Surface-assisted hemagglutination and hemagglutination inhibition assays
Inventors
Kachurin, Anatoly • Wittman, Vaughan • Nguyen, Mike N. • Kachurina, Olga • TAPIA, Tenekua • Dhir, Vipra • Karol, Alexander
Assignees
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Publication Number
US-8962256-B2
Publication Date
2015-02-24
Expiration Date
Abstract
Hemagglutination (HA) and hemagglutination inhibition (HAI) functional assays remain important instruments of analysis of virus-cell interaction and protecting efficacy of virus-specific antibodies and sera. However, they demonstrate limited sensitivity towards many viruses, and require significant volumes of viruses, erythrocytes, sera, and antibodies. The present invention comprises new and significantly more sensitive versions of the HA and HAI assays based on observing agglutination on activated surfaces of specifically opsonized plates and ELISA plates rather than in solution. A version of the new assay that uses ELISA plates additionally allows characterizing the affinity of functional antibodies in the tested sera and fluids, which is not possible in the classical HAI assay. The methods of the present invention can also be used to improve the sensitivity of agglutination methods based on latex beads and to develop agglutination methods using target cells other than erythrocytes.
Core Innovation
The invention provides a surface-assisted hemagglutination/hemagglutination-inhibition assay concept in which HA/HAI readouts are transferred from solution to activated, opsonized culture surfaces or U-bottom ELISA plates. An agglutinating factor is attached to the surface of an activated well bottom so that agglutination of a target object is detected on the surface rather than in the well volume.
Agglutination is detected and quantified by photo-registration and digital image processing using a numerical Hemagglutination Parameter (HAP) derived from two-dimensional agglutination patterns. The agglutinated target objects are quantified as two-dimensional patterns on the well bottom of the plate, yielding a parameter used for titers.
For standardization, the invention uses Linked Standard Dilution/Linked HAP (LSD/LHAP) with automated curve fitting for titers, and it includes a surface-assisted ELISA-plate version supporting functional antibody affinity profiling. The affinity profiling is described in terms of removing or washing dissociable low-affinity antibodies prior to adding erythrocytes, thereby enabling improved measurement sensitivity compared with classical HA/HAI formats.
Claims Coverage
Only one independent claim is explicitly identified in the provided claims list. It covers an antibody functional binding activity method based on surface-attached agglutination detection and a comparison to determine functional binding activity when agglutination is reduced in the presence of the antibody.
Surface-attached agglutinating factor on activated well bottom
Incubating an agglutinating factor in an activated well of a culture plate under conditions achieving attachment of the agglutinating factor to a surface of the well bottom.
Functional binding activity determined by reduced surface agglutination
Adding the antibody and then a target object that is agglutinated by the agglutinating factor, detecting agglutination of the target object on the surface of the well bottom, and determining that the antibody has functional binding activity when agglutination detected with the antibody is less than agglutination detected in the absence of the antibody.
The independent claim defines a method that attaches an agglutinating factor to an activated well bottom, performs antibody-dependent competition with target-object agglutination, detects surface agglutination, and assigns functional binding activity based on reduced agglutination relative to antibody absence.
Stated Advantages
Improved assay sensitivity, with at least about 10 times increased sensitivity when detecting agglutination on an activated well bottom compared with performing the method in a non-activated well where agglutination occurs in the well volume.
Enables functional antibody affinity profiling by removing or washing dissociable low-affinity antibodies prior to adding erythrocytes.
Improved standardization of titers using Linked Standard Dilution/Linked HAP (LSD/LHAP) and automated curve fitting.
Documented Applications
Cross-protection and in vitro immune setups using systems described as “MIMIC®” are discussed in the context of the surface-assisted assay formats and comparisons.
Use with viruses including influenza viruses (e.g., A/Brisbane/59/2007, A/Solomon Islands/3/2006, A/Wisconsin/67/2005, A/California/7/2009, A/New Calcdonia/20/99) and associated standards (e.g., BPL-inactivated virus standards), including measurements using convalescent sera and reference materials such as Fluvirin vaccine.
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