DE19700134A1 - Immune dissociation to improve the immunochemical determination of an analyte - Google Patents

Description

The present invention relates to a method for immunochemical Determination of one or more analytes in a sample using a immobilized specific bioaffect interacting with the analyte Receptor R1 and one also with the analyte bioaffin interacting specific receptor R2, which is usually is marked. The method according to the invention is thereby characterized in that a receptor R3, the one or more than one has specific binding site to the analyte, is added and the resulting immune complexes completely or partially dissociated, afterwards reassociated and then detected. According to another Embodiment in addition to the receptor R3 is a receptor R4 used, which has an affinity for R3 and on the solid phase is immobilized, the resulting immune complexes entirely or partially dissociated, then reassociated and then detected will.

Common immunological procedures for diagnosing diseases that with the formation of specific antibodies against one Disease triggers such as viruses, bacteria, allergens, autoantigens or  Certain medicines are associated based on their ability Antibodies for complex formation with antigenic structures of the trigger.

In certain embodiments of these methods, the general as Heterogeneous immunoassays will be called one on the content on, for example, specific antibodies (analyte antibodies) testing sample with antigenic structures that trigger the disease Brought into contact, these antigenic structures on suitable, known carrier materials are immobilized. Included in the sample Analytical antibodies are used as an immune complex to those at the Carrier materials immobilized antigenic structures of the Disease trigger bound and proven. To prove it Detection antibodies or other specific receptors (e.g. protein A), which are capable of complex binding with the analyte antibody of the sample, be used.

The detection reagent usually has a label that the Amount of the bound antibody makes measurable by measurement.

Common labels are radioactive isotopes, enzymes, fluorescent, phosphorescent or luminescent substances, Substances with stable unpaired electrons, latex particles, Magnetic particles, metal brine and erythrocytes.

These processes are both single-stage and multi-stage Detection methods known. Each process step is common ended with a separation process (washing step).

The very simple technique of the one-step method heterogeneous immunoassays, however, is not for the detection of all Suitable disease markers. For technical reasons, two or multi-stage process steps are applied.  

Multistage processes are also known, which are known as immune complex transfer Enzyme immunoassays (S. Hashida et al., Journal of Clinical Laboratory Analysis 8: 86-95 (1994)). The whole thing is off Solid phase antigen, specific antibody and labeled Conjugate antigen existing immune complex detached from the solid phase. After pipetting onto an antibody-binding solid phase, the entire immune complex fixed and detected.

These methods are very specific, but have the disadvantage that the disease triggers to be demonstrated or directed against them Antibodies, which in the first step with the immobilized, specific A receptor has been received in the following complex Reaction steps in a back reaction known to the person skilled in the art partially detach from the complex again and thus the Withdraw detection reaction, which, for example, increases sensitivity is restricted.

The diagnostic performance of such multi-step procedures will especially severely restricted if the Reverse reaction rate between immobilized receptor and the agent to be detected is high. This is the case with low affinity, for example Antibodies against disease triggers or drugs. The These effects are known to those skilled in the art, in particular in processes for Detection of frequently mutating triggers or Disease markers associated with the immobilized specific receptor show little interaction after mutation.

EP 0 572 845 already disclosed that the back reaction rate by adding another receptor against structural features of the agent to be detected is significantly reduced. This receptor must more than a binding site for the agent to be detected  and must not have immunochemical detection of the agent to disturb.

Despite the significantly reduced back reaction, this method also makes it possible not, special low affinity antibodies against disease triggers or Detect drugs safely and with high sensitivity.

Therefore, the task was to find reagents that have these disadvantages do not have.

Surprisingly, it was found that due to a Immune dissociation and subsequent reassociation step of the already immune complex formed from immobilized specific receptor R1 and thus bioaffine interacting analyte A and the counter specific analyte directed to this analyte R2, which in the Rule is marked, there is a signal increase when in addition a receptor R3 is added. This reduces the share of non-solid-phase-fixed analyte in that it, without the Immune reaction between analyte A and receptor R1 or analyte A and Repressing receptor R2 can bind more than one analyte.

