CH629894A5 - Process for separating the free fractions from the bound fractions in an immunoassay procedure - Google Patents

Description

629,894

2

claims

1. Method for separating the free fractions from the linked fractions in an immunoassay operation in which a solution is brought into contact with a composite comprising a matrix of polysaccharide derivative covalently coupled to an antibody by a bifunctional coupling agent, and the solution containing the free fractions of the reacted composite is separated, characterized in that said bifunctional coupling agent is chosen from those of the following formula:

+ nh, + nh2 He "He

H2e + 1CeO-C- (CH2) „- C-OCeH2e + 1 (I)

where n is an integer having the value 1 to 6 and e is equal to 1 or 2.

2. Method according to claim 1, characterized in that said matrix of Polysaccharide has a maximum average dimension in the wet state of 1 to 18 [x.

3. Method according to claim 1, further characterized in that said method is a solid phase column immunoassay method.

4. Method according to one of claims 1,2 and 3, characterized in that said immunochemical composite has the following formula:

OH

I

polysaccharide-0-CH2-CH-CH2-NH- (CH2) m- matrix

+ nh2 + nh,

He II

NH-C- (CH2) n-C-NH-antibody of primary antibody and in that a labeled antigen is incubated and the sample on said column of primary antibody.

13. Method according to one of claims 1,2 and 3, characterized in that said immunoassay method is a radioimmunoassay method.

14. Method according to claim 1, characterized in that said matrix of Polysaccharide derivative comprises a matrix of activated Polysaccharide coupled to an α, o-diamino-spacer via one of the amino groups of α, "A-diamino-

îc spacer.

15. The method of claim 14, characterized in that said immuno-chemical composite has the following formula:

polysaccharide-0-CH2-CH-CH2- matrix

where m is an integer with the value 1 to 12.

16. Method according to claim 15, characterized in that the said polysaccharide matrix is chosen from the following:

cellulosic polymers, dextran polymers, agarose and their derivatives where m is an integer having the value 4 to 6 and n is an integer having the value 4 to 6.

17. The method of claim 16, characterized in that said antibody is a secondary antibody.

+ nh2 + nh2 Il II

NH- (CH2) m — NH-C— (CH2) n-C — NH-antibody

The subject of the present invention is a method of separation where m is an integer having the value 1 to 12. of free fractions of linked fractions in a printing operation.

5. Method according to claim 4, characterized in that muno-dosing.

said Polysaccharide matrix is chosen from the following: The solid phase radioimmunoassay (RIA) has had cellulosic polymers, dextran polymers, agarosuccess because, because the antigen-antibody reaction and the se and their derivatives where m is an integer having the value 4 to 40 separation of free and bound antigens can be obtained in a

6 and n is an integer having the value 4 to 6. only step, we obtain not only a radioimmunoassay

6. Method according to claim 5, characterized in that simple and rapid but that eliminates a certain number of errors said matrix of Polysaccharide is chosen from the following: handling and others which are inherent in other techniques

cellulosic polymers and their derivatives having a separation dimension. Catt et al., Biochem. J., 100: 31c (1966) maximum ion in the wet state from 10 to 15 | x. 45 originally used as solid phase materials of polymers

7. Method according to claim 5 or 6, characterized in that sprayed carrying reactive thiocyanato groups (-N = C = S)

that said antibody is a secondary antibody. capable of forming covalent bonds with antibodies.

8. Method according to claim 7, characterized in that an antibody coupled to particles of dextran and of cellulose tube containing said composite remains practically immobile activated with cyanogen bromide have become fashionable during the incubation step . 50 continuation of the work of Wide, Porath, and Axen [Wide et al., Bio-

9. Method according to claims 3 and 7, characterized in this ehem. Biophys. Acta, 130: 257 (1966), Axen et al., Nature that a primary immunoassay reaction is carried out outside (Lond.) 214: 1302 (1967) and Wide, Acta Endocrinol. (Copen-

said column, then in that an aliquot part of said hagen is incubated) Suppl. No. 142: 207 (1969)]. Alternatively, Ternynck and reaction mixture on said column of second antibody al., F.E.B.S. Letts. 23:24 (1972) used glutaraldehyde in the solid phase. 55 as a two-stage bi-functional reactive body for coup-

10. Method according to claim 9, characterized in that a 1st of the antibodies to the amido groups of the Polyacrylamide.

excess of the second antibody is present on said H-column is well established that the effectiveness of solid phase adsorbent substances. by affinity, increases a lot when hydro-

11. Method according to claims 3 and 7, characterized in that spacing carbides for separating the ligand from the solid matrix

that the said columns of second antibody in solid phase eo from [Cuatrecasas et al., Proc. Nat. Acad. Sci. U.S., 61: 636

are loaded with a primary antibody, a labeled antigen and (1968)]. It is believed that the spacer increases the flexibility and flexibility of the sample and in that both the so-called primary reaction and mobility of the ligand, which allows free access of the protein to the said incubation step with a solid preparation of a double ligand.

xth antibody continue simultaneously in space vi- Knowing this, Cambiaso et al., Immuno-chem., 12: 273

from the column. 65 (1975), coupled gamma globulin fractions to

12. Method according to claims 3 and 7, characterized in that amino-hexyl derivatives of sepharose-4B activated with glutaraldehy-that the primary antibody is incubated on said second column as indicated in the formula below and they have shown that the solid phase antibody thus producing a column this process provides useful immunosorbents.

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NH

-0-C-NH- (CH2) 6-N = CH- (CH2) 2-CH = N-Protein

It remains to be established whether purified antibodies or an antibody contained in gamma globulin fractions coupled to sepharose by this method will prove to be more effective for the binding of an antigen than the same antibodies coupled to sepharose by means of the cyanogen bromide process. In this regard, Bolton et al., Biochemica and Biophysica Acta, 329: 318 (1973), reported that recovery of antibody activity tends to be higher on solid preparations of antisera against haptens and small peptides activated with cyanogen bromide only for analogous preparations in solid phase of antisera to proteinaceous hormones with high molecular weights:

NH

II

—O — C -NH-Protein

Although it may seem reasonable that solid phase preparations of anti-sera against large molecules produced by methods analogous to those of Cambiaso et al., Above, may provide better isolation of the activity of antibodies as solid preparations produced without a spacer, there are two disadvantages in the chemical processes currently used to covalently bind antibodies to solid matrices. Firstly, in many cases, the exact nature of the chemical reactions is not well established and secondly the immobilized antibodies can be linked to the solid matrix in a statistical manner and thus the possibility that the coupling involves an amino acid residue " essential ”increases.

Already in 1959, Ludwig et al., Abst. 135th Meeting Am. Chem. Soc., 44c (1959) reported the reaction of substituted imido-esters having a and E-amino groups typical of gly-cylglycine and e-amino ~ caproic acid to form amidines:

+ NH2

II

R-C-OET + Pr ± NH3— ”

+ NH2 II

R-C-NH-Pr + ETOH + H +

These authors said that the reaction continues rapidly in an aqueous solution close to a neutral pH even at 1 ° C., and that under these conditions, the reacting body which is the imido-ester does not react with model compounds containing sulfhydryl groups, phenolic groups, imidazole groups or peptide bonds. In addition, they noticed that the net charge of the peptide is unchanged over almost the entire pH range, the amidino function having a pk value close to 12.

