CA2195727A1 - Process for extending the assay range and for avoiding the hook effect in simultaneous sandwich immunoassays, and corresponding test kits - Google Patents
Process for extending the assay range and for avoiding the hook effect in simultaneous sandwich immunoassays, and corresponding test kitsInfo
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- CA2195727A1 CA2195727A1 CA 2195727 CA2195727A CA2195727A1 CA 2195727 A1 CA2195727 A1 CA 2195727A1 CA 2195727 CA2195727 CA 2195727 CA 2195727 A CA2195727 A CA 2195727A CA 2195727 A1 CA2195727 A1 CA 2195727A1
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The present invention relates to simultaneous sandwich immunoassays and test kits which contain polyclonal antibodies which have been purified by means of affinity chromatography. The preliminary purification of the polyclonal antibody by means of affinity chromatography diminishes the hook effect, and extends the analytical range, in the test.
Description
Le A 31 569 - Foreign Countries 2 1 9 5 7 2 7 Pn~cess for ~X~!~ , the assay lange and for avoiding ~e hook effect in lhuleous sandwich i~ ~s~ys, and con~sponding test ki~
Particularly on account oftheir s~ti~f~rtory specificity and sensitivity, immunoassays 5 are frequently ernployed for d~Ptecting proteins in serum samples and urine sarnples for ~i~gncstic purposes in medicine. Ihe so-called sandwich assay is a particularly important vanant of an immlmo~c~y. Tn this assay, two antibodies bind to two di~lclll epitopes of the protein, with one of the antibodies being coupled to a solid phase, for exarnple to support m~t~ such as polystyrene microtitre plates or small 10 beads composed of polymeric particles, in order to effect separation from the liquid phase. Ihe second antibody is used for fl~tecting ~e immllnP. complex which has been formed and, for this purpose, is coupled to a label, for exarnple a dye, a - fluor-PscPn~e or chemil--minPsc~ce label, a radioactive nuclide or an en7yme.
Two types of sandwich immunoassay are known, terrned "one-step" or sim--lt~neous15 assay and "two-step" or seq~lPnti~l immllnoassay, rc~ ;tively. Ihe two types differ in the order in which the two antibody reagents are added. Ihe ~imlllt~neous immunoassay is carried out by mixing the solid-phase antibody (captunng antibody) and the signal-producing antibody simultaneously with the protein-cn"l;.i"i"~ sarnple and then, for the purpose of measuring the signal, s~a alillg the immune complex20 which has been forrned from the liquid phase by binding it to the solid phase.
In the sequential or two-step assay, by contrast, it is only the capturing antibody which is initially in~llk~t~1 with the protein-co"~ g sample. Ihe antibody/antigen complex which is forrned is bound to the solid phase and the excess antigen is removed by washing. It is only after that that the immobilized antibody/antigen 25 complex is incub~tel in the incubation step, with the second, labelled signal antibody.
~he sim-llt~neous "one-step" irmnunoassay is becoming increasingly more important owing to the rapidity of its implPm~nt~tion as con,~aled with the traditional "two-step" assay. In addition, its sensitivity and precision are more favourai,le.
30 A fim~ nt~l problem in qu~ ive imrnunological detPnnin~tion methods which use the simultaneous sandwich immunoassay ensues from the forrn of the reaction curve. From the "0" concentration up to a particular limiting concentration of the TeA31 569 219~)727 analyte, the calibration curve has a positive slope. Wlth further increase in the analyte concentration, the curve passes through a maximum and, after that, declines once again at still higher concenh~ions. As a consequence, the calibration curve has a negative slope for very high concentrations.
5 Analysis of such a calibration curve shows that two diL[~lclll analyte concentrations can be assigned to one measurement signal, which can in turn lead to an erroneous d~ iion of the analyte. Ihis effect is termed the "hook effect".
It is necÇ~ry, therefore, when d~t~ g the analyte using a ~imlllt~n~ous sandwich immlmo~ y, to establish whether t-he measurernent signal is located in the 10 asc~mling or d~c~n-ling branch of the curve in order to avoid an erroneous d~lr.ll.;lli1lion due to this hook effect.
- In t-he region of the positive slope of the calibration curve, for relatively low concentrations, a further difficulty ensues from t-he fact that, as the hook peak is approached, the curve is so flat that it is in practice no longer possible to 15 dis~ le between closely adj~t cnn~ntrations. Ihis further limits the assay range which can be used in practice for ql~ e d~""i~ ions.
A method for recognizing the hook effect which is known from the literature provides for a duplicate d~."-i~ ion of the sample using two di~lcll~ sarnple dilutions so t-hat, in t-he case of a very high cnnt~ntration, the more strongly diluted 20 sarnple generates a higher measurement signal (Schulze and Schwick, Prot. Biol.
Fluids 5 (1958), 15). In addition, ~l~ t~ have been made to recognize the hook effect, and to el;"~;"~l~ it, by analyzing the kinetics of the antibody/antigen reaction (DE 27 24 2722, EP 0 148 463, Hoffrnann et al., Clin. Chem. 30, (1984), 1499).
However, all these abovementioned m~tll~1c suffer from t-he disadvantage ~at they 25 neC~S~it~te ei~er complicated measurement or complicated evaluation of the measurement.
