Immunoassay using at least two pegylated analyte-specific binding agents

Abstract

The disclosure concerns a method and kits for measurement of an analyte in a microparticle-based analyte-specific binding assay. In the assay, the microparticles are coated with the first partner of a binding pair, mixing the coated microparticles and at least two analyte-specific binding agents, each conjugated to the second partner of the binding pair, and a sample suspected of containing the analyte. The second partner of the binding pair is bound to each of the analyte-specific binding agents via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the conjugated analyte-specific binding agents to the coated microparticles. The method also entails separating the microparticles having the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and measuring the analyte bound to the microparticles.

Claims

1. A method for measurement of an analyte in a microparticle-based analyte-specific binding assay, wherein said microparticles are coated with the first partner of a binding pair, said method comprising a) mixing the coated microparticles, at least two analyte-specific binding agents, wherein each of said analyte-specific binding agents is bound to a second partner of the binding pair, and a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to each of said analyte-specific binding agents via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the said analyte-specific binding agents to the coated microparticles, b) separating the microparticles comprising the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and c) measuring the analyte bound to the microparticles.

2. The method of claim 1, wherein said measuring of the analyte bound to the microparticles is based on use of an electrochemiluminescent label.

3. The method of claim 1, wherein said analyte comprises several variants.

4. The method of claim 1, wherein said several variants are different genotypes, isoenzymes, isoforms, serotypes or mutants of said analyte.

5. The method of claim 1, wherein said analyte is an antigen of an infectious agent.

6. The method of claim 1, wherein said analyte is a viral antigen.

7. The method of claim 1 wherein said analyte is a hepatitis virus antigen or a human retroviral antigen.

8. The method of claim 1, wherein said analyte is a hepatitis C virus or a hepatitis B virus or an HIV antigen.

9. The method of claim 1, wherein said analyte is hepatitis C virus core antigen.

10. The method of claim 1, wherein one of said at least two analyte-specific binding agents is an antibody binding to an epitope within the amino acid positions 140 to 172 of SEQ ID NO:1 and one of said at least two analyte-specific binding agents is an antibody binding to an epitope within the amino acid positions 20 to 80 of SEQ ID NO: 1.

11. A kit comprising in separate containers or in separated compartments of a single container unit at least microparticles coated with a first partner of a binding pair and at least two analyte-specific binding agents, each bound to the second partner of said binding pair, wherein said second partner of the binding pair is bound to each of said analyte-specific binding agents via a linker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30).

12. The kit of claim 11, wherein said first partner of a binding pair is avidin or streptavidin, and wherein said second partner of said binding pair is selected from biotin or biotin analogues.

13. The kit of claim 11, wherein said at least two analyte-specific binding agents are viral antigen-specific binding agents.

14. The kit of claim 13 wherein one of said at least two viral antigen-specific binding agents is an antibody binding to an epitope within the amino acid positions 140 to 172 of SEQ ID NO:1.

15. The kit of claim 11, further comprising in a separate container or in a separated compartment of a single container unit a further analyte-specific binding agent which is detectably labeled wherein said further analyte-specific binding agent which is detectably labeled is an antibody binding to an epitope within amino acid positions 100-120 of SEQ ID NO:1.

Description

EXAMPLES

Example 1: Methods

(1) Monoclonal Antibodies

(2) Recombinant HCV core antigen needed for immunization of appropriate animals was obtained using standard techniques known in the art by inserting a DNA fragment encoding the desired antigen amino acid sequence into an E. coli expression plasmid followed by overexpression and purification of the protein. These standard molecular biological methods are described for example in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

(3) Murine or rabbit monoclonal antibodies against HCV core were prepared by standard hybridoma technology or by recombinant nucleic acid techniques as known to the skilled person and in an analogous manner described e.g. in WO2016/091755, respectively.

(4) Monoclonal antibodies against HCV core protein used as capture compounds in the examples below bind to the epitopes aa 157-169 or aa 32-36 or aa 37-46 or aa 65-71 of the HCV core protein (SEQ ID NO:1). Very similar epitopes and their determination have already been disclosed in EP1308507. As detection compound, a monoclonal antibody was chosen capable of binding to the core epitope aa 102-112, an epitope related to epitopes also described in EP0967484 and EP1308507. For the immunization of mice and rabbits, HCV core antigenic sequences of genotype 1a according to Genbank Acc. No: P26664.3 GI:130455 (SEQ ID NO:1), which discloses the complete polyprotein encoded by HCV genotype 1 were used.

