CA1304421C - Affinity separating using immobilized flocculating reagents - Google Patents
Affinity separating using immobilized flocculating reagentsInfo
- Publication number
- CA1304421C CA1304421C CA000570588A CA570588A CA1304421C CA 1304421 C CA1304421 C CA 1304421C CA 000570588 A CA000570588 A CA 000570588A CA 570588 A CA570588 A CA 570588A CA 1304421 C CA1304421 C CA 1304421C
- Authority
- CA
- Canada
- Prior art keywords
- flocculating agent
- particle
- target substance
- column
- particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N2030/524—Physical parameters structural properties
- G01N2030/528—Monolithic sorbent material
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/824—Immunological separation techniques
Abstract
TITLE
AFFINITY SEPARATING USING IMMOBILIZED FLOCCULATING REAGENTS
ABSTRACT OF THE DISCLOSURE
Disclosed are solid supports including an immobilized flocculating agent which can be used as separation and assay media in the field of affinity separations and more particularly, in the field of clinical assays. In a specific embodiment, a flocculating agent such as polyethyleneimine is immobilized within a chromatographic support. The column so formed can then be used as an affinity column for affinity separations or immunoassays.
AFFINITY SEPARATING USING IMMOBILIZED FLOCCULATING REAGENTS
ABSTRACT OF THE DISCLOSURE
Disclosed are solid supports including an immobilized flocculating agent which can be used as separation and assay media in the field of affinity separations and more particularly, in the field of clinical assays. In a specific embodiment, a flocculating agent such as polyethyleneimine is immobilized within a chromatographic support. The column so formed can then be used as an affinity column for affinity separations or immunoassays.
Description
13~ 2~
GEF:tlc TITLE
AFFINrTY SEPARATING USING IMMOBILIZED FLOCCULATING REAGENTS
BACKGROUND OF THE INVENTION
An affinity separation can be defined as any separation achieved by employing the specific binding of one molecule by another. ~ioaffinity separations are affinity separations in which at least one of the components involved in the affinity reaction is biologically active or is of biological interest. Bioaffinity separations generally involve at least one biomacromolecule such as a protein or nucleic ac;d, as one of the components of the binding pair. Examples of such binding pairs include; antigen-ant;body, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding protein-vitamin, binding protein-nucleic acid and others. We will use the terms ligand and binder to represent the two components in of a specific binding pair. The use of these terms is for simplicity and is not intended to limit the scope of the possible applications.
The most common means used to effect the separation of the bound material from that remaining unbound is to attach either the ligand or binder to a solid support, then once binding has occurred, to remove the solid from the liquid environment. This attachment to a solid support can be either covalent or noncovalent and can be either directly to the support or incorporate a linker between the support and ligand or binder.
Affinity separations are commonly used as part of other processes. One example is the area of heterogeneous immunoassays.
Here a bioaffinity separation is used to capture an analyte from a complex mixture, frequently, serum or plasma. After capturing the analyte, the contaminants are washed away and the analyte detected using any number of well-known assay protocols. Some solid supports used in heterogeneous immunoassays are 1/4 inch plastic spheres, the inside of test tubes, the inside of microtitre plate wells, latex particles and magnetic particles.
It is frequently desirable to use small particles in heterogeneous immunoassay applications, these can be either magnetic ~ ~ , , ~3~ 2i particles or nonmagnetic. These particles generally have the advantage of being able to accept a large amount of the ligand or binder attached to the surface. Also, having a relatively high surface area, high binding capacities and rapid capture kinetics are achievable. Rapid capture kinetics are further facilitated if the particles remain suspended in the solution minimizing the diffusion distance required for a target molecule to travel before it encounters the ligand or binder attached to the solid phase.
Separation of such small particles from the liquid solution is often tedious and labor intensive. Centrifugation is one means to accomplish this separation, but is time consuming and labor intensive. Another approach poss;ble when magnetic particles are useJ is to separate them from the liquid under the influence of a magnetic field. This approach requires special particles and a strong magnetic field, and is not easily automated. There remains a need for an improved means of separating small particles of all types from a liquid suspension. This means should be simple, rapid, efficient and easily automated.
Flocculating agents are substances which facilitate and accelerate aggregation of particulates from a suspension and may ultimately lead to precipitation of those aggregates. The largest use of flocculating agents is in the area of water purification to remove both organic and inorganic particulate matter. Polyethylene-imine (PEI) is one example of a flocculating agent. Traditional flocculating agents are potassium sulfate, ferric chloride and ferric sulfate. Currently, polymeric flocculating agents are preferred in water purification applications. A variety of such agents are known in the art. Also, the technique of flocculating negatively charged filter aid particulates or adsorbents and fibers, by means of positively charged polymers is a common practice in the production of filtration media (Hou, U.S. 4,578,150, issued March 25, 1986).
Chromatographic supports with PEI attached have been reported previausly by Alpert et al. lJournal of Chromatography, Volume 185, 375-392 (1979)], Vanecek et al. [Analytical Biochemistry, Volume 121, 156-159 (1982)], and Flasher (EP 0,162,462 published November 27, 1985). These were originally reported for use as HPLC
ion exchange packings and are particularly useful in the purification ~L3~2~
of IgM class monoclonal antibodies.
SUMMARY OF THE INVENTION
This invention relates to the field of affinity separations and more particularly to the field of clinical assays.
- Disclosed are solid supports including an immobilized flocculating agent which can be used as separation and assay media.
Examples are membranes or separation columns with a flocculating agent immobili~ed thereon. As an example, with a conventional chromatographic column, small particles would generally either pass ~ through the column or pack at the top of the column. By I incorporating an immobilized flocculating agent within the chromatographic support, a more eYen dlstribut;on of small particles in the column will result. A particle reagent can be prepared by affixing a ligand or binder to a particle. The particle is then trapped in the flocculating agent column which can be used as a ; standard affinity column, namely, mixtures can be passed through the co1umn whereby a target substance will bind to the ligand or binder.
After the undesired substances are eluted, the target substance can be released by an appropriate change in the eluting buffer. The column can also be used in immunoassays by employing reagent particles which can be easily separated, washed and assayed, by passing an incubated mixture through a column containing a chromato-graphic support immobilized with a flocculating agent whereby the particles will be trapped in the column. The deYeloped substrate is then eluted and measured. The process simplifies enzyme immunoassays that utilize particles as a solid support and facilitates automat;on of the assay.
DETAILED DESCRIPTION OF THE INVENTION
The method of this invention offers great advantages in carrying out affinity separations. Two of the greatest advantages are: the general applicability of the method to a wide variety of particles and processes, and the ease of automation, Other advantages include the opportunity to conserve precious reagents and rapid separations achievable.
Any flocculating agent can be used in the method of this ~ invention, but PEI is preferred. PEI can be attached to suitable j supports in a variety of different ways. PEI can be attached to~i :
:' '~ ''''' `' ` ''~' ' ' ' ' ~3(~
Sephade~ type resins by a periodate oxidation method. In this method, the Sephadex is washed and then oxidized with periodate to generate aldehyde functional groups. The PEI is then added and Schiff bases are allowed to form between the aldehyde groups and the amine groups of the PEI. These Schiff bases are then reduced using sodium cyanoborohydride. The derivatized resin is ready for use after being washed and equilibrated with a suitable buffer.
Another means of attaching PEI to a support is simple adsorbtion. When a porous plastic rod, such as those ava1lable from Porex Technologies (Fairburn, GA) are used, this can be accomplished by soak1ng said rods in a 3% solution of PEI in 30~ isopropanol overnight. The rods are then washed with water and an appropriate buffer and are then ready for use. When polyacrylamide resins, such as those available from Bio-Rad under the trade name Biogel, are used, this can be accomplished by soaking the resin in a 3% solution of PEI in 0.1 M sodium chloride. These are only representative procedures and many alternatives will be recognized by anyone skilled in the art. The PEI derivatized supports of Vanecek et al. and Flasher are also expected to function in this invention~
In addition to the Sephadex, Biogel and porous plastic type supports discussed above any other type of support is also expeoted to function. The support itself should be non-reactive and exhibit low nonspecific b~nding of the target substance and ligand or binder used. The porosity of the support can be adjusted to opt;mize the flow rate obtained with a specific particle. It is frequently preferred to use column packing material with minimal retention of small molecules such as enzyme substrates. 8y using such supports, an enzyme bound to a particle can be assayed while it is trapped on the column. The substrate and product used in said assay can then be read~ly eluted through the column and detected. If these substances were significantly retarded, this would delay complet~on of the assay. Support such as Biogel P-2 is preferred in thls respect.