In 1963, Wofsky et al., Biochem., 2: 104 (1963), amidinated various proteins and found that the reaction was very specific for lysine residues. In addition, they reported that widespread amidination produced very few detectable effects on the biological activity of antibodies. They concluded that lysine is not critically involved in the reactive sites of any of the antibodies tested (anti-bovine serum proteins) (anti-BSA), (anti-benzene arsenate, anti-D-benzoylaminophenyl acetate, anti-SIII, anti-DNP, and anti-ß-lactoside).

Later in 1966, Dutton et al., Biochem. and Biophys. Res. Comm., 23: 730 (1966), have extensively modified anti-DNP antibodies with the crosslinking agent which is diethyl malonimidate dihydrochloride and have similarly reported quantitative retention of activity. the antibody according to the diagram below:

-cih2n + + nh2ci-II II

xo 2-Antibody- + NH3 + QH5-O-C CH2-C-0-C2H5-h>

+ nh2 + nh2

15

Antibody-NH-C-CH2-C-NH-Antibody + 2C2H5OH + 2HC1

Although the work of Ludwig et al., Wofsky et al., And Dutton et al. are known in what would be called in scientific circles a significant number of years, no one has transferred their work in the field of immunoassay procedures. One of the reasons for this may be that qualified technicians prefer not to use loaded derivative dies. For example, Wilchek et al., Molecular and Cellular Biochemi-25 stry Vol. 4, No. 3, p. 181 (1974) report that derived and positively charged arrays cause nonspecific adsorption and therefore are not advantageous for use in immunoassay methods. Therefore, those of ordinary skill in the art may have considered that the use of reactants which are the above imido-esters is disadvantageous in that these reactants would produce positively charged solid phase immuno-chemical composites.

The end point of all competitive binding analyzes involves determining the relative proportion of antigen (or hapten) that is free and antigen (or hapten) that is bound to the saturable binding agent. . The basic separation techniques commonly used, involve the differential migration of bound and free fractions (i.e. chromato-elec-trophoresis on paper, gel filtration), adsorption processes (c ' i.e. carbon, silicate), fractional precipitation (i.e. ammonium sulfate, polyethylene glycol), and a double antibody method. According to Ratcliffe, Br. Med. Bull., 30:32 (1974), an ideal separation technique should meet the following criteria:

45 A. It must completely separate free and linked fractions with a wide range of errors under the conditions used for separation.

B. It should be simple, fast, inexpensive, and use reagents and a readily available device.

n / a C. It should not be affected by plasma or serum.

Furthermore, Ratcliffe notes that for the general clinical application all the manipulations must be carried out in a single tube, they must be suitable for automation and must be applicable to a wide range of antigens or 55 of haptens (it i.e. small peptides and steroids as well as high molecular weight proteins).

Although the double antibody process for the separation of free and bound fractions in radioimmunoassay (RIA) systems is commonly one of the most widely used separation techniques and, under optimal conditions, it satisfies most criteria mentioned above, it nevertheless has certain inherent disadvantages. See Ratcliffe, supra; Seki et al., Endocrinol. Japan, 20: 121 (1973); & Koninckx et al., Acta Endocrinol., 81:43 (1976). As long as a carrier protein has to be added, large amounts of selected precipitation antibodies are required and thus the process is expensive. It takes a considerable time for the immuno-precipitation reaction to reach equilibrium (24 to 48 hours at 40 ° C).

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Finally, because of the possibility of aspecific interference by high re. Similarly, hapten anti-sera coupled by cova-factors present in the serum, it is necessary to carefully evaluate the lence to solid matrices have little or less sensitivity to the assay conditions before choosing a assay system. assay when compared to the antibody system

One of the main disadvantages when using anti-uncoupled when there was a disastrous loss of primary sensitivities in solid phase in RIA systems is that at 5 times when tested antibody-hormones pro-the result of loss of antibody titer and greed that occur at high molecular weight. These authors concluded that often during the coupling stage, one must develop any loss of sensitivity of dosage resulting from the use of longer, more expensive and more complicated systems (ie chemically fixed anti-sera is probably caused by, say, batch dosages). (Zeltner et al., Clin. Chem., 20: 5 steric hindrance of the largest antigens. (1974)). Recently, Den Hollander et al., J. Immunol. Me- Therefore, one of the main disadvantages against indicating u-thods, 1} 2A1 (1972), have developed a new method of using solid preparations of primary antibodies in separations using a second antiserum coupled to a matri- RIA systems is that this process is not universal. (The insoluble approach using cyanogen bromide and they che can not be as good for large molecules and called this separation process the solid phase process of small molecules). Other disadvantages that apply to

double antibody (DASP). Although the titer and avidity of all primary solid phase RIA methods are the second-second antibodies is certainly reduced in these preparations: a precise amount of antibodies of a solid suspension must be pipetted, the reaction of primary antibodies remains unchanged and gel board insoluble, the melan must be mechanically agitated-thus one of the main disadvantages of dosing systems is avoided to ensure proper mixing and a solid centrifugal phase. In fact, this DASP method has advantages. Gation is usually necessary to separate the following fractions compared to the method of separating free and bound fractions.

free and bound of conventional soluble double antibodies: The use of “Sephadex” column in syringes

A. Since solid preparations of plastic precipitate antibodies to bind a labeled thyroxine mixture require little or no protein-carriers, I125 (T4) is required and T4 released from serum proteins by a alkali, less amount of second antibody to precipitate followed by subsequent protein bond analysis with mier antibody. 25 a fixed limiting quantity of globulin binding to the thyroid

B. The DASP process requires less time for the separation (TBG) on each column was introduced recently. Seligson complete ration of free and bound fractions. et al., Clin. Chem. Acta, 38: 199 (1972) and Alexander et al.,

C. Aspecific interference by present factors Clin. Chem., 20: 553 (1974). In this method, the column in serum is completely absent with the Sephadex method also serves to separate free T4 from the T4-TBG complex. DASP precipitation if working in the area of excess. Similar tests have been developed for the Tri-iodothyro-of the second antibody. In fact, once we have established the isotope-labeled condin (T3) in which we use optimal conditions for the precipitation of the immuno-complex, an antibody to T3 as a competitive protein binding agent, frequent reassessment is not necessary. Alexander et al., Clin. Chem., 20: 1353 (1974).

For all the advantages obtained with preparations In the two assays, the column of "Sephadex" serves as solids for the precipitation of the antibody, there remains a destabilization of the mixture of antigens and for labeling while other mechanical advantages common to all solid phase assays, components of the serum are not retained by the gel. Then, in all solid phase assays, it is necessary to shake incubate a fixed amount of competitive protein binding agents the bodies in reaction continuously and in addition, it is necessary first in the empty space of the column and during this time, the anti-stopper the dosing tube then, centrifuge and uncork the sera and the labeled product are distributed again between the before the washing step. Chan et al., Ann. Clin. Biochem., 40 column and the binding agent. Elution with a buffer removes 12: 173 (1975), coupled primary antibodies to "Se-complex binding agent-antigens, while the free antigen remains phadex G25" (brand) activated with cyanogen bromide, one attached to the Sephadex column. Although this technique is dextran gel formed into ultra fine pearls (particle size in easily automated, this system requires precise load of below 10 | i) and it is reported that with small volumes of equal amounts of gel in all columns. Thus, cubation (300 to 400 micro-liters) is required, the reaction of the antibody can continuously agitate the Sephadex by means of an agitator to continue without a vertical rotation being necessary or a magnetic, while transferring the suspension by means of u-other means of continuous agitation. no graduated pipette.

