• The bottoms of the microplates do not become wetted during the AFA process.• “dry AFA) format provides advantages to ELISA assays comparable to “wet” format AFA.

METHODSStudies were primarily performed with a 96-well microtiter plate sandwich ELISA (ABL) that measures

HIV-1 p24 antigen levels in tissue culture samples. Off-the-shelf microplate sandwich ELISA’s for

measuring IGFBP-3 (R&D Systems) and CA-IX (Wiley-Oncogene) were used in a more limited study to

explore the universality to other ELISA’s of the AFA-mediated improvements observed with the HIV-1

p24 ELISA. A one-step direct ELISA in which goat-anti-rabbit-HRP (Pierce, 31460) binds to rabbit IgG

(Pierce, 31235) immobilized in a 96-well microplate (Immulon, Dynex,) was developed in house. It

was primarily used for AFA parameter optimization.

AFA was applied to the first step of the ELISA’s in which antigen in solution binds to immobilized

capture antibody. Sample volumes were 100 µL except for the HIV-1 p24 ELISA which utilizes 125 µL

in the first step of the assay per the kit protocol. AFA was applied to samples with the Covaris “dry”

format prototype instrument and compared to results obtained with Covaris L-Series instruments

operating in the “wet” format. In the latter format, plate bottoms are wetted by water used to (a)

couple ultrasonic energy from the transducer into test samples, and (b) maintain sample temperature

control.

Passive antigen/antibody binding was performed per kit protocols for 1 hr at 37°C for the HIV-1 p24

ELISA; 2 hr at 4-8°C, for the IGFBP-3 ELISA; and 2 hr RT, 800 rpm for the CA-IX ELISA as well as at other

temperatures for comparison to AFA-mediated binding performed at different temperatures.

The L-series instruments utilize transducers that focus the acoustic energy into a line that is

simultaneously applied to all wells of an 8-well column. By slight modulation of the frequency and/or

raster scan of the focal zone, each well receives the identical amount of acoustic energy. Instrument

settings allow an operator to set (1) the Peak Incident Power (PIP), a measure of the amplitude of the

acoustic waves emitted by the transducer (Watts), (2) duty factor (df), the percentage of time that the

transducer is on, (3) cycles per burst (cpb) number of on waves, and (4) duration in seconds.

Temperature control was maintained by the circulation of cooling water from an external chiller.

Temperature of samples was monitored by insertion of a thermocouple wire into control wells. On

completion of the antigen/antibody binding step, samples were processed according to kit protocols

for washing, conjugate binding, reaction with enzyme substrate, and photometric detection.

RESULTS• Coupling of acoustic energy from the transducer to a microtiter plate was achieved

with a dry acoustic waveguide . A schematic of the system is shown in Figure 5.

• Acoustic modeling of the pressure pattern below and above the new dry acoustic waveguide is shown in Figure 6.

• The temperatures of samples during application of AFA in the “dry” format instrument in a typical experiment are shown in Figure 7.

• Comparisons of “dry” AFA-mediated HIV-1 p24 ELISA’s to wet format AFA and passive assays are shown in Figures 8-9.

Figure 7—Temperature of samples during application of AFA in dry format prototypeAFA 10i, 5% df, 200 cpb. 1 second AFA every 22 seconds. Solid Dynex Immulon plate.

Figure 8— Dry AFA enhancement of HIV-1 p24 ELISA. The initial antigen/antibody binding step was performed with the dry AFA instrument set at 10i, 5% df, 200 cpb. AFA was applied for 3 seconds every minute for one hour. Parallel passive binding was performed for one hour at 37°C per the kit protocol. All other assay steps were performed per the kit protocol. Singleton samples.

Figure 9—Dry AFA ELISA performance statistics compared to passive assays

Figure 5—“Dry” format AFA instrument.Cross-section of acoustic assembly perpendicular to long axis of the linear transducer.

Figure 6—Acoustic simulation showing the pressure pattern below and above the Waveguide.

Figure 3—AFA lowers the LOD of microplate ELISA’s

Figure 4—Effect of AFA on Z’ of HIV-1 p24 ELISA

Figure 2 —Rates of AFA-mediated binding of HIV-1 p24 antigen to immobilized capture antibody in a microtiter plate ELISA compared to passive binding. Relative rates of antigen/antibody binding were determined from slopes of binding curves (n ≥3).

PASSIVE BINDING

Column A: 20°C. Column B: 37°C. AFA-MEDIATED BINDING

Column C: 100 PIP, 5% df, 50 cpb. 1 second pulse every 22 seconds. LE220, 18-20°C.

Column D: 175 PIP, 5% df, 200 cpb. 1 second pulse every 22 seconds. L8, 18-20°C.

Column e: 105 PIP, 5% df, 200 cpb. 1 second pulse every 5.5 seconds. L8, 18-20°C.

CONCLUSIONS• Application of Adaptive Focused Acoustics (AFA) to off-the-shelf microplate ELISA’s

with the novel “dry AFA” technology enables superior assay performance of the assays compared to conventional passive prodcedures.

• Key benefits of of the AFA technology are –

- Faster antigen/antibody binding

- Lower NSB

- Lower LOD’s

- Higher Z’ factors

• Commercially available microtiter plate ELISA’s need no adaptation of the passive step to be replaced with an AFA-mediated step.

• The new “dry AFA” technology presented here is compatible with the automated, high speed handling of microplates.

• “dry AFA” instrumentation has the potential to bring significant improvements to HTS and in vitro clinical diagnostic procedures that utilize microplate ELISA’s.

1Biotechnology Consulting Associates, Fitzwilliam, NH, 2Covaris, Inc., Woburn, MA.

DRY AFA PASSIVEIntra-Assay Reproducibility (n = 3) Mean CV(%) 7.9 9.4

Inter-Assay Reproducibility (n=5)

Mean CV(%) 8.4 14.7

Non-Specific Binding (n = 9)

Mean A450 at 0 pg/ml 0.036 0.043

LOD (n = 1) pg/ml HIV-1 p24 0.8 (+/- 0.1) 1.5 (+/- 0.2)

Z’ (n =1)

HIV-1 p24 (pg/ml)

25.0 0.83 0.71

12.5 0.63 0.69

6.25 0.70 0.35

3.12 0.55 0.36

1.56 0.44 0.01