According to a further embodiment, another is in addition to R3 immobilized receptor R4 added, which with R3 bioaffin can interact. R4 reduces by fixation of the immune complex, consisting of analyte A and receptor R3, to the solid phase the proportion of immobilized immune complexes. Here the specific receptor R3 however not as in the above Embodiment, more than a specific binding site to the analyte A have. This additional fixation of the analyte A and thus the receptor R2, which usually bears the label, also becomes the Signal yield increased significantly and ultimately the sensitivity of the Assays for analyte A increased.  

According to a further embodiment, the receptor R4 is on a immobilized in the second solid phase and become the dissociated Immune complexes, for example R3-A-R2, from the first solid phase transferred the second solid phase where the reassociation step and also the detection of R2 is done.

The analyte A to be detected in the sense of this invention can be either for example antibodies induced by a disease trigger, as also be an antigen, such as the disease trigger itself.

The invention therefore relates to a method for the immunological determination of one or more analytes by means of:
of a specific receptor R1, this specific receptor R1 being immobilized on a support and the extent of the binding of the analyte to the specific receptor R1 being determined by means of a further specific receptor R2 which bears a label directly or indirectly, wherein in the method a Dissociation step is carried out, characterized in that a receptor R3, which has one or more binding sites for the agent A to be detected, is added, has no affinity for the immobilized specific receptor R1 and is not labeled and the extent of binding of the Analyte A to the specific receptor R2, which bears a label directly or indirectly, is determined after reassociation.

The invention further relates to a method for the immunological determination of one or more analytes by means of:
of a specific receptor R1, this specific receptor R1 being immobilized on a support and the extent of the binding of analyte A to the specific receptor R1 by means of a further specific receptor R2, which bears a label directly or indirectly, is determined in the method a dissociation step is carried out, characterized in that in addition to the binding factor R3, which in this case does not have to have more than one binding site for the agent to be detected, a further specific immobilized or immobilizable receptor R4 is added, which has an affinity for R3 and the extent the binding of analyte A to the specific receptor R2, which bears a label directly or indirectly, is determined after reassociation.

Another object of the invention is a method for the immunological determination of one or more analytes by means of:
of a specific receptor R1, this specific receptor R1 being immobilized on a support and the extent of the binding of analyte A to the specific receptor R1 by means of a further specific receptor R2, which bears a label directly or indirectly, is determined in the method a dissociation step is carried out, characterized in that, in addition to the binding factor R3, which in this case does not have to have more than one binding site for the agent to be detected, the dissociating immune complexes, for example R3-A-R2, are transferred to a second solid phase, the one contains further specific immobilized or immobilizable receptor R4, which has an affinity for R3 and the extent of the binding of analyte A to the specific receptor R2, which bears a label directly or indirectly, is determined after reassociation.

For the purposes of the present invention, unmarked means that R3 either no label or at least not the label with  the extent of the binding of the analyte to the specific receptor is determined.

The procedures in which the immobilized specific receptors R4 can be used in all of their embodiments Known specialist. It is important that the inventive method in suitable form for all immunochemical processes in which the first step is an immunochemical or comparable bond of an analyte to a preferably immobilized, specific Receptor occurs and in a second, but not necessarily in time separate step, direct or indirect verification is used can be.

These methods are preferred in the sandwich sandwich Processes known to those skilled in the art are used, preferably as Labeling system an enzyme, preferably with a chromogenic or fluorogenic substrate or a luminescent label is used. However, the selected embodiment primarily affects the Not possible uses of the method according to the invention.

Microtitration plates are preferably used as solid phases, Magnetic particles, latex particles or matrix chemical test elements, such as e.g. B. fibers or membranes containing test modules used.

The person skilled in the art is aware that immunochemical processes, as above described for the simultaneous determination of different Analytes, for example HIV 1 (HIV = Human Immunodeficiency Virus), HIV 2, individually or in combination, or HIV 1 and / or HIV 2, combined with HCV (hepatitis C virus or hepatitis C virus antigen), can be used. Analytes in the above sense can both the viral antigens and the antibodies directed against them be. Such embodiments are hereby also included.  

