PROCEDURE FOR ATTACHING SUBSTANCES TO PARTICLES

Abstract: A process for attaching a substance, especially an organic substance, onto one or more microparticles is provided. The process involves providing a quantity of a substance to be attached onto the microparticles, as well as a quantity of microparticles. The organic substance is then directed through a first conduit (12, 206) and the microparticles are sent through a second conduit (16, 212). The first (12, 206) and second (16, 212) conduits meet at a confluence point (26, 214, 305), and it is there that the substance and the microparticles mix such that the substance attaches to, and coats the microparticles. In a preferred embodiment, there is further provided mixing means such as an in-line static mixer (22, 216, 238, 307, 317, 323, 329) for mixing together the substance and the microparticles. The coated microparticles can be utilized in all manners of immunoassay, nucleic acid assay, cell assay and therapeutic injectable applications. The microparticles herein may also be replaced with another organic substance for conjugation or attachment to the first organic substance.

Description: PROCEDURE FOR ATTACHING SUBSTANCES TO PARTICLES BACKGROUND OF THE INVENTION The use of magnetic or nonmagnetic microparticles in immunoassays and other diagnostic procedures is well known. All such methods require the coupling of organic binding elements, such as antibodies, antigens, nucleic acids or other organic compounds to the microparticles used by that particular assay device. The coated microparticles are then utilized in the immunoassays to capture the analyte of interest in the particular sample to be assayed. Typically, the binding of organic substances to microparticles has been accomplished in a batch reaction contained by a reaction vessel. For example, US Patent number 4,554,088 describes methods for coupling antibodies or enzymes to magnetic particles. Such coupling usually requires 16 to 24 hours of reaction time, including constant mixing of the particles and reagents. Other examples of the coating of binding elements to magnetic particles are presented in US Patent number 5,411,863, also requiring several hours for conjugation in batch reactions. Good summaries of various chemical processes useful for coating organic substances to microparticles are available in commercial literature from Rhόne-Poulenc (Paris, France), Seradyn (Indianapolis, IN), Bangs Laboratories (Carmel, IN) or Pierce Chemical (Rockford, IL). The coupling of organic substances to microparticles, particularly magnetic microparticles, in batch reactions can present several problems. First, the high density of magnetic microparticles causes them to settle rapidly to the bottom of the reaction vessel. Constant agitation is therefore required to maintain the semblance of a homogeneous mixture. Secondly, despite the best intentions of the mixing, local concentration variations will occur leading to nonuniformity in the amount of coupling to the particles. This variability in the amount of coupling is particularly serious given the difficulty in demonstrating consistency of coating in a manufacturing setting and therefore proving assay reliability. Batch-to-batch reproducibility wihtin the immunoassay context can also be a problem. Furthermore, mixing in batch reactors is dependent on the size, shape, and volume of the reactor as well as the size and shape of the mixing device, and speed at which it turns or operates. An improvement in the coupling process would therefore be one that can be scaled up or down without changing the parameters of mixing. A need therefore exists for a method of attaching or coating organic substances onto microparticles, including magnetic microparticles, that is more rapid and consistent. There is also a need in the art for a method of coating microparticles which results in an efficient, uniform coating thereof. A further need exists for a process of attaching substances to microparticles which is faster than that currently available in the art. Additionally, a need exists for a more expeditious method of attaching one or more biological or chemical materials to other biological or chemical materials for further use in various biotechnological applications, including immunoassays, as well as for example in injection therapy with radiolabelled or toxin labeled antibodies or proteins. The attendant problems associated with coupling of substances to microparticles are also very much present in the conjugation or attachment of biological and chemical materials to one another. SUMMARY OF THE INVENTION A process for attaching or conjugating a substance onto one or more microparticles, involves providing a quantity of a substance to be attached onto the microparticles. Also provided is a quantity of microparticles. The substance is directed through a first conduit and the microparticles are sent through a second conduit. The first and second conduits come together at a confluence point, and it is there that the substance and the microparticles mix such that the substance attaches to, and coats the microparticles. The process of the invention according to the various embodiments hereinafter described is designed to prepare polymeric and magnetic microparticles for use in immunoassays wherein the extent of binding to a complementary biopolymer is to be measured. Also provided as part of the invention is a method of attaching or conjugating one substance onto one or more other substances, wherein such substances are to be utilized in an immunoassay or in other applications. In this method, a quantity of a first substance is directed through a first conduit and a quantity of a second substance is directed SUBSTTTUTE SHEET (RULE 26) through a second conduit. The first and second substances come together at a confluence point such that the first substance attaches to the second substance. More conduits may then be utilized to attach additional substances onto the bound first /second substance complex. