GB1571988A - Method and apparatus for concentration and purification of antigens and antibodies - Google Patents

Description

(54) METHOD AND APPARATUS FOR CONCENTRATION AND PURIFICATION OF ANTIGENS AND ANTIBODIES (71) We. GENERAL ELECTRIC COMPANY, a corporation organized and existing under the laws of the State of New York. United States of America, of 1 River Road Schenectady l23()5, State of New York. United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed. to be particularlv described in and by the following statement: This invention relates to method and apparatus for concentration and purification of antigens and antibodies. More particularly, this invention relates to concentration and purification of antigens and antibodies where the antigen-antibody reaction takes place at the surface of a substrate.

U.K. Patent Specification No. 1443181 describes and claims a method for separating a protein from a solution containing the protein, which method comprises immersing a substrate in a solution of a protein substantially reactive with the protein to be separated, so as to coat the substrate with a monomolecular layer of the specifically reactive protein immersing the coated substrate in the solution containing the protein to be separated, and removing the substrate therefrom.

This specification also described and claims a medical diagnostic apparatus comprising a substrate member. a plurality of metal globules attached to a surface of said substrate member and a monomolecular layer of protein antigen or antibody overlying at least a portion of said substrate and said metal globules.

U.K. Patent Specification No. 1443182 describes and claims a method for separating a protein from a solution containing the protein, which method comprises immersing a first portion of a continuous belt substrate in the solution containing the protein to be separated, the first portion having on its surface a monomolecular layer of a protein specifically reactive with the protein to be separated. conveying the belt to immerse the first portion of the belt in a solution of weak acid whilst conveying a second portion of the belt having on its surface a monomolecular layer of the specifically reactive protein into immersion in the solution containing the protein to be separated and returning the first portion of the belt from the weak acid solution to the solution containing the protein to be separated.

This specification also describes and claims an apparatus for preparing a purified concentration of a solution of a protein antibody or antigen, the apparatus comprising a continuous belt substrate having thereon. a monomolecular layer of protein specifically reactive with the protein antibody or antigen, a first containment means containing a solution including the protein antibody or antigen, a second containment means containing a solution for severing bonds between the protein antibody and antigen and the specifically reactive protein. and means for alternately immersing the substrate in the solutions in the first and second containment means.

The present is a modification or improvement of the method and apparatus described in U.K. Patent Specification No. 1443182.

Immunological reactions are highlv specific interactions in which an antigen combines with an antibody specific to the antigen to form an immunological complex. Immunological reactions taking place within a biological system such as an animal are vital to the animal in combating disease. In a biological system. the entrv of a foreign protein. i,e., the antigen, causes the biological system to produce the specific antibody proteins to the antigen in a process not fully understood at this time. The antibody protein molecules have available chemical bonding sites which complement those on the antigen molecule and so the antigen and antibody combine to form an immunologically complexed protein.

Antibodies are produced by biological systems in response to invasion thereof by foreign proteins. Hence, the detection of antibodies present in a biological system is of medical diagnostic value in determining the antigens to which the system has been exposed.

Conversely. the detection of certain antigens of a biological system also has medical diagnostic value: examples of diagnostic detection of antigens include detection of HCG protein molecules in urine as a test for pregnancy, and detecction of hepatitis associated antigen molecules in blood of prospective blood donors.

In addition to the immunological reaction which occurs between specific protein antigens and specific protein antibodies resulting in the formation of a protein antigen-protein antibody complex. other immunological complexing reactions between immunologicaly reactive antigens and antibodies are also contemplated by this invention. In addition.

specific reactions between other biological particles, such as enzymes and their substrates, are also among the methods contemplated herein and are embraced bv the term "immunological reaction" as used herein. Furthermore, the terms 'antigen" and "antibody" as used herein are intended to encompass such terms as enzymes. substrates of enzymes, and similar biological particles. As will also be seen, the method is versatile enough to permit substitution of a specific antibody for the corresponding antigen and the antigen for the corresponding specific antibody.

For instance, the following systems include biological particles which are capable of undergoing the immunological reactions described herein: Viruses Bacteria and Bacterial toxins Fungi Parasites Animal tissue and Animal bodv fluids.

With respect to viruses. the antigens are viral cultures. or parts thereof, and the antibody specific thereto can be produced by administration to a living host. Illustrative antigen-antibody complexes in the following virus systems are useful in the herein disclosed procedure: Rubella virus culture (antigen) - Rubella virus antibodv: polio virus culture (antigen) - polio virus antibody: vesicular stomatitis virus (VSV culture (antigen) - VSV antibody.

Regarding bacteria and bacterial toxins, the antigens are the particular bacteria or bacterial toxin. or parts thereof, and the antibody is produced by injection into a living host.

The following are illustrative examples of antigen-antibody pairs which can be used in the present method: tetanus toxoid suspension.

(antigen) - tetanus antibody: diphtheria toxin suspension (antigen) - diphtheria antibody: Neisseria gonorrhoeal suspension (antigen) - gonorrhea antibody: Treponema pallidum suspension (antigen) - syphilis antibody.