(5) In particular, peptides either from amino acid 110-171 as recombinant fusion protein with Escherichia coli SlyD following the procedure disclosed in WO 03/000878 A2, US 2009/0291892 A1, WO 2013/107633 A1 or peptides from amino acid 2-169 as recombinant protein following the procedure described by Boulant, S. et al., 2005, J. Virol. 79:11353-11365 were used for immunization. In an additional approach multiple peptides either from amino acid 82-117 or 9-48 coupled to KLH (keyhole limpet hemocyanin) were used for immunization according to known methods.

(6) Protein Determination

(7) The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide or using the colorimetric BCA method.

Example 2: Synthesis of Activated Biotinylation Reagents

(8) The synthesis of state-of-the-art activated biotin-comprising linkers (biotinylation reagents) like the widely used linker Biotin-DDS is disclosed in EP 632 810.

(9) Biotin-PEGn-NHS-biotinylation reagents (CAS-Nr. 365441-71-0; n=number of ethylene oxide units) were either obtained from IRIS Biotech GmbH or synthesized in house.

(10) In the de novo synthesis the control of discrete number of ethylene oxide units was ensured by the stepwise elongation of shorter PEGs, such as tetraethylene glycol, following the described method from Chen and Baker, J. Org. Chem. 1999, 64, 6840-6873.

(11) In a first step bis-trityl-PEG.sub.n 1 has been obtained (as obvious, n represents the number of ethylene glycol units).

(12) Deprotection of 1 was carried out by stirring in 1M HCl in dioxane for 1 h at room temperature. After evaporation the residue was refluxed in methanol until a clear solution was obtained and the flask was kept at 4° C. overnight. After filtration the solution was extracted with hexane, the methanolic layer evaporated and dried to give the corresponding PEG.sub.n-diol 2 as oil or wax (consistency depending on length/number of units (n) of the PEG).

(13) Next the introduction of the acid function was carried out by sodium catalyzed addition of PEG.sub.n-diol to tert-butyl acrylate according to Seitz and Kunz, J. Org. Chem. 1997, 62, 813-826. This way compound 3 is obtained.

(14) To a solution of HO-PEG.sub.n-COOtBu 3 (1 equivalent) and triethylamine (2.5 equivalents) in methylene chloride methylsulfonyl chloride (2 equivalents) was added drop-wise at 0° C. After stirring for 1 h aqueous work-up and evaporation followed.

(15) The mesylate 4 (1 equivalent) was directly reacted with NaN.sub.3 (2 equivalents) by stirring in dimethylformamide at room temperature for two days. After removal of the solids and dimethylformamide, aqueous work-up with diethylether and Na.sub.2CO.sub.3 followed.

(16) The crude product 5 was purified by column chromatography on silica gel in ethyl acetate/methanol 15/1. Reduction of the azide 5 (1 equivalent) was performed by stirring for 24 h with triphenylphosphine (1.1 equivalents) in tetrahydrofurane/water 4/1 at room temperature. After evaporation the residue was suspended in water and washed with ethyl acetate several times. The water layer was evaporated and dried to give the amine 6 as colorless oil.

(17) Cleavage of the tert-butyl ester was carried out with 5% trifluoroacetic acid in water. Amino-PEG.sub.n-acid 7 was obtained by evaporation with water for several times.

(18) Biotin was introduced by coupling with corresponding N-hydroxysuccinimide ester 8 (1.05 equivalents) with triethylamine (4 equivalents) in dimethylformamide at room temperature overnight.

(19) After evaporation crude product 9 was purified by RP-HPLC in acetonitrile/water.

(20) Finally, N-hydroxysuccinimide ester 10 was formed by reaction with N-hydroxysuccinimide (1.1 equivalents) and ethyl dimethylaminopropyl carbodiimide (1.1 equivalents) in methylene chloride. After completion of the reaction, the reaction mixture was diluted with methylene chloride and washed with water. Evaporation and drying led to pure Biotin-PEG.sub.n-NHS 10 as oil, wax or solid, respectively depending on the number of ethylene oxide units.

(21) ##STR00002## ##STR00003##

Example 3: Labeling of Antibodies

(22) Coupling of biotin and ruthenium moieties, respectively, to antibodies:

(23) Antibodies were obtained and purified according to state-of-the art procedures that are fully familiar to a person skilled in the art.

(24) Prior to labeling, the detection antibody was cleaved by pepsin to obtain a F(ab′).sub.2 fragment and to eliminate the interference prone Fc fragment (the method is described by A. Johnstone and R. Thorpe in Immunochemistry in Practice, Blackwell Scientific 1987). The purified F(ab′).sub.2 fragment was further polymerized with the homobifunctional crosslinker disuccinimidyl suberate (DSS) and applied to a 5400 gel filtration chromatography to gather the optimal size range of the F(ab′).sub.2 polymer (the principle is described in DE3640412).

