AU649397B2 - Immunoreactant carriers having a novel biocompatible intermediate coating and process of making same - Google Patents

Description

OPI DATE 01/05/90 APPLN- ID 46259 89

PCT

AOJP DATE 07/06/90 PCT NUMBER PCT/US89/04467 INTERNATIONAL AFYLUAIIV IN rtU ti-inCU uINj UIN ti riirt. n I UUtr ma ii uIN ILmnti A I(PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/04178 G01N 33/53, 33/534, 33/535 Al GOIN 33/543, 33/548, 33/553 (43) International Publication Date: 19 April 1990 (19.04.90) (21) I*ernational Application Number: PCT/US89/04467 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European pa- (22) International Filing Date: 5 October 1989 (05.10.89) tent), FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), NO, SE (European patent).

Priority data: 255,743 11 October 1988 (11.10.88) US Published With international search report.

(71)Applicant: COULTER CORPORATION [US/US]; 600 Before the expiration of the time limit for amending the West 20th Street, Hialeah, FL 33010 claims and to be republished in the event of the receipt of amendments.

(72)Inventors: KORTRIGHT, Kenneth, H. 5611 Castlegate Avenue, Davie, FL 33331 GUPTA, Ravinder, K.; 9430 N.W. 19th Street, Pembrook Pines, FL 33024 (US).

(74) Agents: CASS, Myron, C. et al.; Silverman, Cass Singer, Ltd., 105 W. Adams Street, 27th Floor, Chicago, IL 60603 (US).

649397 (54)Title: IMMUNOREACTANT CARRIERS HAVING A NOVEL BIOCOMPATIBLE INTERMEDIATE COATING AND PROCESS OF MAKING SAME (57) Abstract An immunoreactant carrier having an antigen or antibody member of a single binding pair covalently bound to a biocompatible medium comprising a protein gel or polysaccharide coating the surface of a carrier body, and the method of producing the immunoreactant carrier, is provided. Where the biocompatible medium is a protein gel, it is coated on the surface of the carrier body by hydrophobic interaction or ccvalent binding. Where the biocompatible medium is a polysaccharide, it is coated on the surface of the carrier by covalent binding. The immunoreactant carrier can be used in an assay involving a member of a ;ingle binding pair such as an antigen or antibody member of a single binding pair in a biological test sample.

WO 90/04178 PCT/US89/04467 -1- Description IMMUNOREACTANT CARRIERS HAVING A NOVEL BIOCOMPATIBLE INTERMEDIATE COATING AND PROCESS OF MAKING SAME TECHNICAL FIELD This invention relates generally to immunoreactant carriers, such as, microspheres or beads for use in immunoassays and more particularly, relates to improvements in immunoreactant carriers having a novel intermediate biocompatible protein or polysaccharide coating to which will be conjugated an antigen or antibody of a single binding pair for immunoassays and the process of making said carriers.

BACKGROUND ART In the field of immunoassays, it is known to employ microspheres or beads as the carrier for one of the members of a single binding pair, such as an antibody or antigen of a single pair of binding members. A common immunoassay procedure is to coat the carrier with a specific labelled antibody selected to bind the antigen to be assayed and introduce the coated carrier to a sample of a biological fluid to be tested.

The resultant complexing of the antibody coated carrier and bound antigen is detected and measured by known procedures to determine the assay resultant.

Another known procedure is to withdraw from a test sample of blood a selected class of blood cells by using magnetic microspheres coated with a monoclonal antibody which binds selectively to the selected class of cells, form the complex of the monoclonal antibody and bound cells and then remove the non-complexed cells from the test sample so as to isolate the bound cells.

U.S. Patent No. 4,743,543 describes a procedure for removing red blood cells from a test sample which thereafter enables study of the remaining white blood cells.

These and other known procedures which use either magnetic or non-magnetic carriers coated with an immunoreactant member intended to bind a second member of a single binding pair require suitable and effective WO 90/04178 PCI'/US89/04467 -2coating of the carrier.

The adsorption of an inert protein such as serum albumin, egg albumin, and lactalbumin on solid carrier particles and the subsequent adsorption of an antigen or antibody on the coated carrier particles is described in U.S. Patent No. 3,551,555.