As for fungi, the antigens are antigenic extracts of fungal suspensions and the antibody is the flingal antibody produced by injection into a living host. Antigen-antibody complexes of fungi systems are illustrated by the following: Aspergillus extract suspension (antigen) - aspergillus fungus antibody: Candida extract suspension (antigen) - candida fungus antibody.

Antigens and antibodies in parasite systems are obtained in a similar fashion to those of fungi. The system Toxoplasma gondii extract (antigen) - Toxoplasma gondii antibody is a typical example.

Bv the term 'polysaccharides' is meant a system wherein the antigen is a carbohydrate antigen. An example of such an antieen-antibodv containing system is pneumococcus poly-saceharides (antigen) - Pneumococcus antibody.

In addition to the typical enzvme - eaxyme substrate reaction which is intended to be covered herein. enzymes themselves. or parts thereof may be utilized as antigens and the antibody is the particular enzyme antibody elaborated by a living host after injection.

Illustrative antigen-anflbody complexes of enzyme systems are: Trypsin extract - trypsin antibody chymotrypsin extract - chymotrypsin antibodv pepsin extract - pepsin antibody ribonuclease extract - ribonucleise antibody thrombin extract - thrombin antibody amylase extract - amvlase 'antibody penicillinase extract - penicillinase antibody With respect to hormones. the antigenic constituent is usually found in a hormone extract and the antibody is the particular hormone antibody elaborated by the living organism after injection. An exemplary antigen-antibody complex is: insulin - insulin antibody As presently practiced, both the collection and purification and the diagnostic utilization of immunol)gically reactive antigens and antibodies rely upon the complexing of the antigen and antibody specific to the antigen.The classic example of these diagnostic uses is the blood typing procedure in which blood samples are mixed with A and B type serum antibodies and blood type is determined by observing any agglutination occurring in the hlood samples. The human chorionic gonadotrophin protein pregnancy test as currently practiced is an inhibition test. The test is performed by mixing a quantity of HCG inti-serum into a urine specimen. A plurality of polystyrene spheres which have been coated with HCG protein are then introduced into the previously prepared urine specimen.

Ibe polystyrene spheres will agglutinate if, but only if, HCG protein is absent from the urine specimen. If HCG protein is absent from a urine specimen, the HCG protein on the polystyrene spheres complexes with the HCG anti-serum previously introduced in the urine specimen and the spheres agglutinate. If, on the other hand. HCG protein is present in the urine specimen in sufficient quantity. it complexes with the previously introduced HCG anti-serum forming a complex which precipitates out of the specimen so that the previously introduced inti-serum is no longer available to complex with the HCG protein on the spheres to cause agglutination thereof.The present HCG protein pregnancy test could be simplified hv adhering HCG anti-serum onto the polystyrene spheres and directly testing a urine specimen. In this case. the polystyrene spheres would agglutinate if. but only if. HCG protein is present in the specimen.

It appears that the reason this simpler procedure has not been employed is that the iviilable HCG anti-seri are complex mixtures containing a large proportion of constituents other than HC'G antibodies. The additional effort required in the prior art to extract the antibodies from the HCG anti-sera made the inhibition test, utilizing sera directly, preferable in the prior art. However, in accordance with one embodiment of this invention, a procedure is provided wherebv HCG antibodies are efficiently separated from sera and.

which pncedure. furthermore, produces diagnostic apparatus wherebv the simpler. direct tesi is perfirm'.ible.

Thee present procedure for obtaining purified concentrations of antibodies comprises the steps of stimulating the production of antibodies in an animal bv introducing the antigen into the animal's system. obtaining blood serum from the animal which contains the antibodies in a dilute forum. and mixing a quantity of the specific antigen into the serum. The mixture of antigen and antibody complexes and precipitates out of the serum solution. The remlining constituents of the serum are drawn off and the anitbody-antigen precipitate is dissolved in an acid which severs the complexing bonds.At this point one has a solution of anitgen and antibody molecules in acid. Since the antibody and antigen molecules have differing physical characteristics. for example. weight. they may be separated from each other by mechanical means. for example. by centrifuging.

It is known that the antil odv-antisTell complexing reaction will take place when an antigen is aelsol heal at a surface. The complexing reaction at a surface has been observed bv means of an ellipsometer.

The present invention provides a method of preparing a purified concentration of an immunologically reactive antibody comprising the steps of immersing a belt of substrate material in an aqueous medium containing a corresponding antigen specifically reactive to said antibody to coat said belt with a monomolecular layer of said antigen, immersing a portion of said belt in an aqueous medium containing said anitbody. and immersing said portion of said belt in a solution of an immunological bond-cleaving reagent other than a yak acid solution and forming a solution of said anitbody in said bond-cleaving reagent solution and leaving said antigen coated on said belt.

lii a further aspect this invention provides a method of preparing a purified concentration of an immunologically reactive antibody comprising the steps of immersing a belt of substrate material comprising a plurality of metallized slides affixed to a flexible belt in an aqueous medium containing a corresponding antigen specifically reactive to said antibody to coat said metallized slides with a monomolecular layer of said antigen, immersing a portion of said belt in an aqueous medium containing said antibody. and immersing said portion of said belt in a solution of an immunological bond-cleaving reagent and forming a solution of said antibody in said bond-cleaving reagent solution and leaving said antigen coated on said belt.