(25) For the attachment of the respective label, in general the lysine ε-amino groups of the antibodies were targeted by N-hydroxy-succinimide activated compounds. At a protein concentration of 10 mg/ml, antibodies were reacted with N-hydroxy-succinimide activated biotinylation reagents (Biotin-DDS or Biotin-PEG24-NHS) and N-hydroxy-succinimide activated ruthenium labeling reagents, respectively. The label/protein ratio of biotinylation or ruthenium labeling reagent was 5-6:1 or 15:1, respectively. The reaction buffer was 50 mM potassium phosphate (pH 8.5), 150 mM KCl. The reaction was carried out at room temperature for 15 minutes and stopped by adding L-lysine to a final concentration of 10 mM. To avoid hydrolytic inactivation of the labels, the respective stock solutions were prepared in dried DMSO (Sigma-Aldrich, Germany). After the coupling reaction, unreacted free biotin or ruthenium label was removed by passing the crude antibody conjugate through a gel filtration column (Superdex 200 HI Load) or by dialysis.

Example 4: Prototype Elecsys HCV Core Antigen Assay

(26) Measurements of an Elecsys HCV core antigen prototype assay were carried out in a sandwich assay format on an automated Cobas® e601 analyzer (Roche Diagnostics GmbH). Signal detection in this analyzer is based on electrochemiluminescence. In this sandwich assay the one or more capture antibody-biotin-conjugates (i.e. analyte-specific binding agents) is/are immobilized on the surface of a streptavidin-coated magnetic bead. The detection-antibody (further analyte-specific binding agent) bears a complexed ruthenium cation as the signaling moiety. In the presence of analyte, the ruthenium complex is bridged to the solid phase and emits light at 620 nm after excitation at the platinum electrode comprised in the measuring cell of the analyzer. The signal output is in arbitrary light units. Measurements were performed with HCV core antigen positive and negative human serum and plasma samples purchased from several sources.

(27) The experimental HCV core antigen assay was conducted as follows. 50 μl of normal human serum of HCV antigen positive sample and 25 μl of a detergent containing pretreatment reagent (PT: 0.25 M KOH, 1.125 M KCl, 1.5% hexadecyltrimethylammoniumchloride (HTAC), 0.75% octylglycoside) were incubated together for 9 minutes to release the antigen followed by the addition of 35 μl of 2 μg/ml of the respective capture antibody-biotin conjugate or a mixture of two different antibody-biotin conjugates (1 μg/ml each) and 40 μl of 1 μg/ml detection antibody ruthenium label conjugate in the same assay buffer R1 and R2 (200 mM potassium phosphate, pH 6.5, 225 mM KCl, 0.5% sodium taurodeoxycholate, 0.3% zwittergent 3-14, 0.1% oxypyrion, 0.01% methylisothiazolinone, 0.2% bovine serum albumin, 0.2% bovine IgG, 50 μg/ml MAK33-IgG1, 50 μg/ml MAK33-F(ab′).sub.2-Poly, 50 μg/ml MAK IgG2b/Fab2a-Poly). After additional 9 minutes incubation time 50 μl streptavidin-coated paramagnetic microparticles were added and incubated for further 9 minutes. Afterwards, the HCV core antigen was detected (via the electrochemiluminescent signal generated in these experiments).

(28) The data in Table 1 and 2 show the actual counts measured in HCV antigen positive samples and in a normal (negative) sample, as well as the recovery relative to the reference for capture antibodies conjugated with the short biotin label Biotin-DDS (Table 1, state of art) and with the long biotin label Biotin-PEG24-NHS (Table 2, invention), respectively.

(29) In this setup the cutoff (decision point above which a sample is regarded as reactive or positive and below which a sample is classified as non-reactive or negative) for all measurements was calculated as three times the signal of a negative (normal) sample in the same experimental setup. For example, if the background signal of a normal (negative) sample shows around 700 counts the cutoff is set at about 2100 counts. As a consequence, all samples showing more counts than 2100 are classified as reactive.

(30) The capture antibody recognizing the HCV core antigen epitope aa 157-169 generates the highest specific signals and was chosen as reference. As can be seen from Table 2, by combining any other capture antibody with the reference antibody to a total concentration of 2 μg/ml (in R1) the signal level is very close to that generated by the reference antibody alone at 2 μg/ml as long as the long biotin label Biotin-PEG24-NHS is used according to the invention.