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of an instrument useful in the practice of the invention according to a first embodiment. Figure 2 is a plan view illustration of a component of the instrument useful in practicing the invention according to the various embodiments. Figure 3 is a side-view illustration of the device set forth in Figure 2. Figure 4 is a schematic diagram of an instrument useful in the practice of the invention according to a second embodiment. Figure 5 is a schematic diagram of an instrument useful in the practice of the invention according to a third embodiment. Figures 6 A, B, C and D are graphs further illustrating the results of the coating process according to one embodiment of the invention in comparison to a more conventional method. Figure 7 is a graph further illustrating the results of the coating process according to another embodiment of the invention. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The invention, according to its various embodiments herein described, provides a process for attaching or conjugating substances, particularly organic substances, to microparticles, including both magnetic and non-magnetic microparticles. More specifically, it provides a process for the continuous coating of organic substances to microparticles, as opposed to batch coating processes currently known in the art. The process generally consists of directing at least two steams of reagents, one containing the organic substance and one containing the microparticles, to a confluence point at which the reagent streams mix. The attachment of the organic substance to the microparticles is effected by the mixing of the streams at the confluence point, and optional subsequent conduits, as well as mixing devices integrated into the conduits downstream of the confluence point. Referring now to the drawings in which like numerals indicate like components throughout the various embodiments, an example of the process of the current invention may be first described with reference to Figure 1. A device 10 includes a first conduit 12 that is connected to a first reagent source 14 and a second conduit 16 that is connected to a second reagent source 18. The first conduit 12 and the second conduit 16 meet at a confluence point 26 and discharge into a third conduit 20. The third conduit 20 may include a mixing device 22 followed by a discharge conduit 24. In this general example of the process, the first reagent source 14 contains the substance, e.g. antibodies, to be bound to the microparticles. The substance may be contained in, or suspended in, a suitable suspending fluid in any suitable container. The suspending fluid may be any type of fluid known to those in the art, such as a buffer solution, typically utilized for storing reagents. The second reagent source 18 contains microparticles, also in a suspending fluid. The container may be any type of device, e.g. bottle, beaker, bucket, etc., suitable for holding the first and second reagents, e.g. the organic substance and microparticles, respectively. The conduits 12, 16, 20, and 24 may be ordinary tubing also readily available to the skilled artisan. The tubing may be of any material, preferably plastic polymer. It may be substantially rigid or flexible, clear or opaque. The internal diameter of the tubing should be such as to permit the easy movement of reagent streams therein. A preferred tubing for use with the process of the invention is referred to as Masterflex silicon tubing (Cole- Parmer Instrument Company, Niles, IL). The confluence point 26 is the junction at which the first and second reagent sources converge, thereby causing the two reagent streams to mix. The confluence point should be configured so as to facilitate the aforesaid mixing and coating of organic substance onto the microparticles. Preferred is a common "T" junction, more desirably model TSO-6 of 3/16 inch internal diameter, available from Value Plastics, Inc., Fort Collins, CO. Referring again to Figure 1 and also to Figures 2 and 3, there is optionally provided as part of the invention the mixing means 22. This mixing means 22 is typically located downstream of the confluence point 26 via the third conduit 20. The mixing means 22 may be attached downstream and in-line from the conduit 20, and upstream from and in-line to the conduit 24. Alternatively, the mixing means 22 may be attached directly inline and downstream of the confluence point 26. The mixing means further facilitates the mixing of the organic substance and the microparticles in the respective reagent streams, thereby in turn facilitating the coating of the organic substances onto the microparticles. The mixing means 22 may be any of those currently available in the art which may be adapted for