CA1215322A - Enzyme immunoassay - Google Patents
Enzyme immunoassayInfo
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- CA1215322A CA1215322A CA000412425A CA412425A CA1215322A CA 1215322 A CA1215322 A CA 1215322A CA 000412425 A CA000412425 A CA 000412425A CA 412425 A CA412425 A CA 412425A CA 1215322 A CA1215322 A CA 1215322A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
ABSTRACT
The present invention is characterized in that, in the conventional solid-phase enzyme immunoassay, all the steps of the immunoassay from the step of immunological reaction of the immunologically reactive component(s) or an immunological complex thereof with a carrier-adsorbed reactant through the step of measuring the activity of enzyme contained in the resulting insolubilized product ,are carried out by applying chromatography with a single column. The method of the present invention has the advantages in that a variety of antigens or antibodies can be assayed by using an identical enzyme or insoluble carrier, and that high sensitivity and accuracy can be attained without difficult operations for the method.
The present invention is characterized in that, in the conventional solid-phase enzyme immunoassay, all the steps of the immunoassay from the step of immunological reaction of the immunologically reactive component(s) or an immunological complex thereof with a carrier-adsorbed reactant through the step of measuring the activity of enzyme contained in the resulting insolubilized product ,are carried out by applying chromatography with a single column. The method of the present invention has the advantages in that a variety of antigens or antibodies can be assayed by using an identical enzyme or insoluble carrier, and that high sensitivity and accuracy can be attained without difficult operations for the method.
Description
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DESCRIPTION
1. Title of Invention Enzyme Immun~a 5 say
DESCRIPTION
1. Title of Invention Enzyme Immun~a 5 say
2. Technical Field This invention relates to a novel immunoassay method for the determination of infinitesimal amounts of substances present in body(or biological) fl~ids, such as serum and cerebrospinal fluid, of mammals.
The term "immunoassay" as used herein refers to a method in which the reaction of an immunologically (serologically) antigenic substance (i.e., a so-called antigen) with a so-called antibody immunologically corresponding to it is applied to the determination of the amount or concentration of antigen or antibody present in the fluid.
The term "immunoassay" as used herein refers to a method in which the reaction of an immunologically (serologically) antigenic substance (i.e., a so-called antigen) with a so-called antibody immunologically corresponding to it is applied to the determination of the amount or concentration of antigen or antibody present in the fluid.
3. Background Art Conventionally, various methods such as bioassay or enzyme activity measurement methods have been available for the determination of infinitesimal amounts of substances present in biological fluids. However, they are not satisfactory because of their troublesome operations and their inadequate sensitivity or accuracy. In contrast, the immunoassay technique which has recently been developed can attain significantly high sensitivity and accuracy without requiring difficult operations. Accordingly, this technique is now being employed in the fields of biochemistry and clinical medicine.
Various kinds of immunoassay methods have been proposed, among which the methods using an antigen or antibody combined (or labeled) with a radioisotope, a fluorescent material or an enzyme have found wide application. Specifically further, the so-called solid-phase modes in these immunoassay methods using the labeling materials have been regarded as being most ad~antageous.
1~53~
In the solid-phase immunoassay method, the so-called carrier-adsorbed reactant is used as a reaction material, which has been prepared by adsorbing, under specific reacti~n conditions, an antibody, a so-called antiantibody (i.e~, an 5 antibody produced by using an antibody as the antigen), or so-called protein A (i.e., a special protein produced by certain microorganisms and ha~ing the ability to react with various antibodies) on a water-insoluble carrier such as pieces of polystyrene or silicone, water-insoluble 10 polysaccharides, etc., to insolubilize the former thereby, said carrier-adsorbed reactants in particular cases being hereinafter referred to as antibody-on-carrier, antiantibody-on-carrier, or protein A-on-carrier, respectively.
Now the general principle of solid-phase immunoassay 15 method using an enzyme as the labeling material is explained hereinbelow before proceeding to the description of the present invention.
At least one member selected from the group consisting of an antigen, an antibody, an enzyme labeled antigen, and an enzyme labeled 20 antibody (these are referred generally to immunologically reactive components hereinafter), or an immunological complex thereof, is reacted immunologically with a carrier-adsorbed reactant selected from the group consisting of an antibody-on-carrier, an antiantibody-on-carrier, and protein A-on-25 carrier either simultaneously ^r successively, to thereby result in an insolubiiizea product formed by the binding of the enzyme labeled antigen (Qr enzyme labeled antibody) or i~s immunological complex to the carrier-adsorbed reactant together with an unbound, soluble portion of the labeled antigen (or labeled antibody~.
30 After the unbound labeled antigen (or labeled antibody) is separated from the insolubilized product, the activity of the enzyme contained in the insolubilized Product (particularly, the absorbance of the reaction mixture of the enzyme with its substrate) is measured. Typically, the 35 same procedures as above are repeated with varying amounts of the antigen (or antibody), and by using the measured values thereby, a calibration curve is constructed which 12~53Z2 represents the relationship between the amounts of antigen (or antibody) initially used and the activity of enzyme attached thereto. Next, the same procedures are carried out with a biological fluid containing an ~nknown amount of 5 the antigen (or antibody)~ to measure the activity of the enzyme contained in the resulting insolubilized product.
Then, the amount or concentration of antigen (or antibody) presënt in the fluid is calculated from the measured value by reference to the calibration curve.
While the ~olid-phase enzyme immunoassay method as above has ad~antageously high sensitivity and accuracy, it has the disadvantage of requiring troublesome operations.
Various kinds of immunoassay methods have been proposed, among which the methods using an antigen or antibody combined (or labeled) with a radioisotope, a fluorescent material or an enzyme have found wide application. Specifically further, the so-called solid-phase modes in these immunoassay methods using the labeling materials have been regarded as being most ad~antageous.
1~53~
In the solid-phase immunoassay method, the so-called carrier-adsorbed reactant is used as a reaction material, which has been prepared by adsorbing, under specific reacti~n conditions, an antibody, a so-called antiantibody (i.e~, an 5 antibody produced by using an antibody as the antigen), or so-called protein A (i.e., a special protein produced by certain microorganisms and ha~ing the ability to react with various antibodies) on a water-insoluble carrier such as pieces of polystyrene or silicone, water-insoluble 10 polysaccharides, etc., to insolubilize the former thereby, said carrier-adsorbed reactants in particular cases being hereinafter referred to as antibody-on-carrier, antiantibody-on-carrier, or protein A-on-carrier, respectively.
Now the general principle of solid-phase immunoassay 15 method using an enzyme as the labeling material is explained hereinbelow before proceeding to the description of the present invention.
At least one member selected from the group consisting of an antigen, an antibody, an enzyme labeled antigen, and an enzyme labeled 20 antibody (these are referred generally to immunologically reactive components hereinafter), or an immunological complex thereof, is reacted immunologically with a carrier-adsorbed reactant selected from the group consisting of an antibody-on-carrier, an antiantibody-on-carrier, and protein A-on-25 carrier either simultaneously ^r successively, to thereby result in an insolubiiizea product formed by the binding of the enzyme labeled antigen (Qr enzyme labeled antibody) or i~s immunological complex to the carrier-adsorbed reactant together with an unbound, soluble portion of the labeled antigen (or labeled antibody~.
30 After the unbound labeled antigen (or labeled antibody) is separated from the insolubilized product, the activity of the enzyme contained in the insolubilized Product (particularly, the absorbance of the reaction mixture of the enzyme with its substrate) is measured. Typically, the 35 same procedures as above are repeated with varying amounts of the antigen (or antibody), and by using the measured values thereby, a calibration curve is constructed which 12~53Z2 represents the relationship between the amounts of antigen (or antibody) initially used and the activity of enzyme attached thereto. Next, the same procedures are carried out with a biological fluid containing an ~nknown amount of 5 the antigen (or antibody)~ to measure the activity of the enzyme contained in the resulting insolubilized product.
Then, the amount or concentration of antigen (or antibody) presënt in the fluid is calculated from the measured value by reference to the calibration curve.
While the ~olid-phase enzyme immunoassay method as above has ad~antageously high sensitivity and accuracy, it has the disadvantage of requiring troublesome operations.
4. Disclosure of the Invention We studied the conventional solid-phase enzyme immunoassay method extensively and invented a very efficient technique thereof, by conducting all ~he steps thereof, from the step of the immunological reaction of immunologically reactive component(s) or an immun~logical complex thereof with a 20 carrier-adsorbed reactant ~ough the step of mea~g the activity of the enzyme contained in the resulting insolubilized product,by applying chromatography with a single column.
According to the method of the present invention, a 25 variety of antigens or antibodies can be assayed by using an identical enzyme or insoluble carrier, and high sensitivity and accuracy can be attained without being affected by undesirable interfering components present in the biological fluid, while no difficult operations are 30 required for the method. In particular, the method of the present invention permits the highly sensitive determination of antibodies in serum which has been resarded as being difficult in the prior art.