Buffers suitable for use in this invention are generally those compatible with the bSological reagent and samples. Phosphate buffers are generally preferred.
As stated above, one of the great advantages of the method of this invention is the ability to use a wide varlety of particles in the method. The preferred particles will, ln general~ be small * trade mark ~304~42~
and have low nonspecific binding of the target molecule and ligand or binder used. While small particles are preferred, larger particles can be used; however, the particles should not be so large as to occlude the column. If larger particles are desired it is possible to adjust the porosity of the column packing material to achieve an acceptable flow rate. Latex particles of the type disclosed by Craig et al. (U.S. 4,401,765 issued August 30, 1983) can be used. Magnetic particles such as those available from Advance Magnetics under the trade name BioMag are also suitable. The preferred part;cle is, however, the stab;llzed chrom;um dioxide part;cle described in U.S.
Patent 4,661,408 issued April 28, 1987 incorporated herein by reference. These particles consist of a core of rutile chromlum dioxide which has been extensively surface reduced, coated with alumina, further coated with silica containing borate and still further coated with a silane to which is attached the desired ligand or binder. These particles have large surface areas 40-100 m2/g, are stable in aqueous solution and easily coupled to the desired ligand or binder.
By ligand is meant an antigen, hapten, nucleic acid, enzyme substrate, vitamin, dye or other small molecule including enzyme effectors and inhibitors; and by binder is meant an antibody, enzyme, nucleic acid, binding protein, synthetic mimics of binding proteins such as polylysine or other molecules capable of specific binding interactions. Ligands or binders can be readily attached to particles by a variety of known methods. Covalent attachment is generally preferred to avoid loss of capacity and degradation of performance over time.
Many assay formats are known in the art of heterogeneous immunoassays. This method is applicable to achieving the required separation in any of the known formats. The following discussion is presented to provide a general outline of how this can be accomplished. The skilled artisan will quickly recognize other means to employ the method of this invention.
The method of this invention can be applied to separation of bound and free label in a competitive immunoassay. The bound label is captured on a small particle and that particle is trapped on a flocculating reagent column. The free label is eluted through the column and is available for detection. If the label is an enzyme~
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1~4~L%~L
then the bound fraction may also be detected by eluting a substrate onto the column, halting the elution to allow the substrate to react with the enzyme then resuming the elution to elute the product which can then be quantitated.
The method of this invention can be applied to separation in a sandwich immunoassay. In this format, a captured antibody is attached to the small particle. This particle is reacted with a sample containing the target substance and a labelled antibody.
After allowing suitable time for reaction, the mixture is eluted through a flocculating reagent column trapping the particles and any labelled antibody bound to the target substance which in turn is bound to the captured antibody. The unbound label may be quantitated, but it is frequently advantageous to assay the bound label. In the case of an enzyme label, this is done essentially as described above for detecting the bound enzyme in the competitive immunoassay.
The method of this invention can also be applied to conducting a bioaffinity separation. A bioaffinity column can be prepared by attaching a ligand or binder to a small particle. This particle is then trapped on a flocculating reagent column. This column can then be used as a standard b;oaffinity column. That is, mixtures can be passed over it and the target substance can bind to the ligand or binder. Once the contaminating substances are eluted, the target molecule can then be released by an appropriate change in eluting buffer. One example of how this might be used is with protein a coated particles. Protein a binds most subclasses of IgG
immunoglobulins. A flocculating reagent column with protein a particles trapped on it would be useful for removing the IgG
immunoglobulins from serum. The IgG can be recovered by suitable changes in the eluting buffer.
The method of this invention can also be applied to affinity column mediated immunoassays (ACMIA). A representative ACMIA assay is described by Freytag et al. [Clin. Chem., Volume 30, 417-420 (1984)] for the aca~ discrete clinical analyzer (E. I. du Po~t de Nemours & Co., Inc., Wilmington, DE). An affinity column is prepared as described above, and then used as described by Freytag et al. It has been found that for a digoxin assay as disclosed by Freytag et al. the amount of ouabain-BSA required is reduced by over ,~,. . ~
.
~3~
300 fold using the method of this invention.
It is also recognized that many chemical precipitates form as small particles. These particles can also be removed by the method of this invention. Such precipitates can be formed as the result of immunoprecipitation, that is, the formation of large aggregates of antigens and antibodies, or the result of chemical precipitation. One example of chemical precipitation is the use of buffered phosphotungst;c reagent to precipitate low density and very low density lipoproteins to allow quantitat;on of high density lipoproteins. One example of immunoprecipitation is the direct reaction of anti-immunoglobulin antibodies with immunoglobulins as used in the nepholmetric or turbidimetric quantitation of immunoglobulins.
Surprisingly, it has been found that columns containing immobilized flocculating reagents prov;de a very simple, efficient and effective means to remove small particles from a liquid suspension. This property makes the use of these columns in bioaffinity separations, and particularly immunoassays, very advantageous.
The following examples further illustrate the invention.
' ' .
' .
4~2~
Competitive D;goxin Assay Using Covalently Attached PEI
(A) Preparation of Column 16.6 g of Sephadex G-10 resin (Pharmacia Biotechnolo~y Products) was heated in boiling water for ten minutes and washed twice with 100 mL of distilled water. To the swelled resin was added 0.66 9 of sodium periodate in 33 mL of distilled water. The mixture was mixed at room temperature in the dark for two hours, washed twice with water and twice with 0.15 M of sodium phosphate (pH 7.8) and made up to a total of 90 mL with said phosphate buffer. The mixture was then swirled to evenly suspend the resin and 60 mL of the suspension was mixed with 2 9 of 30% PEI and 10 mg of sodium cyanoborohydride. The pH of the mixture was adjusted to 5.7 with 6 N
hydrochloric acid. The mixture was mixed at room temperature overnight then washed 3 X 100 mL with phosphate buffer. The resin was packed into columns designed for use in the aca3 discrete clinical analyzer (E. I. du Pont de Nemours & Co., Inc., Wilmington, DE). The column is a plastic tube approximately 5.5 mm in diameter and 88 mm long with rubber stoppers at both ends which allow For automatic sample and diluent entry and eluent exit into an analytical test pack. Such an analytical test pack was described in RE 29725, issued to D. R. Johnson, et al. The columns were substituted for the columns in commercial digoxin packs for the aca~ analyzer.
(B) Preparation of Ouabain-BSA-CrO2 Particles 25i. Reductive Surface Treatment of CrO2 Two hundred and fifty grams of upgraded heated chromium dioxide were mixed with 100 9 of sodium bisulfate in 1750 mL of water. The mixture was milled in a W-250V-B Vertical Belt-Drive Colloid Mill (~reerco Corporatlon, Hudson, N.H~) for 45 minutes and aged in a glass container for one week. The particles were dialyzed against distilled water to remove the excess sodium bisulfite.
; ii. Silica Coating One hundred grams of chromium dioxide particles from above were placed in a 3 liter beaker and 2.5 liters of distilled water were added. The particles were heated to 90C ~ 2C with mechanical stirring. To the mixture was added 5.0 mL of sodium aluminate (40%
solution) and the pH of the suspension was adjusted to 9 by the addition of 5% sulfuric acid. To this mixture was added 150 mL of :~ -~ ' ~3C~
g water contain;ng 25 9 of sodium metasilicate and 6.25 9 of sod~um borate dropwise over a period of one hour. The pH of the mixture was maintained at 9 + 0.5 with the simultaneous dropwise addition of 5%
sulfurlc acid. Vigorous stirrlng was maintalned throughout the reaction. After all the reagents were added~ the mixture was heated at 90C and stirred for an additional 30 minutes before the pH was adjusted to 7 with 5X sulfuric acid and allowed to cool to roo~
temperature. The particles were dialyzed against distilled water.
