CA2104176A1 - Immunochemical method for detecting an analyte - Google Patents
Immunochemical method for detecting an analyteInfo
- Publication number
- CA2104176A1 CA2104176A1 CA002104176A CA2104176A CA2104176A1 CA 2104176 A1 CA2104176 A1 CA 2104176A1 CA 002104176 A CA002104176 A CA 002104176A CA 2104176 A CA2104176 A CA 2104176A CA 2104176 A1 CA2104176 A1 CA 2104176A1
- Authority
- CA
- Canada
- Prior art keywords
- peroxidase
- analyte
- inactive
- specific binding
- binding partner
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Abstract of the disclosure Immunochemical method for detecting an analyte The invention relates to an immunochemical method for detecting an analyte in a sample of a biological mater-ial, which is present in liquid form, in which method the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as the label.
Description
~ ~ v ~
BEHRINGWERKE ~KTIENGESELLSCHAFT 9~/B OlB-Ma 945 Auslandstext Immunoch~mical meth~d for detectin~ a~ analvte The invention relates to an immunochemical method for detecting an analyte in a sample o a blological mater-ial, which is present in liquid form, in wh.ich method the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjuga~e~
directly or indirectly as the label.
Immunological methods ha~e attained great importance, in particular for clinical diagnosis, because of their extraordinary sensitivity and specifici~y. In these methods, as a rule, one of the immunological binding partners is labeled, i.e. is bound to a signal donor.
Common labels are, for example~ radioactive isotopes, fluorescent, phosphorescent or luminescent substances, substances having stable unpaired electrons, erythro-cytes, latex particles, metal sols and enzymes.
Within the group of enz~mes used for th~ labellng, the peroxidase obtained from horseradish (donor: H2O2 oxido-xeducta~e, EC 1.11.1.7) is particularly suitable because it is readily detectable and highly ~table. Reagents, such as, for example, bifunctional N-hydroxysuccinLmide esters, and processes, such as/ for example, periodate activation of the protein-bollnd carbohydrates, for coupling peroxidase to the immunologically active reaction partners are known to the person skilled in the art. These coupling products are designated con~ugates, which are distinguished, in particular, by the fact that both the enzymatic properties of the peroxidase and the receptor properties (e.g. antigen or antibody~ are preserved to the greatest possible extent.
It has e~erged that correct results ar~ obtained for the majority of samples which are investigated by means of such an Lmmunoassay using pero~idase conjugates. However, .. . . . ~
.
in a certai~ number of samples both falsely positive and falsely negative findings do occur. These sexa are designated below as interering factor sera.
These matriX-ba6ed interferences with the enzyme i~muno-assay can be caused by a wide variety of factors. E~fect6caused by rheumatoid factors or heterophilic antibodie6, in this case human anti-mouse antibodies in particular, which are contained in the in~erfering factor sera, axe known to the person skilled in the art. These effects do not, as a rule, depend on the signal dnnor u~ed.
Some exclmpl~s of interferences which are caused dir~ctly by the enzyme label are described in the literature and in patents. Kohno et al. (J. Clin~ Lab. Anal., 5, pp 197-205 (1991)) describe interferences by anti-~-D-galacto-~idase antibodies in an enzyme immunoassay which contains~-D-galactosidase a~ the label enzyme. This interference is elLminated by the addition of recombinant en~ymically inactive, ~-D-galactosidase D
EP 0 209 155 di~closes a further possibilityD false xesults which are caused by interactions of serum components with carbohydrate residues of label enz~mes, in particular peroxidase, can be eliminated by oxidative removal of these sugar~. In this method, the reduction in the enzyme reactivity by about 50 %, and the aggregation of the oxidized enzyme or conjugate, are disadvantageous ~or the function of the en~yme immunoas~ay. In addition, inter-ferences which have their origin in the protein moiety of the pero~id~e are not avoided by khis method.
The invention therefore relates to an immunochemical method for detecting an analyte in a sample of a biologi~
cal material, which i~ present in liquid form, in which method the reaction of the analyte with a specific binding paxtner is detected by a further specific bi.nding .: . .
- ~ . . ., ~ .
. . : . .
partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as th~ label, wherein the reaction mixture additionally contains "inactive peroxidase".
In an advantageous method the "inactive peroxidase" is added to the test mixture before the labeling peroxidase.