The apparatus of this invention for preparing a purified substrate of an immunologically reactive antibody comprises a substrate having thereon a layer of corresponding antigen specifically reactive to said antibody, a first containment means containing an aqueous medium containg said antibody. a second containment means containing a solution of an immunological bond-cleaving reagent other than a weak acid and means for serially immersing said substrate into said aqueous medium and said solution of bond-cleaving reagent in said first and second containment means.

This invention also provides apparatus for preparing a purified concentration of an immunologically reactive antibody comprising a substrate comprising a plurality of metallized slides having thereon a layer of corresponding antigen specifically reactive to said antibody, a first containment means containing an aqueous medium containg said antibody, a second containment means containing a solution of an immunological bond-cleaving reagent and means for serially immersing said substrate into said aqueous medium and said solution of bond-cleaving reagent in said first and second containment means.

The procedures of this invention can, of course, be reversed by starting with an aqueous medium containing the antibody and can thus be utilized to produce purified or concentrated antigen.

The present invention will be further described by way of example only, with reference to the accompanying drawings in which: Figure 1 is a flow chart illustrating the process steps of the various embodiments of this invention.

Figures 2a, 2b and 2c are elevation views of an apparatus illustrating a visually observable immunological reaction.

Figures 3a, 3h, and 3c are elevation views of apparatus useful for the purification and concentration of antigens and antibodies in accordance with one embodiment of this invention.

Figure 4 is a mechanical schematic diagram of apparatus in accordance with an additional embodiment of this invention for concentrating and purifying antigens and antibodies.

Figure 1 is a flow chart illustrating the process steps involved in practicing this invention.

In reading the flow chart from top to bottom, each vertical level represents one time sequential step of the process. The appearance of several steps arrayed horizontally at a given vertical level in the flow chart indicates the alternative prformance of one of the indicated steps at the indicated sequential position in accordance with the various embodiments of this invention.

In accordance with the first embodiment of this invention. the process begins at block .13 of Figure I in which a wafer of substrate material which may be for example. metal. glass.

mica. plastic, fused silica or quartz. possiblv in the form of a metallized glass slide, is immersed in an aqueous medium containing a first biological constituent of interest. This first biologial constituent of interest may be biologically an antigen. which is to be used to purifv its corresponding specifically reacting antibody. The first antigen adsorbs onto the substrate in a monomolecular layer. Any antigen or antibody will adsorb in such monomolecular layer and no further adsorption will take place.That is. the antigen or antibody will attach to the substrate. but will not attach to itself. After a monomolecular laver of antigen has formed over the surface of the substrate. the coated substrate is removed from the aqueous medium containing the first antigen.The time required to completelv coat the substrate is a function of the concentration of the antigen in the aqueous medium and the degree of agitation of the aqueous medium. As an example. a 1 percent bovine serum albumin- solution completely coats a slide in approximately 30 minutes with a monomolecular layer. The next step, illustrated in block 14 of Figure 1 is to immerse the coated substrate in an aqueous medium containing the specifically reacting antibody to the first antigen. This aqueous medium may. and typically does. contain many constituents in addition to the specifically reacting antibody. However. no antigen or antibody other than the specifically reacting antibody will adhere to the first layer on the substrate.Of course. as is known. certain biological substances may adhere by a phenomenon known os non-specific sticking. Such substances are found in for example sera. There are a number of ways to minimize non-specific sticking. the most convenient of which appears to be in diluting the media carrying such substances. In any event, if the specifically reacting antibody is not present the substrate following immersion in the aqueous medium will still contain only a monomolecular layer thereon. If,on the other hand. the specificallv reacting antibody is present in the aqueous medium. immunological complexing betwen the first antigen and its specifically reacting antibody will take place and the substrate will. after a time. have a bimolecular layer thereon.It is to be noted that the steps illustrated in blocks 13 and 14 of Figure 1 are common to all of the embodiments of this invention. The time required for the adhesion of a complete second molecular layer onto the coated substrate is again a function of the concentration of specifically reacting antibody in the aqueous medium. For antibodies in blood serum, this time may be as long as one day or as short as minutes depending upon the concentration. The next step is to immerse the coated substrate in a reagent capable of cleaving immunological bonds. Such a reagent can be a weali acid solution. a base solution or a strong salt solution. as illustrated in block 15 of Figure 1.As further detailed in our Patent Specification No. 1443181, this step can be used for diagnostic purposes by simultaneously observing the coated substrate in an ellipsometer as illustrated in block 22 of Figure 1. While the formation of the specifically reacting layer on the first antigen caoted substrate may take an extended period of time, the immunological bond between the two biological constituents is severed very quickly by the immunological bond-cleaving solution. Accordingly, the observation made with the ellipsometer can be the relatively simple observation of change of film thickness rather than the more complicated measurement of absolute thickness.At the same time. observing the stripping away of the specifically reacting antibody layer by action of the immunological bond-cleaving reagent may be performed much more rapidly than the prior art method of observing the building of the specifically reacting protein layer. Accordingly, a large plurality of test slides may be prepared in accordance with block 13 and each exposed to one of a large plurality of serum samples as indicated in block 14, and then in a short time each test slide may be examined serially in accordance with blocks 15 and 22 to determine which of the serum samples contained the specifically reacting antibody to the first antigen.Since the cleaving reagent will not strip the first antigen from the substrate, those coated substrates which were immersed in aqueous media which did not contain the specifically reacting antibody will exhibit no change of film thickness when immersed in the cleaving reagent and observed in an ellipsometer. On the other hand, those which were immersed in an aqueous medium which did not contain the specifically reacting antibody will exhibit approximately a factor of 2-5 change in thickness when immersed and observed in an ellipsometer. Each observaton can be made in a few minutes thereby providing an efficient.