Dissociation in the sense of the invention includes all chemical and physical methods that enable bioaffine interactions reversible to loosen or lower. Those skilled in the art are chemical Known substances in the immunochemical cleaning of Proteins and antibodies are used, for example Rhodanide, urea or guanidine. Also through immunological Methods such as competition with suitable immunological active substances, dissociation can be brought about. Furthermore, by using acidic or basic solutions Dissociation can be enforced. Physical methods of dissociation are also known to the person skilled in the art: for example Differences in temperature, microwaves or ultrasound.

Reassociation in the sense of the present invention means u. a. the Establishing non-dissociating conditions, for example through Neutralize, dilute or simply by undoing the previously prevailing dissociative conditions, such as eliminating the further above mentioned temperature differences or by switching off of the microwave or ultrasonic generator.

Receptors R3 in the sense of the invention are specific binding partners, the one or more than one bioaffine binding site to the analyte A exhibit. They can also interact with the receptor Enter R4. Binding partner or components of this receptor R3 can be conjugates of antibodies or antibodies themselves. This Antibodies or antibody fragments or conjugates thereof can against A or against A and R4. The R3 receptor can also consist of an antibody or antibody fragment, which is coupled with a structural element against which the Receptor R4 is directed.  

R4 receptors in the sense of the invention are specific binding partners, that have one or more bioaffine binding sites to R3. Receptors or components of this receptor can conjugates of Antibodies / antibody fragments or antibodies / fragments themselves. These antibodies / fragments or conjugates can be used against R3 be directed or have antigenic structures that of Binding sites of the receptor R3 are recognized. R4 can also out consist of a protein which is coupled to a structural element, against which R3 is directed.

The invention also relates to a reagent for use in above procedure.

Preferred combinations of receptors for the specific detection of antigens (analyte) are:

• R1: antibody directed against the analyte
• R2: Labeled antibody against the analyte is directed
• R3: Antibody directed against analyte A.

Preferred combinations of receptors for the specific detection of antigens (analyte) are:

• R1: Antibody directed against analyte A.
• R2: Labeled antibody against the analyte A is directed
• R3: Antibody directed against analyte A.
• R4: Antibody directed against the R3 antibody.

In the case of antigen detection, an antibody is preferably used or an antibody fragment used as receptor R3, which is not  recognizes the same epitope as the solid phase antibody R1 or the Conjugate antibody R2.

Preferred combinations of receptors for the detection of specific antibodies (analyte) are:

• R1: Antigen against which analyte A is directed
• R2: Labeled antigen against which analyte A is directed
• R3: Antibody directed against analyte A.

Preferred combinations of receptors for the detection of specific antibodies (analyte) are:

• R1: Antigen against which analyte A is directed
• R2: Labeled antigen against which analyte A is directed
• R3: Antibody directed against analyte A.
• R4: Antibody directed against the R3 antibody.

Particularly preferred combinations of receptors for the detection of specific antibodies (analyte) are:

• R1: Antigen against which analyte A is directed
• R2: Labeled antigen against which analyte A is directed
• R3: antibody directed against analyte A and carries additional non-antibody epitopes
• R4: Antibody against an additional non-antibody Epitope directed by antibody R3.

Methods for the preparation of such conjugates are known to the person skilled in the art which each from an antibody and an additional one not the body's own antigen (e.g. biotin or digoxigenin), known. Such conjugates can, while maintaining the bioaffine function their starting materials, for example by linking using chemical reagents or by bioaffine interaction will. Hybrid molecules can also be synthesized through chemical synthesis Hybridoma technology or genetic engineering methods are generated.

The reagent according to the invention can be used in a Variety of procedures in human and veterinary diagnostics. As examples should be listed two - or multi-stage tests for the detection of Antibodies of different immunoglobulin classes against structural features viruses (e.g. viruses of hepatitis A, B and C, as well as various HIV Types), of bacterial and parasitic pathogens and of allergic ones Diseases. Further examples are the detection of Pathogens such as viruses (e.g. hepatitis B virus), bacteria, parasites and allergens, as well as markers of diseases (e.g. Tumor markers) in multi-stage detection methods.