- iii. Silane Coating One hundred grams of silica coated chrom;um dioxide particles were suspended in 1.8 liters of d-stilled water in a 2 liter round-bottom flask equipped w~th a mechanical stirrer, a reflux condensor and a temperature sensor. Two hundred mL of aminopropyltriethoxysilane was added and the mixture was stlrred at 55C for 18 hours. The particles were washed three times with 13 liters of distilled water by settllng and decantatlon. The washed particles were suspended ln 10 mM phosphates buffer ~pH 7) at 50 mg/mL.
iv. Preparation of Ouabain-BSA
Five grams of ouabain-octohydrate were dissolved ln 500 mL
of hot distilled water and allowed to cool to room temperature.
7.3 9 of sodium metaperiodate (NaI04) were added to the ouabain solution and stirred for two hours in the dark. The solution was passed through a bed of Dowex*(I-X8) anion exchange resin (prepared by washing 250 9 of the Dowex resin with water until the yellow color d~sappears~. The oxidized ouabain solution was mixed with ~00 mL of 1 M sodium phosphate (pH 7.0) containing 10 9 of BSA. The mixture was stirred for one hour and 0.64 9 of sodium cyanoborohydride (NaCNBH3) added. The mixture was st~rred at room temperature for 48-72 hvurs. The ouabain-~SA conjugate was dia1yzed against running water for 12-24 hours and against 20 volumes of 0.015 M sodium phosphate buffer (pH 7.0) at 4C for 16 hours. The conjugate was stored at 4C.
v. Protein Coupling to CrO2 Particles Ten mL of a 50 mg/mL suspenslon of the silane coated CrO2 part~cles were washed three tlmes with 50 mL o~ 10 mM ph~sphate buffer. To the washed particles 20 mL of 5X glutaraldehyde were added and mixed for three hours at room temperature. The activated * trade mark -. ~ . ., ~304~Z~
particles were washed five times each with 50 mL of phosphate buffer and suspended in 10 mL of the same buffer. A solution of 40 mg of ouabain-bsa conjugate in 10 mL of buffer was added. The mixture was agitated at room temperature for 20 hours. The particles were washed once with 10 mM phosphate buffer and the unreacted aldehyde groups were quenched by the addition of 50 mL of 1 m glycine (pH 8) and mixed for one hour. The protein particles were washed ten times with 50 mL each, of 10 mM phosphate buffer + 0.1% BSA + 0.1% thimerosal, and stored in 50 mL of the same buffer at 4C.
~C) Assay Procedures To 200 ~L of 0.15 M sodium phosphate buffer (pH 7.8) was added 200 ~L of Digoxin antibody-enzyme conjugate reagent (E. I. du Pont de Nemours & Co.~ Inc., Wilmington, DE) and 200 ~L of human serum containing digoxin. The Digoxin antibody enzyme conjugate reagent is a covalent conjugate of F(ab')2 antibody fragment from rabbit anti-digoxin antisera and ~-galactosidase. The mixture was incubated at room temperature for ten minutes and 200 ~L of a 10 mg/mL suspension of ouabain-BSA-CrO2 particles were added.
~ncubation was continued for five more minutes at room temperature.
The mixture was transferred to a sample cup for the aca~ analyzer and 400 ~L of it was pipetted by the instrument and injected into the digoxin pack containing a column prepared as above. The particles were trapped by the column and the enzyme activity of the filtrate was measured by the analyzer automatically using-~-D-galactopyrano-side as substrate. A series of human serum samples containingdigoxin at concentrations from 0 to 5 ng/mL were tested as described.
The 0 ng/mL background (B.G.) and the response difference between 0 and 5 ng/mL (Delta 0,~) were determined and were 18 mA/min and 64 ma/min, respectively.
130~2~
Bound Phase Digoxin Assay Using PEI
Absorbed on Porous Plastic (A) Preparation of Column Fifteen ~m pore size porous plastic rods approximately 5.3 mm in diameter and 80 mm long were obtained from Porex Technologies (Fairburn, GA~. About 20 rods were soaked in a 3X PEI
solution in 30% isopropanol with gently mixing overnight. The columns were rinsed with distilled water and inserted into empty columns described in 1.A. for use on the aca~ analyzer. The column was then washed with 5 mL of 0.15 M sodium phosphate, pH 7.8.
(B) Assay Procedures To a test tube was added 50 ~L of digoxin antibody-enzyme conjugate reagent, 50 ~L of human serum containing digoxin and 50 aL
of 1.15 M sod;um phosphate (pH 7.8) and vortexed. The mixture was incubated at 37C for ten minutes and 50 ~L of a 10 mg/mL suspension of ouabaln-BSA-CrO2 particles prepared in 1.B. was added and mixed well. The mixture was incubated for another two minutes and a 150 ~L
aliquot was injected into a column prepared above and eluted with 2 mL of 0.15 M phosphate. One mL of 0.1 M o nltrophenyl-~-D-galactopyranoside was injected into the column and incubated at 37C
for ten minutes. The column was then eluted with 2 mL of 0.15 M
phosphate and the absorbance at 406 nm was recorded.
(C) Resul~s The B.G. was 307 mA/min and the Delta 0,5 was 200 mA/min.
This procedure was repeated using 50 ~M pore size porous plastic rods. The B.G. was 268 mA/min and the Delta O,S was 162 mA/min.
' 13~4~2~
TSH Sandwich Assay Using_PEI Adsorbed on Bio~el P-2 (A) Preparation of Column A sample of 40 9 of Biogel P-2 (100-200 mesh) and 10 g of PEI were mixed in 300 mL of 0.1 M sodium chloride at room temperature overnight. The resin was washed three times with 300 mL each of 0.1 m sodium chloride and packed into a column for use on the aca~
analyzer as described ;n 1.A. The column was equllibrated with 80 mm NaCL, 0.1% Tween-2C~ pH 6Ø
(B) AntI-TSH-CrO2 Particle Preparatlon .
i. Reductive Surface Treatment of CrO2 Two hundred and fifty grams of upgraded CrO? were m~xed with 100 g of sodium bisulfite in 1750 mL of water. The mixture was milled in a W-250V-B vertical Belt-Drive Colloid Mill (Greerce Corporation, Hudson, NH) for 45 minutes. The particles were washed with water and spray dried. Twenty grams of spray dried CrO2 particles were washed twice w~th 200 mL of distilled water by decantation. The particles were d1spersed ln 200 mL of distilled water containing 20 g of sodium bisulfite and 50 9 of 1/8" glass beads in a 200 mL tissue culture flask. The mixture was rotated at 5 rpm for 48 hours at room temperature. The part~cles were separated from the glass beads and washed three times with 200 mL of 10 mM
sodium phosphate buffer tpH 7) using magnetic separation.
ii. Protein Coupling .
Ten mL of a 5% slurry of silanized CrO2 were washed three times with 50 ml each of 10 mM phosphate~ pH 7.4. After the th~rd was~h, the particles are magnetically separated, the supernatant was aspirated, and to this wet cake 20 mL of 5% glutaraldehyde was added and r~cked for three hours at room temperature. The glutaraldehyde activated particles were washed ten times with 50 mL each of coupling buffer (10 mm potassium phosphate, pH 7.4). After the last wash, the t particles were resuspended in 10 mL buffer To this was added 6 mg of purified ~ subunit specific antibody in 10 mL of coupling buffer and the mixture rocked for 20 hours at 4C. The antibody coupled particles were washed once w~th coupling buffer, then the unreacted aldehyde groups were quenched by reaction with 50 mL of 1 M glycine, pH 8.0 for ten minutes. The partlcle reagent was washed extensively, ten tlmes with 50 mL each of the wash buffer (coupling buffer also * trade mark -~ t "
31~ 4 containing 0.1% BSA), to remove all noncovalently bound antibodies.
