CA1108048A - Sensitized erythrocytes stabilized with pyruvic aldehyde or dimethyl suberimidate - Google Patents

Sensitized erythrocytes stabilized with pyruvic aldehyde or dimethyl suberimidate

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Publication number
CA1108048A
CA1108048A CA304,707A CA304707A CA1108048A CA 1108048 A CA1108048 A CA 1108048A CA 304707 A CA304707 A CA 304707A CA 1108048 A CA1108048 A CA 1108048A
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Prior art keywords
erythrocytes
antigen
composition
stabilized
gonadotrophin
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CA304,707A
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French (fr)
Inventor
Michael A. Hirsch
Douglas S. Irvine
John Krupey
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Wyeth LLC
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American Home Products Corp
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G01N33/555Red blood cell
    • G01N33/556Fixed or stabilised red blood cell

Abstract

ABSTRACT
This invention related to immunologic compositions com-prising pyruvic aldehyde stabilized erythrocytes sensitized with an antigen by coupling of the stabilized erythrocytes to the antigen with bifunctional molecules, and to methods for preparing these compositions. Also disclosed are antiserum compositions which may be utilized with the described stable, sensitized erythrocytes of this invention as reagents in haemagglutination tests, and methods for their preparation. The invention further comprises lyophilization of these compositions and siliconized vials for use with the reagents of this invention.

Description

A~IP-6522 D8~
An early development in haemagglutination inhibition or passive haemagglutination tests was described by Wide and Gemzell, Acta Endocr., 35 26i (1960). They established that the red blood cells ~Rbc) coated by or attached to the antigen must be stablized to be useful in haemagglutination inhibition tests, otherwise the red blood cells would need to be fresh daily. While Wide et al.
stabilized the erythrocytes before attachment of the antigen, others have stabilized following attachment of the antigen.
Stabilization of Rbc was originally described by Boyden ustng formaldehyde, ~. Exp. Med., 93 107 (1951) and the concep+ was extended by Wide, Acta Endocr., supp. 70, 41 tl962), when following pretreatment of formaldehyde treated Rbc with tannic acid, it was shown that hCG could be adsorbed onto the cell surface of the Rbc~
Ling, Brit. J. Haemat., 7 229 (1961), also showed that pyruvic aldehyde could benefically replace formaldehyde and that pretreatment with tannic acid was not necessary for antigen attachment. However,for stabilization, Ling decreed that 48 hours at +4C was necessary. But, the coated erythrocytes are unstable and are also time consuming to prepare.
One approach to improving stability and also of obtaining erythrocytes wtth higher haemagglutination titres was disclosed in U.S. Patents 3,714,345; 3,715,427 and 3,925~541. There the èrythrocytes were treated sequentially with pyruvic aldehyde and then formaldehyde for periods in excess of 12 hours ~or each treatment prior to coating the thus stabilized erythrocytes with an antigen or antibody. The double aldehyde treated erythrocytes were in some cases further subjected to a lengthy freeze-thaw cycle at very low temperatures.

