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1. WO1996040790 - PRESENTATION OF NATIVE ANTIGEN ON BEADS OR OTHER SOLID PHASES

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[ EN ]

PRESENTATION OF NATIVE ANTIGEN ON BEADS
OR OTHER SOLID PHASES

BACKGROUND OF INVENTION
Specific and high affinity molecular recognition is critical for diagnostics. In general, protein molecules that recognize a specific target were generated as antibodies. As a result, antibodies have received a central role in the development of analytical and separation methods that are currently being used. Among these techniques, flow cytometry is unique doe to its capability to perform simultaneous multiparameter analysis and to separate (or sort) unique cell populations from heterogeneous mixtures of cells.
Other techniques similarly utilize this technology. However, in each, the critical parameter is the recognition of the antigen. This is strongly affected by the conformation of the target, and, in general, it is essential to have the target in a corifirmation resembling its native conformation.
As conformation is strongly influenced by the environment of a molecule, it becomes extremely difficult to attach a target to a bead or other structure to facilitate its use in the desired application, yet retain a corifirmation similar to its native conformation.

SUMMARY OF INVENTION
The instant invention presents a rapid simple method for preparing solid phases, preferably beads, with antigens or other substituents presented on the surface in such a manner that the antigens/substituents retain their original functionality and conformation, as well as much of their native structure, to permit their use in a wide array of applications. Specifically, the substituent is isolated from the hydrophobic surface of the bead or other solid phase by coating the surface with a milk protein, alone or in combination with linker molecules, thereby preventing interaction of the substituents with the bead surface.

DETAILED DESCRIPTION OF THE INVENTION
The complexes of the instant invention are formed by attaching the substituent(s) to the solid phase, either directly via surface functionalities or via a linking group, or both. Regardless of the method of attachment used, the bead, other than the substituent binding sites, is coated with a milk protein or a milk protein derivative such as dephosphocasein (as used herein the term "milk" includes whole milk, powdered milk, and modified milk, milk proteins, and milk protein derivatives). This coating, it has been found, serves a two-fold basis preventing binding of the substituents to other sites on the solid phase (i.e., as a blocking agent) and preventing interaction of the bound substituents with the hydrophobic surfaces of the beads. In this way, the substituents retain their natural structure and properties.
The coating is obtained by immersing the solid phase, prior to attaching substituents, in solutions or mixtures containing milk for a period of approximately thirty minutes;
however, it can also be stored for extended periods of time in such solutions. In a preferred embodiment, the solid phase is a bead which can be any of those known to be useful in flow cytometry applications or other applications for antigen presentation. The beads must be capable of forming a stable complex with the substituents. In a further preferred
embodiment, the bead is comprised of polymethylmethacrylate possessing arnino
functionalities on the surface. These beads can often be directly attached to the substituents via the amino functionality. Preferably, activated biotin is attached to the amino groups and the beads are then coated with milk protein. The beads are then admixed with streptavidin creating a streptavidin layer on the bead, which is capable of binding biotinylated
substituents. It is to be understood, however, that other beads and/or surface functionalities can also be used.
The substituents can then be attached to the beads by the appropriate chemical reaction. In a preferred embodiment, the substituent is reacted with biotin to form a biotinylated complex which readily binds to the streptavidin layer on the beads. Thus, beads having the desired type and number of substituents can be easily made, and non-specific attachment would be blocked by the milk protein coating. Further, the milk protein coating serves to reduce and/or eliminate any interactions between the substituents and the hydrophobic bead surface, thereby permitting the substituents to be presented in the form that will exhibit a high degree of functionality. Blocking agents other than milk (or milk protein or its derivatives such as dephosphocasein) are not as efficient in blocking the nonspecific attachment.
The substituents will be chosen based on the ultimate desired use of the solid phase and/or the problem desired to be solved. Thus, substituents will include, but are not limited to, antigens, antibodies, other biologically active molecules, and fluorophores.
Fluorescent proteins are particularly useful with beads which are prepared in accordance with this invention. They produce a specific brightness and emission spectrum which is unaffected by any interactions between the bead surface and the fluorophores. Further, the beads exhibit a high utility as standards for flow cytometry, as the fluorophore coated beads produce the same fluorescence as stained cells.

Examples
The following examples illustrate certain preferred embodiments of the instant invention, but are not intended to be illustrative of all embodiments.