By the way, besides the solid-phase enzyme immunoassay 3~ method, another enzyme immunoassay technique has hitherto been kno~n in which an immunological reaction mixture of immunologically reactive components is subjected to a -~A
12~5322 physicochemical proceduxe such as gel permeation chromatography, affinity chromatography, or the like to separate the free labeled antigen (or labeled antibody) therefrom, followed by measuring the activity ~f the enz~me
According to the method of the present invention, a 25 variety of antigens or antibodies can be assayed by using an identical enzyme or insoluble carrier, and high sensitivity and accuracy can be attained without being affected by undesirable interfering components present in the biological fluid, while no difficult operations are 30 required for the method. In particular, the method of the present invention permits the highly sensitive determination of antibodies in serum which has been resarded as being difficult in the prior art.
By the way, besides the solid-phase enzyme immunoassay 3~ method, another enzyme immunoassay technique has hitherto been kno~n in which an immunological reaction mixture of immunologically reactive components is subjected to a -~A
12~5322 physicochemical proceduxe such as gel permeation chromatography, affinity chromatography, or the like to separate the free labeled antigen (or labeled antibody) therefrom, followed by measuring the activity ~f the enz~me
5 contained in the remainder to determine the concentration of antigen or antibody initially present. However, the known enzyme immunoassay method as above is different in technical conception from the method of the present invention because the chromatography of the former applies not to the 10 immunological reaction but to the physicochemical reaction.
Additionally, the method u~ing phy~icochemical chromatography ha~, in contrast with the method of the present in~ention, the di~advantages in that the kinds of labeling enzymes and adsorbing materials for use therein must be carefully 15 ~elected in particular case~.
The present invention resides in a method of solid-phase enzyme immunoassay for an antigen (or antibody) which consi~ts of the ~tep~ of (a) reacting immunologically either two immunologically reactive components ~elected from the group consist~ng of an antigen, an antibody, an enzyme labeled antigen and an enzyme labeled antibody, or a reaction mixture containing an immunological complex of said components, with a carrier-adsorbed reactant selected from *he group consisting of an antibody-on-carrier, an antiantibody-on-carrier and protein A-on-carrier, simultaneously or successively, to thereby result m an insolubilized product formed by binding of the immunologically reactive component(s) or the immunological complex to the carrier-adsorbed reactant together with an unbound, soluble portion of the labeled antigen (or labeled antibody); (b) separating the unbound labeled antigen (or labeled antibody) therefrom; (c) measuring the activity of the enzyme contained in the insolubilized product by using substrate solution for the enzyme; (d) repeating the above steps (a)-(c) but with varying amounts of the anti~en (or antibody) to thereby construct a calibration curve representing the relati~nship between the amount of antigen (or antibody) initially used and the activity of enzyme attached to the lZ153Z;~
4a insolubilized product; (e) carrying out the same procedures as above with a biological fluid containing an unknown amount of the antigen (or antibody), to thereby measure the activity of the enzyme contained in the resulting insolubilized product; and (f) calculating the amount (concentration) of antigen (or antibody) having been present in the biological fluid by referring the measured value in step (e) to the calibration curve, characterized in that while all the immunolvgical reactions included in steps (a), (d) and (e) are conducted by using purified an*igen, antibody, antiantibody and pr~tein A, under the presence of gelatin and sodium chloride, steps (a)-(c3 are conducted as the immunologically reactive components or the reaction mixture co~taining the immunological complex are/i~ passed through a column packed with the carrier-ad~orbed reactant, whereby the insolubilized product is produced in the co7umn while the unbound labeled antigen (or labeled antibody) is discharged from the column, followed by measuring the acti~ity o~ the enzyme contained in the insolubilized product by pouring a sub~trate solution for the enzyme into the column.
5. Brief Description of the Drawings Fig. 1 is a diagrammatic flow sheet illustrating various modes of operation of the immunological reaction step (i.e., the step preceding the enzyme activity measuring step) involved in the method of the present invention;
Fig. 2 is a graph illustrating the calibration curve obtained in Example 1 which will be gi~en later; and Fig. 3 is a graph illustrating the calibration curve obtained in Example 5 which will also be given later.
Additionally, the method u~ing phy~icochemical chromatography ha~, in contrast with the method of the present in~ention, the di~advantages in that the kinds of labeling enzymes and adsorbing materials for use therein must be carefully 15 ~elected in particular case~.
The present invention resides in a method of solid-phase enzyme immunoassay for an antigen (or antibody) which consi~ts of the ~tep~ of (a) reacting immunologically either two immunologically reactive components ~elected from the group consist~ng of an antigen, an antibody, an enzyme labeled antigen and an enzyme labeled antibody, or a reaction mixture containing an immunological complex of said components, with a carrier-adsorbed reactant selected from *he group consisting of an antibody-on-carrier, an antiantibody-on-carrier and protein A-on-carrier, simultaneously or successively, to thereby result m an insolubilized product formed by binding of the immunologically reactive component(s) or the immunological complex to the carrier-adsorbed reactant together with an unbound, soluble portion of the labeled antigen (or labeled antibody); (b) separating the unbound labeled antigen (or labeled antibody) therefrom; (c) measuring the activity of the enzyme contained in the insolubilized product by using substrate solution for the enzyme; (d) repeating the above steps (a)-(c) but with varying amounts of the anti~en (or antibody) to thereby construct a calibration curve representing the relati~nship between the amount of antigen (or antibody) initially used and the activity of enzyme attached to the lZ153Z;~
4a insolubilized product; (e) carrying out the same procedures as above with a biological fluid containing an unknown amount of the antigen (or antibody), to thereby measure the activity of the enzyme contained in the resulting insolubilized product; and (f) calculating the amount (concentration) of antigen (or antibody) having been present in the biological fluid by referring the measured value in step (e) to the calibration curve, characterized in that while all the immunolvgical reactions included in steps (a), (d) and (e) are conducted by using purified an*igen, antibody, antiantibody and pr~tein A, under the presence of gelatin and sodium chloride, steps (a)-(c3 are conducted as the immunologically reactive components or the reaction mixture co~taining the immunological complex are/i~ passed through a column packed with the carrier-ad~orbed reactant, whereby the insolubilized product is produced in the co7umn while the unbound labeled antigen (or labeled antibody) is discharged from the column, followed by measuring the acti~ity o~ the enzyme contained in the insolubilized product by pouring a sub~trate solution for the enzyme into the column.
5. Brief Description of the Drawings Fig. 1 is a diagrammatic flow sheet illustrating various modes of operation of the immunological reaction step (i.e., the step preceding the enzyme activity measuring step) involved in the method of the present invention;
Fig. 2 is a graph illustrating the calibration curve obtained in Example 1 which will be gi~en later; and Fig. 3 is a graph illustrating the calibration curve obtained in Example 5 which will also be given later.
6. Best Modes for Carrying Out the Invention Firstly, the immunological reaction step, i.e., the step preceding the enzyme activity measuring step, involved in the method of the present invention can be carried out in various modes of operation. These modes of operation are explained hereinbelow by reference to the diagrammatic z 4~
flow sheet shown in Fig. 1. It is to be understood however that, in the following description, no mention is made of the immunological reaction products containing no labeling enzyme because their presence does not exert any influence on the immunoassay in accordance with the present invention.
1,;2~53ZZ
Where mention is made of a smaller or greater amount of an immunologically reacti~e component, it means an amount smaller or greater, respectively, than the reaction equivalent to the given amount of the immunol~gically 5 reactive component to be reacted therewith.
Mode (a): A predetermined amount of a labeled antibody 2-4 is reacted with a smaller amount of an antigen 1 to result in an immunological complex 1-2-4 consisting of the labeled antibody 2-4 and of the antigen 1 together with an unbound, 10 free portion 2-4 of the labeled antibody. The reaction mixture is passed through a c~lumn packed with a carrier 5 having a greater amount of the antibody 2 ads~rbed thereon, whereby an insolubilized product formed by binding of the labeled antibody-antigen complex 1-2-4 to the antibody-on-15 carrier 5-2 is produced, while the unbound labeled antibody 2-4 is discharged from the colùmn.
Mode (b): Contrary to the mode (a), a predetermined amount of a labeled antigen 1-4 is reacted with a smaller amount of an antibody 2 to result in a labeled antigen-antibody 20 complex 2-1-4 and an unbound portion 1-4 of the labeled antigen.
The reaction mixture is passed through a column packed with a carrier 5 ha~ing a greater amount of an antiantibody or protein A 3 adsorbed thereon, whereby an insolubilized product formed by binding of the labeled antigen-antibody comple~ 2-1-4 to the 25 antiantibody-on-carrier or protein A-on-carrier 5-3 is produced, while the unbound labeled antigen 1-4 is discharged from the column.