use in the method of the invention. Desirably, the mixing means 22 is a mixing device such as one or more in-line mixers such as that listed as catalog number FM- 06-09-36, from Conprotec, Inc., Salem, NH, and illustrated in Figures 2 and 3. In operation, the two reagents converge and at least partially mix at the confluence point 26 and flow into the third conduit 20. The two reagents are then thoroughly mixed by the mixing device 22. As the two reagents are mixed, the organic substance becomes attached or conjugated to the microparticle. In theory, there is no particular limit to the number of mixing means or mixing devices 22 which may be included as part of the process. The optimum number of mixing devices may be experimentally determined, as it will vary with the binding affinity between the organic substance and the microparticles. As a practical matter, those who are regularly engaged in this field will probably discover that less than about 25 mixing means, and more desirably between 3 and 15 mixing means should be sufficient. A particularly preferred embodiment of the process of the invention is now set forth with reference to Figure 4. In this embodiment, means are further provided for expediting the movement of the reagent streams towards the confluence point. Preferably, these means are pumping means, and more preferably are pumps that discharge into the associated conduits. The pumping means, and preferably the pumps, are hooked up to the system set forth in Figure 4, and elswhere herein, according to well known methods in the art. The especially preferred use of peristaltic pumps facilitates the replacement of conduit at the conclusion of a particular coating operation. A first pump 202 causes the flow of a first reagent, containing organic substance such as antibodies, preferably in a suspending fluid, from a container or reservoir 204 into a first conduit 206. A second pump 208 causes the flow of a second reagent, containing microparticles, preferably in a suspending fluid, from a container or reservoir 210 into a second conduit 212. The first and second conduits 206 and 212 converge at a confluence point 214 and flow into one or more in-line mixer elements 216. As the two reagents flow through the mixer 216, one or more chemical or physical reactions occur to effect the binding of the antibodies to the microparticles. These chemical and /or physical reactions have been described in the art, and may include for example, covalent bonding. In the embodiment illustrated in Figure 4, there is also optionally provided a third pumping means, preferably a third pump 220 which causes the flow of a third reagent, containing for example, EDAC (1 Ethyl-3(-3-Dimethylaminopropyl)Carbodiimide - Sigma Chemical, St. Louis, MO), from a reservoir 222 to a conduit 224. This third stream of reagent from the third conduit mixes with the mixture of the first and second reagents at a second confluence point 218 and flows into and through a conduit 226. As the mixture of the first, second and third reagents flows through and downstream beyond the conduit 226, the EDAC crosslinks with the antibodies bound to the microparticles, as well as any excess EDAC. Also shown in Figure 4 is a fourth pumping means, or pump 230. Pump 230 is configured to cause the flow of a fourth reagent, for example a buffer solution, from a reservoir 234 to a conduit 228. In this process the fourth pump 230 generally operates to flush the device 200 at the conclusion of its operation (for instance, when the supply of one of the reagents is consumed). The mixture of the first, second, and third reagents flows into another set of one or more in-line mixers 238 and then to a discharge conduit 240. The resulting substance exiting the discharge conduit 240 is a suspension of microparticles coated with antibodies and crosslinked with EDAC. In the exemplary process of Figure 4, the pumps 202, 208, 220, and 230 are most preferably peristaltic pumps such as Watson-Marlow 504u/RL peristaltic pumps. The conduits are cylindrical silicon tubing of about 1.6 mm internal diameter (conduits 206, 212, 224, and 228) or 4.8 mm internal diameter (conduits 226 and 240). The mixers 216 and 238 are Conprotec in-line mixers, with a catalog number FM-06-09-36. In this process, the pumps 202, 208, 220, and 230 are operated to maintain optimum flow through the system. For example, all pumps may be operated simultaneously at a high flow rate sufficient to remove bubbles for a short period of time (about 60 seconds), or to remove air bubbles from the conduits and to prime the inlet and outlet conduits with reagent. During steady state operation, each pump is operated at a steady rate to supply its associated reagent at the desired rate, with pump operation timed to preserve reagent (particularly the first reagent). For example, the following operational sequence may be used, with the assumption that the supply of the first reagent containing the first substance will be exhausted prior to any other reagents, e.g. the microparticles: 1) All pumps are operated at high speed for a time period required to flush air bubbles and prime their associated inlet and outlet conduits with reagent. 2) Pumps 1 and 2 are operated at their normal flow rate (about 10 ml/min) until the leading edge of the mixture of reagents 1 and 2 reaches the second confluence point 218. 