~0 q~, A~IP-6522 ~ 4~ ~

Another approach to obtain antigen sensitized erythrocytes was through the use of bivalent reagents as coupling agents between antigens and erythrocytes, e.g., bis diazotized benzidines illustrated in U.S. Patent 3,236,732; varjous diols ànd quinones described in U.S. Patent 3,322,634; toluene-2,4-diisocyantes described in Immunochemistry, 1, 43 (1964); etc. Since even the coupling coated erythrocytes are still readily susceptible to decomposition, formaldehyde treatment was usually employed prior to coating with antigen or simultaneously as described in U.S. Patent 3,987,159 a where the coupling agent employed was glutaraldehyde. In still a different approach described in U.S. Patent 3,991,175, glutaraldehyde was also utilized as a coupling agent but gelatin was used to stabilize the composition.
The various compositions and methods already noted and numerous others suffer individually from various disadvantages such as instability, lack of reproducibility false positive results, low haemagglutination titres, cost or time of preparation, and others.
It has now been found that the compositions and methods of this invention, demonstrated in part by use in haemagglutination tests, provide a superior grade of stabilized, sensitized cells. The composition of this invention have higher haemagglutinatton titres, are otable for long periods of time, and give reproducible p,atterns and resultsO Another advantage of the herein disclosed invention is the stabili-ty of results obtained. For example, in utilizing a commercially available reagent the haernagglutination patterns change with time. Thus, in a pregnancy test, a negative result, i.e~, complete haemagglutination within 2 hours, will change over 24 hours to show a quasi-inhibition pattern; results recorded from 2 - 24 hours at room temperature will therefore indicate a significant number of ~f~ AIIP-6522 false positi.ve results. I-lowever, utilizing the composition and methods of this invention, results are obtaine-l within 2 hours of tosting ancl will not change for about 11 days thereafter or more. Other advantages will become apparent as the description of the inventiol1 unfolds.
SUh~RY OF T~IE INVENTION
The present invention comprises an immunologic composition o.~ pyruvic aldehyde stabilized erythrocytes sensitiæed with a poly-I)eptide or glycoprotein antigen> said stabilized erythrocytes being l(~ coul)led to saicl antigen with a bifunctional molecule selected from glutaralclehyde, glyoxal, succinaldehyde, hexame-thylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydrochloride, dimethyl suberimidate, cyanuric chloride, tetrazotized o-anisidine, ancl l-ethyl-3-(3-dimethylamlnopropyl)carbodiimide, and the method of producing same.
Another aspect of this invention comprises an antiserum composition and a method for its production. A further aspect of this invention comprises the clisclosed composi-tions in lyophilized forms and their preparati.on and use in haemagglutination tes-ts. Yet another aspec-t of this invention comprises a siliconized vial containing the ~) reagel1t within wl1ich the haemagglutination test is performed.
An important embodiment of this invention relates to an im1null010gical composition comprising pyruvi.c aldehyde or dimethyl s~lbe~ te sta.bilized erythrocytes sensitized with a polypèptide or glycoprotein antigen selected from chorionic gonadotrophin, preg-7~ nant mare's serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotrophin and thyroid stimulating hormone, said stabilized erythrocytes being coupled to said antigen with a bifuJlctional molecule selected from glutaraldehyde, glyoxal, succinaldehyde, hexamcthylene diisocyanate, toluene ~,4-diiso-~O cyanate, diethyl malonimidate dihydrochloride, dimethyl suberimiclate, cyanuric chloricle, tetrazotized o-anisidine~ and l ethyl-3-(3-dime~hyl-~ AIIP-6522 aminopropyl~carbodiimide; with the proviso that when the erythrocytes are dimethyl suberimidate stabilized the b~.functional molecule is other thaIl dimetllyl suberim;date and that when the erythrocytes are pyruvic aldehyde stabilized, the bifuIlctiollal molecule is sclccted from glutaral-dehyde, glyoxal, succinaldehyde, hexame~hylene diisocyanate, toluene ~,4-diisocyanate and dimethyl suberimidate.
A preferred embodiment is an immunological composition comprising pyruvic aldehyde stabilized, the polypeptide or glycoprotein antigen is chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino embryonlc aIltigen, luteini~.ing hormone, follicle stimulating hormone, human meIlopausal gonadotropIlin or thyrokI stimulating hormone, and the bifunctioIlalmolecule is selected from glutaraldehyde, ~lyoxal, succinaldehyde, hexamethy-lene diisocyanate, toluene 2,4-diisocyanate, and dimethyl suberimidate.
Another preferred embodiment is a method for preparing the last-mentioned immunological composition, comprising:
~a) collecting animal blood directly into an isotonic sterile anticoagulant so that a final ratio of blood to anticoagulaIlt is from 0.8:1 to 1.2:1 ~v/v) of the resulting mixture wherein the erytllrocyte concentration packed cell volume of said blood is from about 15`~o to about 25% ~v/v);
~b) separating the erythrocytes from said mixture by centrif~lgation;
~c) washing said erythrocytes with normal saline and then suspending said erythrocytes in normal saline at a concentration of about 10 tD 30~0 ~v/v); r~:
(d) mixing the suspension of erythrocytes with a rnixture o~ pyruvic aldehyde, neutral buffered solution and normal saline to obtain a stabilization mixture;
~e) incubating the stabilization mixture of step (d) at 30 to 55C for one to five hours, and collecting the stabilized erythrocytes by centrifugation followed by was]~ g witll normal saline;

~f) suspending the stabilized erythrocytes in a neutral buffered solution of antigen; the antigen being selected from chorionic gonadotropllin, pregnan~ mare~s serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, folllcle stimulating hormone, human menopausal gonadotrophin and thyroid stimulating hormone;
~g) forming a sensitization mixture ~y thorough agitation of the suspension of stabilized erythrocytes antigen with a solution of a bifunctiollal molecule in saline; the bifunctional molecule being selectecl from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene cliisocyanate, toluene 2,4-diisocyanate and dimethyl suberimidate;
~h) collecting the sensitized erythrocytes by centrifugation and subsequently washing them with neutral buffered saline;
~i) suspending the sensitized erythrocytes in a neutral buffered saline containing normal animal serum wherein the complement of said serum has previously been fixed, said animal s~rum being selected from the group of rabbit, goat, horse and sheep.
DETAII,ED DESCRIPTION OF THÉ INVENTION
The present invention relates to an immunologic composition comprising pyruvic aldehyde stabilized erythrocytes sensitized with a ~() polypepticde or glycoprotein antigen selectecl from chorionic gonadotro-phin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, lut~inizing hormone, -follicle stimulati-lg hormone~ h~nan menopausal ~5 -4b-- A~IP-6522 gonadotrophin, and thyroid stimulating hormone, said stabilized erythrocytes being coupled to said an-i-igen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene-2,4-diisocyanate, bifunctional imido esters including diethylmalonimidate hydrochloride and dimethyl suberimidate, bis diazotized benzidine, cyanuric chloride, water soluble carbodiimides including l~ethyl-3-(3-dimethylamino-propyl)carbodiimide, and tetrazotized o anisidine. However, it is preferred to use as the bifunctional molecule for coupling glutaraldehyde, hexamethylene diisocyanate and dimethyl suberimidate.
The invention further comprises the descr,bed immunologic composition wherein the erythrocy-i-es are dimethyl suberimidate stabilized erythrocytesO In this composition the coupling agent or bifunctional molecule would include all the coupling agents previously described except for the same dimethyl suberimidate.
According to the present invention a composition of stabilized red blood cells (erythrocytes) sensitized with a poly-peptide or glycoprotein such as, human chorionic gonadotrophin (hCG~ is prepared as one component in a two component reagent system. Stabilization of the erythrocytes is firsi accomplished by taking freshly drawn whole blood, generally from sheep, although turkeys,goats or horses can be used, and immediately mixing it with an isotonic sterile anticoagulant, such as Alsever's solution, so that a final ratio of blood to anticoagulant is from 0.8:1 to i.2:1, preferably 1:1, of the resulting mixture~ The erythrocytes are then separated by centrifugation and washed with physiological saline. Pyruvic aldehyde reagent is prepared in physiological saline. A neutral buffered solution, preferably phosphate buffer, and a suspension of washed erythrocytes in saline are added to the pyruvic aidehyde reagent. The suspension is then incubated at 30 to 55C for one to five hours, preferably at 37C fortllree hours, with occasional agitation. Subsequently the stabilized red blood cells ar? t~loroughl~