Mode (c): This mode is the same as the mode (b) above, except that an antigen 1 is added to the reaction system in 30 an amount equivalent to or different from that of the labeled antigen 1-4 (hereinafter referred to briefly as a different amount),whereby the immunological reaction is effected to result in a labeled antigen-antibody complex 2-1-4 and an unb~und, free portion 1-4 of the labeled antigen. Tha reaction mixture 35 is passed *hrough a column packed with a carrier 5 having a greater amount of an antiantibody or protein A 3 adsorbed thereon. Thus, an insolubilized product formed by binding ~2~S3Z;~
of the labeled antigen-antibody complex 2-1-4 to the antiantibody-on-carrier or protein A-on-carrier 5-3 is produced, w~ile the unbound labeled antigen 1-4 is discharged from the column~
Mode (d): A predetermined amount of a labeled antigen 1-4 is mixed ~-ith a different amount of an unlabeled antigen 1, and the mixture is passed through a column packed with a carrier 5 having a smaller amount of an antibody 2 adsorbed thereon. Thus, an insolubilized product formed by binding of the labeled antigen 1-4 to the antibody-on-carrier 5-2 is produced, while the unbound labeled antigen 1-4 is discharged from the column.
M~de (e): A smaller amount of an antigen 1 is ~assed through a column packed ~ith a carrier ~ ha~ing a 15 greater amount of an antibody 2 adsorbed thereon to result in antigen-antibody-on-carrier 5-2-1. Subsequently, a greater amount of a labeled antibody 2-4 is passed through the column, w~ereby an insolubilized product formed by binding of the labeled antibody 2-4 to the antigen-antibody-on-carrier 5-20 2-1 is produced, while the unbound labeled antibody 2-4 is discharged from the column.
Mode (f): A smaller amoun~ of an antibody 2 is passed through a column packed with a carrier 5 having a greater amount of an antiantibody or protein A 3 adsorbed 25 thereon to result in antibody-antiantibody or protein A-on-carrier 5-3-2. Subsequently, a greater amount of a labeled antigen 1-4 i~ passed through the column, whereby an insolubilized product formed by binding of the labeled antigen 1-4 to the antibody-antiantibody-on-carrier or 3 antibody-protein A-on-carrier 5-3-2 is produced, while the unbound labeled antigen 1-4 is discharged from the column.
Secondly, according to the method of the present invention, the enzyme activity measuring step following after the immunological reaction step as explained above, 35 is carried out as follows.
After the immunological reaction step has been accomplished by any one of the modes of operation (a)-(f), ~' ~2~5;~Z
the column is washed with a buffer solution, and the acti~ity of the labeling enzy~e 4 contained in the insolubilized product which has remained in the column is measured by passinS through the column a solution containing a substrate for the enzyme. Typically, the foregoing procedures are pre~iously repeated with ~arying amounts of the antigen 1 or antibody 2, and the measured values thereby are pl~tted against the amounts of antigen 1 or antibody 2 initially used to construct a calibration cur~e. Next, the 10 same procedures as abo~e are conducted by using a biological fluid containing an ul~nown amount of the antigen 1 or antibody 2. Then, the concentration of the antigen 1 or antibody 2 in the fluid is calculated from the measured value by reference to the calibration curve.
In the practice of the present in~ention, the immunological reaction, the reaction of an anti~en or antibody with a labeling material, and the adsorption of an antibody, an antiantibody, or protein A on an insoluble carrier are att~ined at a temperature ranging from about 4C to about 20 40 C, as is usual with biochemical reactions. In most cases, the time required to complete these reactions is 10 minutes or more.
The antigen 1 to be assayed by the method of the present in~ention may be any of the hormones and proteins 25 contained in various biological fluids, the functions of some of said proteins being not elucidated. Specific examples of the antigen include insulin, triiodothyronine, thyroxine, calcitonin, a-fetoprotein, S-100 protein, enolase, calmodulin, secretory immunoglobulin A, and the like. In addition, the 30 blood le~els of externally administered drugs, such as antibiotics, can also be determined. The antibody 2 for use in the method of the present in~ention is an antibody corresponding to the aforesaid antigen 1. Most of these antigens 1, antibodies 2, antiantibodies 3, or biolo~ical 35 fluids containing them are commercially available in `
purified form, and such commercial preparations were used in the examples of the present in~ention gi~en later~
~21~3~
Specific examples of the labeling en3yme for use in the method of the present invention include ~-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase, malate dehydrogenase, and the like. Such an enzyme 4 may be co~pled 5 to an antigen 1 or an antibody 2 by any con~entional procedures. That is, this can be accomplished by using a bi~unctiOnal reagent as the coupling agent such as, glutaraldehyde, carbodiimide, N,N-o-phenylenedimaleimide, m-maleimidobenzoyl-N-hydroxysuccinimide ester, or the like.
1~ Specific examples of the insoluble carrier 5 for use in ~he method of the present invention include polysaccharides s~ch as agarose, dextran, oellulose, etc.; synthetic resins such as polystyrene, polyacrylamide, etc.; and pieces of glass. In carrying out the method of the present in~ention, 1~ it is desirable that the carrier-adsorbed reactant consisting of an antibody 2, antiantibody 3 or protein A 3 adsorbed on the insoluble carrier 5 is in finely spherical or finely fibrous form, so long as the rate of flow of the immunological reaction mixture through the column is not 20 reduced to an undue extent.
The adsorbing reaction of an antibody 2, antiantibody 3 or protein A 3 on an insoluble carrier 5 may be performed by any conventional procedures. For example, this can be accomplished by acti~ating an insoluble polysaccharide with 25 an activating agent such as cyanogen bromide, sodium periodate, epichlorohydrin, 1,1'-carbonyldiimidazole, p-toluenesulfonyl chloride or the like, followed by adding an antibody or the like to the activated polysaccharide.
The amount of antîbody 2, antiantibody 3, or protein A 3 3~ to use for this purpose is suitably in the range of 0.1 to 20 mg per milliliter of the insoluble carrier.
The antibody 2, antiantibody 3 or protein A 3 for use in the method of the present in~ention may be so-called active fragments isolated therefrom. In the case of an 35 anttbody 2 or an antiantibody 3, for example, the active fragments thereof can be isolated by treatment with a protease such as papain, pepsin or the like.
5~Z;Z
In view of the small sample ~olumes usually available for immunoassay, and of the simplification Of operati~ns, and of the attainment of a reasonably short operating time, it is desirable that the capacity of the column f~r use in the 5 method of the present inve~tion is not greater than 1 ml.
~ urthermore, in carrying out the method of the present invention, any interference with the immunological reaction can be prevented by the technique preYiously proposed by us, that is, by the addition of a hydrophobic protein (e.s., ge}atin) together with a salt (e.g., s~dium chloride) to the immunological reaction system.
The method according to the present in~ention i5 more specifically illustrated by the following examples without however limiting it in any way.
15 Example 1 Assay of insulin (1) Preparation of an antibody and a labeled antibody A commercial preparation of swine insulin having been extracted from the pancreas of swine was used as the 20 antigen. A commercial preparation of serum containing an antibody having been produced by injecting the antigen into a guinea pig was treated according to conventional procedures, such as fractionation with ammonium sulfate and DEAE-cellulose chromatography, to isolate the antibody 25 therefrom. The antibody thus obtained was split by addition of pepsin and then subjected to column chromatography using "Sephadex G-150'' whereby the active fragments of the antibody were isolated. These active fragments were reduced by 2-mercaptoethylamine in the usual manner and then reacted 30 with N,N'-o-phenylenedimaleimide to join it to the reduced active fragments. Thereaft,er, the reaction product was further reacted with a commercial preparati,on of ~
galaotosidase to prepare the active fragments labeled with ~-D-galactosidase (hereinafter referred to simply the labeled 35 antibody).
The aforesaid antigen (i.e., s~ine insulin) and its corresponding antibody have generally the ability to react * Trade~ark. "Sephadex" is the trademark for a highly cross-linked -~ dextran, in the form of macroscopic beads. The dextran chains ha~Tefunctional ionic groups attached thereto by ether linka~es.
S3:~2 immunologically with other serum insulins (as antigens) originating from a wide variety of animal species ( including human) and their corresponding antibodies, ~o that they can be used in the assay of insulins (as antigens) or their 5 corresponding antibodies contained in biological fluids of many animal species (e.g., human).
(2) Preparation of an antibody-on-carrier ~ n antibody-on-carrier was prepared by adsorbins the antibody obta~ned in item (1) above on a water-insoluble 10 carrier(C~Br-activated'Sepharose)" in the usual manner.
(3) Co~struction of a calibration curve To l-ml portions of a buffer solution containing 100 ~U/ml of the labeled antibody in the above item (1) were added 0.1 ml each of aqueous solutions containing 15 smaller amounts (i.e., ~ to 320 ~U/ml) of the antigen, and the mixtures were incubated at 37C for 2 hours to result in a labeled antibody-antigen complex together with an unbound portion of the(labeled antibody.