The final reagent was resuspended ~nto 10 mL of the wash buffer containing 0.1% sodium azide as preservative, and stored at 4C~
(C) Assay Procedures To a test tube was added 150 ~L of human serum containing a known amount of TSH and 25 ~L of the antibody conjugate reagent from a Hybritech Tandem~-e TSH lmmunoenzymetric assay kit (Hybritech, Inc., San Diego, CA) and vortexed. The mixture was incubated at 37C
for ten minutes and 25 ~L of ant;-TSH-CR02 partlcles (25 mG/MI) was added vortexed. Incubation was cont;nued for another ten minutes and 150 ~L of the mixture was injected into a column prepared above. The column was washed with 2 mL of 80 mM sodium chloride and 0.1~ Tween-20, pH 6.0 buffer. One mL of 3 mM p-nitrophenylphosphate in 1 M
diethanolamine, 0.5 mM MgC12, pH 8.9 was injected into the column.
The column was incubated at 37C for 30 minutes and eluted with 2 mL
of 50 mM ethylenediamine tetraacetlc acid (EDTA) pH 8.~. The absorbance of the eluent at 406 nm was measured. The O aIulmL
background (B.G.) and the response difference between O aIu/mL and 50 ~IU/mL (Delta 0,50) were determined from those results and were 126 mA/min and 312 mA/min, respectively.
A
:
, ~ ~
13 [)~Z~L
ACMIA Digoxin Assay Using Ouabain-BSA-CrO2 Particles Trapped on PEI-Biogel P-2 as Affinity Column (A~ Preparation of Column PEI-Biogel P-2 columns were prepared as in Example 3.A. To the column was injected 50 to 200 ~L of 10 mg/mL suspensions ouabain-BSA-CrO2 particles. The columns were washed with 3 mL of 0.15 M
sodium phosphate buffer (pH 7.8) and inserted into the commercial digoxin pack for the aca~ analyzer (E. I. du Pont de Nemours and Co., Inc.).
(B)Assay Procedures Five hundred ~L of human serum containing digoxin and 500 aL of antibody-enzyme conjugate (enough for two tests) were mixed in a sample cup and was incubated at room temperature for ten minutes. Four hundred ~L of the mixture was pipetted by the aca~
analyzer and processed as the commercial method.
The test results using different amounts of particles trapped on the column are compared to the commercial method which uses ouabain-BSA-Sephadex G-10 in Table 1:
Digoxin Assay Using Trapped CrO2 particles on ACA
Particles BackgroundSeparation 25(mg) (mA/min)Delta, 0,5 (mA/min) 0.5 88 68 1.0 60 91 2.0 48 84 30Commercial Pack72 79 ~ .
~3l~4~l Affinity Purification of Anti-Digoxin Antibody PEI-Biogel P-2 resin was prepared as in Example 3.A. and packed in a 1.5 X 15 cm column. The column was washed with 100 ml of 10 mM sodium phosphate (pH 7.0) containing 0.9% of sodium chloride (PBS). Three mL of a 50 mg/ml suspensions ouabain-BSA-CrO2 particles prepared in Example 1.B. were loaded on the column. The top of the column was intentionally disturbed to mix the top half of the resin with the CrO2 particles and allowed to pack again. The packed column was washed with 50 mL of PBS, no leakage of the CrO2 particles was observed. Ten mL of rabbit anti-digoxin serum was loaded on the column and the serum was allowed to incubate for 15 minutes. The unbound fraction was eluted with 100 mL of PBS. The bound anti-digoxin was eluted with 2.5 M ammonlum thiocyanate (pH 7.8).
Fractions containing the protein were identified by monitoring the 280 nm absorbance and were pooled and dialyzed against PBS.
Immunodiffusion analysis indicated that all the anti-digoxin was in the bound fractions, no anti-digoxln was observed in the unbound fractions.
~30~L~21 Automated Assay for High_Density Lipoprotein (HDL) Cholesterol The following procedures can be used to perform an assay for high density lipoprotein (HDL) cholesterol which automated the removal of the low density and very low density lipoprotein which are precipitated when the sample is pretreated with phosphotungstic acid.
(A) Preparation of Column PEI-Biogel P-2 columns for the aca~ analyzer are prepared as described in Example 3.A. These columns are then placed in the header af an analytical test pack (as described in Example 1.A.) which contains the reagents necessary to assay for HOL cholesterol.
(B) Assay Procedure Five hundred aL of serum and 100 ~L of a 20 g/L solution of phosphotungstlc acid are mixed in an aca~ analyzer sample cup and allowed to stand for five minutes. Two hundred and fifty ~L of this pretreated sample are then injected into the column described lfrom]
6.A. The column is eluted with 4.75 mL of 10 mM sodium phosphate buffer (pH 7.0). The filtrate is collected in the analytical test pack which can be processed on an aca~ analyzer to determine the quantity of HDL cholesterol present.
GEF:tlc TITLE
AFFINrTY SEPARATING USING IMMOBILIZED FLOCCULATING REAGENTS
BACKGROUND OF THE INVENTION
An affinity separation can be defined as any separation achieved by employing the specific binding of one molecule by another. ~ioaffinity separations are affinity separations in which at least one of the components involved in the affinity reaction is biologically active or is of biological interest. Bioaffinity separations generally involve at least one biomacromolecule such as a protein or nucleic ac;d, as one of the components of the binding pair. Examples of such binding pairs include; antigen-ant;body, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding protein-vitamin, binding protein-nucleic acid and others. We will use the terms ligand and binder to represent the two components in of a specific binding pair. The use of these terms is for simplicity and is not intended to limit the scope of the possible applications.
The most common means used to effect the separation of the bound material from that remaining unbound is to attach either the ligand or binder to a solid support, then once binding has occurred, to remove the solid from the liquid environment. This attachment to a solid support can be either covalent or noncovalent and can be either directly to the support or incorporate a linker between the support and ligand or binder.
Affinity separations are commonly used as part of other processes. One example is the area of heterogeneous immunoassays.
Here a bioaffinity separation is used to capture an analyte from a complex mixture, frequently, serum or plasma. After capturing the analyte, the contaminants are washed away and the analyte detected using any number of well-known assay protocols. Some solid supports used in heterogeneous immunoassays are 1/4 inch plastic spheres, the inside of test tubes, the inside of microtitre plate wells, latex particles and magnetic particles.
It is frequently desirable to use small particles in heterogeneous immunoassay applications, these can be either magnetic ~ ~ , , ~3~ 2i particles or nonmagnetic. These particles generally have the advantage of being able to accept a large amount of the ligand or binder attached to the surface. Also, having a relatively high surface area, high binding capacities and rapid capture kinetics are achievable. Rapid capture kinetics are further facilitated if the particles remain suspended in the solution minimizing the diffusion distance required for a target molecule to travel before it encounters the ligand or binder attached to the solid phase.
Separation of such small particles from the liquid solution is often tedious and labor intensive. Centrifugation is one means to accomplish this separation, but is time consuming and labor intensive. Another approach poss;ble when magnetic particles are useJ is to separate them from the liquid under the influence of a magnetic field. This approach requires special particles and a strong magnetic field, and is not easily automated. There remains a need for an improved means of separating small particles of all types from a liquid suspension. This means should be simple, rapid, efficient and easily automated.
Flocculating agents are substances which facilitate and accelerate aggregation of particulates from a suspension and may ultimately lead to precipitation of those aggregates. The largest use of flocculating agents is in the area of water purification to remove both organic and inorganic particulate matter. Polyethylene-imine (PEI) is one example of a flocculating agent. Traditional flocculating agents are potassium sulfate, ferric chloride and ferric sulfate. Currently, polymeric flocculating agents are preferred in water purification applications. A variety of such agents are known in the art. Also, the technique of flocculating negatively charged filter aid particulates or adsorbents and fibers, by means of positively charged polymers is a common practice in the production of filtration media (Hou, U.S. 4,578,150, issued March 25, 1986).
Chromatographic supports with PEI attached have been reported previausly by Alpert et al. lJournal of Chromatography, Volume 185, 375-392 (1979)], Vanecek et al. [Analytical Biochemistry, Volume 121, 156-159 (1982)], and Flasher (EP 0,162,462 published November 27, 1985). These were originally reported for use as HPLC
ion exchange packings and are particularly useful in the purification ~L3~2~
of IgM class monoclonal antibodies.