Currently, antibodies are covalently attached to Boguslaski et al., Analytical Chemistry, Vol. 47, No 9,1583

variety of solid matrices such as agarose, glass, poly- (1975), have recently reported on a radioimmunoacrylamide and even iron oxide powder. Siegel et al., J. 50 column assay for the digitoxin assay (pm = 765) Clin. Endocrinol. Metab., 37: 526 (1973), Weetall, Chem. which uses a column of immobilized primary antibodies which

Abst., 77: 18064y (1972), Moore et al., Steroids, 20: 199 (1972), acts both as a reaction chamber and as a device and Hersh et al., Clinica Chemica Acta, 63:69 (1975) . of seperation. Radioimmunoassays on anti-column

Although in principle, the first antibody bound to a primary body phase have been reported for solid vitamin B12 represents a simple and versatile separation process, 55 (pm = 1.355), Boguslaski et al., Clinica Chemica Acta, 62: 349 this approach has a number of disadvantages. Les (1975), and insulin (pm ~ 6000), Davis et al., Clinica Chemica two questions to be answered, with regard to Acta, 66: 379 (1976). In view of the work of Bolton et al., Here the solid phase systems using primary antibodies above, this method is not universal because the sensitivity of are: firstly the possible loss in activity of the antibody assay for large molecules (eg TSH ie (title) and the subsequent waste of valuable anti-serum and 6 "thyroid stimulating hormone having a molecular weight of xiii possible reduction in sensitivity which may result) would be restricted because of the properties of ter antibodies with solid systems compared to those which can be obtained solid phase towards large molecules.

nir using the same anti-sera in aqueous solution. Immunochemical composites have been found to contain

As noted above, Bolton et al., Supra, have reported positively charged imido-esters as the isolation of antibody activity tending to be more 6I coupling are excellent means for separate the high fractions in solid preparations of antiserum and bound hapten from the free fractions in an immunoassay method. The present invention relates to a process for the separation of anti-serum with molecular weight protein hormones from the free fractions of the linked fractions in an immunization process.

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no-assay according to which a solution is brought into contact with a ce of Polysaccharide is activated by a cyanogen halide-

composite comprising a matrix derived from a Polysaccharide ne the matrix derived from a Polysaccharide has the formula:

covalently coupled to the antibody by a bifunctional coupler, and the solution containing the free NH moieties is separated from the reacted composite, characterized in that said bi-functional coupler 5 II is selected from those of the following formula: matrix-0-C-NH-Y-NH2 boasts:

in which the matrix is a Polysaccharide matrix such

+ NHZ + NH2 as defined above and in which Y is a spacer. Of

Examples of spacers include :-( CH2) m-, H2e + lCeO-C- (CH2) n-C-OCeH2e + 1

where n is an integer with the value 1 to 6 and e is a - (CH2) b -NH- (CH2) c-, (CH2) d —and an integer equal to 1 or 2. - - ^ - '

The immunochemical composite used in the se-15 / —y process

Ion the present invention comprises a matrix derived from an N = No. n m is an integer having the value 1 to Polysaccharide covalently coupled to an antibody by a bi-functional coupling agent. The matrix of Polysaccharide 12 and preferably 4 to 6, where b and c independently are des can be any matrix to which are attached several whole numbers having the value 1 to 6, preferably 2 or 3, and or hydroxyl groups as well as their derivatives. The matrices of po- 20 ^ is an integer having the value 1 to 10, preferably 2 a

Preferred lysaccharides include polymers celluiosi- preferably Y is the group - (CH2) „1—.

ques, polymeric dextrans, agarose, and their derivatives. As another illustration of a matrix derived from a poly-

cellulosic polymers and their derivatives are the saccharide matrices, if the Polysaccharide matrix is activated by a

Polysaccharide of choice epihalohydrin, the matrix derived from the Polysaccharide has the

According to one embodiment of the invention, the matrix of 25 ^ ormu'e '

Polysaccharide is finely divided and has an average maximum dimension in the wet state of 1 to 18 days, and preferably 10 to 15 [i. According to this embodiment, the Polysaccharide matrix. ''

may be spherical, linear or have any other matrix-0-CH2CHCH2 — NH — Y-NH2 geometric configuration, provided that its maximum dimension 30

male average in the wet state (diameter, side, length) either in 'a1uf "e' with matrix and Y are as defined above, as described above. Several types of polysaccharide matrices '' we covalently couple the matrix

charide are commercially available in this form of a Polysaccharide can be either a primary antibody or finely divided, for example, the trademark Sephadex, a gel of a secondary antibody. As long as the only requirement for dextran in the form of pearls is available in several qualities 35 'to the present invention is that 1 antibody has a residue of having a dry particle diameter of 10 to 40 n thus lysine, practically all primary and secondary antibodies with a diameter below 10 | x. It is also possible to can be covalently coupled to the matrix derived from a reduce the maximum mean dimension in the wet state of the Polysaccharide because all the antibodies possess these resi-matrices of Polysaccharide by chemical techniques, by! Ysine due -Preferably the antibody is a secon-example antibody, by hydrolysis. To hydrolyze this matrix, we put 40 da're "

for example in contact with the Polysaccharide matrix with a ^ ar "secondary antibody", is meant in the present expo-

acid solution, for example, a hydrochloric acid solution, an antibody produced in a living organism in response to sulfuric acid, 3 to 10 N or another suitable acid during the presence of an antigen in this organism when the antigen is sufficient time for example 2 to 24 hours. The antibody itself is then neutralized. By contrast, the term "acid antimixture with a basic solution, for example, a primary solution" means an antibody produced in the same way with 3 to 10 N sodium or potassium hydroxide, etc. and Start from any other antigen. This terminology is well known, then washing and drying according to known techniques. specialists in the field.

Polysaccharide matrices can be activated based on the present invention is the use of imi-

any suitable means known to those in the art. Do-esters as a coupling agent for the composite immuno-examples of the reagents suitable for activating the polychemical matrix. The imido-ester has the general formula:

saccharide include a cyanogen halide, epihalo-

hydrin, haloacetyl halides and divinyl sul- ^ * 2 NH2

phone. See Patty, Industriai Hygiène and Toxicology, Vol. 2. p. ""

634, Interscience, New York, NY (1949), Axen et al., Nature H2e + iCeO-C- (CH2) nC-OCeH2e + 1 (Lond.), 214: 1302 (1967), Rosner et al., Biochem ., 14: 4813 55

(1975), Jagendorph et al., Biochimica and Biophysica Acta, in which n is an integer having the value 1 to 6, of

78: 516 (1963), and Porath et al., Nature NewBiol., 238: 261 preferably 4 to 6, and in which e is an integer equal to 1

(1972), said publications being incorporated in toto here by 0112- ^ use of these imido-esters makes it possible to fix by cova-reference. Advantageously, a halolence of the antibodies to solid supports is used as reagent by cyanogen chiene reactions or an epihalohydrin to activate the 60 known layers which immobilize both the primary antibodies and the polysaccharide matrix. Preferably, the secondary matrix is activated via their remains of lysine of Polysaccharide by an epihalohydrin or one of these. In most cases they are not necessary for the activity.

mixtures and even better we activate the immunological logic Polysaccharide matrix. In addition, the presence of a matrix charged by epichlorohydrin positively does not cause harmful non-specific adsorption.

A spacer a, co-diamino is then coupled to the matrix of 65 on the present immunochemical composite.