While ~e seqllçnti~l assay technique can prevent the hook effect as a rule, it possesses the disadvantages, which have already been mentioned, of lower sensitivity and precision, as well as longer measurement times, when con~cd with a 30 ~imlllt~neous sandwich assay.
A better option for avoiding erroneous illt~ ions in simlll~neous sandwich immlmoassays would be that of altering the shape of the calibration curve to such an I eA31 569 2195 27 ~3 ~
extent that occurrence of the hook effect could be avoided or at least shifted to a higher concentration range. This would result in a wider assay range for the practical test.
It has been reported that the hook effect can be reduced by adding at least one 5 further antibody, which possesses a low affinity for the analyte, in addition to the highly specific antibodies which are norrnally present in an immune test (US 4 595 661). Ihe disadvantage of this method is that two specific antibodies,po~s~cing m~rkP~lly di~ t affinities for the analyte, have to be prepared for each analyte which is to be (l~PtPctPA
10 An object of the present invention was, therefore, to improve a sandwich assay such that, on the one hand, the hook effect can be shifted to conr~ntration ranges which are as high as possible and that, on the other hand, the stP~pnPc~ of the curve thereby - remains so great, up to relatively high analyte concentrations, that closely ~jacf nt analyte concentrations can be readily di~elr~ tPA from each other on the basis of 15 the measurement signals which appertain to them. This would make it possible to extend the assay range for practical use up to relatively high concentrations.
This invention relates to those simlllt~nPous ("one-step") immnrlo~ys in which at least one of the two antibodies of the sandwich assay is a polyclonal antibody. The object according to the invention is achieved by purifying the polyclonal 20 antibody(ies) by means of an affmity-clllull~lographic method, with the antigen corresponding to the antibodies being covalently bound to the separation gel.
In the process according to the invention, the polyclonal antibodies are purified using a cl~oll~lography colurnn which is filled with a gel to which the antigen protein, which is complemPnt~ry to the particular antibody to be purified, is covalently 25 bound. Only the antibodies which are specific for the protein are bound to the column, while all the nonspecific antibodies are eluted llr~hin~1Pred from the column.
The specific antibodies, which are bound to the column, can then be eluted from the column, for exarnple by ~ ngin~ the p~ concenhated and subjected to further ~rocessing. Such a method for purifying antibodies by means of affinity 30 ~;nromatography is described in detail in the literature (P.D.G. Dean, W.S. Johnson and F.A. Middle (Eds.) Affinity Chromatography. A Practical Approach, IRL Press (1985)).
21~5 ~ 27 LeA31 569 A preferred example of the present invention involves a sirnultaneous sandwich immunoassay which contains a monoclonal antibody as the solid-phase antibody anda polyclonal antibody as the signal-generating antibody. According to the process according to the invention which is described here, this polyclonal antibody is 5 purified using a cllloll~ography column which cont~in~ a gel to which the corresponding antigen is covalently bound.
A very particularly pl~rel,~d example of this present invention involves a simultaneous sandwich immunoassay for qu~lli~ti~ely ~ete~ g beta-2-microglobulin. This immlmo~y uses a monoclonal capturing antibody and a 10 polyc!onal signal-ge~ ling antibody. On the basis ofthe process which is described in this application, the polyclonal antibody is purified using a ~ ull~lography column which is filled with a gel to which pure beta-2-microglobulin is covalently bound. Ihis process results in the hook peak being shifted to higher analyte conc~lll.~ions and the calibration curve becoming suitable for a wider concentration 15 range in coll~ison to that achieved using signal-generating antibodies which were not purified by affinity cl~r~ ography.
~he exarnples which are listed below clarify the invention on the basis of a process for qll~ ely d~t~ -g beta-2-microglobulin, without these exarnples in any way limitin~ the totality of the claims in this application.
20 FY~m~e 1 The sandwich immllnnassay for qu~ ti~ely c1~ ;I l;l lg beta-2-microglobulin was carried out on the ~lltom~tefl lmmlm() 1 analyzer (Bayer Diagnostics, Munich). Amonoclonal antibody, A, against beta-2-microglobulin and a polyclonal antibody, B, from goat serum, against beta-2-microglobulin were used.
25 The monoclonal antibody A is used as the capturing antibody, while being labelled with fluorescein isothiocyanate (FITC) in accordance with methods which are known from the literature (e.g. S.S. Wong, Chemistry of Protein Conjugation and Cross-T inkin~ CRC Press, 1991).
Ihe polyclonal antibody B is purified from goat serum by preci~ g with 30 ~mmûnium slllph~te and then using a clllon~ography column to whose separationgel (BioRad Affil-Prep 10 affinity cllroll~alography support) beta-2-microglobulin is covalently bound. A$er it has been isolated, this afflnity-purified antibody is coupled to the enzyme "~lk~line phosphatase" using methods which are known from the Le A 31 569 2 1 9 5 ~ ~ ~ 5 literature (e.g. S.S. Wong, Chernistry of Protein Conjugation and Cross-l inking, CRC Press, 1991). This conjugate serves as the signal-generating antibody reagent.
