Immunocomplex antibodies and non-competitive immunoassay for the detection of testosterone

Abstract

This disclosure relates to anti-immunocomplex antibodies specific for an immunocomplex formed between testosterone and an anti-testosterone primary antibody, as well as a method for the preparation thereof. The disclosure also relates to non-competitive immunoassays and immuno- assay kits for detecting or quantifying testosterone.

Description

[0001] IMMUNOCOMPLEX ANTIBODIES AND NON-COMPETITIVE IMMUNOAS¬

[0002] SAY FOR THE DETECTION OF TESTOSTERONE

[0003] FIELD OF THE INVENTION

[0004] This disclosure relates to anti-immunocomplex antibodies specific for an immunocomplex formed between testosterone and an anti-testosterone primary antibody, as well as a method for the preparation thereof . The disclosure also relates to non-competitive immunoassays and immunoassay kits for detecting or quantifying testosterone .

[0005] BACKGROUND OF THE INVENTION

[0006] Substantial advancements have been made in understanding the relevance of steroid hormones in both health and disease , especially regarding testosterone . Initially, steroid hormones were thought to solely regulate the metabolism, immune function, and reproductive physiology . However, their broader significance now extends to various diseases and chronic conditions , such as polycystic ovary syndrome , hypogonadism, osteoporosis , cancer, diabetes , and cardiovascular disease . Even minor hormonal abnormalities can significantly impact health and disease risk . Thus , precise detection and measurement of hormone levels , even at low concentrations , is essential . For this purpose , immunoassays offer a rapid, straightforward, and high-throughput method in which the performance i s influenced by assay format and the selectivity of the antibody . While non-competitive sandwich enzyme-linked immunosorbent assay (ELI SA) is effective for larger biomolecules , steroid hormones and other low-molecular weight haptens lack available sufficient epitopes for this method . Competitive ELI SA, in turn, although standard, does not offer enough sensitivity and reproducibility to measure haptens . Given the demand for high-performance assays , novel non-competitive immunoassay concepts , especially for detecting testosterone , are needed .

[0007] JP 2023060976 discloses an anti-immunocomplex ( anti- IC) antibody that can specifically bind to an immunocomplex formed between testosterone and an antitestosterone primary antibody . Based on the disclosed data, the half maximal effective concentration (EC50 ) for quantifying testosterone , using the anti- IC antibody, is ca . 3000 pM . The higher the EC50 value , the lower the detection sensitivity . Notably, no data on cross-reactivity of the anti- IC antibody towards free testosterone or to the anti-testosterone antibody as such is given . The anti- IC antibody was obtained by a conventional animal immuni zation procedure .

[0008] There is a need for more sensitive anti- IC antibodies for the detection of testosterone . Ideally, the anti- IC antibody shows specific binding only towards an immunocomplex between testosterone and an anti-testosterone antibody, but not towards the components of the immunocomplex individually, enabling selective detection of the analyte in a non-competitive immunoassay .

[0009] SUMMARY

[0010] The present disclosure relates to an anti-immunocomplex ( anti- IC) antibody specific for an immunocomplex formed between testosterone and an anti-testosterone primary antibody as set forth in independent claim 1 , and to a method for the preparation of the anti- IC antibody as set forth in independent claim 9 . The present disclosure also relates to a non-competitive immunoassay for detecting testosterone in a sample as set forth in independent claim 12 , and to an immunoassay kit for detecting or quantifying testosterone as set forth in independent claim 16 .

[0011] Further aspects , embodiments and details are set forth in the following figures , detailed description, examples , and dependent claims . BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings , which are included to provide a further understanding of the invention and constitute a part of this specification, illustrate embodiments of the invention and together with the description help to explain the principles of the invention . In the drawings :

[0013] Figure 1 is a schematic representation of a method for producing the present anti- IC antibodies .

[0014] Figure 2 is a visual presentation of panning rounds and the path to the testosterone anti- IC Fab according an embodiment described in the Examples . The circled node expresses which fraction produced the exemplified anti- IC antibody .

[0015] Figure 3 shows the immunoreactivity of the phage pools after each panning round . The binding was distinguished between specific signal differences of the bio-Anti-T with and without testosterone in two concentrations ( 5 and 500 nM) . The background ( only primary capture bio-Anti-T ) is subtracted from the signal values .

[0016] Figure 4 shows the primary screening results of anti- IC phages . The screening was done in 96-well plates , where the signal from wells with the IC ( 1 pM testosterone ) was compared with background wells with only the primary capture bio-Anti-T and no testosterone . The heat map shows the signal-to-background ratio for absorbance measured at 405 nm . Wells marked with "X" represent empty cells . Clones B7 , E 6 , F6 , G4 , and G9 were chosen for secondary screening .

[0017] Figure 5 shows the secondary screening results of selected Fab phage clones . Five of the most promi sing clones were analyzed for their affinity to the IC using an anti-phage ELI SA with 10 , 100 and 1000 nM testosterone . The background (wells with no testosterone ) is subtracted from the s ignal values . The error bars represent the standard deviation of three replicate measurements . The signal is measured as europium signal in counts per second (CPS ) .

[0018] Figure 6 shows the screening results of anti- IC Fabs after production as ALP-fusion Fabs . Four of the best-performing testosterone clones from the secondary screening were produced as Fabs and analyzed with an ALP-ELI SA using three different concentrations of testosterone ( 10 , 100 , and 1000 nM) . The results are shown for the best two clones . The background (well s with no testosterone ) is subtracted from the signal values . The error bars represent the standard deviation of three replicate measurements . The signal is measured as absorbance at 405 nm .

[0019] Figure 7 shows the half-maximal effective concentration (EC50 ) of testosterone for clone G9 in an ALP-ELI SA . A standard curve for the detection was done based on 15 concentration points of testosterone ranging between 5 and 50000 pM. The error bars represent the standard deviation of three replicate measurements .

[0020] Figure 8 shows the results of a binding comparison of G9 to two different testosterone-specific primary antibodies : the IC used in the panning (Anti-T) and an IC with an anti-testosterone antibody from another manufacturer ( 77_Fab) .

[0021] Figure 9A illustrates the performance of a TR- F assay by showing the determination of the EC50 value of testosterone for clone G9 . A standard curve for testosterone detection was made with 12 concentration points of testosterone between 0 and 25000 pM . The error bars represent the SD of three replicate measurements .

[0022] Figure 9B illustrates the performance of a TR- F as say by showing the limit of detection (LoD) calculated based on the mean of the background + 3 x SD and the limit of quantification (LoQ) calculated based on the mean of the background + 10 x SD .

[0023] Figure 10A illustrates the matrix effect of the TR-F immunoassay . The effect of increasing plasma concentrations ( 0-40 % ) on assay performance was evaluated using three testosterone concentrations ( 100 , 500 , and 1000 pM) . Signal s from wells containing no testosterone were subtracted from those with testosterone to account for background effects caused by endogenous testosterone in the samples . The error bars represent the SD of the measured signal .

[0024] Figure 10B corresponds to Figure 10A with the exception that increasing serum concentrations were used .

[0025] Figure 11 demonstrates the measurements of circulatory free testosterone directly from plasma . The ability of the TR-F immunoassay to qualitatively discern between male and female samples by measuring plasma samples diluted to 20 % . The error bars represent the SD of three replicate measurements .

[0026] DEFINITIONS

[0027] Before the invention is described, it is to be understood that this disclosure is not strictly limited to any particular compositions , reagents , devices , protocols or methodology described herein, as such may vary . It is al so to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims .

[0028] It is also to be noted that , unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs .

[0029] It is further to be noted that certain features of the disclosure, which are , for clarity, described in the context of separate embodiments , can also be provided in combination in a single embodiment . Conversely, various features of the disclosure , which are , for brevity, described in the context of a single embodiment , can also be provided separately or in any suitable sub-combination .

[0030] As used herein and in the appended claims , the singular forms "a" , "an" and "the" mean one or more . Thus , a singular noun, unless otherwise specified, carries also the meaning of the corresponding plural noun, and vice versa . As such, the terms "a" , "an" , "one or more" and "at least one" can be used interchangeably .

[0031] The term "and / or" in a phase such as "X and / or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning .

[0032] The terms "comprising" , "including" and "having" can be used interchangeably .

[0033] As used herein, the term "antibody" refers to the structure of immunoglobulin G comprising two identical heavy chains ( ~50 - 60 kDa) and two identical light chains ( ~23 kDa) made up of globular structural motifs called Ig-fold domains . In the heavy chain, there are three constant Ig-fold domains (CHI , CH2 , and CH3 ) and one variable Ig-fold domain (VH) , whereas the light chains consist of one constant (CL) and one variable (VL) Ig-fold domain . Each domain consi sts of two antiparallel [3-sheets that are formed of seven-to-nine antiparallel [3-strands connected by loops . The Ig-fold domain is stabili zed by a highly conserved intradomain disulfide bridge formed between cysteine residues in the two anti-parallel [3-sheets . Typically, two or four covalent disulfide bridges in the hinge region located between CHI and CH2 connects the two heavy chains , whi le the heavy and light chains are connected through a covalent disulfide bridge linking the CHI and CL . As used herein, the terms "VH" and "VL" are interchangeable with the terms "VH domain" and "VL domain" , respectively .

[0034] As used herein, the term "complementary-determining region" (CDR) refers to highly variable regions in the variable domains of an antibody . There are three CDRs in each variable domain : CDR-L1 , CDR-L2 , and CRD- L3 in the VL domain, and CDR-H1 , CDR-H2 , and CDR-H3 in the VH domain . All the CDRs are collectively involved in antigen recognition and binding . However, the CDR-H3 is generally considered as the most important CDR engaged in antigen-binding as it i s the most variable in loop length and sequence . As used herein, the terms "CDR" and "CDR loop" are interchangeable .