In a further advantageous method thP "inactive peroxidase~ is added at the same time as the labeling peroxidase.
In a particularly advantageous method the "inactive peroxidase" is removed again before the labeling peroxidase is added.
Advantageously, the "inactive peroxida6e" is added in such a quantity that it is present in the reaction mixture at a concentration of 0.002 to 10 mg/ml.
Preferably, the "inactive peroxidase" is present in a concentration range of a . 02 to 5 mg/ml, particularly preferably in a concentration range o~ 0.2 to 2 mg/ml.
The preferred dstection method is a solid phase Lmmunoassay; a micxotitration plate is very particularly preferred as the solid phase in this con~ext.
The invention furthermore relates to the use of "inactive peroxidase" in an immunochemical detection method.
For the purposes of this invention detection denotes both ~5 qualitativ~ de-tection and quantitative determination.
The invention also relates to a reagent containing "inactive peroxidase" inter al ia O
' : ' , ' ' '. , ' ':
&
The invention is therefore based on an Lmmunochemical process for d~veloping reagents which avoid the inter-actions between serum interfering factors and the marker enzyme peroxida~e without having to tolerate the abov ~ entioned disadvantages.
It has been found, surprisingly, thatl by means of a ~uitable treatment, enzymically inactive peroxidase can ~e prepared, after whose addition to the conjugate~
interfering factor examples, which exhibit interferences with peroxidase conjugates, produce correct results.
It is advantageous that the agent according to the invention is readily available, i.e. horseradish pero~id-ase, which is economical and available in large quarlti-ties, can be used as the starting material and "inactive peroxidase" can be prepared from it, for example by treatment with hydroxymethyl hydroperoxide (Marklund, Arch. Biochem. Biophys. 154, p. 614 - 622 (1973)). It is advantageolls that in this way an inacti~e protein is obtained which is very ~imilar to the native protein with regard to its antigenic character (~ertiary structure, ~uaternary structure and glycosylation).
Without thereby limitin~ the invention to a particular mode of action, a possible explanation for the efficacy of the "inactive peroxidase" is that it captures poten-tial interfering factors which are consequently not ableto bind to the peroxidase.
~he addition of receptors, which are directed against sugar residues or epitopes of the pero~ida~e, has a comparable effect, namely that non-specific reactions with interfering factor ~amples are prevented. ~n this context, a monovalent antibody or a lectin, which reacts with the peroxidase, can, for example, serve as the xecep~or, without the reaction with the receptors leading to any precipitation or agglutination.
- . ~ - .
- . . . . : . . ~
. , . ' - ' ' ' ' ' , , ~ ' :
..
~1~'11 ig - s -Preferred antibodies or lectins are those which do not impair the enzymic activity of the peroxida~e.
The reagents acc~rding to the invention are distin-guished, in particularr by ~he fact that ~hey neither influence the enzymic activity of the peroxida~e nor Lmpair the immunological behavior of the conjugate by aggregation.
The reagents according to the inven~ion may be employed in human and veterinary diagnostics as a buffer additive 10 in a multiplicity of i~ununoassays using peroxidase conjugate~ as an essential test component. Examples which may be cited are one-step or multi-step test~ for the detection of antibodies of ~arious immunoglobulin cla~ses against structural features of viruses ~e.g. ~iru~es of hepatiti~ A, B and C, as well as various HIY types), of ~acterial and parasitic pathogens, and of allergic disease~. Further examples are the detection in one-step or multi-step detection methods of disease-causing agents such as viruses (e.g. hepatitis B ~irus), bacteria, parasite~ or allergens, as well as of disease markers (e.g. tumor markers).
The following examples illustrate ~he subject matter of the invention without limiting it.
~xample 1:
a) Preparation of enzymically inactive pero~ida~e The preparation of the peroxidase, whose enzymic activity wa~ irreversibly inactivated, ~ook place by treating commercial peroxidase with hydrox~methyl hydroperoxide according to the method of Marlclund (Arch. Biochem. Biophys. 154, p. 614-622 (1973~).
According to protein determination, the yield was 60 %. The inactive peroxidas~ wa~ stored ~at a . . .