Ihe two antibody conjugates are used, in an aqueous, protein-cont~ining buffer solution, as the detection reagent for the test on the automated Immuno 1.
Ihe two antibodies and the sarnple are initially incubated on the appliance at 37C
for 21 rni~ in order to form the sandwich complex. After that, small m~nPtic beads are added and the rnixture is incubated at 37C for a further 8 min. ~he m~Ptic beads are coated with antibodies against fluorescein and bind to the fluorescein of the capturing antibody of the irnrnune corn~lex. After the irnmune complex has been separated off and washed, and p-nillu~ ol phosphate has been added, the enzyme alkaline ph~ h~ sP which is bound to the second antibody ofthe immune complex, catalyzes the formation of coloured p-nitrophenolate. Ihe quantity of dye which is formed in the first few mimltes is proportional to the concentration of the analyte.
For comparison, the polyclonal antibody B from goat serum was purified using a colurnn which contained protein A and then coupled, as described above, to ~lk~line phosph~t~ce It is then, as described above, used as the- signal-generating antibody, together with the fluoresceinated monoclonal antibody A, for detPrminin~ beta-2-rnicroglobulin on the Immuno 1.
~he experimental result demonstrates that it is only possible to cletPrmine beta-2-microglobulin qu~ ively in a range of from 0 to 20 mg/l when the polyclonal antibody is purified, before being coupled to alkaline phosph~t~e, using a separation colurnn to whose separation gel beta-2-microglobulin is covalently bound. If, on the other hand, the antibody from goat serum is isolated by protein A ~l~ro~ lography, a calibration curve is obtained which already reaches saturation at an analyte concentration of about 8 mg/l and is no longer able to determine beta-2-microglobulin concentrations qu~"~ ively in the range from 8 to 20 mg/l. The experimental result is not improved by increasing the concentration of the protein A-purified antibody in the reagent.
Fig. 1 shows the result in the forrn of a graph. Calibration cwes were cons~ucted using 6 standard controls. Ihe values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 20 mg/l. ~he calibration curve whose measurement points are indicated by triangles describes the course of the calibration curve for the antibody pair which contains the affinity-purified polyclonal antibody as the signal-generating antibody. The - TeA31569 2195727 calibration curve whose measurement points are indicated by diamonds describes the course of the calibration curve for the antibody pair which contains the protein A-purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis while the y axis gives the change in S absorption per minute which was measured by the analyzer.
e 2 The fluoresceinated antibody A is used as the capturing antibody, as described in Example 1.
The polyclonal antibody C is purified ~om goat serum by precipitation with 10 ~mmonillm slllrh~te and subsequent purifieation by affinity cLloll~ography using a cl~r~ll~ography colurnn which is filled with a gel to which beta-2-microglobulin is covalently bound.
For comparison, the same polyelonal antibody is purified, after having been precipitated from goat serum, using a protein A column.
15 The two antibody fractions are conjugated to ~lk~lin~ phosph~t~ce as deseribed in Example 1. The two conjugates are used as signal-generating antibodies.
Ihe immlmo~ ys are earried out as described in Example 1.
lhe expçrim~nt~l result cl~mn"~l,~es that it is only the affinity-purified polyclonal antibody whieh, when used as the signal-generating antibody conjugate, produces a 20 calibration eurve whieh ean ~ "~ e beta-2-mieroglobulin q~l~"~ ely in the range of from 0 to 20 mg/l. By contrast, if the polyelonal antibody is used in protein A-purified form, the ealibration eurve very rapidly reaehes saturation and is nolonger able to ~ nnin~ analyte concentrations of from 10 to 20 mgll. The experimental result is not improved by increasing the concentration of the protein A-25 purified antibody conjugate.
Fig. 2 shows the result in the form of a graph. Calibration eurves were constructedusing 6 standard controls. Ihe values of these controls are 0, 0.1, l.G, 3.0, 8.0 and 20.0 mg~l. The ealibration curve whose ll~ea~ ent points are indieated by triangles describes the course of the ealibration curve for the antibody pair which cont~in~ the 30 affinity-purified polyclonal antibody as the signal-generating antibody. The calibration curve whose measurement points are indicated by tli~montlc describes the T e A 31 569 2 1 9 5 7 ~ 7 course of the calibration curve for the antibody pair which contains the protein A-purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyzer.
5 ~n~e 3 The monoclonal antibody D is used æ the capturing antibody and is conjugated with FITC æ described in Example 1. The ~lk~line phos~ ce conjugate of the polyclonal antibody B is used as the signal-generating antibody. As described inExarnple 1, the antibody is purified on the one occasion by affinity clllo~ ography 10 and on the other occæion using protein A.
The immlmoac~y is carried out as described in Exarnple 1.
- The c~clilllental result demo~ es that the antibody con~ lion using the affinity-purified, signal-generating antibody yields a calibration curve which rises steeply.
15 If, by co~ , the signal-generating antibody is employed in protein A-purifiedform, a marked hook effect is seen in the calibration curve, with a hook peak already being present at an analyte concentration of about 3-5 mg/l.