Polysaccharide activated above by means of one of the groups Immunochemical composites within the scope of the invention

amino spacer a, co-diamino- thus forming a tion matrix can be prepared by the following procedure. We put in a polysaccharide derivative. To illustrate this point, if matri- contact an activating reagent with the Polysaccharide matrix

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desired in a solution having the desired pH. The pH can generally be from about 7.5 to about 10.0 and the particular pH is chosen depending on the activating reagent and the polysaccharide matrix that is used. The reaction can be allowed to continue at room temperature. The activating reagent is left in contact with the Polysaccharide matrix for a sufficient time, from about 5 minutes to 5 hours, to allow the matrix to be activated. The excess activating reagent is removed from the activated Polysaccharide matrix by washing said matrix with an appropriate medium, for example water, a buffer (for example, sodium bicarbonate), etc. The activated matrix is then suspended in an appropriate medium, for example, an aqueous solution of dimethylformamide. The desired a-cû-diamino-space is then added to the suspended activated polysaccharide matrix and the reaction is allowed to continue for approximately 1 to 10 hours at room temperature. The excess spacer a, co-diamino- is removed from the matrix derived from a Polysaccharide by washing said matrix with an appropriate medium, for example a solution of dimethylformamide, then washing with an appropriate buffer, for example, sodium bicarbonate pad. After this double washing, the matrix derived from a Polysaccharide is suspended in an appropriate buffer, for example, a sodium bicarbonate buffer.

The bi-functional coupling agent or its mixture is dissolved in a basic solution at approximately 4 ° C. If necessary, the pH is adjusted to around 8-9. The matrix derived from a polysaccharide suspended is then brought into contact with the dissolved bi-functional coupling agent and the mixture is stirred by rotation at approximately 4 ° C. for 1 to 5 hours.

After removing excess bifunctional coupler, the matrix derived from a coupled polysaccharide is suspended in a mixture containing an appropriate buffer, for example, sodium bicarbonate buffer and a primary antibody function or secondary. The mixture is stirred for about 10 to about 24 hours in a cold environment. The immunochemical composite is then washed thoroughly with an appropriate buffer, for example, a sodium bicarbonate buffer, and then it is suspended in an appropriate buffer having a pH of approximately 8, for example, a barbital buffer containing about 0.1% gelatin.

The present immunochemical composite prepared according to the above process has the following schematic structure:

NH2 +

II

matrix derived from a polysaccharide-C- (CH2) n-

NH2 +

II

C-NH-antibody where the matrix derived from an Polysaccharide n and the antibody are as defined above. The preferred immunochemical composite within the scope of the invention has the formula:

OH

matrix of I

polysaccharide-0-CH2-CH-CH2-NH- (CH2) m-

+ NH2 + NH2 Il II

NH-C- (CH2) n-C — NH-antibody in which the Polysaccharide matrix m, n and the antibody are as defined above.

The immunoassay method according to the invention involves bringing a solution containing free and bound fractions into contact with the present immunochemical composite by means of techniques well known to those in the art and thus the free fractions are separated linked fractions. See Weir, "Immunology for Undergraduates", Churchill Livingstone, Edinburgh, England 5 (1973) and Ratcliffe, British Medicai Bulletin 30:32 (1974), these publications being incorporated here in toto by reference.

According to a preferred embodiment, the immu-no-dosing process involves a process in which continuous agitation is eliminated and thus the need to plug the tubes. See io Chan et al., Ann. Clin. Biochem., 12: 173 (1975). In another preferred embodiment, the immunoassay method is a solid phase column immunoassay method using the present immuno-chemical composites by well-defined techniques. known to those in the art and thus the free fractions are separated from the bound fractions. See above, Ratcliffe, supra, Boguslaski et al., Clinica Chemica Acta, 62: 349 (1974), Boguslaski et al., Analytical Chemistry, Vol. 47, No. 9.1583 (1975), and Davis et al., Clinica Chemica Acta, 66: 379 (1976). Preferably, the immunoassay methods are RIA procedures, the techniques of which are also well known to those of the art See Skelley et al., "Radioimmunoassay", Clini-cal Chemistry, Vol. 19, No. 2.146 to 186 (1973), this publication being incorporated here in toto by reference.

Another process within the scope of the invention is a suspension process such as those described above except that the composite used in this case is not the present immuno-chemical composite which is described in more detail below but an immunoassay reagent comprising a finely divided matrix of Polysaccharide covalently coupled to a secondary antibody using one of the various techniques known to those skilled in the art. Although the latter method is not as effective as the other methods of the present invention (see examples and discussion below), however, they represent a marked improvement and have clear advantages compared to the antibody methods. known primaries such as those of Chan et al.

In known suspension methods, the primary antibody is attached to a matrix, thereby interfering with the primary antibody-large antigens biological reaction. Furthermore, these known methods require a precise addition of a gel (that is to say composites comprising primary antibodies linked to matrices) which is a constant source of errors. However, the suspension methods within the scope of the invention which use secondary antibodies attached to templates eliminate the two difficulties described above for the known methods.

In general, it is possible to use the immunochemical composites within the scope of the invention in a solid phase column immunoassay method using the following procedure. The present immuno-chemical composites are placed in a coso lonne or other suitable reaction vessel, preferably washed beforehand with an appropriate buffer, thus forming the immuno-chemical apparatus according to the present invention for separating the free fractions from the linked fractions by means of '' a solid phase column immunoassay method. To minimize the cost as well as to improve the flow rate through said column, it is also preferred to add to the column non-activated polysaccharide matrices of various sizes. All loaded columns have an inherent hold-up volume (i.e., the maximum amount of fluid that can be retained by the column without eluting any amount of said fluid).

If it is desired to use the present chemical composites which contain secondary antibodies in a solid phase column immunoassay method, preferably, the primary immunoassay reaction is carried out outside the column. When the primary immunoassay is carried out outside the column, it can be carried out in any suitable volume, preferably a final volume of 400 X. The reaction

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primary immunoassay can take 0.5 to 5 hours, preferably 0.5 to 1 hour, the exact time being dictated by the dosage that is conducted. Then added to the column an aliquot of said reaction mixture approximately equal to the retained volume of said filled column and incubated on the column of solid phase secondary antibody for 0.5 to 1 hour, the exact time being dictated again according to the dosage we drive. After the incubation period, the column is washed with an appropriate amount of an appropriate buffer, said amount being sufficient to separate the free fractions from the bound fractions (i.e. which is at least equal to the volume of retained from the column). The column which now contains only the linked fraction is then closed and then counting is carried out.

The reaction time for the incubation of the columns can be very shortened by using an excess of the second antibody bound and one can even elute immediately through the columns of the second antibody in solid phase, if an excess of the second antibody is used.

The immunoassay method described above using the columns of second solid phase antibodies according to the invention can therefore be separated into three basic steps: the incubation of the reaction of primary antibodies at a station distant from the column, applying an aliquot of the reaction mixture to the column of the second antibody in solid phase and eluting the free fraction with buffer immediately or after a minimum incubation period on the column.

The process described above with the second antibody in solid phase is simple, it is easy to automate, it is precise and versatile. Thus, assays for large molecules (eg TSH) and small haptens (Digoxin, T4, T3) can be carried out as well using the present immuno-chemical composites. In addition, completely separate bound and free fractions are obtained with a large margin of error under conditions used for separation. The process does not interfere with the primary binding reaction, is relatively inexpensive and uses an easily reagents reagent apparatus.