and therefore diagnostically significant. test procedure.

This invention. more importantly; also provides for the concentration and purification of antigens or antibodies and comprises the steps illustrated in Figure 1 at blocks 13, 14. 15, and 23. The substrate is first immersed in aqueous medium containing the available antigen of the antigen-antibody pair dS illustrated in block 13. In the usual case. this will be the antigen. but this invention is not dependent upon the biological identity of the first biological constituent. The coated substrate produced in accordance with block 13 of Figure 1 is then immersed in aqueous medium containing the specifically reacting antibody to the first antigen as shown in block 14.In the usual case, the specificalv reacting biological constituent will be an antibody and the aqueous medium employed in block 14 will be the blood serum of an animal which has been exposed to the antigen. The substrate, now coated with a bimolecular layer, is next immersed in the cleaing reagent as shown in block 14 of Figure 1 which strips the specifically reacting anitbodv layer from the first antigen layer. At this point the substrate is coated with a monomolecular layer of the first biological constituent and a purified solution of specifically reacting antibody in the cleaving reagent has been provided.The next step. as shown in blcok 23 of Figure 1 is to return the substrate with its first layer adhering thereon to the aqueous medium containing the specifically racting antibody to pick up a second layer thereof which is again stripped by the cleaving reagent thereby increasing the concentration of specifically reacting antibody. This process is continued and provides for the collection of a concentration of pure specifically reacting antibody in the cleaving reagent bath. The process can of course be reversed, i.e. started with antibody. to provide purified or concentrated antigen.

A method of visually determining the presence of antigen of antibody during the purification method is illustrated in Figures 2a. 2b or 2c. The substrate which must be a light transmissive substrate such as glass. plastic. fused silica, mica or quartz. and is preferably glass. with microscope slides being a convenientlv available source, is first coated with a plurality of metal globules by evaporating a metal, for example. indium, onto the substrate as indicated in block 12 of Figure 1. The indium may also have a gold overlayer. A detailed discussion of substrate metallization is found in our U.K. Patent Specification No. 1479661.

For example, the indium is evaporated slowlv from a tantalum boat onto the glass substrate in an ordinarv vacuum of about 5 x l(X-5 mm of mercury. Because the indium atoms have high mobility on the surface of the substrate and do not wet the glass substrate significantly. the indium evaporated onto the substrate agglomerates into small particles.

Any metal having similar characteristics so that it will form globules on the substrate when evaporated thereon may be used. In addition to indium, gold, silver, tin, bismuth. and lead have been successfully used. The evaporation of metal is continued until the substrate appears light brown in colour. At this point, the metal globules have diameters on the order of 1()(1() A. The precise size of the globules is not critical but they must have diameters equal to a laree fraction of one of the wavelengths of visible light. The next step is to immerse the globule-covered substrate in an aqueous medium containing a first antigen as illustrated in block 13 of Figure 1. The first antigen again adheres in a monomolecular layer over the substrate and the metal globules thereon. When a monomolecular layer has formed, the coated substrate may be used to test aqueous media for the presence of a specifically reacting antibody to the first antigen by immersing the coated substrate in the aqueous medium as indicated in block 14 of Figure I. If the specifically reacting antibody was present. the sbustrate and metal globules have a bimolecular layer adhering thereto. If the specifically reacting antibody was not present, only a monomolecular layer overlies the substrate and metal globues. The coated substrate is then viewed by either reflected or transmitted light as indicated in block 18 of Figure 1 and a determination is made from the appearance of the coated substrate as to the thickness of the layer adhering thereto and accordingly as to the presence or absence of the specifically reacting antibody.The detection of antibody layers corresponds to variations in the shade of brown which is observed in the coated substrate. These variations are quite pronounced and the detection of layers is therefore a simple straight forward procedure. The particles alone on the substrate appear as a shade of brown, the particles coated with a monomolecular layer appear as a darker shade of brown, and the particles covered with a bimolecular layer appear as a still darker shade of brown. This detection method is based on the fact that electromagnetic radiation is scattered to a large degree by conducting spheres having diameters equal to a large fraction of a wavelength of the incident energy and that in the case of scattering from such spheres, the scattering is strongly influenced by a thin dielectric coating applied to the spheres.