3) Pumps 1, 2, and 3 are operated at their normal flow rate (about 10 ml/min) until reagent 1 is exhausted. The reacted mixture of reagents 1, 2, and 3 is collected at the discharge conduit 240. 4) When reagent 1 is exhausted it is replaced by a buffer solution. A bubble may be introduced to indicate the trailing edge of the mixture of reagents 1, 2, and 3. 5) Pumps 1, 2, and 3 are operated at their normal flow rate until the trailing edge of the mixture of reagents 1, 2, and 3 reaches the second confluence point 218. 6) Pump 4 is operated at about three times the normal flow rate (or 30 ml/min) until the trailing edge of the mixture of reagents 1, 2, and 3 exits the discharge conduit 240. At the conclusion of operation, the system may be operated repeatedly in a similar fashion. Alternatively, and especially if any different reagent is desired, the entire assembly of conduit tubing and in-line mixers may be removed, discarded, and replaced by fresh tubing and mixers. As the skilled artisan should quickly recognize, there is potentially no limitation to the method of the invention for attaching, conjugating or coating biological and chemical materials onto microparticles. The method according to the various embodiments of the invention herein set forth may therefore be further modified, depending upon the myriad setups available to the technician of the components set forth in Figures 1 through 4. For example, as has been illustrated, the method may involve the mixing of more than two reagent streams. It may also involve the utilization of more than one mixing means. The methods according to the several embodiments may be used to coat any of a multitude of microparticle types (for example, those manufactured by Polymer Labs, Nippon Paint, Spherotech, Seradyn, or Bangs Laboratories). These particles may be comprised of polymers, latex, glass, polystyrene, cellulose, or other solid materials, as well as any combination thereof. Magnetically responsive particles may include magnetite or other magnetically responsive material within the particles, either as a core or dispersed within the particle, or layered throughout or over the nonmagnetic portions of the particle as a composite. The list of substances, preferably organic substances, and even more desirably biological materials, to be bound to the particles is essentially limitless. These substances may comprise, for example, antibodies, antigens, haptens, crosslinkers, proteins, single and double stranded nucleic acids, and enzymes, as well as components, fragments or derivatives of any of the foregoing, as well as other organic molecules and substances available or yet to be developed or derived for any immunological test or assay. These substances are utilized to capture and /or detect, as well as measure, various analytes in samples of various fluids to be analyzed. Many of the individual substances may be minute, or even microscopic in size. The substances to be attached to the microparticles may be unlabeled, or labeled with a chemical or biological substance which is capable of producing a signal by visual or instrumental means. Tracers for use in fluorescence polarization immunoassays, as well as in radioactive immunoassays are also contemplated by the various embodiments of the invention. The heretofore tracers and labels thus include all substances known, or to be subsequently developed by the skilled artisan. The fluids to be analyzed in an immunoassay process include biological fluids such as blood, serum, plasma, urine, saliva, perspiration and the like, as well as other fluids, such as water, particularly waste waters. Those skilled in the art should discover that a very high degree of coating consistency may be obtained with the process of the invention. While coating consistency is somewhat difficult to measure directly, considering the small size and number of microparticles involved, it can be assessed through the use of the coated microparticles in various immunoassays. Coating efficiency may also be assessed through a comparison of the same or similar number of microparticles which have been coated with the same substance(s) using a traditional batch coating process. Preferably, the process of the invention according to the embodiments heretofore described will result in a constant stoichiometric attachment of substance onto microparticle. In other words, it is a part of the invention to have a measured ratio of substance to microparticles. For example, the skilled artisan would achieve a ratio of X number of substances coated onto each microparticle. This ratio, in turn, would be substantially constant for the entire batch of coated microparticles. It is also part of the invention that the process according to the several described embodiments result in a faster coating of the microparticles herein, when compared with the known batch coating methods. The skilled technician will discover that the method of the invention should result in at least about a one-third reduction in the time necessary to coat a like quantity or volume of microparticles, and preferably up to at least about a 50% reduction in time can be expected. It is especially desirable that at least a 75% reduction in the time necessary to coat a set quantity of microparticles be achieved with the process of the invention, as compared to the traditional method of batch coating. The time for typical in-line coating, attachment or