With regard to small molecules, the columns of the second solid phase antibodies according to the invention can be used in more than one way. The columns can be loaded with primary antibodies, labeled antigens (or hapten), and the reaction between the sample and both the primary reaction and the antibody reaction in solid phase can be allowed to proceed simultaneously in the free space of the column. After a certain time, the columns are eluted with a buffer to separate the free fractions from the bound fractions.

Another mode of proceeding consists in incubating the primary antibody on the column of second antibody in solid phase and thus of producing columns of primary antibodies on which the primary antibody is biologically linked to the second antibody covalently attached. The labeling product and the sample are then incubated on the columns which serve as a device for separating the free and bound fractions.

In addition to the solid phase column method described above, according to the invention, it is also possible to use the present immunochemical composite composites where said composites contain primary antibodies covalently coupled to a matrix derived from a Polysaccharide.

In addition, another operating mode within the scope of the invention involves a solid phase column immunoassay method in which the composite used is not the present immunochemical composite described above, but a reagent immunoassay comprising a Polysaccharide matrix covalently coupled to a secondary antibody by any of the techniques known to those in the art. Although the latter method is not as effective as the other methods according to the invention, however, it represents a great improvement and presents clear advantages compared to the known solid phase primary column methods. Although there is a loss of antibody activity (titer) when a second antibody is covalently coupled to a matrix derived from a Polysaccharide, the consumption of the second antibody is however reduced s because large amounts of globulins- vehicles are not required as is the case with the dual antibody process in the liquid phase. Another advantage of an immunoassay method using the columns of second antibodies in solid phase is that this method facilitates the difficulty of accurately measuring a precise quantity of a second antibody in a suspension of a gel because in the present method requires only a minimal amount of the second antibody. Levels above this previously established minimum amount have no effect on the sensitivity or accuracy of the results obtained with the present method.

The following examples illustrate the invention without limiting it.

Example 1

3 ml of epichlorohydrin was added to a mixture of 6 g of microcrystalline cellulose type 50 (average particle size = 50 (x) in 30 ml of IN sodium hydroxide with vigorous stirring at room temperature. two hours the excess epichlorohydrin was removed by washing with one liter of water and the activated, washed cellulosic matrix was then suspended in 60 ml of a 50% aqueous solution of dimethylformamide. After activating this suspended matrix, 0.85 g of 1,6-hexanediamine was added, the reaction was allowed to continue with stirring for 2 hours at room temperature and then the excess of 1,6- was removed. hexanediamine by washing with one liter of a 50% aqueous solution of dimethylformamide After washing with one liter of 0.1M sodium bicarbonate, the cellulose matrix derived was suspended in 0.1M sodium bicarbonate to obtain a 1: 1 mixture of derived matrix: sodium bicarbonate. 0.7 was dissolved 35 g (3 moles) of dimethyl adipimidate (DMA) in 0.6 ml of cold 5N sodium hydroxide solution with stirring at 4 ° C. After adding 0.1M of cold sodium bicarbonate, pH 8.5 was adjusted with IN sodium hydroxide. To this solution was added 6 ml of 0.1M sodium bicarbonate containing 0.8 to 1.0 grams of cellulose derivatives and the mixture was stirred by rotating at 4 ° C for two hours.

After removing excess dimethyl adipimidate, the coupled cellulose derivative matrix was suspended in 9 ml of 0.1M sodium bicarbonate (4 ° C) and 1.1 ml of a gamma fraction. anti-rabbit goat globulin (42.82 mg / ml) in 0.1M sodium bicarbonate (4 ° C) and the mixture was stirred in a cold room. The second immobilized antibody was then washed thoroughly with 0.1M sodium bicarbonate and finally suspended in 10 ml of barbital buffer of pH = 8.0 containing 0.1% gelatin.

Another immuno-chemical composite was also prepared by proceeding as in Example 1 except that the bi-55 functional coupling agent used was dimethyl suberimidate dihydrochloride (DMS) instead of the DMA of Example 1.

Example 2

1 g of microcrystalline cellulose type 50 to 60 was added to a solution of 1.0 g of cyanogen bromide in water at room temperature. The pH of the mixture was immediately adjusted to about 11.0 with 2N sodium hydroxide and kept at this pH for 6 to 12 minutes with the addition of 2N sodium hydroxide in a very controlled manner. When the pH 65 had stabilized at about 11, the mixture was allowed to stand for another 5-10 minutes before washing the activated cellulosic matrix with 1.1 liters of 0.1M sodium bicarbonate at 4 ° C to remove the excess cyanogen bromide.

629,894

8

Gêï then suspended the activated cellulosic matrix, washed thoroughly in 10 ml of 0.1M sodium bicarbonate and approximately 47 mg of antilapin goat gamma globulin was added to it in 1.3 ml of sodium bicarbonate 0, 1M. The suspension was then mixed at room temperature overnight.

The following day, the immunochemical composite was washed with 600 ml of 0.1N sodium bicarbonate and 100 ml of barbital buffer (pH = 8.0) containing 0.1% gelatin. Finally, the composite was suspended in about 10 ml of the barbital buffer containing gelatin above.

Example 3

Solid phase preparations of activated microcrystalline cellulose as prepared in Examples 1 and 2 were titrated against 125I-labeled rabbit gamma globulin as follows:

200 X - pH 8.0 barbital buffer containing 3.5% BSA

100 X — 125I-labeled rabbit gamma globulin containing 0.1% normal rabbit serum

100 barbital buffer of pH 8.0

200 X - preparation of the second antibody in solid phase

Each tube was incubated at room temperature for 1 hour with shaking and then centrifuged for 20 minutes at 1000 X g.

The units of activity of the greatest dilution were calculated, that is to say the titer of the second antibody in solid phase from which resulted the maximum binding of labeled antigens. The formula used to calculate the activity units is as follows:

j total volume of preparation X of antibodies in solid phase = Units of activity

title sample size where the size of the sample in the present example is 200 X (0.2 ml) and where the total volume of preparation of secondary antibodies in solid phase = 10 ml. As soon as these titrations of the lines in the presence of 100 X of normal rabbit serum at 0.1% in fact we seek the greatest dilution of the second antibody in solid phase capable of fixing approximately 1 micro-gram of globulin range rabbit. The results of these calculations are shown in Table I. As Table I clearly shows, the titer of the antilapin goat antisera coupled to cellulose derived from DMA were much higher than the preparation of microcrystalline cellulose coupled to bromide of corresponding cyanogen (CNBr).

s Example 4

The cellulose derived as prepared in Example 2 and that derived from DMS of Example 1 were titrated against thyroxine-125! in the presence of approximately 1 micro-gram of rabbit gamma-globulin as follows:

io 20 X - pH 8.0 barbital buffer containing 3.5% BSA

100 X — of thyroxine-125! in pH 8.0 barbital buffer containing 2% BSA

100 X - rabbit antiserum against thyroxine at a dilution of 1 per 1000 in a barbital buffer of pH 8.0

200 X — preparation of the second antibody in solid phase.

Each tube was incubated at room temperature for about V2 hours with shaking and then centrifuged for 20 minutes at 1000 X g. The precipitates were then suspended in 1.0 ml barbital buffer of pH 8.0 containing 2% BSA and centrifuged for 20 minutes at 1000 X g. In these experiments, the labeled thyroxine is immunologically attached to these specific antibodies which are present as fractions of about 1 micro-gamma of rabbit gamma globulin. Thus, in this example, the units of the second antibodies bound to the cellulose matrix can be indirectly measured by calculating the greatest preparation dilution of the second antibody in solid phase to give the maximum binding of labeled thyroxine.