SUMMARY OF THE INVENTION
This invention relates to the field of affinity separations and more particularly to the field of clinical assays.
- Disclosed are solid supports including an immobilized flocculating agent which can be used as separation and assay media.
Examples are membranes or separation columns with a flocculating agent immobili~ed thereon. As an example, with a conventional chromatographic column, small particles would generally either pass ~ through the column or pack at the top of the column. By I incorporating an immobilized flocculating agent within the chromatographic support, a more eYen dlstribut;on of small particles in the column will result. A particle reagent can be prepared by affixing a ligand or binder to a particle. The particle is then trapped in the flocculating agent column which can be used as a ; standard affinity column, namely, mixtures can be passed through the co1umn whereby a target substance will bind to the ligand or binder.
After the undesired substances are eluted, the target substance can be released by an appropriate change in the eluting buffer. The column can also be used in immunoassays by employing reagent particles which can be easily separated, washed and assayed, by passing an incubated mixture through a column containing a chromato-graphic support immobilized with a flocculating agent whereby the particles will be trapped in the column. The deYeloped substrate is then eluted and measured. The process simplifies enzyme immunoassays that utilize particles as a solid support and facilitates automat;on of the assay.
DETAILED DESCRIPTION OF THE INVENTION
The method of this invention offers great advantages in carrying out affinity separations. Two of the greatest advantages are: the general applicability of the method to a wide variety of particles and processes, and the ease of automation, Other advantages include the opportunity to conserve precious reagents and rapid separations achievable.
Any flocculating agent can be used in the method of this ~ invention, but PEI is preferred. PEI can be attached to suitable j supports in a variety of different ways. PEI can be attached to~i :
:' '~ ''''' `' ` ''~' ' ' ' ' ~3(~
Sephade~ type resins by a periodate oxidation method. In this method, the Sephadex is washed and then oxidized with periodate to generate aldehyde functional groups. The PEI is then added and Schiff bases are allowed to form between the aldehyde groups and the amine groups of the PEI. These Schiff bases are then reduced using sodium cyanoborohydride. The derivatized resin is ready for use after being washed and equilibrated with a suitable buffer.
Another means of attaching PEI to a support is simple adsorbtion. When a porous plastic rod, such as those ava1lable from Porex Technologies (Fairburn, GA) are used, this can be accomplished by soak1ng said rods in a 3% solution of PEI in 30~ isopropanol overnight. The rods are then washed with water and an appropriate buffer and are then ready for use. When polyacrylamide resins, such as those available from Bio-Rad under the trade name Biogel, are used, this can be accomplished by soaking the resin in a 3% solution of PEI in 0.1 M sodium chloride. These are only representative procedures and many alternatives will be recognized by anyone skilled in the art. The PEI derivatized supports of Vanecek et al. and Flasher are also expected to function in this invention~
In addition to the Sephadex, Biogel and porous plastic type supports discussed above any other type of support is also expeoted to function. The support itself should be non-reactive and exhibit low nonspecific b~nding of the target substance and ligand or binder used. The porosity of the support can be adjusted to opt;mize the flow rate obtained with a specific particle. It is frequently preferred to use column packing material with minimal retention of small molecules such as enzyme substrates. 8y using such supports, an enzyme bound to a particle can be assayed while it is trapped on the column. The substrate and product used in said assay can then be read~ly eluted through the column and detected. If these substances were significantly retarded, this would delay complet~on of the assay. Support such as Biogel P-2 is preferred in thls respect.
Buffers suitable for use in this invention are generally those compatible with the bSological reagent and samples. Phosphate buffers are generally preferred.
As stated above, one of the great advantages of the method of this invention is the ability to use a wide varlety of particles in the method. The preferred particles will, ln general~ be small * trade mark ~304~42~
and have low nonspecific binding of the target molecule and ligand or binder used. While small particles are preferred, larger particles can be used; however, the particles should not be so large as to occlude the column. If larger particles are desired it is possible to adjust the porosity of the column packing material to achieve an acceptable flow rate. Latex particles of the type disclosed by Craig et al. (U.S. 4,401,765 issued August 30, 1983) can be used. Magnetic particles such as those available from Advance Magnetics under the trade name BioMag are also suitable. The preferred part;cle is, however, the stab;llzed chrom;um dioxide part;cle described in U.S.
Patent 4,661,408 issued April 28, 1987 incorporated herein by reference. These particles consist of a core of rutile chromlum dioxide which has been extensively surface reduced, coated with alumina, further coated with silica containing borate and still further coated with a silane to which is attached the desired ligand or binder. These particles have large surface areas 40-100 m2/g, are stable in aqueous solution and easily coupled to the desired ligand or binder.
By ligand is meant an antigen, hapten, nucleic acid, enzyme substrate, vitamin, dye or other small molecule including enzyme effectors and inhibitors; and by binder is meant an antibody, enzyme, nucleic acid, binding protein, synthetic mimics of binding proteins such as polylysine or other molecules capable of specific binding interactions. Ligands or binders can be readily attached to particles by a variety of known methods. Covalent attachment is generally preferred to avoid loss of capacity and degradation of performance over time.
Many assay formats are known in the art of heterogeneous immunoassays. This method is applicable to achieving the required separation in any of the known formats. The following discussion is presented to provide a general outline of how this can be accomplished. The skilled artisan will quickly recognize other means to employ the method of this invention.
The method of this invention can be applied to separation of bound and free label in a competitive immunoassay. The bound label is captured on a small particle and that particle is trapped on a flocculating reagent column. The free label is eluted through the column and is available for detection. If the label is an enzyme~
......
1~4~L%~L
then the bound fraction may also be detected by eluting a substrate onto the column, halting the elution to allow the substrate to react with the enzyme then resuming the elution to elute the product which can then be quantitated.
The method of this invention can be applied to separation in a sandwich immunoassay. In this format, a captured antibody is attached to the small particle. This particle is reacted with a sample containing the target substance and a labelled antibody.
After allowing suitable time for reaction, the mixture is eluted through a flocculating reagent column trapping the particles and any labelled antibody bound to the target substance which in turn is bound to the captured antibody. The unbound label may be quantitated, but it is frequently advantageous to assay the bound label. In the case of an enzyme label, this is done essentially as described above for detecting the bound enzyme in the competitive immunoassay.
The method of this invention can also be applied to conducting a bioaffinity separation. A bioaffinity column can be prepared by attaching a ligand or binder to a small particle. This particle is then trapped on a flocculating reagent column. This column can then be used as a standard b;oaffinity column. That is, mixtures can be passed over it and the target substance can bind to the ligand or binder. Once the contaminating substances are eluted, the target molecule can then be released by an appropriate change in eluting buffer. One example of how this might be used is with protein a coated particles. Protein a binds most subclasses of IgG
immunoglobulins. A flocculating reagent column with protein a particles trapped on it would be useful for removing the IgG
immunoglobulins from serum. The IgG can be recovered by suitable changes in the eluting buffer.
The method of this invention can also be applied to affinity column mediated immunoassays (ACMIA). A representative ACMIA assay is described by Freytag et al. [Clin. Chem., Volume 30, 417-420 (1984)] for the aca~ discrete clinical analyzer (E. I. du Po~t de Nemours & Co., Inc., Wilmington, DE). An affinity column is prepared as described above, and then used as described by Freytag et al. It has been found that for a digoxin assay as disclosed by Freytag et al. the amount of ouabain-BSA required is reduced by over ,~,. . ~
.
~3~
300 fold using the method of this invention.
It is also recognized that many chemical precipitates form as small particles. These particles can also be removed by the method of this invention. Such precipitates can be formed as the result of immunoprecipitation, that is, the formation of large aggregates of antigens and antibodies, or the result of chemical precipitation. One example of chemical precipitation is the use of buffered phosphotungst;c reagent to precipitate low density and very low density lipoproteins to allow quantitat;on of high density lipoproteins. One example of immunoprecipitation is the direct reaction of anti-immunoglobulin antibodies with immunoglobulins as used in the nepholmetric or turbidimetric quantitation of immunoglobulins.