In this and other examples, "U" represents the unit amount 20 of a hormone having been adopted in the standard bioassay method. Then, each of the reaction mixtures was passed through a column packed with a greater amount of the antibody-on-carrier prepared in item (2) above,whereby the insolubilized product formed by binding of the labeled 25 antibody-antigen complex to the antibody-on-carrier is produced,while the unbound labeled antibody was discharged from the column. After the column was washed with a buffer solution, 0~1 ml of an a~ueous solution containing 10 mg/ml of o-nitrophenyl-~-D-galactoside ~hereinafter referred to 30 o-NPG) was poured into the column, which was then incubated - at 37C for 2 hours to prodùce o-nitrophenol. The column was washed with 2 ml of a 0.1 M solution of Na2C03 and the absorbance at ~20 1~ of the filtrate was measured to determine the acti~ity of enzyme ha~ing been contained thereinD The measured ~alues were plotted against the amounts of antigen initially used to construct a calibration curve as shown in Fi~. 2.
* Trademark for agarose.
1;~153Z2 On the other hand, the above procedure was modified by preparing a series of solutions containing the antigen in the same amounts as before and passing each of these solutions through a column packed with the antibody-on-carrier. After the c~lumn . . .
was washed, the same amount of the labeled antibody was passed therethrough, followed by measurement of the absorbance of filtrate. In this manner, a calibration curve quite similar to that of Fig. 2 was obtained.
(4) Determination of insulin in human serum A human serum sample having an unknown concentration (but falling in the range of 0-320 ~U/ml as defined in item (3); herein-after the same~ of insulin was used as the antigen, and the same procedure as in the former part of item (3) was carried out. That is, 0.1 ml of the sample was reacted with the labeled antibody, and the reaction mixture was passed through a column packed with the antibody-on-carrier. After o-NPG was poured into the column and incubated, the absorbance of the column filtrate was measured.
When calculated from the measured value by reference to the calibration curve shown in Fig. 2, the concentration of insulin in the sample was found to be 92 ~U/ml. For comparison, the same sample was subjected to the similar mode in the conventional solid-phase radioimmunoassay (hereinafter referred to RIA) using a radioisotope as the labeling material, so that the concentration of insulin in the sample was estimated to be 102 ~U/ml.
On the other hand, the same sample was treated following the same procedure as in the latter part of item (3~. Thus, the concentration of insulin in the sample was found to be 95 ~U/ml.
For comparison, the same sample was subjected to the similar mode in the conventional RIA, so that the concentration of insulin in the sample was estimated to be 105 ~U/ml.
Example 2 Assay of anti-thyroxine antibody (1) Preparation of a labeled antigen and an antibody A commercial preparation of thyroxine having been ~21:~3;~Z
isolated from an extract of swine th3~roidglands was used as the antigen. The amino group of this antigen was coupled with the SH group of B-D-galactosidase in the usual manner to form a labeled antigen. On the other hand, a commercial 5 preparation of serum containing an antibody having been produced by injecting the antigen into a rabbit was immediately ~sed as the antibody.
(2) Preparation of an antiantibody-on-carrier Generally, immunoglobulin G (herei~fter referred to ~0 IgG), which is a kind of protein inherently present in the serum of animals, has the ability to react with antigenic substances originating from a ~ariety of animal species and hence the properties of an antibody corresponding to them. Accordingly,IgG has been used widely for the 15 preparation of an antiantibody.
In this example, a commercial preparation of serum containing an antiantibody having been produced by injecting rabbit IgG into a ~oat was treated in the same manner as in item (1) of Example 1 to isolate the antiantibody 20 therefrom. By using the antiantibody so isolated, the same procedure as in item (2) of Example 1 was carried out to prepare an antiantibody-on-carrier.
(3) Construction of a calibration cur~e To 0.5-ml portions of a dilution of the labeled 25 antigen prepared in item (1) abo~e were added 3 ,ul each of serum dilutions containing smaller amounts of the antibody, and after completion of the reaction, each of the reaction mixtures was passed through a column packed with a greater amount of the antiantibody-on-carrier prepared in item (2).
30 Thereafter, the procedure in item t3) of Example 1 was followed to construct a calibration cur~e.
On the other hand, the above procedure was modified by preparing a series of serum dilutions (0.5 ml each) 35 containing the antibody in the same amounts as before and passing each of the dilutions through a column packed with f~
~53;~Z
ehe antibody-on-carrier. After the column was washed, the same amount of the albeled anti8en was passed therethrough, followed by ~easurement of the absorbance of the column filtrates. In this manner, a quite similar calibration curve to the above-described was obtained.
(4) Determination of anti-thyroxine antibody in rabbit serum A rabbit serum sample of unkno~n concentration of anti-thyroxine antibody was treated following the same procedure as in the former part of item (3~ to measure the absorbance of the filtrate. When calculated from the measured value by reference to the calibration curve constructed in item (3) above, the concentration of anti-thyroxine antibody in the sample was fount to be 1.05U/ml. Similarly, the same sample was treated in the same manner as in the latter part of item (3), and the anti-thyroxine antibody concentration was found to be 1.10 U/ml.
Example 3 Assay of anti-insulin antibody.
(1) Preparation of a labeled antigen A commercial preparation of swine insulin was used as the antigen. The same procedure as in item (l) of Example 1 was carried out to label the ~-D-galactosidase to the antigen. On the other hand, a commercial preparation of serum containing an antibody having been produced by iniecting the antigen into a guinea pig was immediately used as the antibody.
(2) Preparation of an antiantibody-on-carrier A commercial preparation of serum containing an antiantibody (i.e., antibody to guinea pig IgG) having been produced by injecting guinea pig IgG into a rabbit was treated in the usual manner to isolate the antiantibody therefrom. By using the antiantibody, the procedure in item (2) of Example 2 was followed to prepare an antiantibody-on-carrier.
(3) Construction of a calibration cur~e To l-ml portions of a buffer solution containing the labeled antigen prepared in item (l) were added a series of serum dilutions containing smaller amounts of the antibody, and after completion of the reaction,-each of the reaction mixtures was passed through a column packed with a greater amount of the antiantibody-on-carrier prepared in item (2).
~2~53Z2 mereafter~ the column was treated follcw~g the ~ pro~re as in the former part of item (3) of Example 2 to mea~ure the absorbance of the column filtrate. The measured ~alues were plotted against the amounts of antibody initially used 5 to construct a calibration curve.
(4) Determination of anti-insulin antibody in guinea pig serum A guinea pig serl2m sample with an unknl)wn concentration of anti-insulin antibo~ was tr~3ated following the s~ne proced~re 10 as in item (3) above to measur0 the absorbance of the filtrate. When calculated from the measured ~alue by reference to the calibration curve in item (3) abo~e, the concentratiOn of anti-insulin antibody in the sample was found to be o.76 ~U/ml.
15 Example 4 Assay of a-fetoprotein, (1) Preparation of a labeled antibody A commercial preparation of a-fetoprotein ha~ing been isolated from a h~ Yerum was used as the antigen. A
20 commercial preparation of serum containing an antibody having been produced by injecting the antigen into a goat was treated in the usual manner to isolate the antibody. By using the antibody, the procedure in item (1) of Example 1 was followed to prepare a labeled antibody.
25 (2) Preparation of an antibody-on-carrier By using Toyopearl HW-55 (trademark) as the insoluble carrier, an antibody-on-carrier was prepared in the usual manner.
(3) Construction of a calibration cur~e To 1-ml portions of a solution containing the labeled antibody prepared in item (1) were added smaller amounts of the antigen, and after completion of the reaction, each of the reaction mixtures ~-as passed through a column packed with a greater amount of the antibody-on-carrier prepared in 35 item (2). Thereafter, the absorbance of the column filtrate was measured. The measured ~alues were plotted against the C amounts of antigen initially used to construct a calibration curve.
(4) Determination of a-fetoprotein in human serum A human serum sample of unknown concentration of a~fetoprotein was treated in the same procedure as in item 5 (3) above, to measure the absorbance of the column filtrate.
W~en calculated from the measured ~alue by reference to the calibration cur~e in item (3) above, the concentration of a-fetoprotein in the sample was found to be 151 ng/ml.
the similar mode in the For comparison, the same sample was subjected to/RIA, so 10 that the concentration of a-fetoprotein in the sample was estimated to be 192 n$/ml.
Example 5 Assay of calcitoninr (1) Preparation of an antibody A commercial preparation of calcitonin having~been isolated from an extract of swine thyroid glands was used as the antigen. A commercial preparation of serum containing an antibody having been produced by injecting the antigen into a rabbit wa~ treated in the same manner as 20in item (1) of Example 1, to thereby isolate the antibody therefrom.
(2) Preparation of a labeled antigen and an antiantibody-on-carrier The procedure in item ~1) of Example 2 was followed 25to prepare a labeled antigen. On the other hand, an antiantibody-on-carrier was prepared by conducting the same procedure as in item (2) of Example 2.
(3) Preparation of antigen solutions having different concentrations An aqueous solution of the antigen ~as diluted -to prepare a series of antigen solutions containing to 150 MRC mU/ml of the antigen ("MRC U" refers to ~he unit amount of calcitonin in the standard bioassay method).