[0035] All numbering of amino acid positions within variable domains as used herein, as well as identification of CDRs , is in accordance with the Rabat numbering scheme unless otherwise specified . The Rabat numbering scheme is well known to those skilled in the art .

[0036] As used herein, the term "framework" ( FR) refers to a non-CDR portion of a variable domain ( [3-sheets and non-hypervariable loops ) . It provides structural support to the antigen-binding site but also affects the CDR loop conformations . In the primary structure of an antibody ( i . e . , its linear amino acid sequence ) , the term "framework" refers to amino acid sequences interposed between the CDRs . Accordingly, there are four framework regions in each variable domain : FR-L1 , FR- L2 , FR-L3 and FR-L4 in the VL domain, and FR-H1 , FR-H2 , FR-H3 and FR-H4 in the VH domain .

[0037] As used herein, the term "conservative sequence variation" refers to an altered amino acid sequence comprising modifications that do not significantly alter the structural or functional properties of the antibody in question as compared to the non-altered, original amino acid sequence . Conservative amino acid sequence variations include variations arising from amino acid substitutions with similar amino acids . As is well known in the art , said similarity may be determined on the basis of similarity in polarity, charge , solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved . For example , nonpolar (hydrophobic) amino acids include alanine (Ala, A) , leucine (Leu, L) , isoleucine (He, I) , valine (Vai, V) , proline (Pro, P) , phenylalanine (Phe, F) , tryptophan (Trp, W) , and methionine (Met, M) ; polar neutral amino acids include glycine (Gly, G) , serine (Ser, S) , threonine (Thr, T) , cysteine (Cys, C) , tyrosine (Tyr, Y) , asparagine (Asn, N) , and glutamine (Gin, Q) ; positively charged (basic) amino acids include arginine (Arg, R) , lysine (Lys, K) , and histidine (His, H) ; and negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E) . Conservative amino acid sequence variations also include variations comprising small amino acid deletions and / or insertions. Preferably, the conservative sequence variations encompassed by the invention can be denoted as "functionally equivalent conservative sequence variations". Those skilled in the art can easily determine whether or not given sequence variations are functionally equivalent or not.

[0038] Antibodies can exist as intact immunoglobulins (i.e., as full-length IgG antibodies) or as any of a number of well-characterized antigen-binding fragments or single-chain variants (scFv) thereof, all of which are herein encompassed by the term "antibody". Said fragments and variants may be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins as is well known in the art.

[0039] As used herein, the term "fragment variable" (Fv) refers to an antibody fragment composed of VL and VH domains .

[0040] As used herein, the term "disulfide-stabilized Fv fragment" (dsFv) refers to an antibody fragment composed of VL and VH domains that are connected to each other through a disulfide bridge.

[0041] As used herein, the term "Fab fragment" refers to a monomeric antigen-binding fragment of an antibody that consists of the VL, CL, VH, and CHI domains. As used herein, the term "Fab ' fragment" refers to an antigen-binding fragment of an antibody that is essentially a Fab fragment with part of the hinge region .

[0042] As used herein, the term "single-chain fragment variable" ( scFv) refers to a recombinant Fv fragment composed of the VL and VH domains connected by a flexible peptide linker, which stabili zes the structure . The VL and VH domains may be connected in either orientation (VL-linker-VH or VH-linker-VL) .

[0043] As used herein, the term "disulfide-stabili zed single-chain fragment variable" (ds-scFv) refers to a recombinant Fv fragment composed of the VL and VH domains connected by a flexible peptide l inker in either orientation (VL-linker-VH or VH-linker-VL) and further stabili zed by an artificial interdomain disulfide bridge .

[0044] As used herein, the term "anti- IC antibody" ( anti- IC antibody) refers to an antibody that specifically binds to an immunocomplex between a primary antibody and its antigen target but does not to a significant extent bind the primary antibody or the antigen target alone . The anti- IC antibody may also be called a secondary antibody . In the context of the present disclosure , the antigen target is testosterone .

[0045] As used herein, the term "primary antibody" refers to an antibody that specifically binds to an antigen target of interest . In the context of the present disclosure , the antigen target of interest is testosterone , the primary antibody thus being an anti-tes- tosterone antibody .

[0046] As used herein, the term "anti-testosterone antibody" refers to a primary antibody that specifically binds to testosterone . The term includes commercially available anti-testosterone antibodies , such as Anti- Testosterone 3201 SPTN-5 (denoted as anti-T in the present Examples ) provided by Medix Biochemica, and Ab00418-l.l Anti-Testosterone [77 Fab] (denoted as 77_Fab in the present Examples) available from Absolute Antibody, and any modifications, variants and fragments thereof provided that their binding specificities remain substantially unaltered. Suitable anti-testosterone antibodies are also disclosed in the detailed description, and further anti-testosterone may be obtained by methods and means known to those skilled in the art.

[0047] As used herein, the term "recombinant expression library" refers to a collection of antibodies or antibody fragments displayed on heterologous host particles (such as on phage particles, on ribosomes or on a cell surface, e.g., on yeast cells, bacterial cells or mammalian cells) , or expressed in vitro. The number of different antibodies or antibody fragments in such a library is typically >1E4, more preferably >1E5, even more preferably >1E6, even more preferably >1E7, even more preferably >1E8, even more preferably >1E9, and most preferably >1E1O. As used herein, the "E" in an expression such as "1E5" refers to the exponent and indicates that the number should be multiplied by 10 raised to the power of the following number. In other words, the expression "1E5", for example, is equivalent to the expression "IxlO5" and equals 100,000. As known in the art, the diversity may depend on the display system in question. For example, it may be difficult to obtain diversity higher than 1E5 in mammalian cell expression libraries.

[0048] A "phage display library" is a protein expression library that expresses a collection of cloned protein sequences, such as antibodies or antibody fragments, as fusions with a phage coat protein. Thus, the phrase "phage display library" refers herein to a collection of phages (e.g., filamentous phages) wherein the phages express an external (typically heterologous) protein. The external protein is free to interact with (bind to) other moieties with which the phages are contacted .

[0049] As used herein, the term "panning" refers to multiple rounds of a screening process in identification and isolation of phage-carrying compounds , such as antibodies , with high aff inity and specif icity to an antigen target . In the panning process , a library, for instance a phage display library is incubated with target molecule , e . g . an antigen target , then phages displaying specificity to the target are bound to the target , while unbound phages are washed out . The speci fic phages are eluted and amplified in bacteria . After several rounds , ampl ified phages can be analysed and further screened . Thus , panning may be employed to select binders from antibody libraries . Several rounds of panning are required to enrich the specific binding subpopulation over the background . Furthermore , the small proportion of specific binders captured at each round of panning may require amplification of these binders by amplification in a host cell , such as a in bacteria .

[0050] As used herein, the term "sample" refers to any biological test sample suspected of containing testosterone and that is to be determined for the presence of testosterone or to be quantified for the concentration of testosterone . Preferred sample types are urine and blood, including whole blood samples , serum samples and plasma samples .

[0051] As used herein, the terms "specific binding" or "specifically binds to" or is "specific for" or similar expressions refer generally to binding where an antibody binds to an antigen target without substantially binding to any non-target antigen . In other words , specific binding refers to the non-random binding of an antibody to an antigen target , such as the binding of an anti- IC antibody to a target immunocomplex, and means binding that is measurably di fferent from a nonspecific interaction ( i . e . , binding of the antibody to non-target antigens ) . Methods for determining whether two or more molecules specifical ly bind are wel l known in the art and include , for example , equil ibrium dialysis , surface plasmon resonance , and the like .

[0052] Specific binding is relatively stable under physiologic conditions , and can be characteri zed by an equilibrium dissociation constant (Kd) , measurable by a variety of techniques known in the art . A smaller Kd denotes a tighter binding affinity . Low-affinity antibodies generally bind their target antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind their target antigen faster and tend to remain bound longer .

[0053] As used herein, the term "no specificity" refers to an instance where an antibody shows no specific detectable binding to a certain molecule . The term "no substantial specificity" as used herein means that a binding reaction, if any, of the antibody with a certain molecule is so minimal that it is of no significance or consequence and is virtually undetectable by routine techniques .

[0054] As used herein, the term "EC50" , also known as half maximal effective concentration, refers to the concentration of the target antigen that elicits a response halfway between baseline and maximum after a specified exposure time . Techniques for measuring the EC50 are available in the art and include , for example , a noncompetitive ELI SA method or a time-resolved fluorescence ( TR-F) assay . The expressions "EC50 concentration" and "EC50 value" are used herein interchangeably, both of which may be called "EC50" for short .

[0055] As used herein, the term "sensitivity refers to the ratio of changes in measured values in response to changes in the quantity or concentration of the analyte in the test sample . The greater the sensitivity ( slope of the calibration curve ) of the test method, the more eas ily subtle changes in the amount or concentration of the analyte can be detected .

[0056] As used herein, the term "limit of detection" (LoD) refers to the lowest s ignal or the lowest corresponding analyte concentration to be determined from the signal , which can be detected with a suff icient degree of confidence , completely distinct from the null ( 0 ) concentration . LoD can be calculated as the mean of the background plus three times the standard deviation ( SD) .

[0057] As used herein, the term "limit of quantification" (LoQ) refers to the lowest signal or the lowest corresponding analyte concentration to be determined form the signal , which can be can be quantified with a sufficient degree of confidence . LoQ can be calculated as the mean of the background plus ten times the standard deviation ( SD) .

[0058] In the context of the present invention, the term "target antigen" refers especially to an immunocomplex between testosterone and an anti-testosterone antibody, whereas the term "analyte" refers especially to testosterone .