Fig. 3 shows the result in the form of a graph. Calibration curves were constructed using 6 standard controls. The values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 20 20.0 mg/l. The calibration curve whose m~r~ points are indicated by trianglesdescribes the course of the calibration curve for the antibody pair which contains the affinity-purified polyclonal antibody æ the signal-generating antibody. The calibration curve whose measur~ points are indicated by ~ mon~ describes the course of the calibration curve for the antibody pair which contains the protein A-25 purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyær.
In the cæe of the affinity-purified, signal-generating antibody, the value for the 20 mg/l control is higher than that for the 8 mg!l control. However, since it lay 30 outside the measurement range, and it was not possible to ascertain any exact value, it wæ not reproduced in the diagrarn.
LeA31 569 2195727 e 4 The monoclonal antibody D is used as the capturing antibody and conjugated with FITC as described in Exarnple 1. The ~Ik~lin~ phosph~t~e conjugate of the polyclonal antibody C is used as the signal-generating antibody. As described in5 Example 2, the antibody is purified by affinity chromatography on one occasion and using protein A on the other.
The immlino~c~y is carried out as described in Example 1.
The experimental result d~no~ les that the antibody collllfill~lion cn~ g the affinity-purified, signal-generating antibody yields a calibration curve which slowly 10 reaches saturation only at high concentrations.
By contrast, if the sigr~ g~ g antibody in protein A-purified form is employed, - a marked hook effect is seen in the calibration curve, with a hook peak already being present at an analyte concentration of about 3-5 mg/l.
Fig. 4 shows the result in the form of a graph. Calibration curves were constructed using 6 standard controls. The values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 20.0 mg/l. The calibration curve whose measurement points are indicated by triangles describes the course of the calibration curve for the antibody pair which contains the affinity-purified polyclonal antibody as the signal-generating antibody. The calibration curve whose measurement points are indicated by tli~mon~l~ describes the 20 course of the calibration curve for the antibody pair which c()nt~in~ the protein A-purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyzer.
FY~n~le 5 25 Capturing antibody: monoclonal antibody A, which is conjugated with FITC as described in Example 1.
~ignal-generating antibody: polyclonal antibody E which is affinity-purified using a column loaded with beta-2-microglobulin, and conjugated with ~lk~line phosphatase, as described in Example 1.
30 The experiment is c~ried out as described in Example 1.
~RA31 569 219~727 Ihe calibration curve which is produced enables beta-2-microglobulin to be det~nined qu~lli~ively up to concentrations of 20 mg/l.
Fig. S shows the result in the form of a graph. Calibration curves were constructed using 6 standard controls. The values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 5 20.0 mg/l. Ihe diagram shown describes the course of the calibration curve for the antibody pair w~Lich contains the affinity-purified polyclonal antibody as the signal-generating antibody. ~he analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyzer.
Particularly on account oftheir s~ti~f~rtory specificity and sensitivity, immunoassays 5 are frequently ernployed for d~Ptecting proteins in serum samples and urine sarnples for ~i~gncstic purposes in medicine. Ihe so-called sandwich assay is a particularly important vanant of an immlmo~c~y. Tn this assay, two antibodies bind to two di~lclll epitopes of the protein, with one of the antibodies being coupled to a solid phase, for exarnple to support m~t~ such as polystyrene microtitre plates or small 10 beads composed of polymeric particles, in order to effect separation from the liquid phase. Ihe second antibody is used for fl~tecting ~e immllnP. complex which has been formed and, for this purpose, is coupled to a label, for exarnple a dye, a - fluor-PscPn~e or chemil--minPsc~ce label, a radioactive nuclide or an en7yme.
Two types of sandwich immunoassay are known, terrned "one-step" or sim--lt~neous15 assay and "two-step" or seq~lPnti~l immllnoassay, rc~ ;tively. Ihe two types differ in the order in which the two antibody reagents are added. Ihe ~imlllt~neous immunoassay is carried out by mixing the solid-phase antibody (captunng antibody) and the signal-producing antibody simultaneously with the protein-cn"l;.i"i"~ sarnple and then, for the purpose of measuring the signal, s~a alillg the immune complex20 which has been forrned from the liquid phase by binding it to the solid phase.
In the sequential or two-step assay, by contrast, it is only the capturing antibody which is initially in~llk~t~1 with the protein-co"~ g sample. Ihe antibody/antigen complex which is forrned is bound to the solid phase and the excess antigen is removed by washing. It is only after that that the immobilized antibody/antigen 25 complex is incub~tel in the incubation step, with the second, labelled signal antibody.
~he sim-llt~neous "one-step" irmnunoassay is becoming increasingly more important owing to the rapidity of its implPm~nt~tion as con,~aled with the traditional "two-step" assay. In addition, its sensitivity and precision are more favourai,le.
30 A fim~ nt~l problem in qu~ ive imrnunological detPnnin~tion methods which use the simultaneous sandwich immunoassay ensues from the forrn of the reaction curve. From the "0" concentration up to a particular limiting concentration of the TeA31 569 219~)727 analyte, the calibration curve has a positive slope. Wlth further increase in the analyte concentration, the curve passes through a maximum and, after that, declines once again at still higher concenh~ions. As a consequence, the calibration curve has a negative slope for very high concentrations.