Cross-linking methods also have been employed to retain a protein on a carrier particle. For example, water-insoluble microcapsules whose circumferential wall contains a protein such as gelatin cross-linked to an antigen or antibody in an acidic solution is disclosed in U.S. Patent No. 4,590,170. This method requires the use of microcapsules negatively charged at an acidic pH.

U.S. Patent No. 4,123,396 describes a procedure for preparing metal containing polymeric microspheres which can be bonded covalent3y to proteins. Crosslinking of the protein and microspheras is preferred so that the stability and size of the microspheres both in aqueous solution and in jrganic solvents can be :naintained.

U.S. Patent No. 4,478,946 teaches carriers such as roughened glass beads to which a film-forming "first layer" protein is cross-linked to the bead and to which a subsequent "second layer" antibody or antigen is covalently coupled. The first layer partially or totally may comprise a protein such as gelatin. Less antibody or antigen is required when the film-forming first layer is composed partially or totally of a protein such as gelatin. Crosslinking methods however are required to retain the first layer on the bead.

One desirable advantage in preparing such an immunoreactant microsphere is to achieve a desirable high ratio of bound antibody to bead surface.

Another desirable advantage is to be able to apply an intermediate biocompatible coating to the microspheres directly, which coating will enable direct conjugation thereto of the antibody or antigen.

Yet another desirable advantage is the ability to -3use reduced quantities of the antibody or antigen to be coated to the microsphere or bead yet achieve the desired immunoassay determinations by means of the herein invention.

All of the desired advantages enumerated and others which will become apparent are derived from the methods embodying the invention as herein disclosed.

An ancillary advantage is to produce the immunoreactant coated microspheres prepared by said methods of the invention for use in immunoassay kits and/or procedures.

DISCLOSURE OF THE INVENTION The invention provides an immunoreactant carrier comprising a biocompatible coating medium retained directly on the surface of the carrier body without an intermediate binding agent and an immunoreactant covalently bound to said coating medium by means of a bifunctional reagent, and the method of producing the carrier.

More particularly, the present invention provides an Lmmunoreactant carrier comprising: a particulate carrier polymer body; a biocompatible medium which is covalently bound to the peripheral surface of said carrier body and which is selected from the group consisting of a protein gel and an amine-derivatized polysaccharide, which medium does not interact or interfere with immunological reactions or components of biological fluid test samples and does not detrimentally influence the immunoreactants involved in an immunological reaction; and an immunoreactant selected from the group consisting of an antibody or an antigen member of a single binding pair, which member is covalently bound to said medium by means of a bifunctional reagent.

940317,q:\oper\ej,46259289,3 -3A- One method of the invention comprises coating the peripheral surface of the carrier body with a biocompatible protein gel by hydrophobic interaction.

After coating the carrier, an immunoreactant member of a single binding pair such as an antigen or antibody, is covalently bound to the coated carrier to form the immunoreactant carrier.

Another method of the invention provides coating the carriers with a biocompatible protein gel or a polysaccharide by covalent binding. The carrier coated with the biocompatible medium then is covalently bound to an immunoreactant member of a single binding pair to form the immunoreactant carrier.

BEST MODE FOR CARRYING OUT THE INVENTION The term "biocompatible" as used herein to describe the intermediate coating medium is intended to mean a medium which does not interact nonspecifically and does not interfere with the immunological reaction or other components of the biological fluid test sample, and does not detrimentally influence the immunoreactants involved in the immunological reaction.

90317,q:\oper\eb46259289,3 WO 90/04178 PCT/US89/04467 -4- The term "immunoreactant" as used herein is intended to mean a substance capable of being one of the reaction members of a single binding pair, as in an antigen or antibody complex formation.

A glossary of reagents employed in practicing the invention is as follows: Glossary of Reagents: Gelatin Solution for Coating Microspheres by Hydrophobic Interaction: Gelatin powder (available from Sigma Chemical or Gelatin capsules (available from Eli Lilly Co.): dissolved in phosphate buffered saline (PBS), pH 7.3, containing 0.1% sodium azide Gelatin Solution for Coating Microspheres by Covalent Binding: Sodium Chloride 0.2M EDAC (2 mg/ml l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride in 0.2M NaC1) Gelatin powder (available from Sigma Chemical or Gelatin capsules (available from Eli Lilly Co.): mg/ml dis olved in PBS, pH 7.3, containing 0.1 sodium azide Glycine (20 mg in 1 ml of water) Water (containing 0.1% sodium azide) Conjugation Procedure A: Phosphate Buffered Saline (PBS) pH 7.3, containing 0.1% sodium azide Bifunctional reagent, such as Sulfo-SMCC (2 mg/ml sulfosuccinimidyl-4-[Nmaleimidomethyl] cyclohexane-l-carboxylate in PBS), available from Pierce Chemical Co.