' 6 concentration of 20-30 g/l in 50 mM Tris, pH 8.0, at h) Sequentially competitive detection of anti-HIV 1 100 ~1 of the sample to be investiqated were added to microtitration plates from the commercial test kit "Enzygno~t Anki-HIV 1~ (Behrin~werke ~G, Marburg, FRG, product number OW B~, which were coated with purified, inactivated HI~ 1 lysatel and the plates were then incubated for 60 min. After that, the addition of 25 ~l of anti-HIV (human)/per-oxidase con~ugate (Behringwerke AG, Marburg, FRG, produc~ number OWSB), together with inactive peroxi~
dase (concentration in the conjugate- 1 mg/Inl ) prepared according to Example 1 a), takes place without any washing step. After a urthex 30 min., the solution is sucked off, the plates are washed and the bound peroxidase is detected by means of the commercially ~vailable chromogen system (Behringwerke AG, Marbur~, FRG, product num~er OUVF/O W G). ~he reaction was then stopped with dilute sulfuric acid after the subs~rate had been allowed to react for 30 min., and ~he reacted chromogen was measured at 450 nm. All the samples were considered to be reactive which exhibited extinction~ below the cu~-off value ~extinction of the negative control * 0.5).
The extinctions which were obtained in testin~ anti-HIV positive and negative sera, as well as interf~r-ing factor ~era, are summarized in Table 1. It can be seen that the power of the test to correctly recosnî~e "normal" anti-HIV p~sitive and negative samples is not affec~ed by the additive according to the invention. ~nti HIV 1 positive interfering factor sera, which react in a falsely negative manner without the addition of inactiY2 peroxi~dase~
-: , .
are correctly found to be positive in the ELISA
according to the invention.
Without the addi~ive according to the invention, interfering factor-containin~ anti-HIV negative sera give extinction values in the te~t which are in some cases above the measurement range. In the E~ISA
according to the invention, the extinction values of these sera are in the range of ~he normal negative ~era.
, .
. . , ~ . . .
::
~ ~ u ~
Table 1:
_ _ _ . .
SampleCon~ugate without C~n~ugate ~ith additive accordirlg adtliti~re accc3rding to the inven~ion~ ~o ~h~ ntic)n __ _ _ cut-off ~574 0 .579 value _ _ ~ _ Controlserum,1.189 1 .12 1 negative 1 . 109 1. 193 _ _ Control ~erum, 0. On7 O . 011 positive O . 009 O . OlS
1 ~ _ Anti--IIIV
neg. ~era ~ 12 1 . 090 1 . 258 # ~5 1 . 247 1 . 1~7 ~0 # 216 1. 198 1 . 111 _ _ Anti-HIV 1 pos. ~era WT 17 0 . 015 0 . 005 WT 19 0 . 058 0 . 051 WT 22 Q . 021 0. OID9 . _ _ Xnterfering factor ~3era 3 0 Anti-~IIV 1 po~itive # 40 l.B64 0.032 # 42 ~ 2.500 ~.060 # 43 > 2.500 ~.05~
# 44 1.023 0.026 # 51 > 2.500 0.053 . _ Int~rfering factor sera 4 0 an~i-EIIV
negative # 00181 > 2,500 1.029 ~ 00202 > 2,500 1.254 _ . : .
45 lReactive ~amples are printed in bold type.
.
, ,. - . : . ' ' . ~:
- , . : ~ - :
.. .. ~ , : :
7~ ~
g ~xample 2:
Semi-~onoclonal sandwich assay for the de~tection of HBsAg 100 ~1 of the serum to be inve~tigated were incubated for 90 min. with 25 ~1 o~ anti-HBs (mou6e)/pleroxidase conju-gate (Behringwerke AG, Marburg, FRG, product number OWNF)in a microtitration plate coated with anti-HBs (sheep) ~Enzygnost HBs~g monoclonal~, Behringwerke AG, Marburg, FRG, product number O~NE). One part of the investigations was carried out without additive according to the inven-tion, another part with the addition of inactive peroxid-ase ~1 mg/ml~, and a iurther part with the addition to the conjugate of the lectin Tetragonolobus purpureas (Sigma, Deisenhofen, FRG) at a concentration of 1 mg/ml.
Subsequently, the plate was washed and the bound enzyme activity was determined as explained in Example 1. All samples were considered to be reactive whose extinctions were at least 0.050 B above the average value of the negative controls.
The results of the experiments are summarized in Table ~.