5 Analysis of such a calibration curve shows that two diL[~lclll analyte concentrations can be assigned to one measurement signal, which can in turn lead to an erroneous d~ iion of the analyte. Ihis effect is termed the "hook effect".
It is necÇ~ry, therefore, when d~t~ g the analyte using a ~imlllt~n~ous sandwich immlmo~ y, to establish whether t-he measurernent signal is located in the 10 asc~mling or d~c~n-ling branch of the curve in order to avoid an erroneous d~lr.ll.;lli1lion due to this hook effect.
- In t-he region of the positive slope of the calibration curve, for relatively low concentrations, a further difficulty ensues from t-he fact that, as the hook peak is approached, the curve is so flat that it is in practice no longer possible to 15 dis~ le between closely adj~t cnn~ntrations. Ihis further limits the assay range which can be used in practice for ql~ e d~""i~ ions.
A method for recognizing the hook effect which is known from the literature provides for a duplicate d~."-i~ ion of the sample using two di~lcll~ sarnple dilutions so t-hat, in t-he case of a very high cnnt~ntration, the more strongly diluted 20 sarnple generates a higher measurement signal (Schulze and Schwick, Prot. Biol.
Fluids 5 (1958), 15). In addition, ~l~ t~ have been made to recognize the hook effect, and to el;"~;"~l~ it, by analyzing the kinetics of the antibody/antigen reaction (DE 27 24 2722, EP 0 148 463, Hoffrnann et al., Clin. Chem. 30, (1984), 1499).
However, all these abovementioned m~tll~1c suffer from t-he disadvantage ~at they 25 neC~S~it~te ei~er complicated measurement or complicated evaluation of the measurement.
While ~e seqllçnti~l assay technique can prevent the hook effect as a rule, it possesses the disadvantages, which have already been mentioned, of lower sensitivity and precision, as well as longer measurement times, when con~cd with a 30 ~imlllt~neous sandwich assay.
A better option for avoiding erroneous illt~ ions in simlll~neous sandwich immlmoassays would be that of altering the shape of the calibration curve to such an I eA31 569 2195 27 ~3 ~
extent that occurrence of the hook effect could be avoided or at least shifted to a higher concentration range. This would result in a wider assay range for the practical test.
It has been reported that the hook effect can be reduced by adding at least one 5 further antibody, which possesses a low affinity for the analyte, in addition to the highly specific antibodies which are norrnally present in an immune test (US 4 595 661). Ihe disadvantage of this method is that two specific antibodies,po~s~cing m~rkP~lly di~ t affinities for the analyte, have to be prepared for each analyte which is to be (l~PtPctPA
10 An object of the present invention was, therefore, to improve a sandwich assay such that, on the one hand, the hook effect can be shifted to conr~ntration ranges which are as high as possible and that, on the other hand, the stP~pnPc~ of the curve thereby - remains so great, up to relatively high analyte concentrations, that closely ~jacf nt analyte concentrations can be readily di~elr~ tPA from each other on the basis of 15 the measurement signals which appertain to them. This would make it possible to extend the assay range for practical use up to relatively high concentrations.
This invention relates to those simlllt~nPous ("one-step") immnrlo~ys in which at least one of the two antibodies of the sandwich assay is a polyclonal antibody. The object according to the invention is achieved by purifying the polyclonal 20 antibody(ies) by means of an affmity-clllull~lographic method, with the antigen corresponding to the antibodies being covalently bound to the separation gel.
In the process according to the invention, the polyclonal antibodies are purified using a cl~oll~lography colurnn which is filled with a gel to which the antigen protein, which is complemPnt~ry to the particular antibody to be purified, is covalently 25 bound. Only the antibodies which are specific for the protein are bound to the column, while all the nonspecific antibodies are eluted llr~hin~1Pred from the column.
The specific antibodies, which are bound to the column, can then be eluted from the column, for exarnple by ~ ngin~ the p~ concenhated and subjected to further ~rocessing. Such a method for purifying antibodies by means of affinity 30 ~;nromatography is described in detail in the literature (P.D.G. Dean, W.S. Johnson and F.A. Middle (Eds.) Affinity Chromatography. A Practical Approach, IRL Press (1985)).
21~5 ~ 27 LeA31 569 A preferred example of the present invention involves a sirnultaneous sandwich immunoassay which contains a monoclonal antibody as the solid-phase antibody anda polyclonal antibody as the signal-generating antibody. According to the process according to the invention which is described here, this polyclonal antibody is 5 purified using a cllloll~ography column which cont~in~ a gel to which the corresponding antigen is covalently bound.
A very particularly pl~rel,~d example of this present invention involves a simultaneous sandwich immunoassay for qu~lli~ti~ely ~ete~ g beta-2-microglobulin. This immlmo~y uses a monoclonal capturing antibody and a 10 polyc!onal signal-ge~ ling antibody. On the basis ofthe process which is described in this application, the polyclonal antibody is purified using a ~ ull~lography column which is filled with a gel to which pure beta-2-microglobulin is covalently bound. Ihis process results in the hook peak being shifted to higher analyte conc~lll.~ions and the calibration curve becoming suitable for a wider concentration 15 range in coll~ison to that achieved using signal-generating antibodies which were not purified by affinity cl~r~ ography.