2-iminothiolane hydrochloride, (2 mg/ml in PBS), available from Pierce Chemical Co.

Immunoreactant >10 mg/ml L-cysteine (5 mg/ml in PBS), lodoacetamide (30 mg/ml in PBS) WO 90/04178 PCT/US89/04467 Bovine Serum Albumin (BSA) (in PBS containing 0.1% sodium azide) 1% 1M Borate pH 9.8 Conjugation Procedure B: 2-iminothiolane hydrochloride (2 mg/ml in

PBS)

Bifunctional reagent, such as Sulfo-SMCC (1 mg/ml sulfosuccinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate in PBS), available from Pierce Chemical Co.

Immunoreactant >10 mg/ml L-cysteine (5mg/ml in PBS), lodoacetamide (20 mg/ml in PBS) Bovine Serum Albumin (BSA) (in PBS containing 0.1% sodium azide) 1% 1M Borate pH 9.8 Preparation of Polysaccharide Solution: Dextran T-40 (Molecular weight 40,000 daltons), or, Dextran T-110 (molecular weight 110,000 daltons), or, Dextran T-500 (molecular weight 500,000 daltons), or, Dextran T-2M (molecular weight 2,000,000 daltons), available from Fluka, Pharmacea; or Ficoll (molecular weight 70,000 daltons), available from Sigma Chemical Co. Potassium Acetate pH 6.5 50 mM Sodium Periodate (0.535 g in 6.25 ml of water) 85.6 mg/ml 1,3-diaminopropane (5 ml in 5 ml of water, adjust to pH 8.7 with glacial acetic acid) ~17ml Sodium Borohydride (0.2 g in 2.5 ml of 0.1mM Sodium Hydroxide), available from Aldrich Chemical Co. 80 mg/ml WO 90/04178 PCT/US89/04467 -6- Covalent Binding of Polysaccharide to Microspheres: Sodium Chloride 200mM Aminoderivatized polysaccharide 3.75 mg/ml EDAC (l-ethyl-3-[3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg/ml in 0.2M NaCI) Water (containing 0.1% sodium azide) Conjugation of Immunoreactant to Polysaccharide Coated Microsphere: The aminopolysaccharide coated beads may be conjugated to immunoreactants by using a bifunctional reagent. Some examples of bifunctional reagents are as follows: Glutaraldehyde Bis (succinimidyl) suberate SPPD (succinimidyl 3-[2-pyridyldithio]propionate SMCC (succinimidyl-4-[N-maleimidomethyl] cyclohexane-l-carboxylate) SIAB (succinimidyl [4-iodoacetyl] aminobenzoate) SMPB (succinimidyl 4-[l-maleimidophenyl]butyrate) The biocompatible intermediate coating medium can be a protein or a polysaccharide. In one preferred embodiment, the intermediate coating medium is a protein, such as gelatin. Commercially available gelatins are graded according to their viscoscity and gel strength (or Bloom level) of their aqueous solutions. Various commercially available porcine and bovine gelatins with Bloom levels between 60 and 300 were tested during preliminary experiments. Gelatin capsules also were testud. It was found that increased Bloom levels of gelatin enabled higher quality coating of the microspheres. Also, bovine gelatin appeared to be preferable to porcine gelatin. Gelatin from capsules was the preferred gelatin of all gelatins tested.

The biocompatible intermediate coating medium can also be a polysaccharide. Polysaccharides tested included Dextran T-40 (molecular weight 40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran WO 90/04178 PCT/US89/04467 -7- T-2M (molecular weight 2,000,000 daltons), Fluka, Pharmacea, and Ficoll (molecular weight 70,000 daltons), Sigma Chemical Co.

Commercially available microspheres tested included divinyl-benzene/polystyrene magnetic microspheres (0.7 micron, 20% magnetic; 0.7 micron, 42% magnetic; and 1.5 micron, 13% magnetic), Seragen Corporation; Non-magnetic mic::ospheres also may be employed.