While the additives according to ~he invention do not in1uence the correctness of the results in the ca~e of "normal" HBsAg positive and negative samples~ HBsAg negative interfering factor-containing sera, which react in a falsely positive manner without conjugate additive according to the invention, are found to be correctly negative both by the addition o inactive peroxidase and by the lectin Tetragonolobus purpureas.
The signal behavior of two HBsAs~ n~3gative samples is depicted in Fig. 1 in relation to the quantity of inactive peroxidase added to the test mixture.
7~ : ~
~abl~ 2: .
_ . .
Sample Con~ugate Con~gate with addi~
~ithout ti~e according ~o additive the in~Jerltion according _ Ito the Inacti-in~rention vated per- I ectin o~ida~e ~ _. . . _ cut-off value 0 . 072 0 . 062 0 .082 _ _ _ ~ _ Control ~erum O . 026 O . 010 O .031 negative 0 . 017 0 . 014 0 .033 _ _. _ _ Control serum 1 . 2 6 6 1. 155 1. 019 pc~iti~Te 1. 279 1. 241 O .994 _ _ _ . _ _ ,_ .
~ g 2 0 neg . sera # 20 0.025 0.016 0.022 # 21 0.028 0.014 ~.036 # 24 O . 031 O . 011 O .û12 . ~
2 5 ~SsAg pos. &era # 88 > 2.500 > 2.500 7 2.50~
# 107 ~ 2,.500 ~ 2.500 ~ 2.,50Q
# 209 ~ 2.500 ~ 2.50~ > 2.~00 3 0 _ .
Interfering factor ~era BsAg negative M 487 0.167 O . 018 O,028 M 640 0.139 0 . 022 0.039 M 64~ 0~187 0 O 009 0 .023 M 674 0 .256 0 . 016 0 .013 M 687 0.272 0.006 0.029 _ _ _ _ .
~eactive samples are printed in bold typeO
.. . . . . . .
BEHRINGWERKE ~KTIENGESELLSCHAFT 9~/B OlB-Ma 945 Auslandstext Immunoch~mical meth~d for detectin~ a~ analvte The invention relates to an immunochemical method for detecting an analyte in a sample o a blological mater-ial, which is present in liquid form, in wh.ich method the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjuga~e~
directly or indirectly as the label.
Immunological methods ha~e attained great importance, in particular for clinical diagnosis, because of their extraordinary sensitivity and specifici~y. In these methods, as a rule, one of the immunological binding partners is labeled, i.e. is bound to a signal donor.
Common labels are, for example~ radioactive isotopes, fluorescent, phosphorescent or luminescent substances, substances having stable unpaired electrons, erythro-cytes, latex particles, metal sols and enzymes.
Within the group of enz~mes used for th~ labellng, the peroxidase obtained from horseradish (donor: H2O2 oxido-xeducta~e, EC 1.11.1.7) is particularly suitable because it is readily detectable and highly ~table. Reagents, such as, for example, bifunctional N-hydroxysuccinLmide esters, and processes, such as/ for example, periodate activation of the protein-bollnd carbohydrates, for coupling peroxidase to the immunologically active reaction partners are known to the person skilled in the art. These coupling products are designated con~ugates, which are distinguished, in particular, by the fact that both the enzymatic properties of the peroxidase and the receptor properties (e.g. antigen or antibody~ are preserved to the greatest possible extent.
It has e~erged that correct results ar~ obtained for the majority of samples which are investigated by means of such an Lmmunoassay using pero~idase conjugates. However, .. . . . ~
.
in a certai~ number of samples both falsely positive and falsely negative findings do occur. These sexa are designated below as interering factor sera.
These matriX-ba6ed interferences with the enzyme i~muno-assay can be caused by a wide variety of factors. E~fect6caused by rheumatoid factors or heterophilic antibodie6, in this case human anti-mouse antibodies in particular, which are contained in the in~erfering factor sera, axe known to the person skilled in the art. These effects do not, as a rule, depend on the signal dnnor u~ed.