Figures 2a. 2b and 2c are a highly magnified view of a portion of apparatus in accordance with the embodiment just described. Figure 2a shows a portion of substrate material 31 having a plurality of globules of evaporated metal 32 attached thereto. After immersion in an aqueous medium containing a first antigen, the slide segment comprising substrate 31 and metal globules 32 is coated with a monomolecular layer of molecules 34 of the first antigen as indicated in Figure 2b generally at 33.If the apparatus indicated generally at 33 is exposed to specifically reacting antibody to the antigen of molecules 34, the apparatus will acquire the appearance indicated generally at 35 in which substrate 31 and metal globule 32 are coated with a bimolecular laver comprising the molecules 34 of th first antigen forming a first monomolecular layer overlying substrate 31 and globules 32 and a second monomoleculir layer of antibody consisting of the molecules 36 of the specifically reacting antibody to the first antigen. immunologically bonded to the molecules of the first antigen or antibody and overlying the first layer, the metal globules and the substrate.

Figures 3a. 31' and 3c are highly magnified sectional elevation views of apparatus useful for diagnostic purposes and for the purification and concentration of antigens and antibodies in accordance with this invention. Indicated generally at 4(x is a substrate 41 coated with a monomolecular laver of antigen molecules 42 which has been prepared as discussed above. Indicated generally at 43 is substrate 41 and antigen layer 42 to which has been immunologically bonded a second monomolecular layer of antibody molecules 44 of th specifically reacting antibody to molecules 42 in accordance with the procedures discussed above. A second substrate 45 has thereon a drop 46 of a cleaving reagent solution.

The mutually facing surfaces of substrates 41 and 45 having thereon respectively a bimolecular layer and a drop of. for example. citric acid in a 0.1 l normal solution, are then physically brought into contact with each other. In accordance with this invention, the weak acid drop 46 severs the immunological bonds between molecules 42 and molecules 44 without affecting the biochemical characteristics of either biological constituent and without severing the adhesion bond between the first antigen molecules 42 and substrate 41.When substrates 41 and 45 are again separated as indicated generally at 47, substrate 41 has adhering thereon a monomolecular layer of first antigen molecules 42 and substrate 45 has adhering thereon a monomolecular layer of specifically reacting antibody molecules 44.

Substrate 41 with molecules 42 thereon may then be used to repeat the process producing another substrate coated with a monomolecular layer of specifically reacting antigen or antibody. while substrate 45 with the purified antibody molecules 44 may be utilized for manx purposes.

Figure 4 is a mechanical schematic diagram of an apparatus in accordance with this invention for concentrating and purifying antigens and antibodies. It will aid in understanding the embodiment of Figure 4 to realize at the outset that in operation. the Figure 4 embodiment is a modification of the Figure 3 embodiment just discussed. In Figure 4. a continuous flexible belt of substrate material 51 which is coated with a monomolecular layer of antigen which specifically reacts with the antibody to be concentrated and purified is first introduced into a container 52 which contains a quantitv of liquid 53 which includes the antibody to be purified.Immunological complexing between antigen and antibody takes place in container 53 and then substrate belt 51. now containing a bimolecular antigen-antibody layer. proceeds to container 58 containing the immunological bond cleaving reagent solution 59 which severs the immunological bond between the two biological constituents thereby collecting the molecules of the second layer in solution 59.

Upon exiting container 58 therefore substrate 51 again has thereon only the original monomolecular layer. Substrate 51 is then returned to container 52 to again pick up a monomolecular layer of the antibody to be collected which is again stripped off in container 5X. Belt 51 is driven through aqueous 53 and 59 by capstans 6() and 63 operating cooperatively with respectively pinch rollers 61 and 61. Capstans 60 and 63 may be driven by nny suitable means which may conveniently be small electric motors coupled to capstans 6() and 63 by speed reducing gear trains.Because the immunological complexing reaction is a much slower reaction than the bond-severing reaction, it is desirable for efficiency that coated substrate belt 51 be in contact with aqueous medium 53 containing the antibody to be collected for a much greater time than it is in contact with the cleaving reagent solution 59. Accordingly. it is desirable that container 52 be substantially larger than container 58 and that a plurality of upper and lower idler rollers 54 and 55, respectively, be provided for causing multiple passage of belt 51 through aqueous medium 53. This provides for a greater length of belt 51 being in contact with aqueous medium 53 at any given time and therefore for any given segment of belt 51 being exposed to aqueous medium 53 for a greater period of time.On the other hand, a single passage of belt 51 through the cleaving reagent solution 59 is quite sufficient to strip the second layer therefrom. Accordingly, a pair of upper idler rollers 56 and a single idler roller 57 are provided for guiding belt 51 through solution 59. A plurality of idler rollers 64 are provided as required to mechanically support belt 51 during its passage between container 52 and 58.Containers 52 and 58 are preferably provided with agitating means 65 and 66, respectively. which fluid-sealingly penetrate the walls thereof for agitating solutions 53 and 59 to ensure the refreshment of the solutions in contact with belt 51. Container 59 is provided with drain pipe 67 controlled by valve 68 for draining solution 53 therefrom when the concentration of the desired antibody therein has been decreased to the point at which efficient collection thereof is no longer possible. A fresh sample of aqueous medium 53 may then be added to container 52 through the open upper end thereof. Container 58 is provided with drain pipe 69 controlled by valve 70 for draining off the desired solution of purified antibody in the cleaving reagent when the desired concentration has been reached.A fresh charge of the cleaving reagent may similarly be added to container 5X through the open upper end thereof. Since as disclosed above with reference to Figure 3, substrate belt 51 may be coated with any desired antigen or antibody, the method and apparatus of Figure 4 is useful for collecting a purified concentration of any arbitrary antigen or antibody desired so long as an immunological reaction involving the desired antigen-antibodv pair exists. Additionally, by slightly modifying its operation, the apparatus of Figure 4 may be used to selectively remove a specific undesired antigen or antibody film a mixture such as a serum.To accomplish this, the apparatus of Figure 4 is simply run tor an extended period of time without refreshment of aqueous medium 53 and with periodic refreshment as may be required of solution 59. In this case, the product obtained at drain pipe 67 is serum 53 from which the undesired antigen or antibody has been eliminated to any required degree depending only on the time of operation of the system.