(4) Construction of a calibration curve To O.1-ml portions of a solution containing the labeled antigen prepared in item (2) above were added 0.5 ml each of solutions containing smaller amounts of the ~Z1~3'~2 ~nt~xX~ ~ item (1) as well as 0.1 ml each of the antiqen solutions prepared in item (3). After completion of the reaction, each of the reaction mixtures ~as pas~ed ~ ough a colt~n.
~hereafter, the column w~s treated foll~w~g the ~ pr~e as in item (3) of Example 2 to measure the absorbance of filtrate. The meastlred ~alues were plotted against the amounts of antigen initially used to construct a calibration curve as shown in Fig. 3.
(5) Determination of calcitonin in a swine thyroid extract A swine thyroid extract sample having an unknown ooncentration of c~citon~ was treated follcw~ the ~ proc~e as in item (3) above to measure the absorbance of filtrate.
When calculated fro~ the measured value by reference to the calibration curve shown in Fig. 3, the concentration ,of 1~ calcitonin in the sample was found to be 75 MRC mU/ml.
Example 6 Assay of thyroxine.
(1) Preparation of a labeled antigen and an antibody A commercial preparation of thyroxine having been 20 isolated from an extract of swine thyroid glands was used as the antigen. The same procedure as in item (1) of Example 2 was conducted to prepare a labeled antigen. On the other hand, a commercial preparation of serum containing an antibody corresponding to the antigen was treated in 25 the same manner as in item (1) of Example 1 to isolate the antibody therefrom.
(2) Preparation of an antibody-on-carrier The same procedure as in item (2) of Example 1 was conducted to prepare an antibody-on-carrier.
30 (3) Preparation of a~tigen solutions having different concentrations An aqueous solution of the antigen was diluted to prepare a series of antigen soltltions having different concentrations.
(4) Construction of a calibration curve To i-ml portions of a solution containing the labeled antigen prepared in item (1) were added the antigen solutions .,~
~5;~ZZ
prepared in item (3). Each of the resulting mixtures was passed throush a column packed with a smaller amo~nt of the antibody-on-carrier, and the column ~as treated in the same procedure as in item (3) of Example 1 to measure the 5 absorbance of the column filtrate. The measured values were plotted against the amounts of antigen initially used to construct a calibration curve.
(5) Determination of thyroxine in a swine thyroid extract A swine thyroid extract samPle with an unknown ~0 concentration of thyroxine was treated f~ w mg the same procedure as in item (3) above to measure the absorbance of the column filtrate. When calculated from the measured value by reference to the calibration curve constructed in (4) above, the concentration of thyroxine in the sample was found to be the similar node m the 15 10.5 ~g/dl. ~or comparison, the same sample was subjected to/
RIA, so that the concentration of thyroxine therein was estimated to be 11.0 ~ug/dl.
flow sheet shown in Fig. 1. It is to be understood however that, in the following description, no mention is made of the immunological reaction products containing no labeling enzyme because their presence does not exert any influence on the immunoassay in accordance with the present invention.
1,;2~53ZZ
Where mention is made of a smaller or greater amount of an immunologically reacti~e component, it means an amount smaller or greater, respectively, than the reaction equivalent to the given amount of the immunol~gically 5 reactive component to be reacted therewith.
Mode (a): A predetermined amount of a labeled antibody 2-4 is reacted with a smaller amount of an antigen 1 to result in an immunological complex 1-2-4 consisting of the labeled antibody 2-4 and of the antigen 1 together with an unbound, 10 free portion 2-4 of the labeled antibody. The reaction mixture is passed through a c~lumn packed with a carrier 5 having a greater amount of the antibody 2 ads~rbed thereon, whereby an insolubilized product formed by binding of the labeled antibody-antigen complex 1-2-4 to the antibody-on-15 carrier 5-2 is produced, while the unbound labeled antibody 2-4 is discharged from the colùmn.
Mode (b): Contrary to the mode (a), a predetermined amount of a labeled antigen 1-4 is reacted with a smaller amount of an antibody 2 to result in a labeled antigen-antibody 20 complex 2-1-4 and an unbound portion 1-4 of the labeled antigen.
The reaction mixture is passed through a column packed with a carrier 5 ha~ing a greater amount of an antiantibody or protein A 3 adsorbed thereon, whereby an insolubilized product formed by binding of the labeled antigen-antibody comple~ 2-1-4 to the 25 antiantibody-on-carrier or protein A-on-carrier 5-3 is produced, while the unbound labeled antigen 1-4 is discharged from the column.
Mode (c): This mode is the same as the mode (b) above, except that an antigen 1 is added to the reaction system in 30 an amount equivalent to or different from that of the labeled antigen 1-4 (hereinafter referred to briefly as a different amount),whereby the immunological reaction is effected to result in a labeled antigen-antibody complex 2-1-4 and an unb~und, free portion 1-4 of the labeled antigen. Tha reaction mixture 35 is passed *hrough a column packed with a carrier 5 having a greater amount of an antiantibody or protein A 3 adsorbed thereon. Thus, an insolubilized product formed by binding ~2~S3Z;~
of the labeled antigen-antibody complex 2-1-4 to the antiantibody-on-carrier or protein A-on-carrier 5-3 is produced, w~ile the unbound labeled antigen 1-4 is discharged from the column~
Mode (d): A predetermined amount of a labeled antigen 1-4 is mixed ~-ith a different amount of an unlabeled antigen 1, and the mixture is passed through a column packed with a carrier 5 having a smaller amount of an antibody 2 adsorbed thereon. Thus, an insolubilized product formed by binding of the labeled antigen 1-4 to the antibody-on-carrier 5-2 is produced, while the unbound labeled antigen 1-4 is discharged from the column.
M~de (e): A smaller amount of an antigen 1 is ~assed through a column packed ~ith a carrier ~ ha~ing a 15 greater amount of an antibody 2 adsorbed thereon to result in antigen-antibody-on-carrier 5-2-1. Subsequently, a greater amount of a labeled antibody 2-4 is passed through the column, w~ereby an insolubilized product formed by binding of the labeled antibody 2-4 to the antigen-antibody-on-carrier 5-20 2-1 is produced, while the unbound labeled antibody 2-4 is discharged from the column.
Mode (f): A smaller amoun~ of an antibody 2 is passed through a column packed with a carrier 5 having a greater amount of an antiantibody or protein A 3 adsorbed 25 thereon to result in antibody-antiantibody or protein A-on-carrier 5-3-2. Subsequently, a greater amount of a labeled antigen 1-4 i~ passed through the column, whereby an insolubilized product formed by binding of the labeled antigen 1-4 to the antibody-antiantibody-on-carrier or 3 antibody-protein A-on-carrier 5-3-2 is produced, while the unbound labeled antigen 1-4 is discharged from the column.
Secondly, according to the method of the present invention, the enzyme activity measuring step following after the immunological reaction step as explained above, 35 is carried out as follows.
After the immunological reaction step has been accomplished by any one of the modes of operation (a)-(f), ~' ~2~5;~Z
the column is washed with a buffer solution, and the acti~ity of the labeling enzy~e 4 contained in the insolubilized product which has remained in the column is measured by passinS through the column a solution containing a substrate for the enzyme. Typically, the foregoing procedures are pre~iously repeated with ~arying amounts of the antigen 1 or antibody 2, and the measured values thereby are pl~tted against the amounts of antigen 1 or antibody 2 initially used to construct a calibration cur~e. Next, the 10 same procedures as abo~e are conducted by using a biological fluid containing an ul~nown amount of the antigen 1 or antibody 2. Then, the concentration of the antigen 1 or antibody 2 in the fluid is calculated from the measured value by reference to the calibration curve.
In the practice of the present in~ention, the immunological reaction, the reaction of an anti~en or antibody with a labeling material, and the adsorption of an antibody, an antiantibody, or protein A on an insoluble carrier are att~ined at a temperature ranging from about 4C to about 20 40 C, as is usual with biochemical reactions. In most cases, the time required to complete these reactions is 10 minutes or more.
The antigen 1 to be assayed by the method of the present in~ention may be any of the hormones and proteins 25 contained in various biological fluids, the functions of some of said proteins being not elucidated. Specific examples of the antigen include insulin, triiodothyronine, thyroxine, calcitonin, a-fetoprotein, S-100 protein, enolase, calmodulin, secretory immunoglobulin A, and the like. In addition, the 30 blood le~els of externally administered drugs, such as antibiotics, can also be determined. The antibody 2 for use in the method of the present in~ention is an antibody corresponding to the aforesaid antigen 1. Most of these antigens 1, antibodies 2, antiantibodies 3, or biolo~ical 35 fluids containing them are commercially available in `
purified form, and such commercial preparations were used in the examples of the present in~ention gi~en later~
~21~3~
Specific examples of the labeling en3yme for use in the method of the present invention include ~-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase, malate dehydrogenase, and the like. Such an enzyme 4 may be co~pled 5 to an antigen 1 or an antibody 2 by any con~entional procedures. That is, this can be accomplished by using a bi~unctiOnal reagent as the coupling agent such as, glutaraldehyde, carbodiimide, N,N-o-phenylenedimaleimide, m-maleimidobenzoyl-N-hydroxysuccinimide ester, or the like.