~he exarnples which are listed below clarify the invention on the basis of a process for qll~ ely d~t~ -g beta-2-microglobulin, without these exarnples in any way limitin~ the totality of the claims in this application.
20 FY~m~e 1 The sandwich immllnnassay for qu~ ti~ely c1~ ;I l;l lg beta-2-microglobulin was carried out on the ~lltom~tefl lmmlm() 1 analyzer (Bayer Diagnostics, Munich). Amonoclonal antibody, A, against beta-2-microglobulin and a polyclonal antibody, B, from goat serum, against beta-2-microglobulin were used.
25 The monoclonal antibody A is used as the capturing antibody, while being labelled with fluorescein isothiocyanate (FITC) in accordance with methods which are known from the literature (e.g. S.S. Wong, Chemistry of Protein Conjugation and Cross-T inkin~ CRC Press, 1991).
Ihe polyclonal antibody B is purified from goat serum by preci~ g with 30 ~mmûnium slllph~te and then using a clllon~ography column to whose separationgel (BioRad Affil-Prep 10 affinity cllroll~alography support) beta-2-microglobulin is covalently bound. A$er it has been isolated, this afflnity-purified antibody is coupled to the enzyme "~lk~line phosphatase" using methods which are known from the Le A 31 569 2 1 9 5 ~ ~ ~ 5 literature (e.g. S.S. Wong, Chernistry of Protein Conjugation and Cross-l inking, CRC Press, 1991). This conjugate serves as the signal-generating antibody reagent.
Ihe two antibody conjugates are used, in an aqueous, protein-cont~ining buffer solution, as the detection reagent for the test on the automated Immuno 1.
Ihe two antibodies and the sarnple are initially incubated on the appliance at 37C
for 21 rni~ in order to form the sandwich complex. After that, small m~nPtic beads are added and the rnixture is incubated at 37C for a further 8 min. ~he m~Ptic beads are coated with antibodies against fluorescein and bind to the fluorescein of the capturing antibody of the irnrnune corn~lex. After the irnmune complex has been separated off and washed, and p-nillu~ ol phosphate has been added, the enzyme alkaline ph~ h~ sP which is bound to the second antibody ofthe immune complex, catalyzes the formation of coloured p-nitrophenolate. Ihe quantity of dye which is formed in the first few mimltes is proportional to the concentration of the analyte.
For comparison, the polyclonal antibody B from goat serum was purified using a colurnn which contained protein A and then coupled, as described above, to ~lk~line phosph~t~ce It is then, as described above, used as the- signal-generating antibody, together with the fluoresceinated monoclonal antibody A, for detPrminin~ beta-2-rnicroglobulin on the Immuno 1.
~he experimental result demonstrates that it is only possible to cletPrmine beta-2-microglobulin qu~ ively in a range of from 0 to 20 mg/l when the polyclonal antibody is purified, before being coupled to alkaline phosph~t~e, using a separation colurnn to whose separation gel beta-2-microglobulin is covalently bound. If, on the other hand, the antibody from goat serum is isolated by protein A ~l~ro~ lography, a calibration curve is obtained which already reaches saturation at an analyte concentration of about 8 mg/l and is no longer able to determine beta-2-microglobulin concentrations qu~"~ ively in the range from 8 to 20 mg/l. The experimental result is not improved by increasing the concentration of the protein A-purified antibody in the reagent.
Fig. 1 shows the result in the forrn of a graph. Calibration cwes were cons~ucted using 6 standard controls. Ihe values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 20 mg/l. ~he calibration curve whose measurement points are indicated by triangles describes the course of the calibration curve for the antibody pair which contains the affinity-purified polyclonal antibody as the signal-generating antibody. The - TeA31569 2195727 calibration curve whose measurement points are indicated by diamonds describes the course of the calibration curve for the antibody pair which contains the protein A-purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis while the y axis gives the change in S absorption per minute which was measured by the analyzer.
e 2 The fluoresceinated antibody A is used as the capturing antibody, as described in Example 1.
The polyclonal antibody C is purified ~om goat serum by precipitation with 10 ~mmonillm slllrh~te and subsequent purifieation by affinity cLloll~ography using a cl~r~ll~ography colurnn which is filled with a gel to which beta-2-microglobulin is covalently bound.
For comparison, the same polyelonal antibody is purified, after having been precipitated from goat serum, using a protein A column.
15 The two antibody fractions are conjugated to ~lk~lin~ phosph~t~ce as deseribed in Example 1. The two conjugates are used as signal-generating antibodies.
Ihe immlmo~ ys are earried out as described in Example 1.
lhe expçrim~nt~l result cl~mn"~l,~es that it is only the affinity-purified polyclonal antibody whieh, when used as the signal-generating antibody conjugate, produces a 20 calibration eurve whieh ean ~ "~ e beta-2-mieroglobulin q~l~"~ ely in the range of from 0 to 20 mg/l. By contrast, if the polyelonal antibody is used in protein A-purified form, the ealibration eurve very rapidly reaehes saturation and is nolonger able to ~ nnin~ analyte concentrations of from 10 to 20 mgll. The experimental result is not improved by increasing the concentration of the protein A-25 purified antibody conjugate.