The above-described operation may be modified so that the cleaving reagent solution 59 is applied on the surface of a second substrate, e.g. a belt. The first substrate, e.g. belt containing the bimolecular layer touches or abuts the second substrate thereby leaving the specifically reacting antibody adhered to the second substrate as a monomolecular layer.

The second substrate can be of the same type and composition as described above for the first substrate.

It should be noted that the configuration of the substrate used herein is not critical to the invention. It can be slide shaped, e.g., in the form of metallized glass slides because of the ready availabilitv of such slides. However. if the belt operation is to be followed. it will be in the form of a belt. For instance. the substrate can comprise a plurality of metallized slides affixed to a flexible belt. The only limitation imposed on the substrate is its abilitv to allow the formation of a monomoleculir laxer thereon. The dimensions or form will be dictated bv the manner in which the procedure is carried out including subsequent analysis and its objective.If it is for collection of a particular immunologically active biological constituent -a belt is preferred.

The following examples. which are to be regarded as illustrative and not limiting. show how antigens and antibodies are purified and are collected according to this invention.

Fxciiiipk' 1 A procedure for isolating hepatitis antibody is carried out as follows: A glass slide is metallized with a layer of indium metal (lobules and immersed in a solution of I mg./ml. of hepatitis-associated antigen in ().X5's saline. The slide is removed and incubated in a moist chamber (plastic box filled with wet sponges) at 230C. until the antigen adheres to the metallized surface (1()-30 minutes). The slide is washed with distilled water and blown dry with a jet of air. The slide can be stored for later use or used immediatelv. The slide is then exposed to a preparation made from rabbit or goat blood serum which contains antibody to hep'ttitis-associated antigen.The slide and antibody solution are incubated at 37"C. in a plastic box attached to a shaker, for approximately 15 minutes. After incubation. the slide is again washed with distilled water. The slide is then immersed in a solution of citric acid (().IN) with agitation. The resulting slide contains monomolecular layer of antigen and is available for reuse and the citric acid solution contains pure antibody, whose isolation is effected using standard techniques.

A:rample 2 A procedure for isolating gonorrhoea antibodies is carried out as follows: A glass slide is metallized with a layer of titanium and the surface layer is then oxidized to produce a cover layer of an oxide of titanium. The slide is immersed in a Neisseria gonorrhoeae extract dissolved in salt water to produce a concentration of I mg.lmi. The slide is incubated in a moist chamber (plastic box filled with wet sponges) at room temperature 23"C) until the antigen adheres to the oxide surface (10-30 minutes). The slide is washed with distilled water and blown dry with a jet of air. The slide can be stored at this point for later use.The slide is then immersed in a preparation made from rabbit or goat blood serum which contains antibody to the Neisseria antigen. Total volume is 2 ml. The slide and antibody solution are incubated at 37"C. in a plastic box attached to a shaker, for approximately 15 minutes. After incubation, the slide is again washed with distilled water. The slide is then immersed in a solution of citric acid (0. IN) with agitation. The resulting slide contains monomolecular layer of antigen and is available for reuse and the citric acid solution contains pure antibody. whose isolation is effected using standard techniques.