[0059] It is to be noted that all ranges disclosed herein are understood to encompass any and all subranges subsumed therein, and every number between the endpoints . For example, a stated range of " 1 to 10" should be considered to include any and all subranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less , including all integers , whole or fractions , contained within the range . It is therefore also to be understood that any ranges disclosed with respect to a certain feature may be used in combination to form a further range relating to that feature . DETAILED DESCRIPTION

[0060] A non-competitive immunoassay for detecting or quantifying testosterone in a sample is provided . The assay utilizes an anti-immunocomplex ( anti- IC) antibody that is also provided herein, and is specific for an immunocomplex formed between testosterone and an antitestosterone primary antibody . The anti- IC antibody is not capable of specific binding to , i . e . has no or no substantial specificity towards , free testosterone or to the anti-testosterone primary antibody when not in complex with testosterone .

[0061] The present anti- IC antibody shows high binding affinity towards an immunocomplex formed between testosterone and an anti-testosterone primary antibody . In some embodiments , the anti- IC antibody has a Kd of at least about 20 nM, alternatively at least about 15 nM, alternatively at least about 10 nM, alternatively at least about 6 nM, alternatively at least about 5 nM, alternatively at least about 4 nM, al-ternatively at least about 3 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, alternatively at least about 0 . 8 nM, alternatively at least about 0 . 6 nM, alternatively at least about 0 . 4 nM, alternatively at least about 0 . 2 nM, alternatively at least about 0 . 1 nM, or less for the immunocomplex formed between testosterone and an anti-testosterone primary antibody .

[0062] The present anti- IC antibody also shows excellent specificity for testosterone , as evidenced by its ability to achieve an EC50 concentration less than 1000 pM, more specifically ranging from about 10 pM to about 1000 pM in an immunoassay . Notably, us ing a comparable calculation method, the EC50 value for the known anti- IC antibody described in JP2023060976 is significantly higher, reaching approximately 3000 pM, which indicates substantially lower sensitivity in the disclosed immunoassay . To be more specific, the EC50 from the JP2023060976 publication was estimated using GraphPad Prism 10 by fitting a curve to the dose-response data and identifying the concentration that yields half of the maximal effect, as defined by the fitting parameters of the software. Arguably, the estimated EC50 may not exactly match the concentration that visually appears to produce a response at 50% of the observed maximum on a raw plot because the fitting accounts for parameters also for the baseline (0%) and hill slope, and the data is normalized to a percentage of the maximum response without subtracting a baseline. Nevertheless, the calculation method shows a clear difference between the performance of the present anti-IC antibody than that of JP2023060976.

[0063] In an embodiment, the (EC50) concentration for quantifying testosterone in a sample, using the present anti-IC antibody, may range from about 100 pM to about 900 pM. In another embodiment, the EC50 may range from about 300 pM to about 800 pM. In a further embodiment, the EC50 may range from about 500 pM to about 700 pM, more specifically from about 570 pM to about 700 pM. In a still further embodiment, the EC50 may be 600 pM. In some other embodiments, the EC50 may range from about 10 pM to about 100 pM, more specifically from about 20 pM to about 80 pM, from about 30 pM to about 70 pM, or from about 40 pM to about 60 pM.

[0064] In an embodiment, the anti-IC antibody has a light chain variable region comprising CDR-L1 of SEQ ID NO:1, CDR-L2 of SEQ ID NO:2, and CDR-L3 of SEQ ID NO : 3 ; and a heavy chain variable region comprising CDR-H1 of SE ID NO: 4, CDR-H2 of SEQ ID NO: 5, and CDR-H3 of SEQ ID NO: 6. In an embodiment, the anti-IC antibody is a mutant of the above-mentioned anti-IC antibody having one or more conservative sequence variations which do not substantially affect binding specificity.

[0065] The CDR regions of the present anti-IC antibodies may be embedded in any appropriate framework. In an embodiment, the light chain framework is that disclosed in SEQ ID NO: 7. In an embodiment, the heavy chain framework is that disclosed in SEQ ID NO: 8. In some further embodiments, one or more of the framework regions set forth in SEQ ID NOs : 7 and 8 may contain one or more conservative sequence variations which do not substantially affect binding specificity. Moreover, the framework may be based on, or essentially correspond to, any framework encoded by a V and a J gene segment. Consensus human framework regions can also be employed, for example, as described in US Patent No. 6,300,064.

[0066] In an embodiment, the anti-IC antibody has a light chain variable region comprising SEQ ID NO: 7, and a heavy chain variable region comprising SEQ ID NO: 8, or is a mutant thereof having one or more conservative sequence variations that do not substantially affect binding specificity.

[0067] In an embodiment, the anti-IC antibody has a light chain polypeptide comprising SEQ ID NO: 9, and a heavy chain polypeptide comprising SEQ ID NO: 10, or is a mutant thereof having one or more conservative sequence variations that do not substantially affect binding specificity.

[0068] The present anti-IC antibodies may be recombinant full-length IgG antibodies, or antigen-binding fragments thereof, such as a Fab, Fab', Fv, dsFv, scFv, ds-scFv fragments.

[0069] In an embodiment, the anti-IC antibody is a full-length IgG antibody comprising CDR-L1, CDR-L2, CDR- L3, CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NOs: 1-6, respectively; a full-length IgG antibody comprising a light chain variable region comprising SEQ ID NO: 7, and a heavy chain variable region comprising SEQ ID NO: 8; a full-length IgG antibody comprising a light chain polypeptide comprising SEQ ID NO: 9, and a heavy chain polypeptide comprising SEQ ID NO: 10; or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity of the mutant anti-IC antibody.

[0070] In an embodiment, the anti-IC antibody is a Fab or a Fab' comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of SEQ ID N0s:l-6, respectively; a Fab or a Fab' comprising a light chain variable region comprising SEQ ID NO: 7, and a heavy chain variable region comprising SEQ ID NO: 8; a Fab or a Fab' comprising a light chain polypeptide comprising SEQ ID NO: 9, and a heavy chain polypeptide comprising SEQ ID NO: 10; or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity of the mutant anti-IC antibody.

[0071] In an embodiment, the anti-IC antibody is an Fv, a dsFv, an scFv, or a ds-scFv comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NOs:l-6, respectively; an Fv, a dsFv, an scFv, or a ds- scFv comprising a light chain variable region comprising SEQ ID NO: 7, and a heavy chain variable region comprising SEQ ID NO: 8; or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity of the mutant anti-IC antibody.

[0072] If the anti-IC antibody is in the form of a scFv or ds-scFv, the VH and VL domains are connected by a flexible linker peptide that is not particularly limited, but is usually 15-20 amino acids long. In some embodiments, the linker may be 12-15 amino acids long or even shorter, while in some other embodiments, the linker may be longer than 20 amino acids, such as 25 or even 30 amino acids. The linker keeps the C-terminus of one variable domain and the N-terminus of the other domain at a distance that favors proper folding and formation of the antigen-binding site.

[0073] Typically, the linker peptide is composed of mainly glycine and serine residues. In an embodiment, the linker is the 15-amino acid (glycine4-serine ) 3 peptide linker set forth in SEQ ID NO : 11 , also expressed as (Gly4-Ser) 3 or (GGGGS ) 3 linker . In an embodiment, the linker is the 20 -amino acid (Gly4Ser)4or (GGGGS H peptide linker set forth in SEQ ID NO : 12 . In some further embodiments , other residues , such as charged residues glutamate (Glu) and / or lysine (Lys ) , can also be incorporated into the linker, for example , to enhance solubility . In some further embodiments , amino acid residues such as alanine (Ala) and / or threonine ( Thr) may be incorporated into the linker as , for example , in the case of the linker GGGGSGAGGSGGGGTGGGGS set forth in SEQ ID NO : 13 . Other possible linker peptides are available in the art , and a skilled person can easily test whether or not a given linker peptide is suitable for use in the present scFvs or ds-scFvs .

[0074] The order, also called orientation, of the VH and VL domains in the present scFvs or ds-scFvs , can be either VL-linker-VH (LH, also expressed as VL-VH) or VH- linker-VL (HL, also expressed as VH-VL) , provided that the speci ficity of the anti- IC scFv or ds-scFv remains essentially non-altered as compared to a corresponding full-length IgG antibody or a Fab, Fab' , Fv or dsFv fragment thereof .

[0075] In an embodiment , the anti- IC antibody is labelled with a detection tag capable of creating a detectable signal . Suitable detection tags are readily available in the art , and are not particularly limited . In an embodiment , the detection tag is a fluorophore , for example a fluorescent protein , such as green fluorescent protein (GFP) , that will emit fluorescence when exposed to light . In an embodiment , the detection tag is an enzyme tag that will generate a coloured or luminescent signal upon contact with a chromogenic or chemiluminescent substrate , respectively . Non-limiting examples of suitable enzyme tags include alkaline phosphatase (AP) and horseradish hydrogen peroxidase (HRP) . Chromogenic and chemiluminescent substrates are readily available in the art for both AP and HRP . Also other detection tags, such as biotin, avidin, and streptavidin may be employed for detection purposes. They can be detected with a biotin, avidin, or streptavidin-binding protein, respectively, that is conjugated to an enzyme tag, fluorophore or other reporter molecule. In an embodiment, the detection tag is a radioactive label. Further detection tags are available in the art.

[0076] The present anti-IC antibodies may also comprise one or more other tags known in the art, such as tags that facilitate purification, isolation, or immobilization. Some non-limiting examples are mentioned later in this specification.

[0077] Techniques for labelling the anti-IC antibody with a selected tag are readily available in the art.