conjugation operation herein described will be about 30 minutes to 24 hours, depending upon the number of microparticles to be coated. Another advantage of the invention according to the embodiments herein set forth is that unlike with traditional batch coating processes, the methods herein may be set up as continuous operations, and are not limited by the size, shape or other parameters of the mixing equipment. With batch coating, for example, the number of microparticles that can be coated is dependent upon the number that will fit into the coating "vat". With the method of the invention, a steady supply of reagents and microparticles is all that is required, and the method can continue itself indefinitely. Once the organic substances have been attached to the microparticles, the coated microparticles in turn may be utilized in any number and manner of immunoassay applications, as well as in other applications, readily known and available to those skilled in the art. Further provided as part of the invention is an additional reagent which may be utilized as part of the process after the organic substance has become attached or bound to the microparticles. EDAC can be used to activate any carboxyl groups present on the bound organic substance, effectively allowing formation of cross-links within and between the substance. This, in turn, increases the long-term stability of the coated microparticles. An amine nucleophile, such as DL-lysine or ornithine or SUBSTTTUTE SHEET (RULE 26) others known in the art, selected because of their reactive epsilon-amino group, may then be added so as to react with any remaining EDAC-activated carboxyl groups to render them inactive. Following this, another substance, such as bovine serum albumin (BSA), gelatin, casein or other non-reactive substance known to the skilled artisan, may then be added to coat any remaining uncoated sites on the microparticles to prevent non-specific adsorption of proteins or other biological materials to the microparticle surface. As part of the invention, the process heretofore described may also be utilized to attach a first substance onto a second substance. Typically, the resulting attached substances will be utilized in immunoassays, but without limitation, applications in nucleic acid assays, cell assays, and therapeutic injectables are also to be included. The list of substances, preferably organic substances, and even more desirably chemical and biological materials, to be bound to one another is essentially limitless. These substances may comprise, for example, antibodies, antigens, haptens, crosslinkers, proteins, single and double stranded nucleic acids, toxins and enzymes, as well as components, fragments or derivatives of any of the foregoing, as well as other organic molecules and substances available or yet to be developed or derived. Many of the individual substances may be minute, or even microscopic in size. The substances to be attached to one another may be unlabeled, or labeled with a chemical or biological substance which is capable of producing a signal by visual or instrumental means. Attachment of a label to the bound first substance /second substance complex may be effected using the process of the invention. Tracers for use in fluorescence polarization immunoassays, as well as in radioactive immunoassays may also be attached according to one or more of the various embodiments of the invention. The heretofore tracers and labels thus include all substances known, or to be subsequently developed by the skilled artisan. By way of example, other adaptations of the process of the invention include enzyme conjugation, linking of two or more proteins, preparation of an immunogen, linking of a drug to a biological substance, fluoresceinating a protein, biotinylating a protein, coupling proteins, coupling nucleic acid to proteins, coupling proteins to cells, attaching fixatives to cells, sensitization of red blood cells, and the like. Labelled proteins can be adapted for use in in vivo diagnostics such as imaging. Those skilled in the art will discover that the process of attaching a substance onto one or more other substances will involve replacing the microparticles heretofore described with one of the substance types described above in any one of the detailed processes as outlined for the microparticles. Thus, in one embodiment, a quantity of a first substance is directed through a first conduit and a quantity of a second substance is directed through a second conduit. The first and second substances come together at a confluence point such that the first substance attaches to the second substance. More conduits may then be utilized to attach additional substances onto the bound first/second substance complex in accordance with the various embodiments described above for the microparticles. Reference may also be taken from the following Examples. The ratios of amounts of substances to be utilized in this manner of the invention would typically be similar to those utilized in traditional batch mixing, or could be readily ascertained by the skilled artisan. Furthermore, it is also part of the invention that the advantages associated with and described for coating of the microparticles are fully applicable to the conjugation or attachment of one substance type to another herein described. Examples The following examples serve to demonstrate various