Fig. 2 shows the result in the form of a graph. Calibration eurves were constructedusing 6 standard controls. Ihe values of these controls are 0, 0.1, l.G, 3.0, 8.0 and 20.0 mg~l. The ealibration curve whose ll~ea~ ent points are indieated by triangles describes the course of the ealibration curve for the antibody pair which cont~in~ the 30 affinity-purified polyclonal antibody as the signal-generating antibody. The calibration curve whose measurement points are indicated by tli~montlc describes the T e A 31 569 2 1 9 5 7 ~ 7 course of the calibration curve for the antibody pair which contains the protein A-purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyzer.
5 ~n~e 3 The monoclonal antibody D is used æ the capturing antibody and is conjugated with FITC æ described in Example 1. The ~lk~line phos~ ce conjugate of the polyclonal antibody B is used as the signal-generating antibody. As described inExarnple 1, the antibody is purified on the one occasion by affinity clllo~ ography 10 and on the other occæion using protein A.
The immlmoac~y is carried out as described in Exarnple 1.
- The c~clilllental result demo~ es that the antibody con~ lion using the affinity-purified, signal-generating antibody yields a calibration curve which rises steeply.
15 If, by co~ , the signal-generating antibody is employed in protein A-purifiedform, a marked hook effect is seen in the calibration curve, with a hook peak already being present at an analyte concentration of about 3-5 mg/l.
Fig. 3 shows the result in the form of a graph. Calibration curves were constructed using 6 standard controls. The values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 20 20.0 mg/l. The calibration curve whose m~r~ points are indicated by trianglesdescribes the course of the calibration curve for the antibody pair which contains the affinity-purified polyclonal antibody æ the signal-generating antibody. The calibration curve whose measur~ points are indicated by ~ mon~ describes the course of the calibration curve for the antibody pair which contains the protein A-25 purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyær.
In the cæe of the affinity-purified, signal-generating antibody, the value for the 20 mg/l control is higher than that for the 8 mg!l control. However, since it lay 30 outside the measurement range, and it was not possible to ascertain any exact value, it wæ not reproduced in the diagrarn.
LeA31 569 2195727 e 4 The monoclonal antibody D is used as the capturing antibody and conjugated with FITC as described in Exarnple 1. The ~Ik~lin~ phosph~t~e conjugate of the polyclonal antibody C is used as the signal-generating antibody. As described in5 Example 2, the antibody is purified by affinity chromatography on one occasion and using protein A on the other.
The immlino~c~y is carried out as described in Example 1.
The experimental result d~no~ les that the antibody collllfill~lion cn~ g the affinity-purified, signal-generating antibody yields a calibration curve which slowly 10 reaches saturation only at high concentrations.
By contrast, if the sigr~ g~ g antibody in protein A-purified form is employed, - a marked hook effect is seen in the calibration curve, with a hook peak already being present at an analyte concentration of about 3-5 mg/l.
Fig. 4 shows the result in the form of a graph. Calibration curves were constructed using 6 standard controls. The values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 20.0 mg/l. The calibration curve whose measurement points are indicated by triangles describes the course of the calibration curve for the antibody pair which contains the affinity-purified polyclonal antibody as the signal-generating antibody. The calibration curve whose measurement points are indicated by tli~mon~l~ describes the 20 course of the calibration curve for the antibody pair which c()nt~in~ the protein A-purified polyclonal antibody as the signal-generating antibody. The analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyzer.
FY~n~le 5 25 Capturing antibody: monoclonal antibody A, which is conjugated with FITC as described in Example 1.
~ignal-generating antibody: polyclonal antibody E which is affinity-purified using a column loaded with beta-2-microglobulin, and conjugated with ~lk~line phosphatase, as described in Example 1.
30 The experiment is c~ried out as described in Example 1.
~RA31 569 219~727 Ihe calibration curve which is produced enables beta-2-microglobulin to be det~nined qu~lli~ively up to concentrations of 20 mg/l.
Fig. S shows the result in the form of a graph. Calibration curves were constructed using 6 standard controls. The values of these controls are 0, 0.1, 1.0, 3.0, 8.0 and 5 20.0 mg/l. Ihe diagram shown describes the course of the calibration curve for the antibody pair w~Lich contains the affinity-purified polyclonal antibody as the signal-generating antibody. ~he analyte concentrations are given on the x axis, while the y axis gives the change in absorption per minute which was measured by the analyzer.
Claims (17)
1. Test kit for a simultaneous sandwich immunoassay containing polyclonal antibodiesl characterized in that these antibodies were purified on the antigen by means of affinity chromatography.
2. Test kit according to claim 1, characterized in that the polyclonal antibody is signal-generating antibody.
3. Test kit according to claim 1 or 2, characterized in that the polyclonal antibody is labelled with a dye, nuclide or enzyme.
4. Test kit according to claim 1, 2 or 3, for detecting beta-2-microglobulin.
5. Use of polyclonal antibodies which are purified on the antigen by means of affinity chromatography for producing immunoassays.