Although the diameter size of the microspheres tested ranged from 0.7 to 1.5 microns, diameter sizes larger or smaller than those tested may be employed.

The immunoreactant may be labelled or unlabelled, depending upon the immunoassay system to be utilized.

Suitable labels include enzymes, radioactive elements or dyes.

The preparation of the immunoreactant carriers involves four basic steps. The carriers are washed in Step I. The washing reagent and procedure will depend upon both the biocompatible medium and the coating method to be used to apply the biocompatible medium on the carrier.

The solution of biocompatible medium is prepared in Step II. Again, reagents and methods will vary depending upon which coating medium is used and which coating method is employed.

In Step III the solution of the biocompatible medium is coated on the carrier and excess codting medium is removed. The coating method may be by either hydrophobic interaction or covalent binding if gelatin is the biocompatible coating medium used. The coating method is by covalent binding if a polysaccharide is used.

The carrier coated with the biocompatible coating medium can be sterilized by known irradiation techniques prior to Step IV.

I-n. Step IV the immunoreactant is covalently bound to the carrier which has been coated with the biocompatible medium by use of a bifunctional reagent.

WO 90/04178 PCT/US89/04467 -8- PREPARATION OF IMMUNOREACTANT CARRIERS: In Step I, 1 trilliliter of a 10% suspension comprised of 0.7 micron, 42% magnetic microspheres is diluted to 4 ml with PBS and 0.1% azide and washed three times using 4 ml of Phosphate Buffered Saline (PBS) containing a bacteriostatic agent such as 0.1% sodium azide and recovered in the conventional manner.

Then, Step II, the gelatin solution is prepared.

Gelatin powder or capsules is weighed and then combined with PBS to obtain a concentration of 10 mg/ml. The solution is heated gently to approximately 50 0 C on a magnetic stir plate until the gelatin solution is clear. The gelatin solution is cooled to room temperature by stirring before further use.

Next, Step III, the gelatin solution is coated onto the microspheres by hydrophobic interaction. 4 ml of the gelatin solution is combined with the washed microspheres, sonicated in a water 'ath at room temperature for 1 minute and roller-mixed for 3 to 16 hours.

Excess gelatin is removed by washing the suspension in the conventional manner four times with 4 ml of PBS and 0.1% azide each wash. The microspheres are resuspended to a volume of 4 ml in PBS and 0.1% azide and stored at 4 0 C until used.

0.7 micron, 20% magnetic microspheres and 1.5 micron, 13% magnetic microspheres also were prepared in this manner. Microspheres thus prepared are ready for Step IV, covalent binding to an immunoreactant by Conjugation Procedure A or Procedure B.

Conjugation Procedure A: 2 ml of a 2.5% suspension of microspheres coated with gelatin by hydrophobic interaction are resuspended to 2 ml in PBS. 27 microliter (ul) of sulfosuccinimidyl-4-[N-malemido-methyl] cyclohexane-lcarboxylate (sulfo-SMCC) is added to the microsphere suspension and this mixture is roller-mixed for 1 hour at room temperature. Then, the mixture is washed four times with 2 ml of PBS each wash in the conventional manner, resuspended to a volume of 2 ml in PBS WO 90/04178 PC7, /US89/0"67 -9and stored at 4°C until used.

Next, the immunoreactant is thiolated usin.y 2imino-thiolane hydrochloride according to the method of R. Jue et al., Biocnemistry 17 5399 (1978). In this method, immunoreactant (2mg) at >10 mg/,l concentration is reacted with 13 ul of 2-iminothiolane hydrochloride for 1 hour at 22 0 C. The thiolated immunoreactant is separated by gel filtration and the protein concentration determined by absorbance at 280 nanometers (nm) in a spectrophotomter. Then, 1 mg of the thiolated immunoreactant is added to 2 ml of the sulfo-SMCC activated microspheres and reacted for 2 hours at room temperature. The reaction is quenched by adding 240 ul of cysteine to the suspension for 15 minutec followed by treatment with 240 ul of Iodoacetamide followed by 240 Al of 1M bora":e buffer. After I hour the microspheres then are blocked for at least 1 hour with 1% bovine serum albumin (BSA), washed 4 times in the conventional manner with 4 ml of 1% BSA each wash and resuspended to a volume of 2 ml in 1% BSA to obtain suspension.