[0078] In an embodiment, the anti-testosterone antibody is a recombinant anti-testosterone IgG antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, an scFv fragment, or an ds-scFv fragment having CDR-L1 of SEQ ID NO: 14, CDR-L2 of SEQ ID NO: 15, CDR- L3 of SEQ ID NO: 16, CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18 and CDR-H3 of SEQ ID NO: 19, or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity of the mutant anti-testosterone antibody.

[0079] In an embodiment, the anti-testosterone antibody is a recombinant anti-testosterone IgG antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, an scFv fragment, or a ds-scFv fragment having a light chain variable region of SEQ ID NQ:20 and a heavy chain variable region of SEQ ID NO: 21. In an embodiment, the anti-testosterone antibody is a recombinant anti-testosterone Fab fragment disclosed by Valjakka et al. (J. Biol. Chem., Vol. 277, No. 46, pp . 44021-44027, 2002, incorporated herein by reference) having a light chain variable region of SEQ ID NO: 20 and a heavy chain variable region of SEQ ID NO:21. In a further embodiment, the anti-testosterone antibody is a mutant recombinant anti-testosterone IgG antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, an scFv fragment, or an ds-scFv fragment having one or more conservative sequence variations as compared to SEQ ID NQ:20 and / or SEQ ID NO:21, provided that its binding specificity remain substantially unaltered as compared to the corresponding non-mutant IgG, Fab, Fab' , Fv, dsFv, scFv, or sd-scFv, respectively.

[0080] In an embodiment, the anti-testosterone antibody is a recombinant anti-testosterone IgG antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, or an scFv fragment, or an ds-scFv fragment having CDR-L1 of SEQ ID NO:22, CDR-L2 of SEQ ID NO:23, CDR-L3 of SEQ ID NO:24, CDR-H1 of SEQ ID NO:25, CDR-H2 of SEQ ID NO:26 and CDR-H3 of SEQ ID NO:27, or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity of the mutant anti-testosterone antibody.

[0081] In an embodiment, the anti-testosterone antibody is a recombinant anti-testosterone IgG antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, an scFv fragment, or a ds-scFv fragment having a light chain variable region of SEQ ID NO:28 and a heavy chain variable region of SEQ ID NO: 29. or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity of the mutant anti-testosterone antibody.

[0082] Since traditional animal immunization is a tedious and problematic procedure with low success rate, especially when antibodies against immunocomplexes comprising small analytes are to be raised, the present disclosure provides a different approach for obtaining anti- IC antibodies disclosed herein . In this approach, said anti- IC antibodies are obtained from a recombinant expression library, such as a scFv or Fab library, by employing any display technique available in the art using immunocomplex ( IC) panning . In an embodiment , the anti- IC antibodies are obtained from a phage library, such as an scFv or Fab phage library, using a phage display based strategy . Alternatively, other display techniques such as ribosome display, bacterial cell surface display, yeast cell surface display or mammalian cell surface display may also be applied .

[0083] An advantage associated with the present approach i s that use of a recombinant expression library allows negative selection, where antibodies recogni zing the primary antibody as such can be removed from the library before selection, i . e . panning, with the immunocomplex of the anti-testosterone antibody and testosterone . This is not possible with immuni zation strategy .

[0084] Accordingly, an expression library may be first preincubated with an immobili zed primary antibody to sort out those antibodies which bind to the primary antibody as such, whereafter non-bound phages are separated and incubated with a mixture of testosterone and an anti-testosterone primary antibody to select those phages that bind to the immunocomplex formed between the immobili zed primary antibody and testosterone , but not to the primary antibody as such . In other words , a method for the preparation of the present anti- IC antibodies may comprise : a) providing an anti-testosterone primary antibody immobili zed on a sol id support or a carrier, such as a microtiter well or a bead; b) performing a negative selection by reacting the immobili zed primary antibody with a recombinant expression library, such as a phage display library; c) collecting a first non-bound fraction of the library; d) contacting the immobili zed primary antibody with testosterone for allowing formation of an immunocomplex between said testosterone and the primary antibody; e ) reacting the immunocomplex obtained in step d) with the non-bound fraction of the library obtained in step c) ; f ) removing a second non-bound library, optionally by washing; g) separating and collecting one or more anti- IC antibodies bound to the immunocomplex ; and h) expressing said one or more anti- IC antibodies in any suitable expression system .

[0085] Typically, the method is repeated two to five times by subj ecting the anti- IC antibodies obtained in step g) to repeated rounds of steps b) to g) or to steps d) to g) prior to carrying out step h) . The method may also be carried out without the negative selection step b) .

[0086] Methods and means for immobili zing the antitestosterone primary antibody on the solid support or the carrier are well known in the art and are not particularly limited . Also conditions , such as the temperature , time , and incubation or wash solutions to be used, suitable for each and every step of the method are well known in the art .

[0087] Each phage particle carries genetic information for the recombinant polypeptide that it displays on its surface . This feature allows for identifying DNA that encodes an antibody exhibiting a desired specificity, by selecting the particular phage which carries the desired antibody from a potentially very complex phage library . DNA from the clone of interest may then be isolated, inserted into a suitable expression vector, and transfected or transformed into a compatible expression host to produce the antibody according to standard recombinant technology . Numerous types of suitable expression vectors are available and include, but are not limited to, plasmids or modified viruses which are maintained in the host cell as autonomous DNA molecules or integrated in genomic DNA. The vector system must be compatible with the host cell employed as is well known in the art. Preferably, DNA encoding an anti-IC antibody according to the present invention is operably linked to one or more heterologous expression control sequences permitting expression of the antibody. Suitable control sequences are readily available in the art and include, but are not limited to, promoter, leader, polyadenylation, and signal sequences.

[0088] In some embodiments, it may be desirable to express the present anti-IC antibody as a fusion to one or more peptide tags or to couple the anti-IC antibody to one or more other tags that facilitate purification, isolation, immobilization, or detection. Non-limiting examples of suitable affinity tags for purification or immobilization purposes include polyhistidine tags (His-tags) , hemagglutinin tags (HA-tags) , glutathione- S-transf erase tags (GST-tags) , and biotin tags. Suitable detection tags are already discussed above. Vectors, other means, and methods for tagging the anti-IC antibodies are readily available in the art.

[0089] Non-limiting examples of suitable host cells include prokaryotic hosts such as bacteria (e.g. E.coli, bacilli) , yeast (e.g. Pichia pastoris, Saccharomyces cerevisiae) , and fungi (e.g. filamentous fungi) , as well as eukaryotic hosts such as insect cells (e.g. Sf9) , and mammalian cells (e.g. CHO cells) . In some embodiments, host cells transfected with an expression vector comprising a polynucleotide encoding for the present anti- IC antibody are cultured under conditions suitable for the production of a present anti-IC antibody followed by recovering the antibody obtained. Expression vectors may be transfected into host cel ls by standard techniques . As used herein , the term "transfection" refers to a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell including, but not limited to , electroporation, nucleof ection, sono- poration, magnetof ection, heat shock, calcium-phosphate precipitation, DEAE-dextran transfection and the like . As used herein , the term "transfection" and all verbal forms thereof is interchangeable with the term "transformed" and all verbal forms thereof , respectively .

[0090] An anti- IC antibody of the invention may also be produced by in vi tro protein expres sion , al so known as in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis . Several cell-free expression systems based on, for instance , bacterial (e . g . E . coli ) , rabbit reticulocyte , CHO, or human lysates are commercially available in the art . In some embodiments , in vi tro protein expression may be performed either in batch reactions or in a di alysis mode .

[0091] Being specific for an immunocomplex enables that the present antibodies are suitable for use in noncompetitive assay formats allowing sensitive and simple detection or quantification of testosterone . Non-competitive assays , also known as reagent excess assays , sandwich, immunometric or two-site assays , generally involve use of two antibodies targeting different epitopes , one antibody for antigen capture and the other labelled for detection . Especially the capture but, in some extent , also the detection antibody can be added in exces s compared to the analyte . At low analyte concentrations , non-occupied capture binding sites are always available, but as a detectable signal is generated only at the occupied binding sites , the signal is directly proportional to the amount of analyte present . The situation is opposite in the reagent limited, competitive assays , where the analyte and a labelled tracer analyte compete for a limited number of binding sites of a single-type anti-analyte antibody used . In general , sandwich format provides considerable benefits in terms of assay robustness , sensitivity, specificity and kinetics . In addition, the working range typically is more extended compared to the competitive assay . For small si zed analytes like testosterone , the competitive assays are generally employed as finding two antibodies with separate epitopes is rare .

[0092] Accordingly, a non-competitive immunoassay for detecting testosterone in a sample is provided . The method comprises :

[0093] - contacting a sample suspected of containing testosterone with an anti-testosterone primary antibody;

[0094] - allowing the anti-testosterone antibody to form a first immunocomplex with testosterone present in the sample , if any, through specific binding to the testosterone ;

[0095] - contacting the first immunocomplex with an anti- IC antibody disclosed herein, labelled with a detection tag capable of generating a detectable signal ;

[0096] - allowing the anti- IC antibody to form a second immunocomplex with the first immunocomplex through specific binding to the first immunocomplex ;

[0097] - removing any non-bound anti- IC antibody; and

[0098] - determining that testosterone is present in the sample , i f a signal generated by the detection tag can be detected .

[0099] In an embodiment of the non-competitive immunoassay disclosed above , the sample is contacted with the anti-testosterone primary antibody and the anti- IC antibody simultaneously .