6. Process for extending the assay range in a simultaneous sandwich immunoassay (one-step assay) by using polyclonal antibodies which are purified against the relevant antigen by means of affinity chromatography.
7. Process according to claim 6, characterized in that at least one polyclonal antibody is used and purified against the antigen by means of affinity chromatography.
8. Process according to claim 6 or 7, characterized in that the capturing antibody is a monoclonal antibody and the signal-generating, second antibody is a polyclonal antibody which is purified against the antigen by means of affinity chromatography.
9. Process according to claim 6, 7 or 8, characterized in that the antigen is beta-2-microglobulin.
10. Process for detecting beta-2-microglobulin using a simultaneous sandwich immunoassay, characterized in that the capturing antibody is a monoclonal, fluoresceinated antibody and the signal-generating antibody is a polyclonal antibody which is purified on beta-2-microglobulin by means of affinity chromatography and is coupled to alkaline phosphatase, and in that the solid-phase immobilization is effected using small magnetic beads to which the antifluorescein antibodies are bound.
11. A test kit for use in a simultaneous sandwich immunoassay for detecting an antigen, which test kit includes a polyclonal antibody that has been purified by affinity chromagraphy on the said antigen.
12. Use of a polyclonal antibGdy that has been purified on an antigen by means of affinity chromatography in an immunoassay for detecting the said antigen.
13. Use according to claim 12, of a purified polyclonal antibody, which polyclonal antibody is labelled to generate a signal in the assay.
14. Use according to claim 12 or 13, in a simultaneous sandwich assay.
15. A process for detecting an antigen in a simultaneous sandwich immunoassay for the said antigen, wherein one of the antibodies used in the immunoassay is a polyclonal antibody that has been purified by affinity chromatography on the said antigen.
16. A polyclonal antibody that has been purified on beta-2-microglobulin by affinity chromatography and is labelled with alkaline phosphatase.
17. A polyclonal antibody as claimed in claim 16, for use in a simultaneous sandwich immunoassay to detect beta-2-microglobulin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1996102491 DE19602491A1 (en) | 1996-01-25 | 1996-01-25 | Methods for increasing the assay area and avoiding the hook effect in simultaneous sandwich immunoassays and corresponding test kits |
| DE19602491.9 | 1996-01-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2195727A1 true CA2195727A1 (en) | 1997-07-26 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2195727 Abandoned CA2195727A1 (en) | 1996-01-25 | 1997-01-22 | Process for extending the assay range and for avoiding the hook effect in simultaneous sandwich immunoassays, and corresponding test kits |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0787986A1 (en) |
| JP (1) | JPH09210998A (en) |
| CA (1) | CA2195727A1 (en) |
| DE (1) | DE19602491A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10064827A1 (en) | 2000-12-22 | 2002-06-27 | Dade Behring Marburg Gmbh | Sandwich assay for detecting analyte, useful e.g. for hormones, with detection or correction of the hook effect by measuring detection signals twice |
| WO2005062048A1 (en) | 2003-12-01 | 2005-07-07 | Dade Behring Marburg Gmbh | Homogeneous detection method |
| JP5798720B2 (en) * | 2010-03-30 | 2015-10-21 | 積水メディカル株式会社 | Immunochromatographic reagent for measuring human C-reactive protein (CRP) |
| JP6908810B2 (en) * | 2017-05-18 | 2021-07-28 | 俊記 内原 | 4 Specific binding reagent for detecting qualitative differences in repeat tau, test methods using this, test kits, and drug screening methods. |
| WO2023074323A1 (en) * | 2021-11-01 | 2023-05-04 | 株式会社日立ハイテク | Immunological measurement method and immunological measurement device |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE2724272A1 (en) | 1977-05-28 | 1978-12-07 | Tan Marinus Johan | Lightweight shoulder mounted reading stand - has angled wire structure supporting book at correct position |
| US4595661A (en) | 1983-11-18 | 1986-06-17 | Beckman Instruments, Inc. | Immunoassays and kits for use therein which include low affinity antibodies for reducing the hook effect |
| DE3347162A1 (en) | 1983-12-27 | 1985-07-04 | Behringwerke Ag, 3550 Marburg | PHOTOMETRIC METHOD FOR DETERMINING CONCENTRATION IN REACTIONS WHICH ARE GIVING OR USING SPREAD CENTERS |
| DE4034509A1 (en) * | 1990-10-30 | 1992-05-07 | Boehringer Mannheim Gmbh | IMMUNOLOGICAL PRECIPITATION METHOD FOR DETERMINING A BINDABLE ANALYTIC AND REAGENT FOR CARRYING OUT THIS METHOD |
-
1996
- 1996-01-25 DE DE1996102491 patent/DE19602491A1/en not_active Withdrawn
-
1997
- 1997-01-13 EP EP97100409A patent/EP0787986A1/en not_active Withdrawn
- 1997-01-21 JP JP9020903A patent/JPH09210998A/en active Pending
- 1997-01-22 CA CA 2195727 patent/CA2195727A1/en not_active Abandoned
Also Published As
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| EP0787986A1 (en) | 1997-08-06 |
| DE19602491A1 (en) | 1997-07-31 |
| JPH09210998A (en) | 1997-08-15 |
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