_ " _ A~IP-6522 washed with saline. The pyruvic aldehyde stabilized cells (PAC) may then be stored at 4C as a 10~ suspension in saline containing 0.1~ sodium azide as a preservative, for at least a year, if desired. To sensitize the stabilized cells with hCG, the pyruvic aldehyde treated cells are washed with dilute saline and then suspended in a neutral buf~ered solutior, preferably phosphate buffer, containing hCG. To this suspension is added glutaraldehyde in normal saline. The mixture is then incubated, for example, at room temperature for about 2 hours under constant agitation and the cells are removed by centrifugation and washed not less than 3 times with neutral buffered saline, preferably phosphate buffered saline. Optionally, the sensitized cells are suspended in a nèutral buffered saline, preferably phosphate buffered saline, containing as little as 0.2% normal rabbit serum in which complement had previously been fixed by heating at 56C for about 30 minutes and from which non-specific agglutinins, interfering proteins, had previously been removed by serial adsorption with an equal volume of pyruvic aldehyde stabilized cells. Alternatively, normal serum from goats, horses or sheep may be used. Sensitized cells may then be stored at 4C. Again alternatively, in place of normal rabbit serum, one could use any properly treated stabilization agent including properly adsorbed, complement fixed serum proteins.
Illustrative of such stabilization agents are gelatin9 dextrans, polyvinyl pyrrolidone, albumins and water soluble carboxymethyl celluloses.

The h~ used for sensitization of the stabilized cells, illustrated above may be of various grades of purity. For example, material which assays, biologicalIy~ at approximately 2,500 I.U./mg may be employed. Alternatively, more purified hCG preparations, for example, a preparation assaying at approximately 10,000 I.U./mg or 14,000-16,000 1.U./mg may be utilized in the sensitization of the aforementioned stabilized red blood cells. Again the beta sub-unit of hCG may also be utilized as the antigen on the red blood cell. It is generally preferable to use the more purified hCG in the stabilized, sensitized erythrocyte compositions of this invention.
While the invention has been illustrated primarily using mammalian erythrocytes, such as those from sheep, these stabilized, sensitized erythrocytes may also be prepared from avian or reptilian red blood cells) which being nucleated settle more rapidly than mammalian erythrocytes. In this way the results from haemagglutination tests may be obtained within 30 minutes or sooner. The speed of the haemagglutination reaction is proportional to the buoyant density of the nucleated ce!l; thus as the buoyant density increases~ less ~0 time is required to obtain results. Furthermore, whereas th~ in invention was illustrated using Alsever's solution as the isotonic sterile anticoagulant, the freshly drawn whole blood may be optionally mixed witn heparin sodiumjEDTA, or sodium oxalate.
The described stabilized, sensitized erythrocyte compositions ~5 together with their antiserum compositions are most useful in haemagglutination and haemagglutination inhibition (passive haemagglutination) methods for determining the presence or absence of a particular antigen or abtibody in the test fluid being examined, usually a body fluid such as urine or serum. Thus determining ~0 the presence OF absence of a pregnant condition in a Temale animal can be based on a haemagglutination method for determining the presence or chorionic gonadotrophin which comprises mixing the erythrocyte ~ P-65~2 composit:ion of this invention with a chorionic gonadotrophin anti-ser~l and with the urine of test subject and, following a suitable incubation period, visually observing the results~ whereby if said gonadotrophin is not present agglutination o-E the erythrocytes occurs upon standing whereas if said gonadotrophin is presellt, no such agglutination occurs. A method for determing the presence of luteinizing hormone in a woman and thereby the day of ovulation may also bc done utilizing the compositions of this invention wherein a urine sample is concentrated by ultrafiltration and luteinizing hormone detected by immunological means~ the antigen being human chorionic gonadotrophin, as described ;n U.S. Patent No. 4~123,510, issued October 31, 197~ of M.L. Givner and U.K. Banik. Similarly, pregnancy in a mare may be determined utilizing as the antigen pregnant mare's serum gonadotrophin and a composition utilizing carcino embryonic antigen would be useful in a blood test for cancer. Other uses for the composition of tllis invention are described therein.
As illustrated below utilizing as the antigen, hCG, it is hypothesized that the superior properties of the compositions of this invention are clue to the coupling of thc stabilizecl erythrocytes to the hCG by mcans of <~ chemical reaction between the amino functiolls of the erythrocytes and the hC~ and the bifunctiollal glutaraldehyde.
The suggested course of reaction of glutaraldehyde is as follvws:

AHP- ~522 (a ) A I do I condensat i ons:
CHO
O~IC CH2 CH2 CH2 3 .OHC CH2 CH2 2 2 >
CHO CHO
2 2 CH2 CH=l-CH2-C=CH-CH2-CH -CH -CHO