Exuinpk ,? - JO Purification procedures according to this invention are carried out by the procedure of Example I substituting the following substrates, antigens, compounds and antibody preparations: Antibody Example Substrate Antigen Source 3 indium hepatitis- rabbit blood metallized associated serum glass 4 indium hepatitis- rabbit blood metallized associated serum glass 5 gold hepatitis- rabbit blood metallized associated serum glass 6 tantalum hepatitis- rabbit blood metallized associated serum glass 7 gold rubella rabbit blood metallized virus serum glass extract 8 gold polio rabbit blood metallized virus serum glass extract 9 gold vesicular rabbit blood metallized stomatitis serum glass virus extract 10 gold Treponema rabbit blood metallized pallidum serum glass suspension (syphilis) In addition to the above specific examples. an isolation procedure may be carried out in accordance with this invention using slide shaped substrates metallized with gold or tantalum upon which are deposited aqueous solutions of the following antigenic preprations: Tetanus toxoid (tetanus) diphtheria toxin (diphtheria) asperigillus extract (fungus) candid extract (fungus) toxoplasma gondii (parasite) nucleoprotein and DNA thyroglobulin (Hashimoto's disease) collagen fractions human chorionic gonadotrophin (pregnancy) insulin keyhole lymphet hemocyanin (antibody response) pencillinase extract (large enzyme) Exmlplr 1/ The procedures of Examples 1-10 can be carried out in a cyclic fashion wherein a substrate in belt form coated with a monomolecular layer of antigen is introduced into a container which contains a second aqueous medium containing the antibody to be purified.

The so-treated belt containing a bimolecular layer is then immersed into a container containing weak acid (().IN citric acid), preferably while agitating. severing the immunological bond leaving the second or outer layer component in the citric acid solution for eventual recovery and the substrate having a monomolecular layer of first antigen which is recvcled to the first aqueous medium and then to the acid solution.

WHAT WE CLAIM IS: 1. A method of preparing a purified concentration of an immunologically reactive antibody comprising the steps of immersing a belt of substraate material in an aqueous medium containing a corresponding antigen specifically reactive to said antibody to coat said belt with a monomolecular layer of said antigen, immersing a portion of said belt in an aqueous medium containing saidantibody. and immersing said portion of said belt in a solution of an immunological bond-cleaving reagent other than a weak acid solution and forming a solution of said antibody in said bond-cleaving reagent solution and leaving said antigen coated on said belt.

2. A method of preparing a purified concentration of an immunologically reactive antibody comprising the steps of immersing a belt of substrate material comprising a plurality of metallized slides affixed to a flexible belt in an aqueous medium containing a corresponding antigen specifically reactive to said antibody to coat said metallized slides with a monomolecular layer of said antigen. immersing a portion of said belt in an aqueous medium containing said antibody. and immersing said portion of said belt in a solution of an immunological bond-cleaving reagent and forming a solution of said antibody in said bond-cleavinp reatent solution and leaving said antigen coated on said belt.

3. A method as claimed in claim I or claim 2 of preparing a purified concentration of an immunologicallv reactive antigen wherein a specifically reactive antibody is substituted for the antigen and the corresponding immunologically reactive antigen is substituted for the antibody.

d A method as claimed in anv one of claims I to 3 wherein said antigen is a protein antigen. said antibody is a protoinantibody specific to the antigen and said aqueous media are solutions.

5. A method as claimed in any one of the preceding claims further including the steps of agitating said aqueous medium containing said immunologically reactive antibody, and agitating said solution of said bond-cleaving reagent.

6. A method as claimed in claim 5 wherein said agitating of said aqueous medium and said bond-cleaving reagent solution is performed continuously.

7. A method as claimed in any one of the preceding claims further including the step of periodically draining and refreshing said aqueous medium containing said immunologically reactive antibody.

S. A method as claimed in claim 7 wherein said purified immunologically reactive antibody is collected by draining said solution of said bond-cleaving reagent and further including the step of refreshing said solution of said bond-cleaving reagent after said draining.

9. A method as claimed in any one of the preceding claims wherein the bond-cleaving reagent solution is applied to the surface of a second belt substrate such that the belt of substrate material touches or abuts the second belt substrate leaving the specifically reacting antibody adhered to the second belt substrate.

1(). A method as claimed in any one of claims 1 to 7 further comprising the step of applying a metal coating to said surface of a second substrate prior to applying said

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (1)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   tantalum upon which are deposited aqueous solutions of the following antigenic preprations: Tetanus toxoid (tetanus) diphtheria toxin (diphtheria) asperigillus extract (fungus) candid extract (fungus) toxoplasma gondii (parasite) nucleoprotein and DNA thyroglobulin (Hashimoto's disease) collagen fractions human chorionic gonadotrophin (pregnancy) insulin keyhole lymphet hemocyanin (antibody response) pencillinase extract (large enzyme) Exmlplr 1/ The procedures of Examples 1-10 can be carried out in a cyclic fashion wherein a substrate in belt form coated with a monomolecular layer of antigen is introduced into a container which contains a second aqueous medium containing the antibody to be purified.

   The so-treated belt containing a bimolecular layer is then immersed into a container containing weak acid (().IN citric acid), preferably while agitating. severing the immunological bond leaving the second or outer layer component in the citric acid solution for eventual recovery and the substrate having a monomolecular layer of first antigen which is recvcled to the first aqueous medium and then to the acid solution.

   WHAT WE CLAIM IS:

   1. A method of preparing a purified concentration of an immunologically reactive antibody comprising the steps of immersing a belt of substraate material in an aqueous medium containing a corresponding antigen specifically reactive to said antibody to coat said belt with a monomolecular layer of said antigen, immersing a portion of said belt in an aqueous medium containing saidantibody. and immersing said portion of said belt in a solution of an immunological bond-cleaving reagent other than a weak acid solution and forming a solution of said antibody in said bond-cleaving reagent solution and leaving said antigen coated on said belt.