embodiments of the invention. These are provided by way of illustration only, and should not be construed as limiting the scope of the invention. Example 1 — Antibody coating of magnetic particles A device similar to that illustrated in Figure 4 was used to coat paramagnetic microparticles (Polymer Labs, model SP1147) with mouse monoclonal antibody to prostate specific antigen (PSA). The microparticles were diluted to 1% solids in a solution of 50 mM 4- morpholineethanesulfonic acid (MES) at pH 6.2, washed in a diafiltration system with a mixture of PBS/Tween at pH 7.4 using a Microgon hollow fiber cartridge, and resuspended in 50 ml of 50 mM MES. PSA antibodies were diluted to a concentration of 7.4 mg/ml in 50 ml of phosphate-buffered saline (PBS) and 6N HC1 was added to bring the pH to 2.1. EDAC was prepared in a 50 ml solution of 50 mM MES at a concentration of 0.5 mg/ml. The device 200 similar to that in Figure 4 was assembled with the antibody solution as reagent 1 in container 204, the microparticle suspension as reagent 2 in container 210, and the EDAC solution as reagent 3 in container 222. The system was operated in the following manner: 1) All pumps were operated at high speed for a time period required to flush air bubbles and prime their associated inlet and outlet conduits with reagent. 2) Pumps 1 and 2 were operated at their normal flow rate (about 10 ml/min) until the leading edge of the mixture of reagents 1 and 2 reached the second confluence point 218. 3) Pumps 1, 2, and 3 were operated at their normal flow rate (about 10 ml/min) until reagent 1 was exhausted. The reacted mixture of reagents 1, 2, and 3 was collected at the discharge conduit 240. 4) When reagent 1 was exhausted it was replaced by a buffer solution of 50 mM MES. A bubble was introduced to indicate the trailing edge of the mixture of reagents 1, 2, and 3. 5) Pumps 1, 2, and 3 were operated at their normal flow rate until the trailing edge of the mixture of reagents 1, 2, and 3 reached the third confluence point 236. 6) Pump 4 was operated at about three times the normal flow rate (or 30 ml/min) until the trailing edge of the mixture of reagents 1, 2, and 3 exited the discharge conduit 240. The microparticles coated by this procedure were confirmed to have been coated with antibodies by mixing with a fluorescein-linked anti- mouse anti-IgG antibody and measuring the mean particle fluorescence in a FACSCAN flow cytometer manufactured by Becton Dickenson. A histogram comparison of two samples prepared conventionally (via batch preparation in Figures 6C and 6D) and two samples prepared using the embodiment of the invention is presented in Figures 6A and 6B. The samples prepared using embodiments of the current invention, although of slightly lower fluorescence (of little significance, particularly in view of the logarithmic scale), possess more uniform fluorescence as indicated by a lower coefficient of variation of fluorescence readings (about 49 percent vs. about 53 percent for the conventional samples). Example 2 — Antibody coating of magnetic particles A further embodiment of the process of the present invention was used to coat magnetic particles with antibodies to the human hormone triiodothyronine (T3). A device 300 similar to that in Figure 5 was assembled. A first pump 301 and second pump 303 supplied the first and second reagent to the first confluence point 305, after which followed an in-line mixer made up of 5 in-line mixer elements 307, for a total mixing distance of about 132.5 cm. A third pump 309 and fourth pump 311 supplied a third and fourth reagent to a second and third confluence point, 313 and 315 respectively. The second and third confluence points were in close proximity, about 3 cm apart. The mixture then flowed through another in-line mixer comprised of 15 inline mixer elements 317 (about 397.5 cm) to a fourth confluence point 319. At the fourth confluence point 319 a fifth pump 321 discharged a fifth reagent, after which there was another in-line mixer with 8 mixer segments 323 (about 212 cm). A sixth pump 325 discharged a sixth reagent to a fifth confluence point 327, after which there was a 6-segment mixer 329 (about 159 cm) and a discharge conduit 331. All of the conduits between the pumps and confluence points consisted of 1.6 mm internal diameter silicon tubing. The in-line mixers were segment combinations of 26.5 cm segments of Conprotec in-line mixers. The confluence points were all polypropylene "T" junctions from Value Plastics, Inc. The pumps were all Watson-Marlow peristaltic pumps. The first reagent consisted of sheep monoclonal antibody to human T3 at a concentration of about 2.0 mg/ml and a pH of about 2.1 suspended in either PBS or MES. The second reagent consisted of Polymer Labs paramagnetic particles (SP1178) suspended in buffer at a concentration of about 0.1% solids. The third reagent was MES buffer, and the fourth reagent was an EDAC solution at about 0.1 mg/ml in a MES buffer. The fifth reagent consisted of DL-lysine-HCl, pH6.0, at about a 4 molar excess above the EDAC concentration. The sixth reagent was 1 percent bovine serum albumin (BSA) in PBS. The device illustrated in Figure 5 was operated in the following manner: 1) All pumps were operated at high speed to remove air bubbles and prime their associated conduit with their reagents. 2) The first and second pumps 301 and 303 were operated at about 10 ml/min, causing mixing of the antibodies and microparticles. 