Conjugation Procedure B: Alternatively, 4 ml of a 2.5% suspension of coated microspheres is treated with 40 ul of 2-iminothiolane hydrochloride at room temperature for 1 hour, washed 4 times in the conventional manner with 4 ml of PBS each 4ash, resuspended to 3.8 ml in PBS and stored at 4 0

C

until used. Then, 1 mg of immunoreactant is treated with 40 Al sulfo- SMCC at 10 mg/ml protein concentration in PBS for 1 hour at room temperature. This treated immunoreactant then is passed through a Sephadex G-50 column in PBS, the protein peak collected and the protein concentration calculated from the A 2 8 0 value. 0.4 mg of the modified immunoreactant is added to 4 ml of the thiolated microspheres. The suspension is roller-mixed at room temperature for 2 hours. The reaction is quenched by adding 120 gl of cysteine per ml of reaction volume to the suspension for 15 minutes.

The free sulphydral groups are capped by adding 120 li WO 90/04178 PCJT/US89/04C467 each of lodoacetamide and 1M Borate per ml of reaction volume tc the suspension, and roller-mixing for minutes at room temperature. The microspheres then are blocked with 1% BSA for 1 hour at room temperature, washed 4 times with 4 ml of 1% BSA each wash, resuspended to 4 ml in 1% BSA and stored at 4 0 C until used.

Alternatively, gelatin is covalently bound on th microspheres. In Step I of this procedure, 1 ml of a i0 10% suspension of 0.7 micron, 42% magnetic microspheres is diluted with 3 ml of 0.2M Sodium Chloride (NaC1), washed once in the conventional manner with 4 ml of 0.2M NaC1 and suspended to a volume of 4 ml with 0.2M NaC1. The microspheres then are treated with 15 pl of l-ethyl-3-[3-dimethylaminopropyl 1 carbodiimide hydrochloride (EDAC) (2 mg/ml) for 15 minutes.

During this treatment period, the gelatin solution is prepared (Step II). Gelatin powder or capsules is weighed and then combined with PBS to obtain a concentration of 20 mg/ml. The solution is heated gently to approximately 50 0 C on a magnetic stir plate until the gelatin solution is clear. The gelatin solution is cooled to room temperature by stirring before further use.

Then, Step III, the microspheres are bathsonicated for 30 seconds, treated with 500 ul of the gelatin solution, bath-soniceted again for 30 seconds, and roller-mixed overnight at room temperature. The reaction is stopped by adding 100 ol of glycine to the treated microspheres for 30 minutes. The microspheres then are washed in the conventional manner four (4) times with 4 ml of water each wash, resuspended to a volume of 4 ml in water containing 0.1% sodium azide to achieve a concentration of 2.5% and stored at 4 0 C until used.

In Step IV, these coated microspheres are covalently bound with an immunoreactant by Conjugation Procedure A.

The carrier also may be coated with a polysac- WO 90/04178 PC~/US89/04467 -11charide. In Step I of this coating procedure, 18 ml of a 10% suspension of 0.7 micron, 42% magnetic microspheres is diluted to 72 ml with 200 mM NaC1 and washed once with 200 ml of 200 mM NaCI.

Then, Step II, the polysaccharide solution is prepared. The method of R. S. Molday and L. L. Molday (FEBS Letters 170, No.2:232 [1984]) for T-40 was adap-ced as herein described. 5 g of dextran is dissolved in 37.5 ml of Potassium Acetate, treated with prepared solution of Sodium Periodate, vigorously stirring during 10 minutes at 20-25 0 C, and allowed to react at 20-25 0 C for 1 hour and 30 minutes. This solution is dialized extensively 5 times against 1 liter of water each time for 1 to 2 hours at 4 0 C. This solution then is added dropwise, with vigorous stirring, to the prepared solution of 1, 3-diaminopropane. The mixture is stirred on a magnetic stir plate at room temperature for 2 hours and 15 minutes and then reduced with prepared solution of Sodium Borohydride for 15 minutes at room temperature. The mixture extensively dialyzed against water at 4 0 c, filverad through a 0.22 micron filter and stored at 4 0

C.