Table I also shows the results of these assays. As clearly shown in Table I, the experiments with DMS compare favorably with the DMA data and the two preparations are much better than their controls with the corresponding cyanogen bromide. Similar improvements are also obtained in the amount of activity units isolated when the present immuno-chemical composites contain primary antibodies instead of secondary antibodies.

Therefore, the present positively charged immuno-chemical composites containing bi-functional imido-esters as coupling agents should be viewed as a great improvement over the prior immuno-chemical composites used in immunoassay procedures.

Table I

Support

*AT. 1 g of microcrystalline cellulose activated with cyanogen bromide

A. 1 g of microcrystalline cellulose activated with dimethyl adipidimate

** B. 1 g of microcrystalline cellulose activated with cyanogen bromide

B. 1 g of microcrystalline cellulose activated with dimethyl suberimidate

Protein concentration of year- Title Unit

antibody containing a fraction of activity

gamma globulin isolated

47 mg gamma globulin 1 / 2.3 115 anti-rabbit goats

47 mg gamma globulin 1 / 16.5 850 anti-rabbit goats

40 mg gamma globulin 1 / 3.2 160 anti-rabbit goat

40 mg gamma globulin 1/16 800 anti-rabbit goat

% increase in isolated activity

739

500

* A - rabbit gamma globulin labeled with 125I ** B - thyroxine labeled with 125I

Example 5

9 g of microcrystalline cellulose type 50 (average particle size = 50 µt) was added to 30 ml of 6N hydrochloric acid solution and the mixture was stirred for 4 h at room temperature. After 4 hours of reaction time, the mixture was neutralized with a solution of sodium hydroxide.

9

629,894

6N dium and the hydrolyzed cellulose was washed with 1200 ml of water. The packed cellulose was further washed with 300 ml of methanol, then with 100 ml of diethyl ether. The gel residue was suspended in 100 ml of ether and dried under reduced pressure.

Example 5 was repeated several times, only varying the reaction time. The reaction time was varied from 4 h to 6.24 and 64 h. Microscopic examination showed that type 50 microcrystalline cellulose which had been hydrolyzed for 4 h and 6 h had an average wet length of approximately 15-20 | x and that those hydrolyzed during 24 and 64 h had an average wet length of approximately 10-12 | x.

Example 6

3 ml of epichlorohydrin was added to a mixture of 6 g of microcrystalline cellulose type 50 in 30 ml of IN sodium hydroxide with vigorous stirring at room temperature. After 2 h, the excess epichlorohydrin was removed by washing with 11 of water. The activated cellulosic matrix was then suspended and washed in 60 ml of a 50% aqueous solution of dimethylformamide. To this suspended activated matrix was added 0.85 g of 1,6-hexanediamine. The reaction was allowed to continue with stirring for 2 h at room temperature, then the excess 1,6-hexanediamine was removed by washing with 11 of a 50% aqueous solution of dimethylformamide. After washing with 11 of 0.1M sodium bicarbonate, the derivative cellulose matrix was suspended in 0.1M sodium bicarbonate to obtain a 1: 1 mixture of derivative matrix: sodium bicarbonate.

0.80 g (3 mmol) of dimethyl suberimidate (DMS) was dissolved in 0.6 ml of cold 5N sodium hydroxide solution with stirring at 4 ° C. After adding 0.1 M cold sodium bicarbonate, the pH was adjusted to 8.5 with IN sodium hydroxide. To this solution was added 6 ml of 0.1 M sodium bicarbonate containing 0.8-1.0 g of derived cellulose and the mixture was stirred at 4 ° C for 2 h.

After removing the excess DMS, the coupled cellulose matrix, suspended, was suspended in 10 ml of 0.1 M sodium bicarbonate (4 ° C) and 1.3 ml of an anti goat gamma globulin fraction - rabbit (36-38 mg / ml) in 0.1 M sodium bicarbonate (4 ° C) and the mixture was stirred while turning in a cold room. The immobilized secondary antibody was then washed thoroughly with 0.1 M sodium bicarbonate and finally suspended in a barbital buffer of pH = 8.0, containing 0.1% gelatin to obtain a final volume of 25 ml.

Example 6 was repeated, only changing the size of the microcrystalline cellulose used. The various other sizes of microcrystalline cellulose used were type 20 microcrystalline cellulose and type 50 microcrystalline cellulose which had been hydrolyzed for 4 h, 6 h, 24 h and 64 h.

Example 7

1 g of microcrystalline cellulose type 50 was added to a solution of 1.0 g of cyanogen bromide (CNBr) in water at room temperature. The pH of the mixture was immediately adjusted to about 11.0 with 2N sodium hydroxide and kept at this pH for 6 to 12 min by adding 2N sodium hydroxide in a controlled manner. was stabilized at about 20, the mixture was allowed to stand for a further 5-10 min before washing the activated cellulosic matrix with 1.11 0.1 M sodium bicarbonate at 4 ° C to remove excess CNBr.

The activated cellulosic matrix was then suspended, washed thoroughly in 10 ml of 0.1 M sodium bicarbonate and about 47 mg of antilapine goat gamma globulin was added thereto in 1.3 ml of sodium bicarbonate 0 , 1 N. The suspension was then mixed at room temperature overnight.

The following day, the immunochemical composite was washed with 600 ml of 0.1 N sodium bicarbonate and 100 ml of barbital buffer (pH = 8.0) containing 0.1% gelatin. Finally, the composite was suspended in the barbital buffer containing gelatin above to obtain a final solution of 25 ml.

Example 7 was repeated, but by modifying the size of the microcrystalline cellulose used. The various other sizes of microcrystalline cellulose used were type 20 semi-crystalline cellulose and type 50 microcrystalline cellulose which had been hydrolyzed for 4,6,24 and 64 h.

Example 8

Preparations of microcrystalline cellulose type 50, solid with the second antibody, prepared as in Examples 6 and 7 against thyroxine-12SI, were titrated in the presence of approximately 1 μg of rabbit gamma globulin as follows:

20 X - barbital buffer of pH = 8.0 containing 3.5% BSA

100 X - thyroxine-125I in a barbital buffer of pH = 8.0 containing 2% BSA

100 X - rabbit antiserum against thyroxine at a dilution of 1 per 1000 (containing approximately 1 (ig of rabbit IgG)

200 X - preparations of second antibody in solid phase.

In the experiments where the reactants were shaken, each tube was incubated at room temperature for 1 hr with shaking and then centrifuged for 20 min at 100 X g. The precipitates were suspended in 1.0 ml of pH-8.0 barbital buffer containing 3.5% BSA and centrifuged again for 20 min at 1000 X g. However, in the experiments where the reactants were not agitated, each tube was incubated at 37 ° C. for 1 h h and before centrifugation, 1 ml of buffer was added to the barbital of pH = 8 containing 3, 5% BSA. Then, each tube was centrifuged at 1000 X g for 20 min.

In both of these experiments, the labeled thyroxine is immunologically attached to these specific antibodies which are present as a fraction of approximately 1 microgram of rabbit gamma globulin. Thus, it is possible to indirectly measure the units of the second antibody attached to the various cellulose matrices by calculating the greatest dilution of each of the preparations of antibodies which precipitate in the solid phase to give the maximum binding of labeled thyroxine.