The present invention is further illustrated by the following Examples which, however, do not restrict the general teaching should mean and explained by the claims.  

example 1 1a) Preparation of the solid phase

Type B microtitration plates (from Nunc, Roskilde, Denmark) are also used 100 µl per well of coating solution (600 µg / l recombinant gp41 [Behringwerke AG, Marburg, FRG] and 10 mg / ml monoclonal antibody against biotin [Behringwerke AG, Marburg, FRG] in 50 mM Sodium carbonate buffer, pH 9.5) incubated for 24 hours at 4 ° C. The The microtitration plates are then deepened three times with 300 µl each Wash solution (50 mM Tris, 0.1% Tween 20, pH 7.2). The above Silica gel dried microtitration plates are approximately in the absence of air Stable for 1 year.

1b) Preparation of the conjugate

10 mg of HIV 1-gp41 peptide (IAF Biochem, Laval, Canada) in 1 ml Glacial acetic acid / water (50: 50, v / v) dissolved. After neutralization with 5N Sodium hydroxide solution is mixed with a 10-fold molar excess of N-γ- Maleimidobutyrylsuccinimid added and 1 hour at room temperature incubated. The unreacted heterobifunctional reagent is replaced by Gel filtration (Sephadex G-25) with 100 mM sodium phosphate, 5 mM Nitriloacetic acid, pH 6.0 separated.

10 mg horseradish peroxidase (Boehringer Mannheim, Mannheim, Germany) are in 10 ml of 10 mM sodium phosphate, 100 mM sodium chloride, pH 8.0 with 100-fold molar excess of 2-iminothiolane for 1 hour Incubated at room temperature. Then free modification reagent by gel filtration (Sephadex G-25) with 100 mM sodium phosphate, 5 mM Nitriloacetic acid, pH 6.0 removed.

Both eluates (SH-activated peroxidase and maleimide-modified HIV 1-peptide) are combined and incubated overnight at room temperature.  

After stopping with 1/10 vol 100 mM N-ethyl-maleimide the conjugate by gel filtration (Sephadex G-25) of unreacted HIV 1 peptide cleaned. After concentration (2 mg / ml) the conjugate is at -20 ° C stored.

1c) Production of the biotinylated antibody

10 mg monoclonal antibody against human immunoglobulin G [Behringwerke AG, Marburg, FRG] are 10 mM in 10 ml Sodium phosphate, 100 mM sodium chloride, pH 8.0 with 10 times molar Excess of N-hydroxysuccinimide-X-biotin in 10 mM sodium phosphate, 100 mM sodium chloride, pH 8.0 incubated for 1 hour at room temperature. Then free modification reagent is obtained by gel filtration (Sephadex G-25) with 100 mM Tris, 5 mM nitriloacetic acid, pH 7.0 away.

Example 2 Use of the reagent according to the invention 2a) Enzyme immunoassay for the detection of HIV 1 antibodies (Reference system I)

An enzyme immunoassay for the detection of anti-HIV 1 is carried out as follows:
25 μl of sample buffer (0.3 M Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) are incubated with 100 μl of human serum at 37 ° C. for 30 minutes in the wells of the microtitration plates produced according to Example 1a. After washing four times with 50 mM PBS, 0.1% Tween 20, 125 ul of the conjugate prepared according to Example 1b (1: 1000 in 0.1 M Tris / HCl, 1 mM glycine, 0.2% albumin, 0.4% Pluronic F64, 1 mg / l monoclonal Antibodies against human immunoglobulin G, pH 8.1) are pipetted in. The 30-minute incubation (+ 37 ° C) is ended with 4 further washing steps. The bound peroxidase activity, which correlates directly with the number of bound anti-HIV 1 antibodies, is determined by adding H ₂ O ₂ / tetramethylbenzidine. The substrate conversion is stopped after 30 minutes at room temperature by adding 0.5 M sulfuric acid. The absorbance is determined at 450 nm.