Surprisingly, it has been found that columns containing immobilized flocculating reagents prov;de a very simple, efficient and effective means to remove small particles from a liquid suspension. This property makes the use of these columns in bioaffinity separations, and particularly immunoassays, very advantageous.
The following examples further illustrate the invention.
' ' .
' .
4~2~
Competitive D;goxin Assay Using Covalently Attached PEI
(A) Preparation of Column 16.6 g of Sephadex G-10 resin (Pharmacia Biotechnolo~y Products) was heated in boiling water for ten minutes and washed twice with 100 mL of distilled water. To the swelled resin was added 0.66 9 of sodium periodate in 33 mL of distilled water. The mixture was mixed at room temperature in the dark for two hours, washed twice with water and twice with 0.15 M of sodium phosphate (pH 7.8) and made up to a total of 90 mL with said phosphate buffer. The mixture was then swirled to evenly suspend the resin and 60 mL of the suspension was mixed with 2 9 of 30% PEI and 10 mg of sodium cyanoborohydride. The pH of the mixture was adjusted to 5.7 with 6 N
hydrochloric acid. The mixture was mixed at room temperature overnight then washed 3 X 100 mL with phosphate buffer. The resin was packed into columns designed for use in the aca3 discrete clinical analyzer (E. I. du Pont de Nemours & Co., Inc., Wilmington, DE). The column is a plastic tube approximately 5.5 mm in diameter and 88 mm long with rubber stoppers at both ends which allow For automatic sample and diluent entry and eluent exit into an analytical test pack. Such an analytical test pack was described in RE 29725, issued to D. R. Johnson, et al. The columns were substituted for the columns in commercial digoxin packs for the aca~ analyzer.
(B) Preparation of Ouabain-BSA-CrO2 Particles 25i. Reductive Surface Treatment of CrO2 Two hundred and fifty grams of upgraded heated chromium dioxide were mixed with 100 9 of sodium bisulfate in 1750 mL of water. The mixture was milled in a W-250V-B Vertical Belt-Drive Colloid Mill (~reerco Corporatlon, Hudson, N.H~) for 45 minutes and aged in a glass container for one week. The particles were dialyzed against distilled water to remove the excess sodium bisulfite.
; ii. Silica Coating One hundred grams of chromium dioxide particles from above were placed in a 3 liter beaker and 2.5 liters of distilled water were added. The particles were heated to 90C ~ 2C with mechanical stirring. To the mixture was added 5.0 mL of sodium aluminate (40%
solution) and the pH of the suspension was adjusted to 9 by the addition of 5% sulfuric acid. To this mixture was added 150 mL of :~ -~ ' ~3C~
g water contain;ng 25 9 of sodium metasilicate and 6.25 9 of sod~um borate dropwise over a period of one hour. The pH of the mixture was maintained at 9 + 0.5 with the simultaneous dropwise addition of 5%
sulfurlc acid. Vigorous stirrlng was maintalned throughout the reaction. After all the reagents were added~ the mixture was heated at 90C and stirred for an additional 30 minutes before the pH was adjusted to 7 with 5X sulfuric acid and allowed to cool to roo~
temperature. The particles were dialyzed against distilled water.
- iii. Silane Coating One hundred grams of silica coated chrom;um dioxide particles were suspended in 1.8 liters of d-stilled water in a 2 liter round-bottom flask equipped w~th a mechanical stirrer, a reflux condensor and a temperature sensor. Two hundred mL of aminopropyltriethoxysilane was added and the mixture was stlrred at 55C for 18 hours. The particles were washed three times with 13 liters of distilled water by settllng and decantatlon. The washed particles were suspended ln 10 mM phosphates buffer ~pH 7) at 50 mg/mL.
iv. Preparation of Ouabain-BSA
Five grams of ouabain-octohydrate were dissolved ln 500 mL
of hot distilled water and allowed to cool to room temperature.
7.3 9 of sodium metaperiodate (NaI04) were added to the ouabain solution and stirred for two hours in the dark. The solution was passed through a bed of Dowex*(I-X8) anion exchange resin (prepared by washing 250 9 of the Dowex resin with water until the yellow color d~sappears~. The oxidized ouabain solution was mixed with ~00 mL of 1 M sodium phosphate (pH 7.0) containing 10 9 of BSA. The mixture was stirred for one hour and 0.64 9 of sodium cyanoborohydride (NaCNBH3) added. The mixture was st~rred at room temperature for 48-72 hvurs. The ouabain-~SA conjugate was dia1yzed against running water for 12-24 hours and against 20 volumes of 0.015 M sodium phosphate buffer (pH 7.0) at 4C for 16 hours. The conjugate was stored at 4C.
v. Protein Coupling to CrO2 Particles Ten mL of a 50 mg/mL suspenslon of the silane coated CrO2 part~cles were washed three tlmes with 50 mL o~ 10 mM ph~sphate buffer. To the washed particles 20 mL of 5X glutaraldehyde were added and mixed for three hours at room temperature. The activated * trade mark -. ~ . ., ~304~Z~
particles were washed five times each with 50 mL of phosphate buffer and suspended in 10 mL of the same buffer. A solution of 40 mg of ouabain-bsa conjugate in 10 mL of buffer was added. The mixture was agitated at room temperature for 20 hours. The particles were washed once with 10 mM phosphate buffer and the unreacted aldehyde groups were quenched by the addition of 50 mL of 1 m glycine (pH 8) and mixed for one hour. The protein particles were washed ten times with 50 mL each, of 10 mM phosphate buffer + 0.1% BSA + 0.1% thimerosal, and stored in 50 mL of the same buffer at 4C.
~C) Assay Procedures To 200 ~L of 0.15 M sodium phosphate buffer (pH 7.8) was added 200 ~L of Digoxin antibody-enzyme conjugate reagent (E. I. du Pont de Nemours & Co.~ Inc., Wilmington, DE) and 200 ~L of human serum containing digoxin. The Digoxin antibody enzyme conjugate reagent is a covalent conjugate of F(ab')2 antibody fragment from rabbit anti-digoxin antisera and ~-galactosidase. The mixture was incubated at room temperature for ten minutes and 200 ~L of a 10 mg/mL suspension of ouabain-BSA-CrO2 particles were added.
~ncubation was continued for five more minutes at room temperature.
The mixture was transferred to a sample cup for the aca~ analyzer and 400 ~L of it was pipetted by the instrument and injected into the digoxin pack containing a column prepared as above. The particles were trapped by the column and the enzyme activity of the filtrate was measured by the analyzer automatically using-~-D-galactopyrano-side as substrate. A series of human serum samples containingdigoxin at concentrations from 0 to 5 ng/mL were tested as described.
The 0 ng/mL background (B.G.) and the response difference between 0 and 5 ng/mL (Delta 0,~) were determined and were 18 mA/min and 64 ma/min, respectively.
130~2~
Bound Phase Digoxin Assay Using PEI
Absorbed on Porous Plastic (A) Preparation of Column Fifteen ~m pore size porous plastic rods approximately 5.3 mm in diameter and 80 mm long were obtained from Porex Technologies (Fairburn, GA~. About 20 rods were soaked in a 3X PEI
solution in 30% isopropanol with gently mixing overnight. The columns were rinsed with distilled water and inserted into empty columns described in 1.A. for use on the aca~ analyzer. The column was then washed with 5 mL of 0.15 M sodium phosphate, pH 7.8.
(B) Assay Procedures To a test tube was added 50 ~L of digoxin antibody-enzyme conjugate reagent, 50 ~L of human serum containing digoxin and 50 aL
of 1.15 M sod;um phosphate (pH 7.8) and vortexed. The mixture was incubated at 37C for ten minutes and 50 ~L of a 10 mg/mL suspension of ouabaln-BSA-CrO2 particles prepared in 1.B. was added and mixed well. The mixture was incubated for another two minutes and a 150 ~L
aliquot was injected into a column prepared above and eluted with 2 mL of 0.15 M phosphate. One mL of 0.1 M o nltrophenyl-~-D-galactopyranoside was injected into the column and incubated at 37C
for ten minutes. The column was then eluted with 2 mL of 0.15 M
phosphate and the absorbance at 406 nm was recorded.