The activity units were calculated as described above except that the total volume of the secondary antibody preparation in solid phase is 25 ml. The results of these calculations are shown in Tables II and III. As clearly shown in Tables II and III, the titer of anti-rabbit goat antiserum coupled to finely divided cellulose derived from DMS was much higher than the corresponding preparation of finely divided microcrystalline cellulose coupled to CNBr. In fact, the titer of this finely divided cellulose derived from DMS which did not undergo agitation was even higher than the preparations coupled to the shaken CNBrs.

5

10

15

20

25

30

35

40

45

50

55

60

629,894

10

Table II Support

Concentration Protein titer of (agitation)

the fraction of antibodies containing gamma globulin

Isolated activity units (agitation)

Title (without Units% Agitation Units) of activity (agitation)

isolated (without Units (without agitation) agitation)

1 g of microcrystalline cellulose 47 mg of gamma 1 / 1.6 type 50 activated with CNBr globulin goat anti-rabbit

212

1 g of microcrystalline cellulose do. type 20 activated with CNBr

1 g of microcrystalline cellulose do. type 50 hydrolyzed by an acid for 4 h and activated with CNBr

1 / 3.7

1/5

462

625

1 / 1.6

1 / 2.9

200

362

43.29% 57.92%

1 g of microcrystalline cellulose type 50 hydrolyzed with an acid for 6 h activated with CNBr do.

1/5

625

1 / 2.9

362

57.92%

1 of type 50 microcrystalline cellulose hydrolyzed with an acid for 24 h activated with CNBr do.

1/5

625

1 / 3.4

425

68.00%

1 g of microcrystalline cellulose do. 1 / 5.5 688 1 / 3.4 425 61.77%

type 50 hydrolyzed with an acid for 64 h activated with CNBr

Tabelau III

Support Concentration in Title Units Title (without Units% Protein units of the agitation activity) agitation activity

fraction of isolated antibodies isolated (without Units containing agitation) (without agitation)

gamma globulin

1 g of microcrystalline cellulose 49 mg of gamma 1/5 750 1/2 250 33.33%

type 50 activated with anti-rabbit goat globulin DMS

1 g of microcrystalline cellulose do. 1 / 11.5 1,438 1 / 5.1 638 44.37%

type 20 activated with DMS

1 g of microcrystalline cellulose do. 1 / 11.5 1,438 1 / 7.0 875 60.85%

type 50 hydrolyzed with an acid for 4 h activated with DMS

1 g of microcrystalline cellulose do. 1 / 11.5 1,438 1 / 7.0 875 60.85%

type 50 hydrolyzed in an acid for 6 h activated with DMS

1 g of microcrystalline cellulose do. 1 / 11.5 1,438 1 / 7.0 875 60.85%

type 50 hydrolyzed in acid for 24 h activated with DMS

11

629,894

Tabelau III Support

Protein Title Concentration of (shaking)

antibody fraction containing gamma globulin

1 g of microcrystalline cellulose type 50 hydrolyzed in an acid for 64 h activated with DMS

do.

1 / 11.5

Isolated activity units (agitation)

1438

Title (without shaking units) of activity

isolated (without agitation)

1 / 7.0

875

Units

(agitation)

Units

(without agitation)

60.85%

Table IV Support

Type 50 microcrystalline cellulose

Type 20 microcrystalline cellulose

Type 50 microcrystalline cellulose hydrolyzed with acid for 4 h

Type 50 microcrystalline cellulose hydrolyzed with acid for 24 h

Type 50 microcrystalline cellulose hydrolyzed with acid for 64 h

(shaking) Activity units with DMS Activity units with CNBr

750 212

1438 462

1438 625

1438 625

1438 625

= 3.54 = 3.11 = 2.30

= 2.30

= 2.09

(without shaking) Activity units with DMS Activity units with CNBr

250 0

638,200

875,362

875,425

875,425

= Undefined

= 3.19

= 2.42

= 2.06

= 2.06

Activity units with DMS (without agitation) Activity units with CNBr (agitation)

250 212

638,462

875,625

875,625

875,625

= 1.18 = 1.38 = 1.40

= 1.40

= 1.27

Table V

Dosing time Control serum Control serum at 37 ° C "Beckman" from "Beckman",

"Digoxin" thyroxine

Control sera “Beckman” control serum, tri-iodo-thyronine

Control serum Control serum "Beckman", "Beckman",

TSH

1/2 diluted TSH

"Digoxin" y2 h

(ng / ml)

Tri-iodo-thyronine 2 h (ng / dl)

Thyroxine '/ d h

(Hg / dl)

Hormone stimulating 6 h human thyrotropin (international micro-units / ml)

2.75

(2.0-2.7) *

86

(90-110)

12.3 (12-20)

222

(165-225)

85

(70-90)

5.0 (6-7)

77.0 (40-75)

35.2

* The values in brackets are those obtained with the known double antibody process.

629,894

12

Table V (continued)

Sample serams

Lederle I

Lederle II

Ortho

I

Ortho n

"Digoxin" (ng / ml)

1.44 (0.94-1.78)

> 6.0

0.62 (0.6-1.2)

4.25 (3.2-5.2)

Tri-iodothyronine (ng / dl)

116 (98-118)

474 (409-457)

-

-

Thyroxine (Hg / dl)

7.2 (6.4-9.6)

16.1 (13.6-22)

-

-

Hormone stimulating human thyrotropin (international micro-units / ml)

3.7 (3—4)

2.6

(2.3-2.95)

7.2 (3.9-7.9)

32.2 (23-39)

Example 8 was repeated, except that the size was varied as a limiting factor to determine the amount of activity isolated,

of the microcrystalline cellulose used. As above, this In addition, we can see that although we do not get the degree

variation involved the use of microcrystalline cellulose of activity present when there is agitation, the activity units

type 20 and type 50 microcrystalline cellulose which was stirred isolated using any of the present hydrolyzed for 4,6,24 and 64 h. The results of this immunochemical composite data greatly exceed the quan-

are also shown in Tables II and III. 25 units of isolated activity units with agitation using only-

Looking at Table II, we notice several things. an antibody directly coupled to a matrix through the

In the column titled “Isolated activity units (agitation)”, medial of the process with CNBr. The reason for this great am-

as long as the vial is shaken, all the magnitudes of enhancement is that the present composites bind the antibody to it must also be mixed and effectively suspended at some distance from the surface of the

also and therefore the exposure of the second antibody fixed by matrix and thus they decrease the effect and the influence of the covalent size to CNBr to the antigen is the same. However, there is matrix on the biological reaction. According to the present inven-clear that the size of the matrix has an effect on the biological activity, the antibody is separated from the surface of the matrix but only from the second antibody covalently coupled to CNBr. This couple also the antibody to a matrix derived through

column indicates three basic groups, namely the cellulose microse of a coupling agent which, as noted above,

type 50 crystalline, micro-crystalline cellulose type 20 and 3s in most cases does not have an adverse effect on the activity

type 50 microcrystalline cellulose which has been immunologically hydrolyzed from the antibody.

for 4 hours or more. This column shows that more can be isolated In addition to the advantages just mentioned, the best effi-

of activity by shaking a CNBr-activated matrix that can be achieved from the present immunological chemical composites

to isolate using a microcrystalline cellulose of type 50 requires using larger sizes of matrix in one which has been hydrolyzed for 4 h. 40 process without agitation, which makes it possible to use a centrifuge