"X"

CHO CHO

1 2 T ICHO ~ CH
C~2CH~ ~CH ~H~ 2 etc ~8~ AHP-6522 red cell (~) Cross-linking reactions: ~ /

CH0 C~H
~H-CH-CH -C=CH-CH -Red Cell-NH +hCG-NH + "X"
2 2 hCG-NH ~ ICHO CIH
(PAC) fH- ~ -CH

hCG-NH NH red cell The reactlon with hexamethylene diisoeyante proceeds in 1~ the ~ollowing fashion:

Red Cell-NH2+ 0=C=N-(CH2)6-N=C=0 + NH-hCG

Red Cell-NH-C-NH-(CH2)6-NH-C-NH-NH-hCG

urea derivative ~ 4 ~ Al-IP-6522 In order to conduct the passive haemagglutination tests contemplated by th:is invention it is necessary -to prepare an anti-ser~lm composition to be used in r.onjection with the antigen sensitized erythrocytes previously described. Depending O]l the test hCG which assays biologically as low as 2500 I.lJ./mg may be employed.
Altei~natively, more purified hCG preparations that assay at approxi-mately 1OJOOO I.U./mg, 15,000 I.U./mg or hig]ler may be empl~yed. It is preferrecl to utilize highly purified hC(; and most preferred to use the ~-s~lb~mit o hCG in preparing the an-tiserum of this invention to obtain greater specificity. An example of highly ~urified ~-IICG
and a method of producing same is reported in copending application Calladiall Patent Application Serial No. 304J688 (corresponcllng to l).S~
Patent No. 4,123,343~ issued October 31, 1978) of Krupey and Welchner, file~ of even date herewith.
The antiserum compositions are generally prepared by immunizing a host animal with the desired antigen thereby producing antibodies to thEIt antigen which may be obtained in the serum separated from the host ani~nal. Suitable animals for preparing the antiserum compositiolls include rabbits, goats, horses and shcep. ~ diffi.culty in ~() the past has been that the hCG antigen has had other antigen impurities presellt, thus mdesirable antibodies were also produced. A haemaggluti-nat:ioll method utilizing such an antiserum composition would thus possess ~mdesirable cross reactivity.
The antisera composition of this invention is diluted either in microtitre slides or ln a tube system titration to determine the maximum dilution of antisera which can still agglutinate the red blood cell. The dilution is ~hen adjusted when utilized in a method for determining pregnancy so that tilc anti-hCC seruM llas a sensitivi.ty to hC(, of about 100-150 m I.U./test. The acceptable antisera will ~0 also have a cross reactivity of less tharl 25% particularly against.
other glycoprotein horlDorle antigens. 'rllus an antisera sensitive -lL-~ ~ AHP-6522 to 100 m I.U./test of hCG would at the same time have a sensitivity to 400 m I.U./test or more of hLH or hCG. Ihis highly specific antiser~ compos.ition obtained using highly purifiedhCG as the antigen, ~ill detect early pregnancy via the presence of small amounts ofhCG and also minimizes the possibility of obtaining false positives in the passive haemagglutination inhibition test.
A method and apparatus for use of the herein described reagents is described in M.L. Givner et al., IJ.S. Patent No. 4,123,509, issued October 31, 1978 cmd in ~I.L. Givner and G. Schilling~ U.S. Patent No. 4,033,723, issued July 5, 1977.
While the compositions of th:is invention may be utilized in aqueous compositions, it is preferred to lyophilize these compositions for use in the particular haemagglutination method under consideration. The compositions may be lyophilized into two separate pellets by methods known to those skilled in the art such as that described in U.S. Patent 3,862,302. That disclosure, however, does not teach how to make lyophili~ed compositions containing in either tlle antiserum or the stabilized erythrocytes enough buffer for comple~ing the interfering calcium ions present in urine. Rather it involves the use of a third lyophilized pellet containing the neccssary buffers or as in a commercially available pregnancy test a separate buffer composition which is reconstituted at the time of its use .

~ AHP-6522 The compositions of this invention may also be lyophilized into a single layered cakc, as dcscribed in U.S. Patent 3,269,905.
Also while a suitable chelating agent may be included within the lyophili2e~ compositions of this invention to remove quantities of calcium present in a urine test sample, the compositions of this inven-tion without added chelating agent may be utilized with urine and/or ultraconcentrated urine which has been passed through a filter in which is placed a styrene divinyl benzene copolymer containing imide acetate functional groups (CIIF~LEX* 100 or DOWEX* CIIEI.ATING RESIN ~-l).
The physical characteristics of the vial ln which the immunologic reagents e.g., the Rbc preparation described in this invention together with a suitable antiserum and appropriate buffers whicll are to be used in passive haemagglutination test, must be properly defined. Pyrex* culture tubes have been subjected to various physical pretreatments prior to utilization of the immunologic reagents, described herein in passive haemagglutination tests. We have found that round bottomed siliconized glass tubes having an I.D. of 12-16 mm is preferable in order to obtain reproducible flocculation times and reproducib]e agglutination patterns.
The invention is further illustrated in the followlng e~amples:

In the examples, ratios and percentages designate a volume to volume relationship unless otherwise noted.