2b) Enzyme immunoassay for the detection of HIV 1 antibodies (System I according to the invention)

An enzyme immunoassay for the detection of anti-HIV 1 is carried out as follows:
25 μl of sample buffer (0.3 M Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) are incubated with 100 μl of human serum at 37 ° C. for 30 minutes in the wells of the microtitration plates produced according to Example 1a. After washing 4 times with 50 mM PBS, 0.1% Tween 20, 100 ul of the dissociation buffer (5 mM glycine 1 HCl, pH 2.5) are added for 30 min at RT. 25 μl of conjugates prepared according to Example 1b (1: 200 in 0.5 M Tris / HCl, 1% albumin, 2% Pluronic F64, 5 mg / l monoclonal antibody against human immunoglobulin G, pH 8.1) are then pipetted in without washing. The 30-minute incubation (+ 37 ° C) is ended with 4 further washing steps. The bound peroxidase activity, which correlates directly with the number of bound anti-HIV 1 antibodies, is determined by adding H ₂ O ₂ / tetramethylbenzidine. The substrate conversion is stopped after 30 minutes at room temperature by adding 0.5 M sulfuric acid. The absorbance is determined at 450 nm.

Anti-HIV 1 positive and anti-HIV 1 positive samples with unusual low reactivity and anti-HIV negative sera were found in both Reference system as well as in the enzyme immunoassay according to the invention examined.  

The results (extinction units) of the investigation can be found in the Table 1.

Table 1

There is significant difference in the signal formation of the two test systems to be recognized especially with low affinity samples (e.g. 9111/46). The Sensitivity to these samples is almost double the reference system I increased. High affinity samples for Coating antigen, which is already high in reference system I  Dilution can be detected, experienced in system I according to the invention no signal increase. Anti-HIV negative sera react in both Test systems comparable.

Example 3 Use of the reagent according to the invention 2a) Enzyme immunoassay for the detection of HIV 1 antibodies (Reference system II)

An enzyme immunoassay for the detection of anti-HIV 1 is carried out as follows:
25 μl of sample buffer (0.3 M Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) are incubated with 100 μl of human serum at 37 ° C. for 30 minutes in the wells of the microtitration plates produced according to Example 1a. After washing 4 times with 50 mM PBS, 0.1% Tween 20, 125 μl of the conjugate according to Example 1b (1: 1000 in 0.1 M Tris / HCl, 1 mM glycine, 0.2% albumin, 0.4% Pluronic F64, 1 mg / l according to the example 1c) pipetted in biotinylated monoclonal antibody against human immunoglobulin G, pH 8.1). The 30-minute incubation (+ 37 ° C) is ended with 4 further washing steps. The bound peroxidase activity, which correlates directly with the number of bound anti-HIV 1 antibodies, is determined by adding H ₂ O ₂ / tetramethylbenzidine. The substrate conversion is stopped after 30 minutes at room temperature by adding 0.5 M sulfuric acid. The absorbance is determined at 450 nm.

2b) Enzyme immunoassay for the detection of HIV 1 antibodies (System II according to the invention)

An enzyme immunoassay for the detection of anti-HIV 1 is carried out as follows:
25 μl of sample buffer (0.3 M Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) are incubated with 100 μl of human serum at 37 ° C. for 30 minutes in the wells of the microtitration plates produced according to Example 1a. After washing 4 times with 50 mM PBS, 0.1% Tween 20, 100 ul of the dissociation buffer (5 mM glycine / HCl, pH 2.5) are added for 30 min at RT. Then, without washing, 25 μl conjugates prepared according to Example 1b (1: 200 in 0.5 M Tris / HCl, 1% albumin, 2% Pluronic F64, 5 mg / l according to Example 1c) are biotinylated monoclonal antibodies against human immunoglobulin G, pH 8.1 ) pipetted in. The 30-minute incubation (+ 37 ° C) is ended with 4 further washing steps. The bound peroxidase activity, which correlates directly with the number of bound anti-HIV 1 antibodies, is determined by adding H ₂ O ₂ / tetramethylbenzidine. The substrate conversion is stopped after 30 minutes at room temperature by adding 0.5 M sulfuric acid. The absorbance is determined at 450 nm.

Anti-HIV 1 positive and anti-HIV 1 positive samples with unusual low reactivity and anti-HIV negative sera were found in both Reference system as well as in the enzyme immunoassay according to the invention examined.