1~ Specific examples of the insoluble carrier 5 for use in ~he method of the present invention include polysaccharides s~ch as agarose, dextran, oellulose, etc.; synthetic resins such as polystyrene, polyacrylamide, etc.; and pieces of glass. In carrying out the method of the present in~ention, 1~ it is desirable that the carrier-adsorbed reactant consisting of an antibody 2, antiantibody 3 or protein A 3 adsorbed on the insoluble carrier 5 is in finely spherical or finely fibrous form, so long as the rate of flow of the immunological reaction mixture through the column is not 20 reduced to an undue extent.
The adsorbing reaction of an antibody 2, antiantibody 3 or protein A 3 on an insoluble carrier 5 may be performed by any conventional procedures. For example, this can be accomplished by acti~ating an insoluble polysaccharide with 25 an activating agent such as cyanogen bromide, sodium periodate, epichlorohydrin, 1,1'-carbonyldiimidazole, p-toluenesulfonyl chloride or the like, followed by adding an antibody or the like to the activated polysaccharide.
The amount of antîbody 2, antiantibody 3, or protein A 3 3~ to use for this purpose is suitably in the range of 0.1 to 20 mg per milliliter of the insoluble carrier.
The antibody 2, antiantibody 3 or protein A 3 for use in the method of the present in~ention may be so-called active fragments isolated therefrom. In the case of an 35 anttbody 2 or an antiantibody 3, for example, the active fragments thereof can be isolated by treatment with a protease such as papain, pepsin or the like.
5~Z;Z
In view of the small sample ~olumes usually available for immunoassay, and of the simplification Of operati~ns, and of the attainment of a reasonably short operating time, it is desirable that the capacity of the column f~r use in the 5 method of the present inve~tion is not greater than 1 ml.
~ urthermore, in carrying out the method of the present invention, any interference with the immunological reaction can be prevented by the technique preYiously proposed by us, that is, by the addition of a hydrophobic protein (e.s., ge}atin) together with a salt (e.g., s~dium chloride) to the immunological reaction system.
The method according to the present in~ention i5 more specifically illustrated by the following examples without however limiting it in any way.
15 Example 1 Assay of insulin (1) Preparation of an antibody and a labeled antibody A commercial preparation of swine insulin having been extracted from the pancreas of swine was used as the 20 antigen. A commercial preparation of serum containing an antibody having been produced by injecting the antigen into a guinea pig was treated according to conventional procedures, such as fractionation with ammonium sulfate and DEAE-cellulose chromatography, to isolate the antibody 25 therefrom. The antibody thus obtained was split by addition of pepsin and then subjected to column chromatography using "Sephadex G-150'' whereby the active fragments of the antibody were isolated. These active fragments were reduced by 2-mercaptoethylamine in the usual manner and then reacted 30 with N,N'-o-phenylenedimaleimide to join it to the reduced active fragments. Thereaft,er, the reaction product was further reacted with a commercial preparati,on of ~
galaotosidase to prepare the active fragments labeled with ~-D-galactosidase (hereinafter referred to simply the labeled 35 antibody).
The aforesaid antigen (i.e., s~ine insulin) and its corresponding antibody have generally the ability to react * Trade~ark. "Sephadex" is the trademark for a highly cross-linked -~ dextran, in the form of macroscopic beads. The dextran chains ha~Tefunctional ionic groups attached thereto by ether linka~es.
S3:~2 immunologically with other serum insulins (as antigens) originating from a wide variety of animal species ( including human) and their corresponding antibodies, ~o that they can be used in the assay of insulins (as antigens) or their 5 corresponding antibodies contained in biological fluids of many animal species (e.g., human).
(2) Preparation of an antibody-on-carrier ~ n antibody-on-carrier was prepared by adsorbins the antibody obta~ned in item (1) above on a water-insoluble 10 carrier(C~Br-activated'Sepharose)" in the usual manner.
(3) Co~struction of a calibration curve To l-ml portions of a buffer solution containing 100 ~U/ml of the labeled antibody in the above item (1) were added 0.1 ml each of aqueous solutions containing 15 smaller amounts (i.e., ~ to 320 ~U/ml) of the antigen, and the mixtures were incubated at 37C for 2 hours to result in a labeled antibody-antigen complex together with an unbound portion of the(labeled antibody.
In this and other examples, "U" represents the unit amount 20 of a hormone having been adopted in the standard bioassay method. Then, each of the reaction mixtures was passed through a column packed with a greater amount of the antibody-on-carrier prepared in item (2) above,whereby the insolubilized product formed by binding of the labeled 25 antibody-antigen complex to the antibody-on-carrier is produced,while the unbound labeled antibody was discharged from the column. After the column was washed with a buffer solution, 0~1 ml of an a~ueous solution containing 10 mg/ml of o-nitrophenyl-~-D-galactoside ~hereinafter referred to 30 o-NPG) was poured into the column, which was then incubated - at 37C for 2 hours to prodùce o-nitrophenol. The column was washed with 2 ml of a 0.1 M solution of Na2C03 and the absorbance at ~20 1~ of the filtrate was measured to determine the acti~ity of enzyme ha~ing been contained thereinD The measured ~alues were plotted against the amounts of antigen initially used to construct a calibration curve as shown in Fi~. 2.
* Trademark for agarose.
1;~153Z2 On the other hand, the above procedure was modified by preparing a series of solutions containing the antigen in the same amounts as before and passing each of these solutions through a column packed with the antibody-on-carrier. After the c~lumn . . .
was washed, the same amount of the labeled antibody was passed therethrough, followed by measurement of the absorbance of filtrate. In this manner, a calibration curve quite similar to that of Fig. 2 was obtained.
(4) Determination of insulin in human serum A human serum sample having an unknown concentration (but falling in the range of 0-320 ~U/ml as defined in item (3); herein-after the same~ of insulin was used as the antigen, and the same procedure as in the former part of item (3) was carried out. That is, 0.1 ml of the sample was reacted with the labeled antibody, and the reaction mixture was passed through a column packed with the antibody-on-carrier. After o-NPG was poured into the column and incubated, the absorbance of the column filtrate was measured.
When calculated from the measured value by reference to the calibration curve shown in Fig. 2, the concentration of insulin in the sample was found to be 92 ~U/ml. For comparison, the same sample was subjected to the similar mode in the conventional solid-phase radioimmunoassay (hereinafter referred to RIA) using a radioisotope as the labeling material, so that the concentration of insulin in the sample was estimated to be 102 ~U/ml.
On the other hand, the same sample was treated following the same procedure as in the latter part of item (3~. Thus, the concentration of insulin in the sample was found to be 95 ~U/ml.
For comparison, the same sample was subjected to the similar mode in the conventional RIA, so that the concentration of insulin in the sample was estimated to be 105 ~U/ml.
Example 2 Assay of anti-thyroxine antibody (1) Preparation of a labeled antigen and an antibody A commercial preparation of thyroxine having been ~21:~3;~Z
isolated from an extract of swine th3~roidglands was used as the antigen. The amino group of this antigen was coupled with the SH group of B-D-galactosidase in the usual manner to form a labeled antigen. On the other hand, a commercial 5 preparation of serum containing an antibody having been produced by injecting the antigen into a rabbit was immediately ~sed as the antibody.
(2) Preparation of an antiantibody-on-carrier Generally, immunoglobulin G (herei~fter referred to ~0 IgG), which is a kind of protein inherently present in the serum of animals, has the ability to react with antigenic substances originating from a ~ariety of animal species and hence the properties of an antibody corresponding to them. Accordingly,IgG has been used widely for the 15 preparation of an antiantibody.
In this example, a commercial preparation of serum containing an antiantibody having been produced by injecting rabbit IgG into a ~oat was treated in the same manner as in item (1) of Example 1 to isolate the antiantibody 20 therefrom. By using the antiantibody so isolated, the same procedure as in item (2) of Example 1 was carried out to prepare an antiantibody-on-carrier.
(3) Construction of a calibration cur~e To 0.5-ml portions of a dilution of the labeled 25 antigen prepared in item (1) abo~e were added 3 ,ul each of serum dilutions containing smaller amounts of the antibody, and after completion of the reaction, each of the reaction mixtures was passed through a column packed with a greater amount of the antiantibody-on-carrier prepared in item (2).
30 Thereafter, the procedure in item t3) of Example 1 was followed to construct a calibration cur~e.
On the other hand, the above procedure was modified by preparing a series of serum dilutions (0.5 ml each) 35 containing the antibody in the same amounts as before and passing each of the dilutions through a column packed with f~
~53;~Z
ehe antibody-on-carrier. After the column was washed, the same amount of the albeled anti8en was passed therethrough, followed by ~easurement of the absorbance of the column filtrates. In this manner, a quite similar calibration curve to the above-described was obtained.