[0100] Also provided is a non-competitive immunoassay for quantifying testosterone in a sample . The method comprises : - contacting a sample suspected of containing testosterone with an anti-testosterone primary antibody;

[0101] - allowing the anti-testosterone antibody to form a first immunocomplex with testosterone present in the sample , if any, through specific binding to the testosterone ;

[0102] - contacting the first immunocomplex with an anti- IC antibody disclosed herein, labelled with a detection tag capable of generating a detectable signal ;

[0103] - allowing the anti- IC antibody to form a second immunocomplex with the first immunocomplex through specific binding to the first immunocomplex ;

[0104] - removing any non-bound anti- IC antibody; and

[0105] - measuring the intensity of the detectable signal ; and

[0106] - correlating the measured intensity of detectable signal to the quantity of testosterone in the s amp 1 e .

[0107] Correlating the measured intensity of the detectable signal to the quantity of testosterone in the sample may be based on previous measurements of standard solutions of known testosterone concentrations . In other words , quantitating testosterone may involve use of a calibration curve , i . e . a standard curve . Generating calibration curves is well known in the art , and can in this case be carried out by plotting a known testosterone concentration in a given standard solution on the x-axis versus a corresponding signal intensity on the y-axis . Any unknown concentration of testosterone may then be determined by comparing the measured signal intensity to a corresponding intensity on the calibration curve , achieved in the presence of known testosterone concentrations .

[0108] In an embodiment of the non-competitive immunoassay for quantifying testosterone disclosed above , the sample is contacted with the anti-testosterone primary antibody and the anti-IC antibody simultaneously.

[0109] The present non-competitive immunoassays may be employed in any available non-competitive immunoassay format as is readily understood by those skilled in the art. Non-limiting examples of suitable immunoassays include enzyme linked immunoabsorbent assays (ELISA) , im- munof luorometric assays (IFMA) , fluorescent immunosorbent assays (FIA) , such as time-resolved immunofluo- rometric assays (TR-IFMA) , chemiluminescence immunoassay (CLIA) , radioimmunoassay (RIA) , open sandwich immunoassays (OS) and microsphere-based immunoassays (MIA) .

[0110] Depending on the assay type employed, either the primary anti-testosterone antibody or the present anti-IC antibody, or both, may be conjugated or otherwise associated with a detection tag selected from the group including, but not limited to, optical agents such as fluorescent labels including a variety of organic and / or inorganic small molecules or a variety of fluorescent proteins and derivatives thereof, phosphorescent labels, chemiluminescent labels, and chromogenic labels; radioactive labels such as radionuclides that emit gamma rays, positrons, beta or alpha particles, or X-rays, and enzymes such as alkaline phosphatase (AP) , or (horseradish) hydrogen peroxidase (HRP) . Said association can be direct, e.g. through a covalent bond, or indirect, e.g. via a secondary binding agent, chelator, or linker. Techniques for conjugating or otherwise associating the selected detection tag to antibodies are well known and antibody labelling kits are commercially available from dozens of sources.

[0111] In some embodiments, the anti-IC antibody is labelled with detection tag. In some other embodiments, the anti-IC antibody is recognized by a further antibody (e.g. a species-specific antibody) comprising a detection tag. In some still other embodiments, the anti-IC antibody comprises a tag that is recognizable by a further antibody comprising a detection tag. In some still further embodiments, the anti-IC and said further antibodies are both labelled with the same tag, e.g. for improving sensitivity in assays where the immunocomplex to be detected is expected to be rare. The anti-IC antibody and said further antibody may also be labelled with different tags.

[0112] In an embodiment, the anti-IC antibody is labelled with a detection tag which is a reactive enzyme, such as AP or HRP. In such an embodiment, a chromogenic or chemiluminescent substrate compatible with the selected reactive enzyme must be added into the reaction for generating a detectable signal which is then to be detected or its intensity measured. Typically, the substrate is added after removal of any non-bound anti-IC antibodies .

[0113] An immunoassay provided herein may be a solidphase immunoassay, such as a lateral flow assay or a conventional sandwich assay carried out on a solid surface, e.g. a microtiter plate. In these assay formats, either the primary anti-testosterone antibody or the anti-IC antibody is immobilized on the solid surface. Preferably, the antibody to be immobilized is the primary anti-testosterone antibody. A detectable sandwich between the analyte and the primary and secondary antibodies forms if the sample to be analysed contains testosterone. Said solid-phase immunoassay may be either heterogeneous or homogeneous. In the heterogeneous assay, any free analytes or antibodies are physically separated from immunocomplexes formed, e.g. by washings, while no such separation is necessary in homogeneous assays making homogeneous assays preferable.

[0114] The present anti-IC antibodies are suitable for use not only in homogeneous solid-phase assay formats but also in homogeneous immunoassays carried out in solution. Such in-solution immunoassays are particularly advantageous because no immobilization or washing steps are required, making them simple and easy to perform. Thus, in some preferred embodiments, the immunoassay is liquid-based homogeneous immunoassay.

[0115] Exact LoD values for an immunoassay may generally depend on different variables, such as the type of the immunoassay and, in the present case, properties of the anti-testosterone antibody, as is readily understood by those skilled in the art. Nevertheless, in some embodiments, the present immunoassay has a LoD ranging from about 0.5 pM to about 50 pM. In some embodiments, the immunoassay has an LoD ranging from about 0.5 pM to about 1 pM. In some embodiments, the present immunoassay has an LoD ranging from about 1 pM to about 5 pM. In some embodiments, the present immunoassay has an LoD of ranging from about 5 pM to about 10 pM. In some embodiments, the present immunoassay has an LoD ranging from about 10 pM to about 15 pM. In some embodiments, the immunoassay has an LoD ranging from about 15 pM to about 20 pM. In some embodiments, the present immunoassay has an LoD ranging from about 20 pM to about 25 pM. In some embodiments, the present immunoassay has an LoD ranging from about 25 pM to about 30 pM. In some embodiments, the present immunoassay has an LoD ranging from about 30 pM to about 35 pM.

[0116] In an embodiment, the present immunoassay is a TR-F assay having an LoD raging from about 15 pM to about 20 pM.

[0117] Exact LoQ values for an immunoassay may also vary depending on different variables, just like the LoD as is readily understood by those skilled in the art. Nevertheless, in some embodiments, the present immunoassay has a LoQ ranging from about 10 pM to about 70 pM. In some embodiments, the immunoassay has an LoQ ranging from about 26 pM to about 30 pM. In some embodiments, the present immunoassay has an LoQ ranging from about 30 pM to about 36 pM. In some embodiments, the present immu-noassay has an LoQ of ranging from about 36 pM to about 40 pM. In some embodiments, the present immunoassay has an LoQ ranging from about 40 pM to about 46 pM. In some embodiments, the immunoassay has an LoQ ranging from about 46 pM to about 50 pM.

[0118] In an embodiment, the present immunoassay is a TR-F assay having an LoQ raging from about 36 pM to about 40 pM.

[0119] In yet another aspect, an immunoassay kit for detecting or quantifying testosterone in a sample is provided. The kit comprises at least an anti-IC antibody disclosed herein and an anti-testosterone antibody, wherein at least one of the antibodies is labelled with a detection tag. The anti-IC and anti-testosterone antibodies may, independently from each other, be intact immunoglobulins or any antigen-binding fragments thereof, such as Fab, Fab', Fv, dsFv, scFv or ds-scFv fragments. In some still further embodiments, the antibodies may be provided in dried form.

[0120] In some further embodiments, either the anti- IC antibody or the anti-testosterone antibody, preferably the anti-testosterone antibody is immobilized on a solid surface.

[0121] In some embodiments, the kit may also comprise one or more other components for carrying out the immunoassay, such as blots (e.g., nylon or nitro-cellulose) , microtiter plates, reaction vials, lateral flow strips, appropriate standards or calibrators (i.e. testosterone) for preparing a calibration curve., and reagents such as buffers, detection reagents (e.g. labels, chromogenic substrates, labelled further antibodies recognizing the present anti-IC antibodies, etc.) , and wash solutions. Depending on the selected detection tag, or other particulars of the immunoassay in question, the kit may also comprise additional reagents such as stabilizers or agents that enhance or enable generation of a detectable signal, or intensify the signal generated. In an embodiment , the detection tag is a reactive enzyme , such as AP or HRP , and the kit also comprises a substrate capable of creating a detectable signal upon reaction with the reactive enzyme . Further components may al so be included, as is readily understood by those skilled in the art . For example , if the selected reactive enzyme is HRP, hydrogen peroxide may be included in the kit as an oxidi zing agent .

[0122] Typically, the kit also includes instructions for use , or direction to an outs ide source of instruction such as a website .

[0123] Any disclosed detail , advantage , embodiment , etc . relating to any aspect of the present invention also apply to other aspects of the intention unless clearly indicated otherwise . For example , features described in the context of the present anti- IC antibody apply to the immunoassay kit as appropriate , and vice versa .

[0124] It is further to be noted that certain features of the disclosure which are described in the context of separate embodiments , can also be provided in any combination j ust as if each and every combination was individually and explicitly disclosed .

[0125] It is obvious to a person skil led in the art that with the advancement of technology, the basic idea of the invention may be implemented in various ways . The invention and its embodiments are thus not limited to the examples described above . Instead, they may vary within the scope of the claims . EXAMPLES

[0126] Example 1 . Preparation of anti-IC antibodies

[0127] A method for producing anti- IC antibodies of the present disclosure is illustrated schematically in Figure 1 . An embodiment of the method is described in more detail below .

[0128] 1 . Materials and methods

[0129] 1 . 1 Biotinylation of Anti-T

[0130] As the anti-testosterone antibody, the 3201 SPTN-5 antibody (Anti-T ) purchased from Medix Biochem- ica (Espoo, Finland) was used . The Anti-T was biotinylated to be used in the immunoassays . The biotinylation was done using EZ-Link NHS-PEG4 -Biotin ( Thermo Fisher Scientific, USA) according to the manufacturer' s instructions . Non-reacted NHS-PEG4 -Biotin was removed using a Vivaspin 500 centrifugal concentrator column (Mw 10 000 ) ( Sartorius , Germany) . All centrifugations were done at 12 000 g for 5 minutes at room temperature . The biotinylation product was centrifuged in the column and PBS was added up to 100 pL . After centrifugation, the flowthrough was discarded and the washing was repeated two times . The remaining solution was collected from the filter by pipetting . The protein concentration was measured with Nanodrop and 0 . 1 % DTPA-BSA was added as a preservative .