(C) Resul~s The B.G. was 307 mA/min and the Delta 0,5 was 200 mA/min.
This procedure was repeated using 50 ~M pore size porous plastic rods. The B.G. was 268 mA/min and the Delta O,S was 162 mA/min.
' 13~4~2~
TSH Sandwich Assay Using_PEI Adsorbed on Bio~el P-2 (A) Preparation of Column A sample of 40 9 of Biogel P-2 (100-200 mesh) and 10 g of PEI were mixed in 300 mL of 0.1 M sodium chloride at room temperature overnight. The resin was washed three times with 300 mL each of 0.1 m sodium chloride and packed into a column for use on the aca~
analyzer as described ;n 1.A. The column was equllibrated with 80 mm NaCL, 0.1% Tween-2C~ pH 6Ø
(B) AntI-TSH-CrO2 Particle Preparatlon .
i. Reductive Surface Treatment of CrO2 Two hundred and fifty grams of upgraded CrO? were m~xed with 100 g of sodium bisulfite in 1750 mL of water. The mixture was milled in a W-250V-B vertical Belt-Drive Colloid Mill (Greerce Corporation, Hudson, NH) for 45 minutes. The particles were washed with water and spray dried. Twenty grams of spray dried CrO2 particles were washed twice w~th 200 mL of distilled water by decantation. The particles were d1spersed ln 200 mL of distilled water containing 20 g of sodium bisulfite and 50 9 of 1/8" glass beads in a 200 mL tissue culture flask. The mixture was rotated at 5 rpm for 48 hours at room temperature. The part~cles were separated from the glass beads and washed three times with 200 mL of 10 mM
sodium phosphate buffer tpH 7) using magnetic separation.
ii. Protein Coupling .
Ten mL of a 5% slurry of silanized CrO2 were washed three times with 50 ml each of 10 mM phosphate~ pH 7.4. After the th~rd was~h, the particles are magnetically separated, the supernatant was aspirated, and to this wet cake 20 mL of 5% glutaraldehyde was added and r~cked for three hours at room temperature. The glutaraldehyde activated particles were washed ten times with 50 mL each of coupling buffer (10 mm potassium phosphate, pH 7.4). After the last wash, the t particles were resuspended in 10 mL buffer To this was added 6 mg of purified ~ subunit specific antibody in 10 mL of coupling buffer and the mixture rocked for 20 hours at 4C. The antibody coupled particles were washed once w~th coupling buffer, then the unreacted aldehyde groups were quenched by reaction with 50 mL of 1 M glycine, pH 8.0 for ten minutes. The partlcle reagent was washed extensively, ten tlmes with 50 mL each of the wash buffer (coupling buffer also * trade mark -~ t "
31~ 4 containing 0.1% BSA), to remove all noncovalently bound antibodies.
The final reagent was resuspended ~nto 10 mL of the wash buffer containing 0.1% sodium azide as preservative, and stored at 4C~
(C) Assay Procedures To a test tube was added 150 ~L of human serum containing a known amount of TSH and 25 ~L of the antibody conjugate reagent from a Hybritech Tandem~-e TSH lmmunoenzymetric assay kit (Hybritech, Inc., San Diego, CA) and vortexed. The mixture was incubated at 37C
for ten minutes and 25 ~L of ant;-TSH-CR02 partlcles (25 mG/MI) was added vortexed. Incubation was cont;nued for another ten minutes and 150 ~L of the mixture was injected into a column prepared above. The column was washed with 2 mL of 80 mM sodium chloride and 0.1~ Tween-20, pH 6.0 buffer. One mL of 3 mM p-nitrophenylphosphate in 1 M
diethanolamine, 0.5 mM MgC12, pH 8.9 was injected into the column.
The column was incubated at 37C for 30 minutes and eluted with 2 mL
of 50 mM ethylenediamine tetraacetlc acid (EDTA) pH 8.~. The absorbance of the eluent at 406 nm was measured. The O aIulmL
background (B.G.) and the response difference between O aIu/mL and 50 ~IU/mL (Delta 0,50) were determined from those results and were 126 mA/min and 312 mA/min, respectively.
A
:
, ~ ~
13 [)~Z~L
ACMIA Digoxin Assay Using Ouabain-BSA-CrO2 Particles Trapped on PEI-Biogel P-2 as Affinity Column (A~ Preparation of Column PEI-Biogel P-2 columns were prepared as in Example 3.A. To the column was injected 50 to 200 ~L of 10 mg/mL suspensions ouabain-BSA-CrO2 particles. The columns were washed with 3 mL of 0.15 M
sodium phosphate buffer (pH 7.8) and inserted into the commercial digoxin pack for the aca~ analyzer (E. I. du Pont de Nemours and Co., Inc.).
(B)Assay Procedures Five hundred ~L of human serum containing digoxin and 500 aL of antibody-enzyme conjugate (enough for two tests) were mixed in a sample cup and was incubated at room temperature for ten minutes. Four hundred ~L of the mixture was pipetted by the aca~
analyzer and processed as the commercial method.
The test results using different amounts of particles trapped on the column are compared to the commercial method which uses ouabain-BSA-Sephadex G-10 in Table 1:
Digoxin Assay Using Trapped CrO2 particles on ACA
Particles BackgroundSeparation 25(mg) (mA/min)Delta, 0,5 (mA/min) 0.5 88 68 1.0 60 91 2.0 48 84 30Commercial Pack72 79 ~ .
~3l~4~l Affinity Purification of Anti-Digoxin Antibody PEI-Biogel P-2 resin was prepared as in Example 3.A. and packed in a 1.5 X 15 cm column. The column was washed with 100 ml of 10 mM sodium phosphate (pH 7.0) containing 0.9% of sodium chloride (PBS). Three mL of a 50 mg/ml suspensions ouabain-BSA-CrO2 particles prepared in Example 1.B. were loaded on the column. The top of the column was intentionally disturbed to mix the top half of the resin with the CrO2 particles and allowed to pack again. The packed column was washed with 50 mL of PBS, no leakage of the CrO2 particles was observed. Ten mL of rabbit anti-digoxin serum was loaded on the column and the serum was allowed to incubate for 15 minutes. The unbound fraction was eluted with 100 mL of PBS. The bound anti-digoxin was eluted with 2.5 M ammonlum thiocyanate (pH 7.8).
Fractions containing the protein were identified by monitoring the 280 nm absorbance and were pooled and dialyzed against PBS.
Immunodiffusion analysis indicated that all the anti-digoxin was in the bound fractions, no anti-digoxln was observed in the unbound fractions.
~30~L~21 Automated Assay for High_Density Lipoprotein (HDL) Cholesterol The following procedures can be used to perform an assay for high density lipoprotein (HDL) cholesterol which automated the removal of the low density and very low density lipoprotein which are precipitated when the sample is pretreated with phosphotungstic acid.
(A) Preparation of Column PEI-Biogel P-2 columns for the aca~ analyzer are prepared as described in Example 3.A. These columns are then placed in the header af an analytical test pack (as described in Example 1.A.) which contains the reagents necessary to assay for HOL cholesterol.
(B) Assay Procedure Five hundred aL of serum and 100 ~L of a 20 g/L solution of phosphotungstlc acid are mixed in an aca~ analyzer sample cup and allowed to stand for five minutes. Two hundred and fifty ~L of this pretreated sample are then injected into the column described lfrom]
6.A. The column is eluted with 4.75 mL of 10 mM sodium phosphate buffer (pH 7.0). The filtrate is collected in the analytical test pack which can be processed on an aca~ analyzer to determine the quantity of HDL cholesterol present.