*Trade mark ~ A~IP-6522 EXAMPL~ 1 Whole sheep blood was collected directly into Alserver's solution so tha~ the final ratio of sheep blood to Alserver's solution was l:l. (For the purpose of this invention it has been establized -that the Rbc concentration or packed cell volume (pcv) of such a mixture must be in the range of 15-25% (i.e., a hematocrit of 30-50~0) and preferably, 15 to 25%, o~ more preferably 22%.) The cells from 100 ml of such a mixture were L0 collectcd by centrifugatioIl and washed three ~imes with Ilorl~al, saliIle solution, i.e. 0.9% (w/v) sodium chloride in distilled water. The preferred volume of saline per wash is 320 ml but may be in the range of 100-500 ml. After washing, tlle cells are suspended in normal saline solution at a concentration of 20%. A mixture of pyruvic aldehyde (64 ml of 25% a~ueous solution) and normal saline (120 ml) was adjusted to p~I 7.0 using a 10% (w/v) aqueous sodium carbonate solution. To this mi~ture of pyruvic aldehyde and saline was added the above-mentioned cell suspension (lO0 ml at 20%), followed by phosphate buffer ~28 ml., 0.15 M, p}I 8.0). This stabilization mixture was then incubate(I at 37-C for 3 hours, the mixture being shaken vigorously once every 30 minutes.

$~3 The cells were then collected by centrifugation and ~ashed 4 tilIIes with normal saline solution utilizing preferably 320 ml of saline per wash. The stabilized cells may then be stored as a suspension, preferably lO~, in normal saline con-taining, for example, 0.1% (w/v) sodiuIll a~ide as a preservative.This is stable for at least a year at -~4C.

Stabilized cells as described in Example 1 (10 ml of about a 10o suspenslon) were collected by ccntrifugation and I0 ~ashcd 3 times with normal saline solution, preferably 40 ml for each ~ash. ~lternatively, one may of course use unstored cells directly from Example 1. These washed cells were then suspended in a solution of hCG ~100 ~g in 100 ~1 of 0.5 M phosphate bufEer~
contained in 0.15 M phosphate buffer (8.65 ml, pH 7.4). To this suspension was added a glutaraldehyde reagent (1.75 ml), prepared by dilutiTIg a 25% aqueous solution of glutaraldehyde (1 ml) in normal saline (9 ml).
TIle sensitization mixture was thoroughly agitated and then gently mixed for 2 hours at room temperature. The cells were collected by cen-trifugation and washed, preferably 4-5 times, witIl 0.15 M phospIlate buffered saline (pl-I 7.4).
The sensitized cells are then suspended in ().15 M phosphate bufered saline (3~ ml, pll 7.4) containing 0.2~ of normal rabbit serum in \~hich complement had previousLy been fixed by incubating at 56~C for 30 minutes and from which non-specific agglutinins had been absorbed by triple trea~ment with stabilized cells (~xample 1).
The removal of non-specific agglutinins is illustrated in Example 3.

EXAMPI.E 3 Prior to use for the removal of non-specific agglutinins from normal rabbit serum, stabilized cells as prepared in E~ainple l, ~Yere washed ~Yith norInal saline (5 volumcs saline: l volume of s ln% cell suspension) and resuspended in saline solut:ion at 10%.
E~ual volumes of rabbit serum and a 10% suspension of stabilized red blood cells were then incubated at room temperature for 30 minutes. TIIe cells were collec~ed by centrifugati.on, d:iscarded and the process repea~ed 2 more times, using collected cells as opposecI to a 10% suspension.
Alternatively, the last adsorption may be allowed to proceed at t4C for 18 hours. Under the conditions describe(I in this example, the normal rabbit serum has been diluted l:l with saline.
It should be understood, however, that for this adsorption it is possiblc to util:ize collectcd cells as opposed to 10% slIspcnsion whieh would not result in a dilution of the normal rabbit serum.

Utilizing the methods of Example 2, but with pregnant mare's ser~ gonadotrophin as the antigen, a stable sensitizecl composition 20 W.IS also prepared alld satisfactorily evaluated.
F:XAMPLE 5 LyopIIilized cakes were prepa~ed as follows. A suspension of the stabilized, sens:itized red blood cells (S-PAG(:) was washed ~bout 3 times with a 0.15 M phosphate buffer solution adjusted to a pH of 7.4~ The washed S-PAGC were then resuspended as a 0.415% (v/v) s-Ispenslon in lyophilizatioIl buffer, I,L3, (L13 used was lOg sucrose, lO ml of 1% ~w/v) merthiolate, l() ml of NRS triply adsorbed, ~16-~ AHP-6522 and c~.s. to l liter witll 0~15 M phosphate buffered saline containing about 0.2% EDTA ~w/v) to a p~I of 7.0). 300 ~l of the cell suspension were then pipetted into differeJlt siliconized vials already in a -test -tube rack, and the rack containing thc vials was immersed in an acetone-dry ice bath at about -70C and fro7,en.
Dilutions of antisera and NRS, ~w}lich had been pretitrated and adjusted to give a predetermined sensitivity) were made with LB such that the roper concentra-tioIl of antisera in NRS is containcd in a 200 ~1 aliq~Iot, was then pipetted onto the frozen cell layers in the batI~.
The frozen reagents may then be stored at about -lOO~C
until lyophilization or lyophilized immediately. I,yophili~ation of these reagents was then done in a freeze drier for at least 18 hours at about 75-200 microns Hg, while gradually reaching ambient temperatures. The vacuum was not permitted to be less than 75 microIls to avoid spontaneous haemagglutination. T]le lyophilized reagents wheIl stored at 4C are stable for at least 9 months.