[0131] The biotinylation was verified by coating 50 , 100 and 150 ng of the biotinylated Fab (bio-anti-T ) on a yellow streptavidin plate in triplicates . The total volume was 200 pL per well for all assay steps and the incubations were done for one hour with low shaking at room temperature . After each incubation, the wells were washed four times . After incubation, 50 ng anti-mouse IgG Eu antibody was added . After incubation, 200 pL enhancement solution was added and the TR-F was measured with Hidex Sense after 10 minutes . 1.2 Panning

[0132] A synthetic Fab library where Fabs are displayed as fusions with a coat protein of the filamentous VCSM13 phage, constructed at the Biotechnology unit, University of Turku, Finland, was employed.

[0133] The Fab library was enriched for anti-IC Fabs for testosterone with a total of four phage display panning rounds. The panning conditions for each round are described in Table 1. The second and third panning rounds were made on the phage stocks of the preceding panning rounds. The fourth round was done in round 3B (see Table 1 and Figure 2) .

[0134] 1.2.1 Conjugating biotinylated anti -testosterone (Anti- T) on beads

[0135] As the anti-testosterone antibody, the 3201 SPTN-5 antibody (anti-T) purchased from Medix Biochemica (Espoo, Finland) was used.

[0136] For panning round 1, Tosyl-activated paramagnetic beads (Dynal, Norway) described in Leivo et al. (2019) were used. For the rest of the panning rounds, 200 pg streptavidin (SA) coated Dynabeads MyOne Streptavidin Cl beads (Thermo Fisher Scientific) or avidin coated Dynabeads M-270 Epoxy beads (Thermo Fisher Scientific) were used. The beads were washed two times with 0.5 mL TBT-0.05 using a Dynal magnet. The beads were resuspended in 40 pL TBT-0.05. 500 ng of biotinylated anti-testosterone antibody was added to half of the prewashed beads. The beads were incubated on rotation for 30 minutes at room temperature. The beads were washed three times with 0.8 mL TBT 0.05. After removal of the supernatant, the beads were suspended in 40 pL TBT 0.05. Table 1. Panning selection conditions. The fraction column refers to the fractions seen in Figure 2.

[0137] *Beads from Leivo et al. (2019) were used. Beads were coated with Anti-T amount unknown. Stock concentration was also unknown.

[0138] 1.2.2 Immunocomplex selections

[0139] Panning was done by incubating the conjugated beads with the phage library, testosterone (Sigma-Aldrich) and anti-estradiol S16-Fab (Biotechnology unit, University of Turku) as described in Table 1 overnight or for two hours at +4 °C. S16-Fab was added as a negative selection to deplete unspecific phages. The beads were washed two times with 0.8 mL TBT-0.05 and once with 0.8 mL TBS using a Dynal magnet. New tubes were used in every wash. The supernatant was removed. To elute the phages from the beads, they were suspended in 200 pL trypsin diluted to 60 pg / mL in TBS. The solution was incubated on rotation at room temperature for 30 minutes. The beads were collected with the Dynal magnet and the eluate was transferred to fresh tubes.

[0140] 1.2.3 XLl-Blue cell culture

[0141] XLl-Blue cells were inoculated in 3 mL Super Broth (SB) medium [30 g / 1 tryptone, 20 g / 1 yeast extract, 10 g / 1 MOPS ( 3-N-orpholinopropanesulf onic acid) , pH 7.0 in ultrapure H2O] with 0.2% glucose and 10 ug / mL tetracycline and incubated overnight shaking at 300 rpm at +30 °C. 10 and 50 pL of overnight grown cells were inoculated in 20 mL SB (0.2% glucose, 10 pg / mL tetracycline) and incubated at +37 °C shaking at 300 rpm overnight. In the morning, the media was changed to fresh media and the cells were incubated at +37 °C shaking at 300 rpm until OD(600) was 0.5.

[0142] 1.2.4 Infection and plating

[0143] 5x volume of OD 0.5 XLl-Blue cells were added on the phage eluate. The solution was mixed gently by inverting the tube. In addition, 180 pL XLl-Blue cells was incubated in a separate tube to be used as a contamination control. In addition to the contamination control, controls with 5 pL phage stock from panning round 1 were used to infect 180 pL cells each. The cells were incubated for 1.5 h at +37 °C. 100 pL of the controls were plated on 10 cm Luria Agar (LA) plates (10 g / 1 tryptone, 5 g / 1 yeast extract, 10 g / 1 NaCl, 15 g / 1 agar) with 0.5% glucose, 10 pg / mL tetracycline, and 25 ug / ml chloramphenicol. 100 pL of 10-3 and 10-4 dilutions of the infected cells were also plated on LA plates. For the first panning round, the remaining infection solutions were plated on large petri dishes and for the consecutive rounds on medium round petri dishes. The plates were left to dry and then incubated overnight at +30 °C. The panning output was determined by counting the colonies on the dilution plates.

[0144] 1.2.5 Phage production

[0145] The cells were collected from the large petri dishes with 7 mL SB medium. The cells were suspended in 20 mL SB (1% glucose, 10 pg / mL tetracycline, 25 pg / mL chloramphenicol) so that OD600 was 0.1. Incubation was done at +37 °C and 300 rpm until the cells had grown to logarithmic phase (OD(600) 0.5) . 5E10 VCS M13 helper phages were added to each cell solution and incubated at +37 °C for 30 minutes. The cells were then cooled on ice and collected by centrifugation for 10 minutes at 4000 rpm and +4 °C. The cell pellet was resuspended in 20 mL glucose-free SB medium (10 pg / mL tetracycline, 25 pg / mL chloramphenicol) . Incubation was done for 30 minutes at 300 rpm and +30 °C. To induce the phage production, 50 pg / mL kanamycin and 100 pM IPTG was added. The cells were incubated overnight at 300 rpm and +26 °C.

[0146] 1.2.6 Phage stock preparation

[0147] The cultures were centrifuged for 10 minutes at 12 000 g in +4 °C (Beckman Coulter Avanti J-26 XP, rotor JA 25.50) . The supernatant was transferred to fresh centrifuge tubes. 1 / 5 volume of 20% PEG / 2.5 M NaCl was added and the vials were vortexed. The solutions were incubated on ice for 45 minutes to let the phages precipitate . The phages were centrifuged for 20 minutes at 10 000 g in +4 ° C and the supernatant was removed . The tubes were centrifuged briefly and the remaining supernatant was removed . The pellets were suspended in 1 mL TBS . The suspension was centrifuged for 5 minutes at 16 000 g and +4 ° C to pellet residual cells . The supernatant was transferred to fresh microcentrifuge tubes . One fifth volume of PEG / NaCl was added, mixed and incubated on ice for 5 minutes to let the phages precipitate . The tubes were centrifuged for 5 minutes at 10 000 g and +4 ° C and the supernatant was discarded . The rest of the supernatant was removed by centrifuging the tubes briefly . The pellet was suspended in 0 . 5 mL TSA / BSA and stored at +4 ° C .

[0148] 1 .2 . 7 OCCA phage titer assay

[0149] The phage titer was quantified for panning rounds 2 and 3 with a homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes , as described in Lehmusvuori et al . ( 2012 ) .

[0150] 1 . 3 Screening

[0151] Hyperphages were from Progen (Germany) and anti-phage antibody Nl -Eu anti-VCSM13 was used for detection of phages was made inhouse (Biotechnology unit , University of Turku, clone 9E7 ) . Streptavidin and rabbit anti-mouse (RAM) coated 96-well microtiter plates , immunoassay buffers and Delfia Enhancement solution used in the immunoassays were purchased from Kaivogen / Uniogen ( Finland) . The alkaline phosphatase substrate para-ni- trophenylphosphate (pNPP) was from Sigma-Aldrich . Hidex Sense microplate reader from Hidex ( Finland) was used for the absorbance and time-resolved fluorescence ( TR- F) measurements . For the cloning, the transformation reagents ( Tango buffer, Sfil, T4 DNA ligase , T4 DNA ligase buffer) were from Thermo Fisher Scientific . 1 .3. 1 Phage immunoreactivity

[0152] The immunoreactivity of the phages obtained from panning rounds 2 -4 was determined with a phage immunoreactivity assay . Yellow low fluorescence plates were prewashed (Wallac) and 500 ng capture antibody was added . 10 nM and 1 pM testosterone was added in triplicates . Kaivogen assay buffer red was added up to 200 pL . Background wells consisted of only assay buffer and only capture antibody, respectively . The plates were incubated on low shaking for 1 h at room temperature . The plates were washed four times and 1E8 phages from the phage stocks prepared in section 1 . 2 . 7 were added to each well . The plates were incubated on low shaking for 1 h at room temperature . The plates were washed four times and 4 ng of anti-phage antibody Nl -Eu anti-VCSM13 9E7 was added to each well . The plates were incubated on low shaking for 1 h at room temperature . The plates were washed four times and 200 pL Enhancement Solution was added . The plates were incubated for 10 minutes on low shaking and the time-resolved fluorescence was measured with Hidex Sense .