3) When the leading edge of the particle /antibody mixture reached the third confluence point 315, the fourth pump 311 was operated at about 10 ml/min. The first and second pumps 301 and 303 continued to operate at about 10 ml/min. 4) As the leading edge of the particle /antibody /EDAC mixture reached the fourth and fifth confluence points 319 and 327, the associated pumps (the fifth pump 321 and sixth pump 325, respectively) were operated at about 10 ml/min. All pumps except the third pump 309 were operating at this stage, and the coated microparticles were collected from the discharge conduit 331. 5) When the supply of antibody was exhausted, a buffer solution was substituted for the first reagent. A bubble was introduced to indicate the trailing edge of the mixture of reagents 1 and 2 (the antibodies and microparticles). 6) When the trailing edge of the particle /antibody mixture reached the second confluence point 313, the first, second and fourth pumps 301, 303, and 311 were deenergized and the third pump 309 was operated at about 30 ml/min. The fifth and sixth pumps 321 and 325 continued to operate and coated microparticles continued to be collected from the discharge conduit 331. 7) When the trailing edge of the particle /antibody/ EDAC /DL-lysine/ BSA mixture was discharged from the discharge conduit 331, all pumps were stopped. The utility of the process in coating microparticles was demonstrated by staining with fluorescently labeled goat anti-sheep IgG and measuring the fluorescence in the FACSCAN flow cytometer. Two preparations of microparticles coated by this process yielded mean fluorescence intensity levels of 480 and 418 (arbitrary fluorescence units), indicating the amount of bound IgGfor antibody preparations suspended in PBS and MES, respectively. A conventional batch coating method for identical particles and antibodies yielded a mean fluorescence intensity level of 256, indicating significantly more efficient coating in the process described here. Utilization of these particle preparations in a chemiluminescence-based immunoassay also indicates the increased efficiency of coating. Particles coated by the process described here yielded mean light levels of 325970 and 319384 relative light units (RLU), as opposed to 162484 RLU for the conventionally coated particles. Example 3 -- Demonstration of coating uniformity The usefulness of the process of the invention in maximizing the uniformity of microparticle coating was demonstrated by coating paramagnetic microparticles with mouse monoclonal antibody to human thyroid stimulating hormone (TSH). A device similar to that in Figure 5 was assembled in a manner similar to that described in Example 2. The first reagent consisted of monoclonal antibody to human TSH at a concentration of about 2.0 mg/ml and a pH of about 2.1. The second reagent consisted of Polymer Labs paramagnetic particles (SP1147) suspended in buffer at a concentration of about 0.1% solids. The third reagent was MES buffer, and the fourth reagent was an EDAC solution at about 12 mg/ml. The fifth reagent consisted of DL-lysine-HCl, pH 6.0, at about a 4 molar excess above the EDAC concentration. The sixth reagent was 1 percent bovine serum albumin (BSA) in PBS. The device illustrated in Figure 5 was operated in a manner similar to that described in Example 2. Particles coated with the above procedure were tested for antibody activity by staining with fluorescently labeled goat anti-mouse IgG and measuring the fluorescence in a flow cytometer. The results, shown in Figure 7, show the fluorescence activity for these particles compared to particles coated in a conventional batch apparatus. The particles treated by the process of the invention displayed a 5 percent higher fluorescence level, 830 versus 788 arbitrary units, indicating greater levels of coating. More importantly, the method of the present invention yielded a 24 percent smaller variation in fluorescence, a coefficient of variation of 3.37 percent versus 4.44 percent for the conventional method and a standard deviation of 28 v. 35, indicating more uniform coating. Lastly, this process of the invention accomplished the coating of 50 ml of microparticle suspension in about 12 minutes, as opposed to about 60 minutes for the conventional method of microparticle coating a like quantity of microparticles. Example 4 - Conjugation of a First Substance to a Second Substance To further substantiate the improvement the method of the invention brings to the art, an enzyme immunoassay conjugate was prepared using the in-line mixing method herein described, and compared in the Abbott AxSYM® HAVAb 2.0 immunoassay to a reference conjugate prepared by conventional batch production means. A device similar to that set forth in Figure 4 was assembled and operated, but excluding the third and fourth pumping means and second in-line mixing element. Antibody (Human anti- HAV IgG) at 10 mg/ml in PBS, 6 mM EDTA, pH 7.0, was first thiolated by incubation with a final concentration of 0.02 M dithiothreitol (DTT) for 30 minutes at room temperature, then purified by Sephadex G-25 gel filtration (Pharmacia, Uppsala, Sweden). The enzyme to be conjugated to the antibody, alkaline phosphatase, was modified by the addition of a 30 carbon atom heterobifunctional linker containing a maleimide group, capable of