Lyophili7,ed pellets were prepared as follows:
A suspension o~ S-P~GC was washed as in Example 5 and 7 thcn resuspended as a 2.5% suspension in LB concentr~ted by a factor of 5 in all its components. This latter suspension was constantly agitated while a proportioning pump repeatedly delivered about 50~1 of the S-PAGC s~Isl)ension into a liquid N2 bath at -196C and the pellets were harvested in a petri dish.

The antisera pellets were prepared similarly but may optionally have incorporated within the LB 2.5% ~w~v) of polyvinyl pyrroliclone or do~tran. The pellcts may be stored as the cakes are stored ln E~ample 4 or transferred immediately to the freeze drier.
Pellets llave been dried satisfactorily at about, 50-200 microns ~Ig for about 18 hours, with the temperature finally reaching room temperature or in some cases 3.~C. The pellets were stored in a dessicator at room temperature until packing into siliconized vials. These pellets have been found to maintain their sensitivity and physical characteristics in long term stability studies at both 30C and 4C.

Pretreatment with silicone is most beneficial as e~emplified in E~cample No. 8. The method used for pretreatment with silicone is described under e), whilst other pretreatments, usecl for comparative purposes, are described in a) to cl).
a~ Untreated tubes used clirectly from the manufacturer.
b) Pyrolyzed tubes prepared by heatlng at 560C (not more than 5 minutes).
c) BSA coatecl tubes i) w~sh in 1% ~w/v) bovine serum albumin with agitation, 2n sec.
ii) rinse in running ~listilled water, 3 times.
iii) dry overnight at 60C.
d) BSA-coated acid washed tubes i) chromic-sulfuric acid wash, overnight soak.
ii) rinse in running tap water, 3 times.
iii) soak in 95% ethanol - one hour, 2 changes.
iv) rinse in running tap water, 2 times.

A~IP-6522 v) rinse in running distilled water, 3 times.
vi) dry overnight at lOO~C. ~.
vii~ wash in 1% BSA ~w/v) with agitation, 2~ sec.
viii) rinse in running distilled water, 3 times.
ix) dry overnight at 60C.
e) Siliconized tubes i) wasll in 1% ~v/v3 SILICLAD* ~Clay Ad~ms)s a silane in aqueous alcohol solution, with agitation, 20 sec.
ii) r.inse in running distilled water, 6 times.
iii) dry overnight at 100C.
E~AMPLE 8 The following results were observed with the pretreated tubes of Example 7.
i) Untreated, pyrolized, BSA-treated and acid washed BSA-treated vials lead to collapsed cell matts, or matts of rough appearance, when a haemagglutinstion pattern should result using the reagents of this invention.
ii) With the vials pretreated as in ~i) above, the transition from haemagglutillation to inhibition of haemagglutination is irregular :iii) Vials pretreatecl with silicone demonstrate smooth haemagglutination patterns.
iv) In vials pretreated with silicone, the transition from haemagglutination to inhibition of haemagglutination, in the presence of increasing amount of antigen, is regular and the degree to which haemagglutination is inhibited is proportional to the increasing amount of antigen present.
A method for preparing the antiserum compositon of this invention is illustrated in the following example.

*Trade mark ~19-Antiqen ~-hCG subunit Initial Challenqe Solution (ICS) Tubercle bacillus 5.0 mg ~-hCG 200 mg saline 2 ml complete Freund's adjuvant 2 ml Subsequent Challenae Solution (SCS) ~-hCG 1.0 mg saline 5 ml complete Freund's adjuvant 5 ml Booster Solution (BS) ~-hCG 100 ~9 saline I ml Each of the ICS and SCS are homogenized well until the suspensions are thick and creamy.
Immunization Protocol Day I - Three month old virgin female New Zealand white rabbits are injected with 2.0 ml of "ICS" at 30-50 intradermal sites.
Five hundred ~1 of crude Bordetella pertussis vaccine is injected at a separate intradermal site.

~5 Day 14 - Each rabbit is bied from the marginal ear vein.
Day 15 - Each rabbit is injected with a total of 1.0 ml "SCS'~
in the hind foot pads.
Days 22, 29, 36, 43 - Each rabbit is injected with a total of 1.0 ml "SCS" in multiple intradermal sites.
Day 49 - Each rabbit is injected with 1.0 ml "BS" intravenously in the marginal ear vein.
Day 56 - Each rabbit is bled from the marginal ear vein.
Thereafter, one ml of "BS" is injected intravenously every 0 6 weeks and the rabbits bled 5-7 days after each booster. The total time necessary to produce an antiserum using this combined multiple intradermal site-foot pad method of immunization will vary and is considered complete when the antibody titre which is constantly being monitored reaches a plateau.
The antiserum is separated from the rabbit blood and complement may be fixed and the suspension adsorbed as described with the S-PAGC composition in Example 2 and Example 3, a single adsorption usually being sufficient. Undiluted antisera or diluted antisera may be stored preferrably at -100C in small aliquots.

