Methods for enhancing binding interactions between members...

Details Methods for enhancing binding interactions between members... Methods for enhancing binding interactions between members...

1999-01-29

2003-04-22

Brumback, Brenda (Department: 1642)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

Carrier is inorganic

C435S007100, C435S007500, C435S007900, C435S007920

Reexamination Certificate

active

06551843

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the fields of specific binding pair interactions, bioentity separations and the isolation of rare substances from biological fluids. Methods are provided for enhancing such bioseparations, preferably via enhanced magnetic loading onto target entities, thereby facilitating biochemical and diagnostic analysis of target entities so isolated.
BACKGROUND OF THE INVENTION
There are a substantial number of manufacturing, analytical and laboratory processes and procedures which involve specific binding pair interactions. Many laboratory and clinical procedures are based on such interactions, referred to as bio-specific affinity reactions. Such reactions are commonly utilized in diagnostic testing of biological samples, or for the separation of a wide range of target substances, especially biological entities such as cells, viruses, proteins, nucleic acids and the like. It is important in practice to perform the specific binding pair interactions as quickly and efficiently as possible. These reactions depend on classical chemical considerations such as temperature, concentration and affinity of specific binding pair members for one another. In the ideal, separations employing specific binding partners which rapidly form multiple non-covalent bonds are utilized. The use of such binding partners is important, particularly when the concentration of one of the specific binding pair members to be isolated is extremely low, as often is the case in biological systems. Of course, concentration considerations are relevant in other separation processes, such as in water purification, or in applications where it is necessary to remove trace contaminants or other undesirable products.
Various methods are available for binding, separating or analyzing the target substances mentioned above based upon complex formation between the substance of interest and another substance to which the target substance specifically binds. Separation of the resulting complexes from solution or from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the ligand substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation, may be employed for this purpose. However, processes relying on magnetic principles are preferred.
Magnetic particles generally fall into two broad categories. The first category includes particles that are permanently magnetizable, or ferromagnetic; and the second comprises particles that demonstrate bulk magnetic behavior only when subjected to a magnetic field. The latter are referred to as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamagnetic. However, materials exhibiting bulk ferromagnetic properties, e.g., magnetic iron oxide, may be characterized as superparamagnetic when provided in crystals of about 30 nm or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter due to strong particle-particle interaction.
Magnetic particles can be classified as large (1.5 to about 50 microns), small (0.7-1.5 microns), and colloidal or nanoparticles (<200 nm). The latter are also called ferrofluids or ferrofluid-like particles and have many of the properties of classical ferrofluids. Liberti et al pp 777-790, E. Pelezzetti (ed) “
Fine Particle Science and Technology
, Kluver Acad. Publishers, Netherlands,
Small magnetic particles are quite useful in analyses involving bio-specific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g., proteins), provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents.
In addition to the small magnetic particles mentioned above, there is a class of large magnetic particles (>1.5 microns to about 50 microns) which also have superparamagnetic behavior. Such materials include those invented by Ugelstad (U.S. Pat. No. 4,654,267) and manufactured by Dynal, (Oslo, Norway). Polymer particles are synthesized, and through a process of particle swelling, magnetite crystals are embedded therein. Other materials in the same size range are prepared by performing the synthesis of the particle in the presence of dispersed magnetite crystals. This results in the trapping of magnetite crystals thus making the materials magnetic. In both cases, the resultant particles have superparamagnetic behavior, readily dispersing upon removal of the magnetic field. Unlike magnetic colloids or nanoparticles referred to above, such materials, as well as small magnetic particles, because of the mass of magnetic material per particle are readily separated with simple laboratory magnetics. Thus, separations are effected in gradients as low as a few hundred gauss/cm, to up to about 1.5 kilogauss/cm. Colloidal magnetic particles (below approximately 200 nm) require substantially higher magnetic gradients for separation because of their diffusion energy, small magnetic mass/particle and stokes drag.
U.S. Pat. No. 4,795,698 to Owen et al. relates to polymer-coated, sub-micron size colloidal superparamagnetic particles. The '698 patent describes the manufacture of such particles by precipitation of a magnetic species in the presence of a biofunctional polymer. The structure of the resulting particles, referred to herein as single-shot particles, has been found to be a micro-agglomerate in which one or more ferromagnetic crystallites having a diameter of 5-10 nm are embedded within a polymer body having a diameter on the order of 50 nm. These particles exhibit an appreciable tendency not to separate from aqueous suspensions for observation periods as long as several months. Molday (U.S. Pat. No. 4,452,773) describe a material which is similar in properties to those described in the '698 patent of Owen et al. produced by forming magnetite and other iron oxides from Fe
+2
/Fe
+3
via base addition in the presence of very high concentrations of dextran. Materials so produced have colloidal properties. This process has been commercialized by Miltenyi Biotec, (Bergisch Gladbach, Germany). Those products have proved to be very useful in cell separation assays.
Another method for producing superparamagnetic colloidal particles is described in U.S. Pat. No. 5,597,531. In contrast to the particles described in the '698 patent, these latter particles are produced by directly coating a biofunctional polymer onto pre-formed superparamagnetic crystals which have been dispersed by sonic energy into quasi-stable crystalline clusters ranging from about 25 to 120 nm. The resulting particles, referred to herein as direct-coated or DC particles, exhibit a significantly larger magnetic moment than the nanoparticles of Owen et al. or Molday et al. having the same overall size.
Magnetic separation techniques utilize magnetic field generating aparatus to separate ferromagnetic bodies from the fluid medium. In contrast, the tendency of colloidal superparamagnetic particles to remain in suspension, in conjunction with their relatively weak magnetic responsiveness, requires the use of high-gradient magnetic separation (HGMS) techniques in order to separate such particles from a fluid medium in which th

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