Example 1 - Preparation of streptavidin coated beads
Polymethacrylate amino beads (6.1 microns PMMA-RNH2, Bangs Laboratories, Inc., Carmel, IN) were washed once with 10 volumes PBS + 0.1% sodium azide, twice with three volumes PBS +0.1% SDS, and twice with PBS. The washed beads were then suspended (3% w/v) in PPBS containing 1.5mM NHS-XX-Biotin, and incubated, with mixing, a room temperature for one hour. The beads were then washed twice with three volumes of PBS, and subsequently, suspended and stored overnight in a PBS +0.1% sodium azide + 2% nonfat dry milk.
3

SUBSUME SHEET (RUiE 26) The beads were then suspended in PBS, 2.5% w/v, and co-mixed with streptavidin (0.4mg/ml) at room temperature for two hours, and subsequently, washed 5 times with 6 volumes of PBS + 0.1% sodium azide +0.5% BSA. The beads were ultimately stored as a 2.5% suspension in this buffer.

Example 2 - Preparation of biotinylated hCG and hLH
Samples of each protein (human chorionic gonadotropin and human luteinizing hormone) were suspended at 1 mgl/ml in PBS, and admixed for 2 hours at room temperature with 0. lmM NHS-LC-Biotin. Subsequently, unreacted biotin was removed on a Sephadex PD10 column, and the biotin content was assayed by binding to a polystyrene bead coated with the appropriate antibody and staining with FITC labeled streptavidin as compared with staining with an appropriate FITC labeled second step.

Example 3 - Attachment of biotinylated protein to streptavidin beads
Streptavidin beads (Example 1), at a suspension of 2.5% in PBS, were admixed with biotinylated hCG or hLH (Example 2) and incubated at room temperature for two hours.

The beads were then washed twice in 5 volumes PBS +0.1% sodium azide +0.5% BSA, and suspended at 2.5% w/v in the same buffer.
The beads present the protein in an unhindered conformation and can be used to bind and/or sort antibodies for hCG or hLH.

Example 4 - Preparation of fluorophore-coated beads
Beads were prepared as in Example 1, except that the streptavidin coating step was omitted. These biotinylated beads were reacted with excess streptavidin conjugates of fluorescein isotheocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll - a protein (Per CP), PE/Cy5 reactive dye (Biological Detection Systems), or allophycyanin (APC).
The resultant SA-FITC, SA-PE, and SA-Per CP beads were used to compensate a FACScan; using the same settings, standard Calibrite™ beads, and lysed whole blood were also run. The results are shown in Figure 1. As shown, it can be seen that the delta channel values of these beads are very close to those of stained cells, while these emissions of the Calibrite™ beads are significantly different.

Example 5 - Use of other blocking agents
SA-PE beads were prepared as in Example 4, except that BSA, Tween 20, gelatin, and Tetronic 908 were used in place of the milk protein. These compounds were much less efficient in blocking the non-specific absoφtion, resulting in a much higher interfering signal on a flow cytometer, then when milk was used. The milk protein derivative,
dephosphocasein was found to be at least as efficient a blocker as whole milk protein.

Example 6 - Preparation of Beads with CD4
Streptavidin coated amino beads were prepared using the above procedure (Example

1) and reacted with biotinylated CD4 (prepared as described in Example 2). The beads were titrated and found to have approximately 10^ molecules. A control lot of streptavidin coated polystyrene beads (prepared by coating the bead directly with streptavidin, without any blocking) were similarly reacted with biotinylated CD4.
Separate samples of both lots of beads were stained with either Leu3a-PE or L200-PE

(antibodies specific for Region I of the CD4 molecule) and L120-PE (an antibody specific for Region IV). When analyzed on a flow cytometer, the results demonstrate that the streptavidin coated polystyrene beads exhibit a fluorescent signal for the LI 20 which is 5 times that of the Leu3a/L200 signal; with beads prepared using the methods of this invention yielded nearly identical signals.
Normal blood cells such as T-helper lymphocytes express CD4 on their cell surface. Staining with Leu3a-PE, L200-PE or L120-PE gives approximately equal signals for all three antibodies, indicating that the coated beads prepared according to the methods of this invention preserve the natural functionality of the CD4 molecule.

While not wishing to be bound by theory, it is postulated that the method of the instant invention prevents non-specific binding of the CD4, making Region I of the CD4 more accessible to the antibody, thereby yielding a higher signal.
It is apparent that many modifications and variations of this invention as hereinabove set forth may be made without departing from the spirit and scope thereof. The specific embodiments are given by way of example only and the invention is limited only by the terms of the appended claims.

SUBS UTESHEI HaE26