Claims (40)

The embodiments of this invention in which an exclusive property or privilege is claimed are defined as follows:
1. An immunologic composition comprising pyruvic aldehyde or dimethyl suberimidate stabilized erythrocytes sensitized with a polypeptide or glycoprotein antigen selected from chorionic gonadotro-phin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotrophin and thyroid stimulating hormone, said stabilized erythrocytes being coupled to said antigen with a bifunctional mole-cule selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydro-chloride, dimethyl suberimidate, cyanuric chloride, tetrazotized o-anisidine, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; with the proviso that when the erythrocytes are dimethyl suberimidate stabilized the bifunctional molecule is other than dimethyl suberimi-date, and that when the erythrocytes are pyruvic aldehyde stabilized the bifunctional molecule is selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate and dimethyl suberimidate.
2. A composition as claimed in claim 1 wherein the erythrocytes are pyruvic aldehyde stabilized, the polypeptide or glycoprotein antigen is chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotrophin or thyroid stimulating hormone, and the bifunctional molecule is selected from glutaraldehyde, glyozal, succinaldehyde, hexamethylene diiso-cyanate, toluene 2,4-diisocyanate, and dimethyl suberimidate.
3. The composition of claim 1, wherein said erythrocytes are selected from mammalian, avian, and reptilian erythrocytes.
4. The composition of claim 1 or 2 in which the antigen is chorionic gonadotrophin.
5. The composition of claim 1 or 2 in which the antigen is human chorionic gonadotrophin.

?
6. The composition of claim 1, wherein said antigen is chorionic gonadotrophin and said bifunctional molecule is glutaral-dehyde.
7. The composition of claim 1, wherein said antigen is chorionic gonadotrophin and said bifunctional molecule is hexamethylene diisocyanate.
8. The composition of claim 1, wherein said antigen is chorionic gonadotrophin and said bifunctional molecule is dimethyl suberimidate.
9. The composition of claim 1, wherein said stabilized, antigen sensitized erthrocytes have been treated with phosphate buffered saline containing a serum protein in which complement has previously been fixed and from which non-specific agglutinins has previously been removed by serial adsorption with pyruvic aldehyde stabilized erythrocytes.
10. The composition of claim 1, wherein said stabilized antigen sensitized erythrocytes have been treated with phosphate buffered saline containing a member selected from gelatin, dextrans, polyvinylpyrrolidine, and water soluble carboxymethyl celluloses which have previously been treated by adsorption with pyruvic aldehyde stabilized erythrocytes.
11. The composition of claim 1 or 2 which is lyophilized.
12. The composition of claim 6 which is lyophilized and wherein said chorionic gonadotrophin is human chorionic gonadotrophin,
13. The composition of claim 7 which is lyophilized and where-in said chorionic gonadotrophin is human chorionic gonadotrophin,
14. The composition of claim 8 which is lyophilized and where-in said chorionic gonadotrophin is human chorionic gonadotrophin,
15. The composition of claim 1 which is lyophilized and wherein said antigen is pregnant mare's serum gonadotrophin and said bifunctional molecule is glutaraldehyde.
16. A method for preparing an immunologic composition of animal erythrocytes sensitized with an antigen, as claimed in claim 2, useful in passive haemagglutination tests, which comprises:
a) collecting animal blood directly into an isotonic sterile anticoagulant so that a final ratio of blood to anticoagulant is from 0.8:1 to 1.2:1 (v/v) of the resulting mixture wherein the erythrocyte concentration packed cell volume of said blood is from about 15% to about 25% (v/v);
(b) separating the erythrocytes from said mixture by centrifugation;
(c) washing said erythrocytes with normal saline and then suspending said erythrocytes in normal saline at a concentration of about 10 to 30% (v/v);
(d) mixing the suspension of erythrocytes with a mixture of pyrovic aldehyde, neutral buffered solution and normal saline to obtain a stabilization mixture;
(e) incubating the stabilization mixture of step (d) at 30 to 55°C for one to five hours, and collecting the stabilized erythrocytes by centrifugation followed by washing with normal saline;
(f) suspending the stabilized erythrocytes in a neutral buffered solution of antigen; the antigen being selected from chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human meno-pausal gonadotrophin and thyroid stimulating hormone;
(g) forming a sensitization mixture by thorough agitation of the suspension of stabilized erythrocytes antigen with a solution of a bifunctional molecule in saline; the bifunctional molecule being selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate and dimethyl suberimidate;
(h) collecting the sensitized erythrocytes by centrifugation and subsequently washing them with neutral buffered saline;
(i) suspending the sensitized erythrocytes in a neutral buffered saline containing normal animal serum wherein the complement of said serum has previously been fixed, said animal serum being selected from the group of rabbit, goat, horse and sheep serum
17. The method of claim 16, wherein said neutral buffered solution in step (d) is phosphate buffer having a pH 8.0, said neutral buffered solution in step (f) is phosphate buffer having a pH 7.4, and the neutral buffered saline in step (i) is phosphate buffered saline having a pH 7.4.
18. The method of claim 16,wherein the animal erythrocytes are sheep, goat, horse or turkey erythrocytes.
19 A method as claimed in claim 16, wherein the neutral buffered saline in step (i) is phosphate buffered saline having a pH
of about 7.4.
20. A method as claimed in claim 16, wherein in step (a) the isotonic sterile anticoagulant is Alserver's solution, the final ratio of blood to Alserver's solution is about 1:1 and the erythrocyte concentration cell volume is from about 20% to about 25%.
21. A method as claimed in claim 20, wherein in step (c) the concentration of the erythrocytes in normal saline is about 20%.
22. A method as claimed in claim 21, wherein in step (d) the erythrocytes are mixed with a buffered mixture of pyruvic aldehyde and normal saline, buffered at about pH 8Ø
23. A method as claimed in claim 22, wherein in step (e) the incubation is effected at about 37°C for about 3 hours.
24. A method as claimed in claim 23, wherein in step (f) the neutral buffered solution is a phosphate buffered solution.
25. A method as claimed in claim 24, wherein after agitating the suspension of stabilized erythrocytes antigen with the solution of bifunctional molecule in step (g), the sensitization mixture is mixed for about a further 2 hours at room temperature before collecting the sensitized erythrocytes.
26. A method as claimed in claim 25,wherein in step (i) the neutral buffered saline is phosphate buffered saline and the animal is rabbit.
27. A method as claimed in claim 26, wherein the antigen is human chorionic gonadotrophin.
28. A method as claimed in claim 26 or 27, wherein the bifunc-tional molecule is glutaraldehyde, hexamethylene diisocyanate or dimethyl suberimidate.
29. A method as claimed in claim 16 or 17, wherein the erythrocytes are mammalian, avian or reptile erythrocytes.
30. A method as claimed in any one of claims 16, 17 or 18, wherein the composition is lyophilized.
31. A method for determining the presence of human chorionic gonadotrophin (hCG) human body fluid in which comprises mixing the com-position of claim 1 with a highly purified hCG antiserum and with ultra-filtration concentrated urine of said human whereby if hCG is not present agglutination of the erythrocytes occurs upon standing whereas if hCG is present no such agglutination occurs.
32. The method of claim 31, wherein said antiserum is a .beta.-hCG specific antiserum.
33. The method of claim 31, wherein said antiserum is adjusted to have a sensitivity to hCG of 100-150 m.I.U. per test and a cross reactivity to other glycoprotein hormone antigens of less than 25 percent.
34. An antiserum composition to hCG for use in a haemag-glutination test with the immunological composition of claim 1, which has been prepared using a highly purified hCG as the immunizing antigen said composition having a cross reactivity to other glycoprotein hormone antigens of less than 25 percent.
35. The antiserum composition of claim 34 which has been lyophilized.
36. The composition of claim 34, wherein said highly purified hCG is highly purified .beta.-hCG.
37. The composition of claim 36 which has been lyophilized.
38. The method of claim 31 which is conducted within a siliconized, round bottom, glass vial having an inner diameter of about 12-16 millimeters.
39. The method of claim 38, wherein each of said compositions have been lyophilized into pellets.