Some exclmpl~s of interferences which are caused dir~ctly by the enzyme label are described in the literature and in patents. Kohno et al. (J. Clin~ Lab. Anal., 5, pp 197-205 (1991)) describe interferences by anti-~-D-galacto-~idase antibodies in an enzyme immunoassay which contains~-D-galactosidase a~ the label enzyme. This interference is elLminated by the addition of recombinant en~ymically inactive, ~-D-galactosidase D
EP 0 209 155 di~closes a further possibilityD false xesults which are caused by interactions of serum components with carbohydrate residues of label enz~mes, in particular peroxidase, can be eliminated by oxidative removal of these sugar~. In this method, the reduction in the enzyme reactivity by about 50 %, and the aggregation of the oxidized enzyme or conjugate, are disadvantageous ~or the function of the en~yme immunoas~ay. In addition, inter-ferences which have their origin in the protein moiety of the pero~id~e are not avoided by khis method.
The invention therefore relates to an immunochemical method for detecting an analyte in a sample of a biologi~
cal material, which i~ present in liquid form, in which method the reaction of the analyte with a specific binding paxtner is detected by a further specific bi.nding .: . .
- ~ . . ., ~ .
. . : . .
partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as th~ label, wherein the reaction mixture additionally contains "inactive peroxidase".
In an advantageous method the "inactive peroxidase" is added to the test mixture before the labeling peroxidase.
In a further advantageous method thP "inactive peroxidase~ is added at the same time as the labeling peroxidase.
In a particularly advantageous method the "inactive peroxidase" is removed again before the labeling peroxidase is added.
Advantageously, the "inactive peroxida6e" is added in such a quantity that it is present in the reaction mixture at a concentration of 0.002 to 10 mg/ml.
Preferably, the "inactive peroxidase" is present in a concentration range of a . 02 to 5 mg/ml, particularly preferably in a concentration range o~ 0.2 to 2 mg/ml.
The preferred dstection method is a solid phase Lmmunoassay; a micxotitration plate is very particularly preferred as the solid phase in this con~ext.
The invention furthermore relates to the use of "inactive peroxidase" in an immunochemical detection method.
For the purposes of this invention detection denotes both ~5 qualitativ~ de-tection and quantitative determination.
The invention also relates to a reagent containing "inactive peroxidase" inter al ia O
' : ' , ' ' '. , ' ':
&
The invention is therefore based on an Lmmunochemical process for d~veloping reagents which avoid the inter-actions between serum interfering factors and the marker enzyme peroxida~e without having to tolerate the abov ~ entioned disadvantages.
It has been found, surprisingly, thatl by means of a ~uitable treatment, enzymically inactive peroxidase can ~e prepared, after whose addition to the conjugate~
interfering factor examples, which exhibit interferences with peroxidase conjugates, produce correct results.
It is advantageous that the agent according to the invention is readily available, i.e. horseradish pero~id-ase, which is economical and available in large quarlti-ties, can be used as the starting material and "inactive peroxidase" can be prepared from it, for example by treatment with hydroxymethyl hydroperoxide (Marklund, Arch. Biochem. Biophys. 154, p. 614 - 622 (1973)). It is advantageolls that in this way an inacti~e protein is obtained which is very ~imilar to the native protein with regard to its antigenic character (~ertiary structure, ~uaternary structure and glycosylation).
Without thereby limitin~ the invention to a particular mode of action, a possible explanation for the efficacy of the "inactive peroxidase" is that it captures poten-tial interfering factors which are consequently not ableto bind to the peroxidase.
~he addition of receptors, which are directed against sugar residues or epitopes of the pero~ida~e, has a comparable effect, namely that non-specific reactions with interfering factor ~amples are prevented. ~n this context, a monovalent antibody or a lectin, which reacts with the peroxidase, can, for example, serve as the xecep~or, without the reaction with the receptors leading to any precipitation or agglutination.
- . ~ - .
- . . . . : . . ~
. , . ' - ' ' ' ' ' , , ~ ' :
..
~1~'11 ig - s -Preferred antibodies or lectins are those which do not impair the enzymic activity of the peroxida~e.
The reagents acc~rding to the invention are distin-guished, in particularr by ~he fact that ~hey neither influence the enzymic activity of the peroxida~e nor Lmpair the immunological behavior of the conjugate by aggregation.