[0153] 1 .3.2 Phage screening

[0154] For the screening, colonies were picked from the panning output plates from the fourth panning round . The bacteria were grown in 96-wel l plates with 150 pL SB medium ( 25 pg / mL chloramphenicol , 10 ug / mL tetracycline , 1 % glucose ) . The plates were covered with breathable sealing tape ( Thermo Fisher Scientif ic) and incubated at +37 ° C, 700 rpm with 70 % moisture (Multitron shaker, Infers HT, Switzerland) . After a few hours , 100 pL from the primary culture was used to inoculate a secondary culture with 100 pL SB ( 25 pg / mL chloramphenicol , 10 ug / mL tetracycline , 1 % glucose ) . Incubation was done as described above until the cultures looked cloudy . Each well was infected with 7E7 efu of hyperphages and the plates were mixed gently and incubated at +37 ° C without shaking for 45 minutes . The cultures were then grown overnight at +26 ° C, 700 rpm and 70 % moisture .

[0155] For the screening immunoassay, the plates were first centrifuged at 4000 rpm for 30 minutes . 100 pL of the supernatant was transferred to a 500 pL 96-well plate with 400 pL TY buffer solution RED ( assay buffer, AB) in each well . Streptavidin-coated 96-well microtitration plates were prewashed (Kaivogen plate washer, wash solution) and 50 ng of biotinylated Anti-T and 1 pM testosterone in 100 pL AB was added to each well . The total volume of each well was 100 pL in each step in the assay . The plates were incubated for one hour at room temperature with low shaking . After four washes , 50 pL from the phage-AB plate was added and the volume was adj usted to 100 pL with AB . The plate was incubated and washed as mentioned before . 12 . 5 ng of anti-VCSM13 antibody diluted in AB was added and the plates were incubated and washed as before . After addition of enhancement solution, the plates were incubated for 15 minutes with low shaking and the time-resolved fluorescence was measured with Hidex sense .

[0156] 1 .3.3 Second screening

[0157] A second screening was performed on the five best anti- IC phages from the first screening . The assay was performed in a s imi lar way as the f irst screening, but three testosterone concentration points were used ( 10 nM, 100 nM and 1000 nM) in combination with 100 ng biotinylated capture bio-Anti-T per well . Also, only 20 pL of the phage-AB mix was used per well . The rest of the assay was done as described in 1 . 3 . 2 .

[0158] 1 .3. 4 Cloning into pLK06FT vector

[0159] The four best binders from the second screening were cloned into pLK06FT expression vector to be produced as fusion proteins with bacterial alkaline phosphatase. To do this, the clones were grown from glycerol preps and minipreps were done with the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) . The minipreps were digested with the restriction enzyme Sfil. The digestion reaction consisted of 500 ng miniprep DNA, lx Tango buffer and 5 U Sfil. The volume was adjusted to 20 pL with ultrapure water and the reaction was incubated at +50 °C for two hours. The insert was purified with GeneJet PGR Purification Kit (Thermo Fisher Scientific) according to the manufacturer' s instructions. The optional step of adding NaAcetate was done. The purified fragment was ligated into a Sfil- digested pLK06FT vector. The ligation reaction consisted of 50 ng insert fragment, 150 ng purified template vector, lx T4 DNA ligase buffer and 2.5 U T4 DNA ligase, and ultrapure water up to 50 pL . The reaction was incubated at room temperature for one hour and then inactivated by heating at +65 °C for 10 minutes.

[0160] Electrocompetent XL1 Blue cells were thawed on ice and 1 pL of the ligation reaction was added to 20 pL of cells. 30 pL cold ultrapure water was added and the components were mixed gently by pipetting. The solution was moved to pre-chilled Gene Pulser cuvettes (Bio-Rad Laboratories, USA) and transformation was performed through electroporation with Gene Pulser Xcell (Bio-Rad Laboratories) (200 Q, 25 pFd and 1.25 kV) . After electroporation, the cells were suspended in 500 pL SOC medium (composition) and moved to microcentrifuge tubes. The cells were incubated at +37 °C, 250 rpm for 30 minutes and then diluted 1:10 and 1:100 in SOC media. 100 pL of each dilution was plated on LA plates with 0.5% glucose and 100 pg / mL ampicillin. As a contamination control, 100 pL of SOC medium was plated. The plates were incubated at +37 °C overnight. Two colonies per clone were picked and inoculated in 5 mL SB medium (0.5% glucose, 100 pg / mL ampicillin) . After an overnight incubation in +26 °C and 300 rpm, the cells were suspended in 5 mL of fresh SB (0.2% glucose, 100 pg / mL ampicillin) at an OD(600) of 0.1. When OD(600) was 0.6, 100 pM IPTG was added and the cells were incubated at +26 °C overnight. The cells were centrifuged for 30 minutes at 4000 rpm and +4 °C and the supernatant was diluted 1:5 in AB. The supernatant-AB mix was used to determine the affinity of the clones to the Anti-T-testosterone immunocomplex .

[0161] 1.3.5 3rd screening - ALP-ELISA

[0162] A screening assay was done to assess the clones in their final form. The total volume of each well was 100 pL in each step in the assay. Clear streptavidin wells were prewashed and 100 ng of biotinylated Anti-T was added. 10 nM, 100 nM and 1000 nM testosterone was added in triplicates and three wells were left without testosterone. After a 30-minute incubation at low shaking in room temperature, the plate was washed four times and 100 pL of the supernatant-AB mix was added. The plate was incubated for one hour at room temperature with low shaking and washed four times. 1 mg / mL pNPP in pNPP buffer (500 mM Tris, 200 mM NaCl, 10 mM MgC12, pH 9) was added and the plate was incubated at 37 °C until yellow color was visible. The absorbance was measured at 405 nm with Hidex Sense. The two best clones were selected for Sanger sequencing and compared with each other using AlignX (results not shown) .

[0163] 1.3.6 EC50 of clone G9

[0164] The half maximal effective concentration (EC50) , i.e., the concentration of testosterone needed for half-maximal response, was determined with an assay similar to the one described in section 1.2.5. The assay was done with 50 ng capture antibody (Anti-T) per well and 15 different concentrations of testosterone (0, 3, 6, 12, 24, 49, 98, 195, 391, 781, 1563, 3125, 12500, 25000 and 50000 pM) . 100 ng of G9 Fab was used as the secondary antibody. The EC50 value was determined by normalizing the data followed by a nonlinear regression using GraphPad Prism version 10.1.1 (GraphPad Software, Boston, Massachusetts USA) .

[0165] 1.3.7 Binding comparison of G9 to two different ICs

[0166] The binding of G9 to the IC used in the panning was compared with its binding to the IC of testosterone and an anti-testosterone antibody from another manufacturer. The antibody in question was Ab00418-l.l Anti- Testosterone [77 Fab] by Absolute Antibody (United Kingdom) (77_Fab) . Despite the name, the antibody is a full- size mouse IgGl . The assay was performed with an ALP- ELISA as described in section 1.3.5, with slight modifications. 50 ng of Anti-T antibodies were used, and 0.1, 10 and 1000 nM testosterone. Purified G9 (from section 1.4) was added to bind to the ICs.

[0167] 1.4 Production of G9

[0168] 1.4.1 Cell culture and protein production

[0169] Cells were inoculated from glycerol preps in 5 mL SB medium (1 % glucose, 100 pg / mL ampicillin) and incubated overnight at 30C shaking at 300 rpm. The preculture was diluted 1 / 100 in 500 mL SB medium (1 % glucose, 100 pg / mL ampicillin) and incubated at 37 shaking at 250 rpm overnight until CD (600) was 0.8. The protein production was induced by adding 100 pM IPTG and the cells were incubated at +26 °C for 6 hours at 250 rpm and then at +10 °C at 50 rpm until cell collection. The cells were collected by centrifugation for 30 minutes at 4000 rpm at +4 °C. The supernatant was discarded and the cell pellets were stored at -20 °C until protein purification. 1.4.2 Cell lysis

[0170] The cell pellets were thawed and suspended in 4 mL lysis buffer (PBS with 0.4 mg / mL lysozyme, 25 U / mL nuclease, pH 7.4) . The cells were incubated in rotation for 30 minutes at room temperature and then frozen for 20 minutes in -70 °C. The cells were thawed for 10 minutes in a +40 °C water bath. To pellet the cells, they were centrifuged for 15 minutes at 13 000 rpm in + 4 °C (PrismR, Labnet, Finland) . The supernatant was transferred to fresh tubes.

[0171] 1.4.3 Protein purification

[0172] The lysate was incubated with 250 pL Ni-NTA for 30 minutes in rotation in +4 °C. The solution was added to Ni-NTA columns and the flowthrough was discarded. The resin was washed with 1 mL PBS and the proteins were eluted with 1 mL elution buffer (PBS with 500 mM imidazole) . The protein concentrations were measured with Nanodrop (Thermo Fisher Scientific) . The purity of the samples was verified with an SDS PAGE (results not shown) .

[0173] 2. Results

[0174] 2.1 Enrichment of high-affinity anti-IC Fabs

[0175] 2.1.1 Panning output and phage titer

[0176] The panning output and the output to input ratio was determined by comparing the number of colonies on the panning output plates (section 1.2.5) with the number of phages used in the panning. The phage titer for the phage stocks was measured after the second and third panning round to be able to decide on an exact phage input amount for the next round. The TR-F values were compared with a standard curve made alongside the measurement. The results can be seen in Table 2. Table 2. Panning output and titer of phage stocks. The fraction column refers to the fractions shown in Figure 2. unknown not measured

[0177] 2.1.2 Immunoreactivity of phage stocks

[0178] The aim of the phage display panning was to obtain high-affinity anti-IC Fabs for testosterone. The immunoreactivity of all phage stocks was analyzed after the last round. The immunoreactivity was analyzed using bio-Anti-T in streptavidin wells and 0, 10 and 1000 nM testosterone (Figure 3) .