?
40. The method of claim 39,wherein said compositions together have been lyophilized into one stratified cake.
CA304,707A 1977-06-14 1978-06-02 Sensitized erythrocytes stabilized with pyruvic aldehyde or dimethyl suberimidate Expired CA1108048A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4780312A (en) * 1985-06-04 1988-10-25 National Institute Of Immunology Birth control vaccine

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5665044A (en) * 1979-10-31 1981-06-02 Dainippon Ink & Chem Inc Carbon fiber-reinforced resin composition
JPS56135549A (en) * 1980-03-26 1981-10-23 Dainippon Ink & Chem Inc Polyarylene sulfide resin composition
EP0039195B1 (en) * 1980-04-28 1986-06-18 Montefiore Hospital and Medical Center Antibody detection process
US4543339A (en) * 1982-03-16 1985-09-24 Oneill Christopher Early pregnancy detection by detecting enhanced blood platelet activation
GB2138132B (en) * 1983-04-12 1987-02-18 Robin Royston Amos Coombs Storage-stable antibody-linked erythrocytes
EP0134868A1 (en) * 1983-08-26 1985-03-27 Anda Biologicals Unicellular organisms activated by glutaraldehyde as solid carriers for sensitizing agents and uses of sensitized unicellular organisms
DE3428984A1 (en) * 1984-08-07 1986-02-20 Bayer Ag, 5090 Leverkusen METHOD FOR THE PRODUCTION OF HIGH MOLECULAR POLYARYL SULFIDES, BRANCHED IF NEEDED
US4900685A (en) * 1987-01-29 1990-02-13 Cytosignet, Inc. Analyte detection in particulate-containing samples
US5494800A (en) * 1987-01-29 1996-02-27 Cytosignet, Inc. Analyte detection in particulate-containing samples
JPH01107154A (en) * 1987-10-20 1989-04-25 Seitetsu Kagaku Co Ltd Processed red blood-corpuscle and its manufacturing method
US4910294A (en) * 1988-06-20 1990-03-20 Idemitsu Petrochemical Company Limited Two-stage process for production of polyarylene sulfides with lithium compound
US11722295B2 (en) 2020-04-30 2023-08-08 Musarubra Us Llc Methods, apparatus, and articles of manufacture to securely audit communications

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3639558A (en) * 1968-02-19 1972-02-01 Louis Csizmas Immunological reagent particles having proteinaceous materials covalently bonded thereto
US3991174A (en) * 1970-07-20 1976-11-09 Rafa Laboratories Ltd. Method of determining concentration of luteinizing hormone in body fluid
FR2187909A1 (en) * 1972-06-08 1974-01-18 Merieux Inst Stable antigens - by freeze-drying antigen bonded to red blood cells
US3914400A (en) * 1973-05-21 1975-10-21 Us Health Stable antigen-erythrocytes for measuring antibodies against toxoplasma organism

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4780312A (en) * 1985-06-04 1988-10-25 National Institute Of Immunology Birth control vaccine

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