(4) Determination of anti-thyroxine antibody in rabbit serum A rabbit serum sample of unkno~n concentration of anti-thyroxine antibody was treated following the same procedure as in the former part of item (3~ to measure the absorbance of the filtrate. When calculated from the measured value by reference to the calibration curve constructed in item (3) above, the concentration of anti-thyroxine antibody in the sample was fount to be 1.05U/ml. Similarly, the same sample was treated in the same manner as in the latter part of item (3), and the anti-thyroxine antibody concentration was found to be 1.10 U/ml.
Example 3 Assay of anti-insulin antibody.
(1) Preparation of a labeled antigen A commercial preparation of swine insulin was used as the antigen. The same procedure as in item (l) of Example 1 was carried out to label the ~-D-galactosidase to the antigen. On the other hand, a commercial preparation of serum containing an antibody having been produced by iniecting the antigen into a guinea pig was immediately used as the antibody.
(2) Preparation of an antiantibody-on-carrier A commercial preparation of serum containing an antiantibody (i.e., antibody to guinea pig IgG) having been produced by injecting guinea pig IgG into a rabbit was treated in the usual manner to isolate the antiantibody therefrom. By using the antiantibody, the procedure in item (2) of Example 2 was followed to prepare an antiantibody-on-carrier.
(3) Construction of a calibration cur~e To l-ml portions of a buffer solution containing the labeled antigen prepared in item (l) were added a series of serum dilutions containing smaller amounts of the antibody, and after completion of the reaction,-each of the reaction mixtures was passed through a column packed with a greater amount of the antiantibody-on-carrier prepared in item (2).
~2~53Z2 mereafter~ the column was treated follcw~g the ~ pro~re as in the former part of item (3) of Example 2 to mea~ure the absorbance of the column filtrate. The measured ~alues were plotted against the amounts of antibody initially used 5 to construct a calibration curve.
(4) Determination of anti-insulin antibody in guinea pig serum A guinea pig serl2m sample with an unknl)wn concentration of anti-insulin antibo~ was tr~3ated following the s~ne proced~re 10 as in item (3) above to measur0 the absorbance of the filtrate. When calculated from the measured ~alue by reference to the calibration curve in item (3) abo~e, the concentratiOn of anti-insulin antibody in the sample was found to be o.76 ~U/ml.
15 Example 4 Assay of a-fetoprotein, (1) Preparation of a labeled antibody A commercial preparation of a-fetoprotein ha~ing been isolated from a h~ Yerum was used as the antigen. A
20 commercial preparation of serum containing an antibody having been produced by injecting the antigen into a goat was treated in the usual manner to isolate the antibody. By using the antibody, the procedure in item (1) of Example 1 was followed to prepare a labeled antibody.
25 (2) Preparation of an antibody-on-carrier By using Toyopearl HW-55 (trademark) as the insoluble carrier, an antibody-on-carrier was prepared in the usual manner.
(3) Construction of a calibration cur~e To 1-ml portions of a solution containing the labeled antibody prepared in item (1) were added smaller amounts of the antigen, and after completion of the reaction, each of the reaction mixtures ~-as passed through a column packed with a greater amount of the antibody-on-carrier prepared in 35 item (2). Thereafter, the absorbance of the column filtrate was measured. The measured ~alues were plotted against the C amounts of antigen initially used to construct a calibration curve.
(4) Determination of a-fetoprotein in human serum A human serum sample of unknown concentration of a~fetoprotein was treated in the same procedure as in item 5 (3) above, to measure the absorbance of the column filtrate.
W~en calculated from the measured ~alue by reference to the calibration cur~e in item (3) above, the concentration of a-fetoprotein in the sample was found to be 151 ng/ml.
the similar mode in the For comparison, the same sample was subjected to/RIA, so 10 that the concentration of a-fetoprotein in the sample was estimated to be 192 n$/ml.
Example 5 Assay of calcitoninr (1) Preparation of an antibody A commercial preparation of calcitonin having~been isolated from an extract of swine thyroid glands was used as the antigen. A commercial preparation of serum containing an antibody having been produced by injecting the antigen into a rabbit wa~ treated in the same manner as 20in item (1) of Example 1, to thereby isolate the antibody therefrom.
(2) Preparation of a labeled antigen and an antiantibody-on-carrier The procedure in item ~1) of Example 2 was followed 25to prepare a labeled antigen. On the other hand, an antiantibody-on-carrier was prepared by conducting the same procedure as in item (2) of Example 2.
(3) Preparation of antigen solutions having different concentrations An aqueous solution of the antigen ~as diluted -to prepare a series of antigen solutions containing to 150 MRC mU/ml of the antigen ("MRC U" refers to ~he unit amount of calcitonin in the standard bioassay method).
(4) Construction of a calibration curve To O.1-ml portions of a solution containing the labeled antigen prepared in item (2) above were added 0.5 ml each of solutions containing smaller amounts of the ~Z1~3'~2 ~nt~xX~ ~ item (1) as well as 0.1 ml each of the antiqen solutions prepared in item (3). After completion of the reaction, each of the reaction mixtures ~as pas~ed ~ ough a colt~n.
~hereafter, the column w~s treated foll~w~g the ~ pr~e as in item (3) of Example 2 to measure the absorbance of filtrate. The meastlred ~alues were plotted against the amounts of antigen initially used to construct a calibration curve as shown in Fig. 3.
(5) Determination of calcitonin in a swine thyroid extract A swine thyroid extract sample having an unknown ooncentration of c~citon~ was treated follcw~ the ~ proc~e as in item (3) above to measure the absorbance of filtrate.
When calculated fro~ the measured value by reference to the calibration curve shown in Fig. 3, the concentration ,of 1~ calcitonin in the sample was found to be 75 MRC mU/ml.
Example 6 Assay of thyroxine.
(1) Preparation of a labeled antigen and an antibody A commercial preparation of thyroxine having been 20 isolated from an extract of swine thyroid glands was used as the antigen. The same procedure as in item (1) of Example 2 was conducted to prepare a labeled antigen. On the other hand, a commercial preparation of serum containing an antibody corresponding to the antigen was treated in 25 the same manner as in item (1) of Example 1 to isolate the antibody therefrom.
(2) Preparation of an antibody-on-carrier The same procedure as in item (2) of Example 1 was conducted to prepare an antibody-on-carrier.
30 (3) Preparation of a~tigen solutions having different concentrations An aqueous solution of the antigen was diluted to prepare a series of antigen soltltions having different concentrations.
(4) Construction of a calibration curve To i-ml portions of a solution containing the labeled antigen prepared in item (1) were added the antigen solutions .,~
~5;~ZZ
prepared in item (3). Each of the resulting mixtures was passed throush a column packed with a smaller amo~nt of the antibody-on-carrier, and the column ~as treated in the same procedure as in item (3) of Example 1 to measure the 5 absorbance of the column filtrate. The measured values were plotted against the amounts of antigen initially used to construct a calibration curve.
(5) Determination of thyroxine in a swine thyroid extract A swine thyroid extract samPle with an unknown ~0 concentration of thyroxine was treated f~ w mg the same procedure as in item (3) above to measure the absorbance of the column filtrate. When calculated from the measured value by reference to the calibration curve constructed in (4) above, the concentration of thyroxine in the sample was found to be the similar node m the 15 10.5 ~g/dl. ~or comparison, the same sample was subjected to/
RIA, so that the concentration of thyroxine therein was estimated to be 11.0 ~ug/dl.
Claims (16)
1. A method of solid-phase enzyme immunoassay for an antigen (or antibody) which consists of the steps of (a) reacting immunologically either two immunologically reactive components selected from the group consisting of an antigen, an antibody, an enzyme labeled antigen and an enzyme labeled antibody, or a reaction mixture containing an immunological complex of said components, with a carrier-adsorbed reactant selected from the group consisting of an antibody-on-carrier, an antiantibody-on-carrier and protein A-on-carrier, simultaneously or successively, to thereby result in an insolubilized product formed by binding of the immunologically reactive component(s) or the immunological complex to the carrier-adsorbed reactant together with an unbound, soluble portion of the labeled antigen (or labeled antibody); (b) separating the unbound labeled antigen (or labeled antibody) therefrom; (c) measuring the activity of the enzyme contained in the insolubilized product by using substrate solution for the enzyme; (d) repeating the above steps (a)-(c) but with varying amounts of the antigen (or antibody) to thereby construct a calibration curve representing the relationship between the amount of antigen (or antibody) initially used and the activity of enzyme attached to the insolubilized product; (e) carrying out the same procedures as above with a biological fluid containing an unknown amount of the antigen (or antibody), to thereby measure the activity of the enzyme contained in the resulting insolubilized product; and (f) calculating the amount (concentration) of antigen (or antibody) having been present in the biological fluid by referring the measured value in step (e) to the calibration curve, characterized in that while all the immunological reactions included in steps (a), (d) and (e) are conducted by using purified antigen, antibody antiantibody and protein A, under the presence of gelatin and sodium chloride, steps (a)-(c) are conducted as the immunologicall reactive components or the reaction mixture containing the immunological complex are/is passed through a column packed with the carrier-adsorbed reactant, whereby the insolubilized product is produced in the column while the unbound labeled antigen (or labeled antibody) is discharged from the column, followed by measuring the activity of the enzyme contained in the insolubilized product by pouring a substrate solution for the enzyme into the column.