Aminoderivatives of the following polysaccharides were prepared in this manner: Dextran T-40, Dextran T- 110, Dexiran T-500, Dextran-2M and Ficoll.

Next, Step III, the aminoderivatized polysaccharide is covalently bound to the microspheres by use of a carbodiimide reagent. The washed microspheres are resuspended to 72 ml in 200 mM NaC1 containing the aminoderivatized polysaccharide at 3.75 mg/ml. 450 ul of DAC then is added to the suspension and the suspension roller-mixed overnight at room temperature. The microspheres are washed 3 times with 72 ml of water and resuspended to 72 ml in water containing 0.1% sodium azide.

Other microspheres coated with a polysaccharide by this method included 1.5 micron, 13% magnetic microspheres and 0.7 micron, 20% magnetic microspheres.

In Step IV, the polysaccharide coated micrcspheres WO 90/04178 PCT/US89/04467 -12are covalently bound to an immunoreactant by either Conjugation Procedure A or Conjugation Procedure B. A preferred method is by using thiolated immunoreactant and SMCC-treated microspheres as p.z-eviouslv descrcibed in Conjugation Procedure A.

Claims (16)

1. An immunoreactant carrier comprising: a particulate carrier polymer body; a biocompatible medium which is covalently bound to the peripheral surface of said carrier body and which is selected from the group consisting of a protein gel and an amine-derivatized polysaccharide, which medium does not interact or interfere with immunological reac- tions or components of biological fluid test samples and does not detrimentally influence the immunoreactants in- volved in an immunological reaction; and an immunoreactant selected from the group con- sisting of an antibody or an antigen member of a single binding pair, which member is covalently bound to said medium by means of a bifunctional reagent.

2. An immunoreactant carrier according to claim 1 wherein said amine-derivatizod polysaccharide is selected from the group consisting of an amine-derivatized dextran or an amine-derivatized Ficcll®.

3. The carrier according to claim 1 wherein said carrier body is a microsphere or a bead.

4. The carrier according to claim 3 wherein said carrier body is magnetic. The carrier according to claim 3 wherein said carrier body is polystyrene.

6. The carrier according to claim 1 wherein said im- munoreactant is labelled with one selected from the group consisting of a dye, an enzyme and a rad oactive element.

7. A method of preparing an immunoreactant carrier which has covalently bound thereto either an antigen or an antibody member of a single binding pair, said method comprising: coating the peripheral surface of a particulate carrier polymer body with one selected from the group consisting of a protein gel and an amine-derivatized polysaccharide by covalently binding said gel or polysaccharide to said surface, and 940317,q:\oper\ejb46259.28.13 14 covalently binding one member of said single binrtng pair to said protein gel or said polysaccharide coating said carrier body.

8. The method of claim 7 wherein said amine- derivatized polysaccharide is selected from the group consisting of an amine-derivatized dextran or an amine derivatized Ficoll@.

9. The method according to claim 8 wherein said car- rier is labelled with a detectable label selected from the group consisting of an enzyme, a dye and a radioac- tive element. The process for making an immunoreactant carrier having a biocompatible medium coated thereon, said proc- ess comprising: washing a particulate carrier body and suspend- ing the same in an aqueous solution; adding said biocompatible medium, which is selected from the group consisting of a protein gel and an amine-derivatized polysaccharide, to said carrier sus- pension of step and covalently coupling said medium to the peripheral surface of said carrier body by a chemical reaction between said medium and said surface to form said immunoreactant carrier; stopping the coupling reaction of step if necessary, by the addition of a coupling reaction stop- ping reagent; and separating and washing che immunoreactant car- rier of step which has a biocoimpatible medium coated on the carrier bodr; whereby the immunoreactant carrier of step may either be used upon completion of step for coupling with a member of a single antigen/antibody binding pair or may be stored for future use by suspension in aqueous 1% sodium azide and stored at about 4 0 C for subsequent use.

11. The process according to claim 10 -herein said amine-derivatized polysaccharide is selected from the group consisting of an amine-derivatized dextran or an amine-derivatized Ficoll@.

12. The process according to claim 10 wherein said carrier body contains functional groups reactive with the protein gel or the polysaccharide.

13. The process according to claim 10 wherein said carrier body is reacted with l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride prior to reaction with said protein gel or said polysaccharide.