The results (extinction units) of the investigation can be found in the Table 2.  

Table 2

There is significant difference in the signal formation of the two test systems to be recognized especially with low affinity samples (e.g. 9111/39). The Sensitivity to these samples is doubled over that Reference system II increased. High affinity samples for Coating antigen, which is already in high use in reference system II Dilution can be detected, experienced in System II according to the invention another signal increase. Anti-HIV negative sera react comparable to both test systems.

Claims (11)

1. A method for determining an analyte, wherein

• (1) a sample optionally containing the analyte to be determined is brought into contact with a receptor R1 immobilized on a solid phase, which has a binding affinity for analyte A, so that an immune complex R1-A is formed;
• (2) one or more washing steps are carried out;
• (3) a labeled receptor R2, which has at least one binding site with binding affinity for analyte A and an unlabeled receptor R3, which has more than one binding site with binding affinity for analyte A, are added, the addition of receptors R2 and R3 in any Sequence can take place, and initially dissociating conditions that lead to the cleavage of immune complexes and then non-dissociating conditions that allow the reassociation and new formation of immune complexes are brought about, so that immobilized immune complexes containing A, R1, R2 and R3 are formed;
• (4) the amount of the label immobilized on the solid phase, which is a measure of the amount of the analyte immobilized on the solid phase, is appropriately detected.

2. The method according to claim 1, wherein (1) on the solid phase in addition, a receptor R4 is immobilized, which at least has a binding site with binding affinity for the receptor R3, wherein (2) the receptor R3 has at least one binding site Has affinity for analyte A and wherein (3) immune complexes containing on the one hand A, R2 and R3 and on the other hand R1 and / or R4 arise. 3. A method for determining an analyte, wherein

• (1) a sample optionally containing the analyte to be determined is brought into contact with a receptor R1 immobilized on a first solid phase, which has a binding affinity for analyte A, so that an immune complex R1-A is formed;
• (2) one or more washing steps are carried out;
• (3) a dissociation step is carried out which leads to the cleavage of the immune complex R1-A;
• (4) a labeled receptor R2, which has at least one binding site with binding affinity for analyte A and an unlabeled receptor R3, which has at least one binding site with binding affinity for analyte A, are added, the addition of receptors R2 and R3 in any order can take place and whereby non-dissociating conditions which allow the new formation of immune complexes are brought about and wherein the analyte A and the receptors R2 and R3 are brought into contact with a second solid phase on which a receptor R4 is immobilized which has at least one binding site with Binding affinity for the receptor R3, so that immobilized immune complexes containing A, R2, R3 and R4 arise and
• (5) the amount of the label immobilized on the solid phase, which is a measure of the amount of the analyte immobilized on the solid phase, is appropriately detected.

4. The method according to claim 1, wherein R1 and R3 each against the Antibodies directed to analytes or antibody fragments are and R2 is a labeled antibody directed against the analyte or a labeled antibody fragment directed against the analyte is. 5. The method according to claim 2 or 3, wherein R1 and R3 each antibodies directed against the analyte or Antibody fragments are, R2 a labeled, against the analyte directed antibody or labeled against the analyte directed antibody fragment and R4 a directed against R3 Antibody or antibody fragment directed against R3. 6. The method of claim 5, wherein R3 recognizes another epitope as R1 or R2. 7. The method of claim 1, wherein the analyte is an antibody, R1 an antigen against which the analyte is directed, R2 a labeled one Antigen against which the analyte is directed and R3 against the Antibody directed against or against the analyte is directed antibody fragment. 8. The method of claim 2 or 3, wherein the analyte Antibody, R1 an antigen against which the analyte is directed, R2  a labeled antigen against which the analyte is directed, R3 antibody directed against the analyte or against the Analyte-directed antibody fragment and R4 an against R3 directed antibody or directed against R3 Antibody fragment is. 9. The method of claim 8, wherein R3 is not an additional one the body's own epitope and R4 against the additional does not antibody's epitope is directed by R3. 10. The method according to one or more of the preceding claims for the determination of antibodies against HIV. 11. Diagnostic agent for performing the method according to one or several of claims 1 to 10.