The reagents according to the inven~ion may be employed in human and veterinary diagnostics as a buffer additive 10 in a multiplicity of i~ununoassays using peroxidase conjugate~ as an essential test component. Examples which may be cited are one-step or multi-step test~ for the detection of antibodies of ~arious immunoglobulin cla~ses against structural features of viruses ~e.g. ~iru~es of hepatiti~ A, B and C, as well as various HIY types), of ~acterial and parasitic pathogens, and of allergic disease~. Further examples are the detection in one-step or multi-step detection methods of disease-causing agents such as viruses (e.g. hepatitis B ~irus), bacteria, parasite~ or allergens, as well as of disease markers (e.g. tumor markers).
The following examples illustrate ~he subject matter of the invention without limiting it.
~xample 1:
a) Preparation of enzymically inactive pero~ida~e The preparation of the peroxidase, whose enzymic activity wa~ irreversibly inactivated, ~ook place by treating commercial peroxidase with hydrox~methyl hydroperoxide according to the method of Marlclund (Arch. Biochem. Biophys. 154, p. 614-622 (1973~).
According to protein determination, the yield was 60 %. The inactive peroxidas~ wa~ stored ~at a . . .
' 6 concentration of 20-30 g/l in 50 mM Tris, pH 8.0, at h) Sequentially competitive detection of anti-HIV 1 100 ~1 of the sample to be investiqated were added to microtitration plates from the commercial test kit "Enzygno~t Anki-HIV 1~ (Behrin~werke ~G, Marburg, FRG, product number OW B~, which were coated with purified, inactivated HI~ 1 lysatel and the plates were then incubated for 60 min. After that, the addition of 25 ~l of anti-HIV (human)/per-oxidase con~ugate (Behringwerke AG, Marburg, FRG, produc~ number OWSB), together with inactive peroxi~
dase (concentration in the conjugate- 1 mg/Inl ) prepared according to Example 1 a), takes place without any washing step. After a urthex 30 min., the solution is sucked off, the plates are washed and the bound peroxidase is detected by means of the commercially ~vailable chromogen system (Behringwerke AG, Marbur~, FRG, product num~er OUVF/O W G). ~he reaction was then stopped with dilute sulfuric acid after the subs~rate had been allowed to react for 30 min., and ~he reacted chromogen was measured at 450 nm. All the samples were considered to be reactive which exhibited extinction~ below the cu~-off value ~extinction of the negative control * 0.5).
The extinctions which were obtained in testin~ anti-HIV positive and negative sera, as well as interf~r-ing factor ~era, are summarized in Table 1. It can be seen that the power of the test to correctly recosnî~e "normal" anti-HIV p~sitive and negative samples is not affec~ed by the additive according to the invention. ~nti HIV 1 positive interfering factor sera, which react in a falsely negative manner without the addition of inactiY2 peroxi~dase~
-: , .
are correctly found to be positive in the ELISA
according to the invention.
Without the addi~ive according to the invention, interfering factor-containin~ anti-HIV negative sera give extinction values in the te~t which are in some cases above the measurement range. In the E~ISA
according to the invention, the extinction values of these sera are in the range of ~he normal negative ~era.
, .
. . , ~ . . .
::
~ ~ u ~
Table 1:
_ _ _ . .
SampleCon~ugate without C~n~ugate ~ith additive accordirlg adtliti~re accc3rding to the inven~ion~ ~o ~h~ ntic)n __ _ _ cut-off ~574 0 .579 value _ _ ~ _ Controlserum,1.189 1 .12 1 negative 1 . 109 1. 193 _ _ Control ~erum, 0. On7 O . 011 positive O . 009 O . OlS
1 ~ _ Anti--IIIV
neg. ~era ~ 12 1 . 090 1 . 258 # ~5 1 . 247 1 . 1~7 ~0 # 216 1. 198 1 . 111 _ _ Anti-HIV 1 pos. ~era WT 17 0 . 015 0 . 005 WT 19 0 . 058 0 . 051 WT 22 Q . 021 0. OID9 . _ _ Xnterfering factor ~3era 3 0 Anti-~IIV 1 po~itive # 40 l.B64 0.032 # 42 ~ 2.500 ~.060 # 43 > 2.500 ~.05~
# 44 1.023 0.026 # 51 > 2.500 0.053 . _ Int~rfering factor sera 4 0 an~i-EIIV
negative # 00181 > 2,500 1.029 ~ 00202 > 2,500 1.254 _ . : .
45 lReactive ~amples are printed in bold type.
.