In the column entitled "Isolated activity units (without runaway with less than g. The possibility of using a centrifuge

agitation) ", the possibility of suspending a gaseous particle having a lower g value is important because and therefore the size of the matrix comes into play as well as the activity when working with the present biological immunological composites of the second antibody covalently coupled. We can now use laboratory centrifuges found in this column four subdivisions or conventional cellulose 45 and thus avoid the high cost that otherwise would be required for micro-crystalline type 50, the micro-crystalline cellulose of the type using a known method, for example that of pe 20, micro-crystalline cellulose of type 50 which has been hydrolyzed- Chan et al.

seed for 4 h and 6 h and microcrystalline cellulose type 50 As very clearly shown in Table IV, we obtain that it was hydrolyzed for 24 and 64 h; and the activity is found to increase by about 27 to 40% using the maximum biological present in the latter case. so immunochemical composites within the scope of the invention in

By analogously comparing the results of the Table a process without agitation compared to the process with agitation where

III, we see the marked improvement in the present composites; the antibody is directly coupled to a matrix via

munochemical compared to the immunochemical composites of the CNBr process.

as in Table II. The column entitled “Units of iso- activity Table V compares the efficiency of the use of pre-leashes (agitation)” has two basic subgroups instead of three which 55 composite immuno-chemical sents compared to processes appear in Table II. That is, double cellulose with previous antibodies for various immuno-do-

microcrystalline type 50 differs from microcrystalline cellulose. As indicated in Table V, the results obtained in line type 20 and higher. This difference implies that only the present method and the present immuno-composites are used.

can not obtain more biological activity than that which is isolated chemical advantageously compare to the known method using a microcrystalline cellulose type 20. However using the said double known antibody method. It is noteworthy

Dant, when examining the column entitled "Units of activity quer that the radioimmunoassay of thyroxine by the method

isolated (without stirring) "we see that the column is divided into known double antibodies takes about 2 h, while a radio

three groups, namely microcrystalline cellulose type 50, the immunoassay using the present immuno-chemical composites

micro-crystalline cellulose type 20 and micro-critical cellulose takes only half an hour.

type 50 stalline which has been hydrolyzed for 4,6,24 or 64 h. 65

We can therefore conclude that when using a micro cellulose Example 9

crystalline or a similar Polysaccharide having a dimension 3 ml of epichlorohydrin have been added to a mixture of 6 g maximum of 18 n or less, the size of the matrix is not microcrystalline cellulose type 50 (particle size

13

629,894

mean = 50 n) in 30 ml of 1N sodium hydroxide, shaking vigorously at room temperature. After 2 h, the excess epichlorohydrin was removed by washing with 11 of water. The activated cellulosic matrix was then suspended, washed in 60 ml of a 50% aqueous solution of dimethylformamide. 0.85 g of 1,6-hexanediamine was added to this suspended activated matrix. The reaction was allowed to continue with stirring for 2 h at room temperature, then the excess 1,6-hexanediamine was separated by washing with 11 of a 50% aqueous solution of dimethylformamide. After washing with 1 L of 0.1 M sodium bicarbonate, the cellulose derivative matrix was suspended in 0.1 M sodium bicarbonate to obtain a 1: 1 mixture of derivative matrix: sodium bicarbonate.

0.80 g (3 mmol) of dimethyl suberimidate (DMS) was dissolved in 0.6 ml of cold 5 N sodium hydroxide solution with stirring at 4 ° C. After adding 0.1 M of cold sodium bicarbonate the pH was adjusted to 8.5 with IN sodium hydroxide. To this solution was added 6 ml of 0.1 M sodium bicarbonate containing 0.8 to 1.0 g of cellulose resin and the mixture was stirred at 4 ° C for 2 h.

After separation of the excess DMS, the coupled cellulose-derived matrix was suspended in 9 ml of 0.1 M sodium bicarbonate (4 ° C) and 1.3 ml of anti-goat gamma globulin fraction rabbit (36-38 mg / ml) in 0.1 M sodium bicarbonate (4 ° C) and the mixture was stirred in a cold room. Oli then thoroughly washed the second immobilized antibody with 0.1 M sodium bicarbonate and finally it was suspended in a barbital buffer of pH = 8.0 containing 0.1% gelatin to obtain a volume final 25 ml.

Example! 0 Column preparation process:

Each empty column is washed beforehand with 1 ml of barbital buffer containing 0.1% gelatin and 2% BSA. Fibrous cellulose and type 50 microcrystalline cellulose are washed in separate tubes. The cellulose fractions are centrifuged and 1: 1 (by volume) mixtures of fibrous cellulose: buffer and microcrystalline cellulose are prepared. : tam-pon with the said buffer being a barbital buffer containing 0.1% gelatin. Then add equal volumes of mixtures of fibrous cellulose and microcrystalline cellulose to obtain a final mixture which can be used as support for all columns. Each column contains the equivalent of 1.4 ml of the above mixture plus 200 X of the appropriate dilution of solid phase precipitation antibodies. This gives a holding volume of 300 [xl. Then, each column loaded is washed beforehand with 1 ml of said barbi-15 tal buffer containing 0.1% gelatin and 2% BSA. The columns are then closed with a cap for storage.

Example II

The primary radioimmunoassay is conducted in a final volume of 400 X at a location remote from the column. The primary reaction can be carried out for 0.5 to 5 hours and preferably 0.5 to 1 hour. An aliquot of the primary reaction mixture (300 X) is then applied to the column of the second antibody in solid phase. This mixture is allowed to incubate for 0.5 to 1 hour. The solid phase column is then washed with 1 ml to 3 ml of said barbital buffer containing 0.1% gelatin and 2% BSA 30 to separate the free fractions from the bound fractions. The column which now contains only the bound fractions is then closed with a cap, then subjected to counting.

Data obtained by conducting various tests according to the general methods outlined in the above examples is shown in Table VI.

Table VI

37 ° C primary dosing time away from the column (h)

Ordinary temperature incubation time on the column (h)

Control sera

Lederle Lederle Control Serum Control Serum Control Serum I II "Beckman" from "Beckman" "Beckman"

TSH thyroxine, diluted V2

"Digoxin"

'h l / n

1.23

3.48

-

-

(ng / ml)

(0.8-1.3) *

(3-3.8)

Thyroxine

'h

Go

8.33

17.37

20.05

7.58

-

(Hg / dl)

. 6.4-9.6)

(13.6-22)

(12-20)

(6-7)

Tri-iodo-

2

V:

110.60

517.57

106.70

93.45

_

thyronine

(98-118)

(409-457)

(90-110)

(70-90)

Hormone stimulating

5

1

_

59.36

30.92

human thyro-tropine

(40-75)

(international micro-units / ml)

* The values in brackets are those obtained with the known double antibody process

629,894

Table Vf (continued)

"Digoxin" (ng / ml)

Thyroxine (jig / dl)

Tri-iodo-thyronine (ng / dl)

Human thytrotropin stimulating hormone (international micro-units / ml)

37 ° C

primary dosing time away from column (h)

'/ 2

72 2

14

Ordinary temperature, incubation time on the column (h)

'/ 2

V2

> / 2

Control sera Ortho "Beckman" control serum by I tri-iodothyronine

245.06 (165-225)

Ortho n

7.95

(3.9-7.9)

31.63 (23-39)

Table VI compares the effectiveness of the use of the present immuno-chemical composites in a solid phase column assay with the known dual antibody method in various immunoassay assays. As indicated in Table VI, the results obtained using the present method on solone in solid phase and the present immuno-composites

chemicals compare favorably to the known method using the so-called conventional double antibody method.

The present solid phase column immunoassay method not only has all the advantages of solid phase immunoassay methods but eliminates the need for centrifugation and is also suitable for automation.

VS