(31) In contrast to this and as can be seen from Table 1, analogous mixtures of capture antibodies conjugated with the short biotin label Biotin-DDS known in the state of art exhibit a strong signal decline relative to the reference antibody used alone. The signal recovery relative to the reference is—depending on the respective antibody combinations—only around 40% or maximum 75%, whereas the recovery using the long linker biotin label according to the invention is at least 92%, mean values 91%, 95% and 96%, respectively (Table 2). This might indicate a stronger competition of biotinylated capture antibodies with the streptavin-coated solid phase conjugated with short biotin labels than for longer ones.

(32) In order to assess the reliable recognition of different genotypes of HCV core antigen the prototype Elecsys HCV core antigen assay was performed using one or two capture antibodies conjugated with the long biotin label Biotin-PEG24-NHS. The antibody concentration of each antibody in R1 was further optimized for the mixture variant. Seroconversion panel of HCV genotype 1 and 3 were purchased from ZeptoMetrix Corporation. The data in Table 3 clearly exhibit the advantage of a mixture of capture antibodies over the single antibody variant. Using two antibodies binding to the HCV core antigen (invention) linked to the long biotin label Biotin-PEG24-NHS instead of one antibody, the signal increases for most seroconversion samples so that the assay reliably detects HCV genotype 1 and 3. The approach according to the current invention of using at least two analyte-specific binding agents conjugated to the long biotin label leads to a higher signal and thus to an improved sensitivity, in particular to an improved assay sensitivity for HCV core antigen detection.

(33) TABLE-US-00002 TABLE 1 Prior art linker Biotin DDS for analyte-specific binding agents, HCV core antigen detection Conjugation with Biotin- DDS (reference) HCV core antigen epitope 157-169 157-169 157-169 157-169 of mAb 65-71 65-71 32-36 32-36 37-46 37-46 concentration of mAb in 2 μg/ml 2 μg/ml 2 μg/ml 2 μg/ml 1 μg/ml 1 μg/ml 1 μg/ml R1 each each each recovery recovery recovery relative relative relative to to to sample ID counts counts counts counts counts reference counts reference counts reference normal sample #960064560 1′019 660 956 516 856 1′082 630 HCV antigen positive #217293 64′294 22′516 19′087 6′812 49′536 77% 50′000 78% 26′018 40% samples #205104 479′104 183′722 150′828 47′726 335′293 70% 370′837 77% 203′624 43% #205085 1′322′663 499′147 367′297 128′315 861′295 65% 896′309 68% 491′133 37% #205081 68′063 28′008 26′054 8′910 53′318 78% 54′913 81% 29′032 43% #9174627 85′874 31′323 27′807 10′824 58′827 69% 61′679 72% 36′095 42% mean 72% 75% 41%

(34) TABLE-US-00003 TABLE 2 Long PEG linker (invention) for analyte-specific binding agents, HCV core antigen detection Conjugation with Biotin-PEG24-NHS (reference) HCV core antigen 157-169 157-169 157-169 157-169 epitope of mAb 65-71 65-71 32-36 32-36 37-46 37-46 concentration 2 μg/ml 2 μg/ml 2 μg/ml 2 μg/ml 1 μg/ml 1 μg/ml 1 μg/ml of mAb in R1 each each each recovery recovery recovery relative relative relative to to to sample ID counts counts counts counts counts reference counts reference counts reference normal sample #960064560 1′080 1′103 970 960 1′083 1′040 1′143 HCV antigen #217293 131′763 62′704 64′032 35′384 123′366 94% 122′084 93% 122′477 93% positive samples #205104 892′491 594′608 481′780 288′334 863′767 97% 771′143 86% 832′111 93% #205085 127′615 79′954 71′631 43′148 128′918 101% 117′921 92% 121′642 95% #205081 2′018′050 1′427′999 1′100′971 725′673 1′947′496 97% 1′784′309 88% 1′910′317 95% #9174627 139′911 77′536 81′413 57′186 128′076 92% 132′503 95% 137′170 98% mean 96% 91% 95%

(35) TABLE-US-00004 TABLE 3 HCV Seroconversion panels HCV core antigen epitope of mAb 157-169 157-169 65-71 concentration of mAb in R1 2 μg/ml 1.25 μg/ml 0.75 μg/ml counts counts normal sample   649   698 #960064560 seroconversion HCV panel genotype 10003_6 3   764 1′290 10003_7 4′008 30′745  10003_11 1′506 9′062 10003_12 1′582 7′277 10010_1 1 5′321 3′876 10010_3   904 1′296 10030_7 1 1′096 1′275 10030_8 4′101 5′987 10032_4 3   720   954 10032_5 1′293 11′724  10032_10   853 3′189 10032_13   797 2′992 10032_15   755 2′221