, ,. - . : . ' ' . ~:
- , . : ~ - :
.. .. ~ , : :
7~ ~
g ~xample 2:
Semi-~onoclonal sandwich assay for the de~tection of HBsAg 100 ~1 of the serum to be inve~tigated were incubated for 90 min. with 25 ~1 o~ anti-HBs (mou6e)/pleroxidase conju-gate (Behringwerke AG, Marburg, FRG, product number OWNF)in a microtitration plate coated with anti-HBs (sheep) ~Enzygnost HBs~g monoclonal~, Behringwerke AG, Marburg, FRG, product number O~NE). One part of the investigations was carried out without additive according to the inven-tion, another part with the addition of inactive peroxid-ase ~1 mg/ml~, and a iurther part with the addition to the conjugate of the lectin Tetragonolobus purpureas (Sigma, Deisenhofen, FRG) at a concentration of 1 mg/ml.
Subsequently, the plate was washed and the bound enzyme activity was determined as explained in Example 1. All samples were considered to be reactive whose extinctions were at least 0.050 B above the average value of the negative controls.
The results of the experiments are summarized in Table ~.
While the additives according to ~he invention do not in1uence the correctness of the results in the ca~e of "normal" HBsAg positive and negative samples~ HBsAg negative interfering factor-containing sera, which react in a falsely positive manner without conjugate additive according to the invention, are found to be correctly negative both by the addition o inactive peroxidase and by the lectin Tetragonolobus purpureas.
The signal behavior of two HBsAs~ n~3gative samples is depicted in Fig. 1 in relation to the quantity of inactive peroxidase added to the test mixture.
7~ : ~
~abl~ 2: .
_ . .
Sample Con~ugate Con~gate with addi~
~ithout ti~e according ~o additive the in~Jerltion according _ Ito the Inacti-in~rention vated per- I ectin o~ida~e ~ _. . . _ cut-off value 0 . 072 0 . 062 0 .082 _ _ _ ~ _ Control ~erum O . 026 O . 010 O .031 negative 0 . 017 0 . 014 0 .033 _ _. _ _ Control serum 1 . 2 6 6 1. 155 1. 019 pc~iti~Te 1. 279 1. 241 O .994 _ _ _ . _ _ ,_ .
~ g 2 0 neg . sera # 20 0.025 0.016 0.022 # 21 0.028 0.014 ~.036 # 24 O . 031 O . 011 O .û12 . ~
2 5 ~SsAg pos. &era # 88 > 2.500 > 2.500 7 2.50~
# 107 ~ 2,.500 ~ 2.500 ~ 2.,50Q
# 209 ~ 2.500 ~ 2.50~ > 2.~00 3 0 _ .
Interfering factor ~era BsAg negative M 487 0.167 O . 018 O,028 M 640 0.139 0 . 022 0.039 M 64~ 0~187 0 O 009 0 .023 M 674 0 .256 0 . 016 0 .013 M 687 0.272 0.006 0.029 _ _ _ _ .
~eactive samples are printed in bold typeO
.. . . . . . .
Claims (12)
1. An immunochemical method for detecting an analyte in a sample of a biological material, which is present in liquid form, in which method the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as the label, wherein the reaction mixture additionally contains "inactive peroxidase".
2. The method as claimed in claim 1, wherein it is a solid-phase enzyme immunoassay.
3. The method as claimed in claim 1, wherein the "in-active peroxidase" is added to the test mixture before the labeling peroxidase.
4. The method as claimed in claim 1, wherein the "in-active peroxidase" is added at the same time as the labeling peroxidase.
5. The method as claimed in claim 1, wherein the "in-active peroxidase" is removed again before the addition of the labeling peroxidase.
6. The method as claimed in at least one of claims 1 - 4, wherein the "inactive peroxidase" is present at a concentration of 0.002 to 10 mg/ml of test mixture.
7. The method as claimed in claim 1, wherein the "in-active peroxidase" is present in a concentration range of 0.02 to 5 mg/ml.
8. The method as claimed in claim 1, wherein the "in-active peroxidase" is present in a concentration range of 0.2 to 2 mg/ml.