reacting with thiols, and an N-hydroxy-succinimide ester which reacts with amines. A 7 mg/ml solution of alkaline phosphatase in PBS, 1 mM zinc chloride, 5 mM magnesium chloride, pH 7.0 was incubated for 15 minutes at room temperature with the heterobifunctional linker in dimethyl formamide. The modified enzyme was then immediately purified by Sephadex G-25 gel filtration in the same buffer, and conjugated to the thiolated antibody within 4 hours. The first reagent consisted of 3 ml of the thiolated antibody at 0.9 mg/ml in PBS, 6 mM EDTA, pH 7.0. The second reagent consisted of 3 ml of the hetero-bifunctional linker attached to alkaline phosphatase at 1.2 mg/ml in PBS, 1 mM zinc chloride, 5 mM magnesium chloride, pH 7.0. The device was operated as in the examples above using a flow rate of 40 ml /minute. The total reaction time was 5 seconds. The enzyme-antibody conjugate prepared above was then used in the Abbott AxSYM® HAVAb 2.0 immunoassay and compared to a reference conjugate prepared by conventional means. The conjugate prepared by the above process gave comparable performance to the reference conjugate in the assay as determined by the negative control signal: 492.4 counts /second /second (c/s/s) vs. 493.2 c/s/s for the reference conjugate, the index control signal: 494.4 c/s/s vs. 508.4 c/s/s for the reference conjugate, and positive control signal: 43.2 c/s/s vs. 33.9 c/s/s for the reference conjugate. The enzyme-antibody conjugate prepared above was also analyzed by HPLC using a BioRad SEC 400 column to determine the molecular size distribution of the resultant conjugate. The conjugate prepared by the method herein had a molecular weight and distribution comparable to that of a conventionally prepared conjugate, with similar amounts of unreacted (free) protein. These data demonstrate that enzyme-antibody conjugates can be prepared by the process described here, resulting in reagents usable in immunoassays, while also reducing reaction time 360-fold, from 30 minutes for conventional conjugate preparation to 5 seconds as related above, and scale up issues associated with reacting one protein solution with another. While the invention has been described in each of its various embodiments, it is expected that certain modifications thereto may be effected by those skilled in the art without departing from the true spirit and scope of the invention as set forth in the specification and accompanying claims.

Claims: CLAIMS: What is claimed is: 1. A process for attaching at least one substance onto one or more microparticles, comprising: a) providing a first quantity of a substance to be attached, and a second quantity of microparticles; b) directing said first quantity through a first conduit and said second quantity through a second conduit, said first and second quantities being directed so as to contact each other such that said substance attaches to said microparticles. 2. The process as claimed in claim 1, further comprising pumping means for moving at least one of said quantities through said conduits. 3. The process as claimed in claim 2, wherein said pumping means is a powered pump. 4. The process as claimed in claim 3, wherein said powered pump is a peristaltic pump. 5. The process as claimed in claim 1, further comprising at least one mixing means for mixing together said quantities of substance and microparticles. 6. The process as claimed in claim 5, wherein said mixing means is an in-line mixer. 7. The process as claimed in claim 5, wherein said mixing means is at least two in-line mixers. 8. The process as claimed in claim 1, wherein said first and second conduits converge at a confluence point. 9. The process as claimed in claim 8, wherein said confluence point is a "T" junction. 10. The process as claimed in claim 1, further comprising the step of providing a third substance through a third conduit for attachment to one of said microparticles or said first substance. 11. The process as claimed in claim 10, further comprising the step of providing a fourth substance through a fourth conduit for attachment to one of said microparticles, said first substance or said third substance. 12. The process as claimed in claim 1, wherein said substance is selected from the group consisting of biological and chemical materials. 13. A process for attaching at least one substance onto one or more other substances, comprising: a) providing first and second substances; b) directing said first substance through a first conduit and said second substance through a second conduit, said first and second substances being directed so as to contact each other such that said first substance attaches to said second substance; wherein said first and second substances are selected from the group consisting of biological and chemical materials. 14. The process as claimed in claim 13, wherein said biological and chemical materials further comprise those materials utilized in immunoassays, nucleic acid assays, cell assays and therapeutic injectables.

Inventors: COHEN LARRY M, EBLE KIM S, JOHNSON PAUL A, MANEY PETER J, OSTROW DAVID H, RUBIN BRIAN L, STECKEL ERIC W, WITTMAN CRAIG A, COHEN, LARRY, M., EBLE, KIM, S., JOHNSON, PAUL, A., MANEY, PETER, J., OSTROW, DAVID, H., RUBIN, BRIAN, L., STECKEL, ERIC, W., WITTMAN, CRAIG, A.
Applicants: ABBOTT LAB, ABBOTT LABORATORIES
Application year: 1997
Main classification: G01N33
IPC codes: B01J19, G01N33