2. A method of solid-phase enzyme immunoassay for an antigen as claimed in claim 1, wherein the steps (a) and (b) are carried out in such a way that a predetermined amount of a labeled antibody (2-4) is reacted with a smaller amount of the antigen (1) to result in a reaction mixture containing an immunological complex (1-2-4) and an unbound, soluble portion of the labeled antibody (2-4), which reaction mixture is then passed through a column packed with a greater amount of the carrier-adsorbed reactant consisting of an antibody (2) and a carrier (5) so as to produce an insolubilized product (5-2-1-2-4) in the column.
3. A method of solid-phase enzyme immunoassay for an antibody as claimed in claim 1, wherein the steps (a) and (b) are carried out in such a way that a predetermined amount of a labeled antigen (1-4) is reacted with a smaller amount of the antibody (2) to result in a reaction mixture containing an immunological complex (2-1-4) and an unbound, soluble portion of the labeled antigen (1-4), which reaction mixture is then passed through a column packed with a greater amount of the carrier-adsorbed reactant consisting of an antiantibody or protein A (3) and a carrier (5) so as to produce an insolubilized product (5-3-2-1-4) in the column.
4. A method of solid-phase enzyme immunoassay for an antigen as claimed in claim 1, wherein steps (a) and (b) are carried out in such a way that a predetermined amount of a labeled antigen (1-4) is reacted with a smaller amount of an antibody (2) under the presence of a different amount of the antigen (1) to result in a reaction mixture containing an immunological complex (2-1-4) and an unbound portion of the labeled antigen (1-4), which reaction mixture is then passed through a column packed with a greater amount of the carrier-adsorbed reactant consisting of an antiantibody or protein A (3) and a carrier (5) so as to produce an insolubilized product (5-3-2-1-4) in the column.
5. A method of solid-phase enzyme immunoassay for an antigen as claimed in claim 1, wherein steps (a) and (b) are carried out in such a way that a mixture of a predetermined amount of a labeled antigen (1-4) and a different amount of the antigen (1) is passed through a column packed with a greater amount of the carrier-adsorbed reactant consisting of an antibody (2) and a carrier (5) so as to produce an insolubilized product (5-2-1-4) in the column.
6. A method of solid-phase enzyme immunoassay for an antigen as claimed in claim 1, wherein steps (a) and (b) are carried out in such a way that a smaller amount of the antigen (1) is passed through a column packed with a greater amount of the carrier-adsorbed reactant consisting of an antibody (2) and a carrier (5) to produce an insolubilized intermediate (5-2-1) in the column, and subsequently a greater amount of a labeled antibody (2-4) is passed through the said column so as to produce an insolubilized product (5-2-1-2-4) in the column.
7. A method of solid-phase enzyme immunoassay for an antibody as claimed in claim 1, wherein steps (a) and (b) are carried out in such a way that a smaller amount of the antibody (2) is passed through a column packed with a greater amount of the carrier-adsorbed reactant consisting of an antiantibody or protein A (3) and a carrier (5) to produce an insolubilized intermediate (5-3-2) in the column, and subsequently a greater amount of a labeled antigen (1-4) is passed through the said column so as to produce an insolubilized product (5-3-2-1-4) in the column.
8. A method as claimed in claim 2 where m the antigen (1) constituting one of the immunologically reactive components is a member selected from the group consisting of insulin, tri-iodothyronine, thyroxine, calcitonin, .alpha.-fetoprotein, S-100 protein, enolase, calmodulin and secretory immunoglobulin A.
9. A method as claimed in claim 2 wherein the insoluble carrier (5) constituting the carrier-adsorbed reactant is a member selected from the group consisting of agarose, dextran, cellulose, polystyrene, polyacrylamide, and pieces of glass.
10. A method as claimed in claim 8 wherein the insoluble carrier (5) constituting the carrier-adsorbed reactant is a member selected from the group consisting of agarose, dextran, cellulose, polystyrene, polyacrylamide, and pieces of glass.
11. A method as claimed in claim 2,3 or 4 wherein the labeling enzyme (4) constituting the labeled antigen or labeled antibody is a member selected from the group consisting of .beta.-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase and malate dehydrogenase.
12. A method as claimed in claim 8 wherein the labeling enzyme (4) constituting the labeled antigen or labeled antibody is a member selected from the group consisting of .beta.-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase and malate dehydrogenase.
13. A method as claimed in claim 9 wherein the labeling enzyme (4) constituting the labeled antigen or labeled antibody is a member selected from the group consisting of .beta.-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase and malate dehydrogenase.
14. A method as claimed in claim 10 wherein the labeling enzyme (4) constituting the labeled antigen or labeled antibody is a member selected from the group consisting of .beta.-D-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase and malate dehydrogenase.
15. A method as claimed in claim 9, wherein the carrier-adsorbed reactant is in finely spherical or finely fibrous form, so long as the rate of flow of the immunologically reactive component(s) or the immunological reaction mixture thereof through the column is not reduced to an undue extent.
16. A method as claimed in claim 9, wherein the capacity of the column for use in the method is not greater than 1 ml.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15681281A JPS5856696A (en) | 1981-09-30 | 1981-09-30 | Enzyme immunoassay using column |
| JP156,812/1981 | 1981-09-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1215322A true CA1215322A (en) | 1986-12-16 |
Family
ID=15635861
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000412425A Expired CA1215322A (en) | 1981-09-30 | 1982-09-29 | Enzyme immunoassay |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS5856696A (en) |
| CA (1) | CA1215322A (en) |
| DE (1) | DE3235516A1 (en) |
| FR (1) | FR2519764B1 (en) |
| GB (1) | GB2109931B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL73577A (en) * | 1983-12-12 | 1989-10-31 | Miles Lab | Method and reagent system for detecting dna or rna sequences in a test medium containing single stranded dna or rna using antibodies to intercalation complexes with double stranded nucleic acid |
| JPS60162956A (en) * | 1984-02-04 | 1985-08-24 | Olympus Optical Co Ltd | Automatic immunological analysis method |
| GB8422452D0 (en) * | 1984-09-05 | 1984-10-10 | Serono Diagnostics Ltd | Assay |
| EP0455735B1 (en) * | 1989-01-30 | 1994-09-14 | Epitope, Inc. | Avidin-biotin assisted immunoassay |
| AU8654891A (en) * | 1990-08-10 | 1992-03-02 | Purdue Research Foundation | Matrix sequential addition immunoassay |
| GB2270976A (en) * | 1992-09-18 | 1994-03-30 | Marconi Gec Ltd | Immunoassay/separation process using an auxiliary species on a support |
| GB9511586D0 (en) * | 1995-06-08 | 1995-08-02 | Tepnel Medical Ltd | Immunological determinations |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4039652A (en) * | 1973-10-11 | 1977-08-02 | Miles Laboratories, Inc. | Column method of immunoassay employing an immobilized binding partner |
| SE387746B (en) * | 1974-05-29 | 1976-09-13 | Pharmacia Diagnostics Ab | PROCEDURE FOR VISUAL DISPLAY OF ANTIBODIES IN AN AQUATIC SAMPLE |
| GB1572220A (en) * | 1976-10-07 | 1980-07-30 | Mochida Pharm Co Ltd | Immunochemical process of measuring physiologically active substances |
| US4098876A (en) * | 1976-10-26 | 1978-07-04 | Corning Glass Works | Reverse sandwich immunoassay |
| US4318980A (en) * | 1978-04-10 | 1982-03-09 | Miles Laboratories, Inc. | Heterogenous specific binding assay employing a cycling reactant as label |
| US4200625A (en) * | 1978-07-12 | 1980-04-29 | Becton, Dickinson And Company | Immobilized binder for automated assay |
| DE2930706A1 (en) * | 1979-07-28 | 1981-02-05 | Medac Klinische Spezialpraep | METHOD FOR DETECTING POTENTIAL-SPECIFIC ANTIBODY |
| JPS5767860A (en) * | 1980-10-15 | 1982-04-24 | Fuji Photo Film Co Ltd | Material for multilayer analysis |
-
1981
- 1981-09-30 JP JP15681281A patent/JPS5856696A/en active Pending
-
1982
- 1982-09-22 DE DE19823235516 patent/DE3235516A1/en not_active Ceased
- 1982-09-24 GB GB08227285A patent/GB2109931B/en not_active Expired
- 1982-09-24 FR FR8216134A patent/FR2519764B1/en not_active Expired
- 1982-09-29 CA CA000412425A patent/CA1215322A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| FR2519764B1 (en) | 1987-06-19 |
| FR2519764A1 (en) | 1983-07-18 |
| JPS5856696A (en) | 1983-04-04 |
| GB2109931B (en) | 1985-03-06 |
| GB2109931A (en) | 1983-06-08 |
| DE3235516A1 (en) | 1983-04-21 |
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