14. The immunological carrier prepared according to claim 10 having a biological antibody or antigen attached thereto by reaction of the product of step with said biological antigen or antibody. A process for making an immunoreactant carrier having a gelatin coating thereon, said process comprising: washing a particulate carrier body with aqueous sodium chloride and suspending said carrier body in the same; reacting the suspended carrier body of step (a) with l-ethyl-3-(3-dimethylaminopropyl)carbodi. i .ide hy- drochloride and sonicating the same; adding a gelatin gel to the sonicated prod- uct of step and sonicating the product of step and mixing the same overnight to covalently couple said gela- tin to the peripheral surface of said carrier body by a reaction between said gelatin and the product of step adding glycine to the product of step to stop said coupling reaction; and washing the product of step with water to obtain said immunological carrier; whereby the product obtained after step may ei- ther be used immediately for coupling with a member of a single binding pair or may be suspended in 1% aqueous 16 sodium azide and stored at about 4 0 C for subsequent use.

16. A process for making an immunological carrier having an amine-derivatized polysaccnaride coating thereon, said process comprising: washing a particulate carrier body with aqueous sodium chloride and suspending said carrier body in the same; reacting the suspended carrier body of step (a) with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; adding a solution containing an amine- derivatized polysaccharide to the product of step (b) followed by the addition of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and mixing the resulting mixture overnight to covalently couple said polysaccharide to the peripheral surface of said carrier body by a reaction between said polysaccharide and the product of step and washing the product of step witn water to obtain said immunological carrier.

17. The process according to claim 16 wherein said amine-derivatized polysaccharide is selected from the group consisting of an amine-derivatized dextran or an amine-derivatized Ficoll@. INTERNATIONAL SEARCH REPORT International Application No. PC'rIUS8 9 /04467 1. CLASSIFICAT.1-N OF SUBJECT MATTER (it several classification symbols apply, indicate all) According to International Patent classif'ication (IPC) or to both National Classification and [PC IPC G01N 33/53,534,535,543-548,553 U.S. Cl. 435/7; 436/518,525,529,531,532,534,547; 422/57,58 11. FIELDS SEARCHED Minimum Documentation Searched I Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 9 Ill. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation of Document, ti with indication, where appropriate, of the relevant passages t2 Relevant to Claim No. 13 X A, 4,452,773 (MOILDAY), 5 June 1984, see 1-2,4-8,10,12- Y Abstract, columns 3-4, and column 5, lines 12-36. 14,16-18,27-31

23-26 x 1 4,253,844 (LDhE'), 3 March 1981, see 1 and Y column 1 la.55 to column 2, Jhne 65. 2-3,6-9,12-16, 19-23,25,27 X A, 4,210,418 (BRON), 1 July 1980, see Y column 1, line 63 to column 2 line 26, column 2, 1 and lines 40-55, column 3, lines 29-39, column 4, lines 2-3, 6-7, 9,111-

50-56, colummn 5, lines 31-67, and column 6, lines 13,15-16,19-23, 7-26. 25,27-28,31-32 A, 3,639,558 (CSIZMAS), 1 February1 1972, Y see Abstract, column 2, lines 40-48,coluimn 3, lines 2-3,6-16,19-23, 15-30, column 4, lines 31-41, column 7, lines 20- 25,27-28,31-32 47 and lines 56-63. Speciat categories ol cihed documents- %0 later document published atear the international riling date document defining the general state of the art which I% not or priority data and not in conflict with the application but considered to be of piarticular relevance cited to understand the principle or theory underlying the invention earlier document but published on or alter the International document of particular relevance; the claimed invention filin datecannot be considered novel or cannot be considered to document which may throw doubts on priority clairm(f) or involve an inventive step which Is cited to establish the publication date ot another document of particular reldiance: the claimed invention citation or other special reason (as specified) cannot be considered to Involve an inventive %teo when the documrent reivtring to an Oral disclosure. use, exhibition or document is combined with one or more other such docu- other means manta, such combination belnv obvious to ax person skilled "P document published prior to the international filing date but in the art. lter than the pfloiity date ctaimed document member of the same patent family WV. CERTIFICATION Oate of the Actual Completion ul the International Search Date of Meiling of this International Search Report 16 February 1990 07 MAR 1990 International Searching Authority Signature I Authoricd Dill e Form PCTRSD21O#aewtd ShW) (llsw.1 147)