Claims (23)
1. The method of performing an affinity separation comprising:
forming a solid support with a flocculating agent attached thereto;
forming a reaction mixture comprising a sample suspected of containing a target substance and a reagent which contains a ligand or binder which will react with the target substance to form a particulate suspension, said solid support being non-reactive and exhibiting low non-specific binding of the target substance and ligand or binder;
allowing the target substance to bind to said ligand or binder; and, eluting the reaction mixture through said solid support so as to trap the particulate suspension in the flocculating agent on the solid support and elute any unbound substances from the support.
forming a solid support with a flocculating agent attached thereto;
forming a reaction mixture comprising a sample suspected of containing a target substance and a reagent which contains a ligand or binder which will react with the target substance to form a particulate suspension, said solid support being non-reactive and exhibiting low non-specific binding of the target substance and ligand or binder;
allowing the target substance to bind to said ligand or binder; and, eluting the reaction mixture through said solid support so as to trap the particulate suspension in the flocculating agent on the solid support and elute any unbound substances from the support.
2. The method of Claim 1 wherein the flocculating agent is polymeric.
3. The method of Claim 2 wherein the flocculating agent is polyethyleneimine.
4. The method of Claim 1 wherein the flocculating agent is selected from the group comprising polyethyleneimine, potassium sulfate, ferric choloride and ferric sulfate.
5. The method of Claim 1 wherein the solid support is a chromatographic column.
6. The method of Claim 5 wherein the flocculating agent is polymeric.
7. The method of Claim 6 wherein the flocculating agent is polyethyleneimine.
8. A method of detecting the presence of a target substance in a sample, said method comprising:
Attaching a captured antibody for the target substance to a particle to form an antibody couple particle:
reacting the antibody coupled particle with a sample containing the target substance and a labelled antibody to form a mixture comprising unreacted labelled antibody and a complex of said particle, capture antibody, target substance and labelled antibody, and unreacted labelled antibody;
forming a chromatographic column with a flocculating agent attached thereto:
eluting the mixture through the column thereby trapping the particles and any labelled antibody bound to the target substance which in turn is bound to the capture antibody: and, measuring the labelled antibody associated with the particles trapped on the flocculating agent immbolized in the column or the unreacted labelled antibody to thereby detect the presence of target substance in the sample.
Attaching a captured antibody for the target substance to a particle to form an antibody couple particle:
reacting the antibody coupled particle with a sample containing the target substance and a labelled antibody to form a mixture comprising unreacted labelled antibody and a complex of said particle, capture antibody, target substance and labelled antibody, and unreacted labelled antibody;
forming a chromatographic column with a flocculating agent attached thereto:
eluting the mixture through the column thereby trapping the particles and any labelled antibody bound to the target substance which in turn is bound to the capture antibody: and, measuring the labelled antibody associated with the particles trapped on the flocculating agent immbolized in the column or the unreacted labelled antibody to thereby detect the presence of target substance in the sample.
9. The method of Claim 8 wherein the flocculating agent is polymeric.
10. The method Claim 9 wherein the flocculating agent is polyethyleneimine.
11. The method of Claim 8 wherein the flocculating agent is selected from the group comprising polyethyleneimine, potassium sulfate, ferric chloride and ferric sulfate.
12. The method of Claim 10 wherein the particle is a stabilized chromium dioxide particle.
13. A method of conducting a competitive immunoassay for determining the presence of a target substance in a sample, said method comprising:
Forming a particle reagent by affixing a ligand or binder for the target substance onto a particle;
forming a reaction mixture comprising a sample suspected of containing the target substance and a labelled antibody for the target susbstance;
providing a chromatographic column with a flocculating agent attached thereto;
eluting the reaction mixture through the chromatographic column whereby said particles having any target susbstance in the sample and labelled antibody bound thereto are trapped within the column and any unbound labelled antibody passes through the column: and, measuring either the bound or unbound labelled antibody to determine the presence of the target substance.
Forming a particle reagent by affixing a ligand or binder for the target substance onto a particle;
forming a reaction mixture comprising a sample suspected of containing the target substance and a labelled antibody for the target susbstance;
providing a chromatographic column with a flocculating agent attached thereto;
eluting the reaction mixture through the chromatographic column whereby said particles having any target susbstance in the sample and labelled antibody bound thereto are trapped within the column and any unbound labelled antibody passes through the column: and, measuring either the bound or unbound labelled antibody to determine the presence of the target substance.
14. The method of Claim 13 wherein the flocculatng agent is polymeric.
15. The method of Claim 14 wherein the flocculating agent is polyethyleneimine.
16. The method of Claim 13 wherein the flocculating agent is sleected from the group comprising polyethyleneimine, potassium sulfate, ferric chloride and ferric sulfate.
17. The method of Claim 15 wherein the particle is a stabilized chromium dioxide particle.
18. A method of performing a bioaffinity separation, said method comprising:
Forming a chromatographic column with a flocculating agent attached thereto forming a particle reagent by affixing a ligand or binder to a particle;
eluting the particle reagent through the chromatographic column thereby trapping the particles on the flocculating agent within the column;
passing a mixture containing a target substance through the column whereby the target substance can bind to the ligand or binder affixed to the particle reagent: and, eluting any unbound substance from the column.
Forming a chromatographic column with a flocculating agent attached thereto forming a particle reagent by affixing a ligand or binder to a particle;
eluting the particle reagent through the chromatographic column thereby trapping the particles on the flocculating agent within the column;
passing a mixture containing a target substance through the column whereby the target substance can bind to the ligand or binder affixed to the particle reagent: and, eluting any unbound substance from the column.
19. The method of Claim 18 wherein the flocculating agent is polymeric.
20. The method of Claim 19 wherein the flocculating agent is polyethyleneimine.
21. The method of Claim 18 wherein the flocculating agent is selected from the group comprising polyethyleneimine, potassium sulfate, ferric chloride and ferric sulfate.
22. The method of Claim 20 wherein the particle is a stabilized chromium dioxide particle.
23. A method of performing a bioaffinity separation, said method comprising:
Forming a solid support with a flocculating agent immobilized thereon;
forming a particle reagent by affixing a ligand or binder to a particle;
passing the particle reagent over the solid support thereby trapping the particle reagent on the flocculating agent immobilized on the solid support;
passing a mixture containing a target substance over the solid support whereby the target substance can bind to the ligand or binder affixed to the particle reagent; and, washing any unbound substances from the solid support.
Forming a solid support with a flocculating agent immobilized thereon;
forming a particle reagent by affixing a ligand or binder to a particle;
passing the particle reagent over the solid support thereby trapping the particle reagent on the flocculating agent immobilized on the solid support;
passing a mixture containing a target substance over the solid support whereby the target substance can bind to the ligand or binder affixed to the particle reagent; and, washing any unbound substances from the solid support.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US7424287A | 1987-07-16 | 1987-07-16 | |
| US074,242 | 1987-07-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1304421C true CA1304421C (en) | 1992-06-30 |
Family
ID=22118519
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000570588A Expired - Lifetime CA1304421C (en) | 1987-07-16 | 1988-06-28 | Affinity separating using immobilized flocculating reagents |
Country Status (5)
| Country | Link |
|---|---|
| US (3) | US5302532A (en) |
| EP (1) | EP0367795A4 (en) |
| JP (1) | JPH01503808A (en) |
| CA (1) | CA1304421C (en) |
| WO (1) | WO1989000446A1 (en) |
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-
1988
- 1988-06-02 WO PCT/US1988/001779 patent/WO1989000446A1/en not_active Application Discontinuation
- 1988-06-02 JP JP63506308A patent/JPH01503808A/en active Pending
- 1988-06-02 EP EP19880906663 patent/EP0367795A4/en not_active Withdrawn
- 1988-06-28 CA CA000570588A patent/CA1304421C/en not_active Expired - Lifetime
-
1989
- 1989-04-14 US US07/337,992 patent/US5302532A/en not_active Expired - Lifetime
-
1993
- 1993-10-29 US US08/145,709 patent/US5422284A/en not_active Expired - Fee Related
- 1993-10-29 US US08/145,289 patent/US5447870A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01503808A (en) | 1989-12-21 |
| WO1989000446A1 (en) | 1989-01-26 |
| US5302532A (en) | 1994-04-12 |
| EP0367795A1 (en) | 1990-05-16 |
| EP0367795A4 (en) | 1992-01-15 |
| US5422284A (en) | 1995-06-06 |
| US5447870A (en) | 1995-09-05 |
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