   2. A method of preparing a purified concentration of an immunologically reactive antibody comprising the steps of immersing a belt of substrate material comprising a plurality of metallized slides affixed to a flexible belt in an aqueous medium containing a corresponding antigen specifically reactive to said antibody to coat said metallized slides with a monomolecular layer of said antigen. immersing a portion of said belt in an aqueous medium containing said antibody. and immersing said portion of said belt in a solution of an immunological bond-cleaving reagent and forming a solution of said antibody in said bond-cleavinp reatent solution and leaving said antigen coated on said belt.

   3. A method as claimed in claim I or claim 2 of preparing a purified concentration of an immunologicallv reactive antigen wherein a specifically reactive antibody is substituted for the antigen and the corresponding immunologically reactive antigen is substituted for the antibody.

   d A method as claimed in anv one of claims I to 3 wherein said antigen is a protein antigen. said antibody is a protoinantibody specific to the antigen and said aqueous media are solutions.

   5. A method as claimed in any one of the preceding claims further including the steps of agitating said aqueous medium containing said immunologically reactive antibody, and agitating said solution of said bond-cleaving reagent.

   6. A method as claimed in claim 5 wherein said agitating of said aqueous medium and said bond-cleaving reagent solution is performed continuously.

   7. A method as claimed in any one of the preceding claims further including the step of periodically draining and refreshing said aqueous medium containing said immunologically reactive antibody.

   S. A method as claimed in claim 7 wherein said purified immunologically reactive antibody is collected by draining said solution of said bond-cleaving reagent and further including the step of refreshing said solution of said bond-cleaving reagent after said draining.

   9. A method as claimed in any one of the preceding claims wherein the bond-cleaving reagent solution is applied to the surface of a second belt substrate such that the belt of substrate material touches or abuts the second belt substrate leaving the specifically reacting antibody adhered to the second belt substrate.

   1(). A method as claimed in any one of claims 1 to 7 further comprising the step of applying a metal coating to said surface of a second substrate prior to applying said

   bond-cleaving reagent solution thereto.

   11. A method as claimed in claim 9 or claim 10 wherein said second belt substrate is a plurality of metallized slides affixed to a flexible belt.

   12. A method as claimed in claim 11 or any one of claims 3 to 8 when dependant on claim 2 wherein the metal is indium, gold, silver, tin or lead.

   13. A method as claimed in claim 11 or any one of claims 3 to 8 when dependant on claim 2 wherein the substrate comprises indium on a glass slide.

   14. A method as claimed in claim 11 or any one of claims 3 to 8 when dependant on claim 2 wherein the substrate comprises gold on a glass slide.

   15. A method as claimed in claim 11 or any one of claims 3 to 8 when dependant on claim 2 wherein the substrate comprises a glass slide coated with a thin indium layer and overcoated with a gold layer.

   16. A method as claimed in claim 11 or any one of claims 3 to 8 when dependant on claim 2 wherein the metallized slide also includes a metal oxide layer intermediate the metal of the slide and said monomolecular layer.

   17. A method as claimed in claim I I or any one of claims 3 to 8 when dependant on claim 2 wherein said metal is titanium and said oxide is an oxide of titanium.

   18. An apparatus for preparing a purified concentration of an immunologically reactive antibody comprising a substrate having thereon a layer of corresponding antigen specifically reactive to said antibody. a first containment means containg an aqueous medium containing said antibody, a second containment means containing a solution of an immunological bond-cleaving reagent other than a weak acid and means for serially immersing said substrate into said aqueous medium and said solution of bond-cleaving reagent in said first and second containment means.

   19. An apparatus for preparing a purified concentration of an immunologically reactive antibody comprising a substrate comprising a plurality of metallized slides affixed to a flexible belt. the metallized slides having thereon a layer of corresponding antigen specifically reactive to said antibody. a first containment means containg an aqueous medium containing said antibody, a second containment means containing a solution of an immunological bond-cleaving reagent and means for serially immersing said substrate into said aqueous medium and said solution of bond-cleaving reagent in said first and second containment means.

   2(1. An apparatus as claimed in claim 18 wherein the sbustrate comprises gold on a glass slide.

   21. An apparatus as claimed in claim 18 wherein the substrate comprises a glass slide coated with a thin indium layer and over-coated with a gold layer.

   22. An apparatus as claimed in claim 18 wherein the metallized slide also includes a metal oxide layer intermediate the metal of the slide and said monomolecular layer.

   23. An apparatus as claimed in claim 22 wherein said metal is titanium and said oxide is an oxide of titanium.

   24. A method as claimed in claim 1 substantially as hereinbefore described in Example 10.

   25. A purified concentration of an immunologically reactive antibody when produced by a method as claimed in any one of claims 1 to 15 and 24.

   26. An apparatus as claimed in claim 18 substantially as hereinbefore described with reference to and as illustrated in the accompanying drawings.

   '7. A purified concentration of an immunological reactive antibody when produced using an apparatus as claimed in any one of claims 18 to 23.