9. Use of "inactive peroxidase" in immunochemical detection methods.
10. A reagent for use in immunochemical detection methods containing "inactive peroxidase", inter alia.
11. The method as claimed in claim 2, wherein the solid phase is a microtitration plate.
12. An immunochemical method for detecting an analyte in a sample of a biological material, which is present in liquid form, in which method the reaction of the analyte with a specific binding partner is detected by a further specific binding partner to which horseradish peroxidase is coupled (conjugate) directly or indirectly as the label, wherein the reaction mixture additionally contains at least one antibody directed against an epitope of the peroxidase, or an appropriate lectin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4227102A DE4227102C2 (en) | 1992-08-17 | 1992-08-17 | Immunochemical method for the detection of an analyte |
| DEP4227102.9 | 1992-08-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2104176A1 true CA2104176A1 (en) | 1994-02-18 |
Family
ID=6465672
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002104176A Abandoned CA2104176A1 (en) | 1992-08-17 | 1993-08-16 | Immunochemical method for detecting an analyte |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0583689B1 (en) |
| JP (1) | JP3398189B2 (en) |
| AT (1) | ATE203328T1 (en) |
| AU (1) | AU679921B2 (en) |
| CA (1) | CA2104176A1 (en) |
| DE (2) | DE4227102C2 (en) |
| ES (1) | ES2161221T3 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112114128A (en) * | 2020-09-24 | 2020-12-22 | 武汉生之源生物科技股份有限公司 | Sealing agent and preparation method thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1767942A4 (en) * | 2004-06-14 | 2008-07-16 | Kyowa Medex Co Ltd | Immunoassay in which non-specific reaction is suppressed and reagent therefore |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1231067A (en) * | 1983-08-16 | 1988-01-05 | Bioclones (Proprietary) Limited | Monoclonal antibody to peroxidase and hybridoma producing it |
| GB8514288D0 (en) * | 1985-06-06 | 1985-07-10 | Amersham Int Plc | Enzyme assay of body fluids |
| DE3525911A1 (en) * | 1985-07-19 | 1987-01-29 | Boehringer Mannheim Gmbh | CONJUGATE FOR ENZYME IMMUNE DETERMINATION |
| US4810635A (en) * | 1986-04-16 | 1989-03-07 | Miles Inc. | Specific binding assays employing label analog to reduce sample interferences |
| DE3720983A1 (en) * | 1987-06-25 | 1989-01-05 | Hoechst Ag | IMMUNOMETRIC DETERMINATION PROCEDURE |
| IT1230673B (en) * | 1987-09-04 | 1991-10-29 | Sclavo Spa | METHOD FOR THE IMMUNOLOCALIZATION OF ANTIGENS WITH THE USE OF DIRECT ANTIBODIES AGAINST EPITOPES OF A NON-GLUCIDIC NATURE |
-
1992
- 1992-08-17 DE DE4227102A patent/DE4227102C2/en not_active Expired - Fee Related
-
1993
- 1993-08-05 ES ES93112533T patent/ES2161221T3/en not_active Expired - Lifetime
- 1993-08-05 AT AT93112533T patent/ATE203328T1/en not_active IP Right Cessation
- 1993-08-05 DE DE59310190T patent/DE59310190D1/en not_active Expired - Fee Related
- 1993-08-05 EP EP93112533A patent/EP0583689B1/en not_active Expired - Lifetime
- 1993-08-16 JP JP20213993A patent/JP3398189B2/en not_active Expired - Fee Related
- 1993-08-16 AU AU44628/93A patent/AU679921B2/en not_active Ceased
- 1993-08-16 CA CA002104176A patent/CA2104176A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112114128A (en) * | 2020-09-24 | 2020-12-22 | 武汉生之源生物科技股份有限公司 | Sealing agent and preparation method thereof |
| CN112114128B (en) * | 2020-09-24 | 2023-08-29 | 武汉生之源生物科技股份有限公司 | Sealing agent and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0583689B1 (en) | 2001-07-18 |
| ES2161221T3 (en) | 2001-12-01 |
| EP0583689A1 (en) | 1994-02-23 |
| DE4227102C2 (en) | 2002-11-14 |
| DE59310190D1 (en) | 2001-08-23 |
| AU4462893A (en) | 1994-02-24 |
| JPH06160386A (en) | 1994-06-07 |
| DE4227102A1 (en) | 1994-02-24 |
| ATE203328T1 (en) | 2001-08-15 |
| JP3398189B2 (en) | 2003-04-21 |
| AU679921B2 (en) | 1997-07-17 |
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| FZDE | Discontinued |