[0179] 2 .2 Screening

[0180] From the panning output plates of the fourth panning rounds, colonies were picked for screening. The primary screening was done with an ALP-assay, and the results can be seen in Figure 4. Five of the testosterone clones (B7, E6, F6, G4 and G9) showed higher signals than the other clones, with absorbance signal to background ratios between 3.7 and 24.9. The clones were chosen for secondary screening.

[0181] The secondary screening was done on the above- mentioned best phage clones. Three different testosterone concentrations (10, 100 and 1000 nM) were used to assess the affinity of the clones to the ICs. An increase in binding could be seen with increasing concentrations of testosterone (Figure 5) . The clones with the highest signal increase at the lowest concentration was E6 and G9, which were then cloned into the production vector .

[0182] The clones were cloned into a pLK06FT vector with alkaline phosphatase as a fusion protein and transformed into XLl-Blue cells. After production and purification, the affinity of the Fab binders was assessed with an ALP-ELISA, using 10 nM, 100 nM and 1000 nM testosterone (Figure 6) . Clones E6 and G9 had a high signal-to-background ratio, 4.6 and 4.9, respectively, even with the lowest concentration of testosterone. The responses were also very similar, so Sanger sequencing was done to determine if there were any differences in the Fab sequences. The sequencing result showed that the Fabs had the same DNA sequence and were thus the same clone that had enriched during the panning. Due to this, only one clone was chosen for EC50 determination.

[0183] 2.3 EC50 of G9

[0184] The half maximal effective concentration, EC50, for Fab G9 was determined with an ALP-ELISA. 15 different concentrations of testosterone were used. Based on the regression analysis, the EC50 value was 570.2 pM (Figure 7) .

[0185] 2.4 Binding comparison of G9 to two different ICs

[0186] Fab G9 was analysed for its binding to an IC between testosterone and 77_Fab. The binding was compared to the binding to the IC used in the panning (Anti- T-testosterone) . The binding was higher to the 77_Fab- testosterone IC than to the IC used in the panning (Figure 8 ) .

[0187] Example 2. Plate-based UNCP assay

[0188] UCNP conjugation with G9

[0189] Fab G9 is conjugated to PAA-coated upconverting nanoparticles (UCNPs) as described in (Raiko et al., 2021) . Plate-based assay test

[0190] The functionality of the UCNPs is tested in a platebased assay. The total volume is 200 pL per well for all assay steps and the incubations are done for one hour with low shaking at room temperature, followed by four washes with a plate washer. One hour before use, the UCNPs are diluted in dilution buffer (0.2% milk powder, 0.08% native mouse IgG, 0.05% denatured mouse IgG, 1 mM KF, 0.05% PAA) . 100 ng of biotinylated anti-T is coated on a white streptavidin plate and incubated. Then, the UCNP dilutions are bath sonicated and added to the wells, along with testosterone in different concentrations (0, 0.1, 10 and 1000 nM in triplicates) . After incubation and washing off unbound UCNPs, the upconver- sion luminescence (UCL) is measured with a luminescence spectrometer with parameters described in Raiko et al. (2021) [for example Plate Chameleon microplate reader (Hidex, Finland) equipped with a laser] . When the signal increases with increased concentrations of testosterone, the functionality of the UCNPs is verified.

[0191] After functionality verification, the same assay is performed with multiple known testosterone concentration points. Based on the signals emitted from the known testosterone standards, a standard curve is drawn. The same assay principle is applied for measuring testosterone in patient serum samples. Instead of adding testosterone, the patient sample is added. The signals emitted from the measurements are compared to the standard curve and converted into a concentration. The concentration of testosterone in the patient sample can thus be determined.

[0192] Example 3. Lateral flow assay

[0193] Preparation of lateral flow strips

[0194] Conjugate pads (16 mm) are blocked with blocking buffer (10 mM borate buffer pH 7.5, 0.1% Tween-20, 0.5% BSA, 50 mM EDTA, 0.05% PAA, 0.22 pM filtered) and dried in +35 °C overnight. Test lines (0.5 mg / mL streptavidin) and control lines (0.5 mg / mL anti-ALP) in printing buffer (PB buffer, pH 7.3, 100 mg / mL amaranth) are printed on nitrocellulose membrane (25 mm) using an in-house line dispenser (Olo Health Oy, Turku, Finland) . The lines are printed 5 mm apart, starting 10 mm from the front end of the membrane. After printing, the cards are dried overnight in +35 °C. The conjugate pad and membrane along with a 34 mm wide wicking pad are attached to backing plastic and covered with cover tape overlapping with the conjugate pad. 4.8 mm wide strips are cut with BioDot CM5000 guillotine cutter (BioDot, USA) and stored in room temperature protected from humidity and light .

[0195] Dipstick testosterone assay

[0196] G9 UCNPs prepared as disclosed in Example 2 are diluted in assay buffer (AB, 0.05 M Tris pH 7.5, 500 mM NaCl, 2 mM KF, 1.4 % BSA. 0.05 % PAA, 0.04 / NaN3) one hour before use and bath sonicated right before being used in the assay. 100 ng biotinylated anti-T is preincubated with 45 pL of standards with known testosterone concentrations between 0 and 3.47 nM (concentrations covering the whole reference range for testosterone) in assay buffer in a 96-well microtitration plate in triplicates. Incubation is done for 15 minutes with low shaking. The lateral flow (LF) strips are dipped in the wells for 9 minutes and then transferred to wells with 20 pL AB. After 5 minutes, the strips are dipped in 20 pL UCNP dilution (100 ng per well) for 9 minutes. After the incubation, the strips are washed by dipping in 70 pL AB. When dry, the upconversion luminescence (UCL) is measured with Ronia (Revvity, Turku, Finland) .

[0197] Based on the signals emitted from the known testosterone standards, a standard curve is drawn. The same assay principle is applied for measuring testosterone in patient serum samples . Instead of pre-incu- bating biotinylated anti-T with testosterone , biotinylated anti-T is pre-incubated with the patient sample . The signals emitted from the measurements are compared to the standard curve and converted into precise concentrations . The concentration of testosterone in the patient sample can thus be determined .

[0198] Example 4 . Homogenous TR-FRET assay

[0199] Homogeneous immunoassays without washing or separation steps are attractive detection tools for their simplicity, rapidity, and minimal instrumentation requirement . The two antibodies involved in the immunocomplex-based recognition of a low molecular weight analyte are brought into close interaction, making the f luorescence / Forster resonance energy transfer ( FRET ) process an excellent basis for signal generation in a homogeneous assay . By labeling the Fab antibody pair with suitable donor and acceptor labels , the energy transfer due to the binding of the anti-immune complex Fab can be detected as the acceptor fluorescence . TR- FRET , using lanthanide ion-containing chelate or UCP as the donor compound, can improve assay sensitivity as compared to the standard FRET proces s . The energy from a donor fluorophore is excited to a higher energy state and transferred to an acceptor fluorophore via intermo- lecular dipole-dipole coupling, occurring only if the donor and acceptor are close enough to each other ( 1-10 nm) and the fluorescence spectra of the donor and the acceptor are matched . By labeling the Fab antibody pair with suitable donor and acceptor labels , the energy transfer due to the binding of the anti-testosterone complex Fab can be detected as the acceptor fluorescence . This assay ' s simplicity makes it suitable for different rapid on-site tests for challenging samples , thus demonstrating an immune complex assay ' s potential for detecting testosterone directly from, e . g . , whole blood or other unprocessed biofluids .

[0200] Example 5. Time-resolved fluorescence (TR-F) assay

[0201] The performance of the present anti- IC antibodies in a TR-F assay format was studied as described below .

[0202] 5. 1 . Materials and methods

[0203] Fab G9 was labeled with a 50 -fold molar excess of N1 -EU3+chelate in 50 mM carbonate buffer (pH 9 ) . After an overnight incubation at room temperature in the dark, the labeled antibodies were separated from the free chelate by fast protein liquid chromatography ( FPLC) . The labeling degree was 2 Eu3+molecules per Fab molecule .

[0204] All TR-F immunoassays had a similar setup : 77 Fab ( 50 - 100 ng) was conj ugated on streptavidin wells and incubated shaking for one hour . After washing, the sample (diluted to 20 % in assay buffer) or free testosterone was added and the reaction was incubated shaking for one hour . After another wash, Eu-labeled G9 ( 50 - 100 ng) was added and incubated shaking for one hour . The TR-F was measured after incubating 10 minutes with DEL- FIA Enhancement Solution, using a standard europium protocol with the excitation wavelength of 340 nm and emission wavelength of 615 nm . The final volume in each well was 200 uL .

[0205] The half-maximal effective concentration (EC50 ) for clone G9 was determined using 12 different concentrations of testosterone ranging between 0 and 25 000 pM . 77 Fab ( 100 ng) was conj ugated on streptavidin wel ls and after a 1 h incubation testosterone and Eu-labeled G9 ( 100 ng) were added to each well . The TR- F was measured after incubating 10 minutes with DELFIA Enhancement Solution . The limit of detection and limit of quantitation were calculated based on the mean of the background + 3 x SD and 10 x SD, respectively . 5.1.1 Plasma and serum experiments

[0206] To reduce the effect of endogenous free testosterone on the experiments using real samples, the samples were stripped of hormones as described in Sikora et al. (2016) . Briefly, the samples were incubated with dextran-coated charcoal for 12 hours at +4 °C, whereafter the charcoal was pelleted by centrifugation at 10 000 g for 15 minutes and the sample was filtered with a 0.22 urn filter .

[0207] The effect of the sample matrix on the assay performance was analyzed using separate pools of charcoal-stripped serum and plasma samples. The samples were diluted to 2.5, 5, 10, 20, and 40% in assay buffer and spiked with 100-1000 pM testosterone in triplicates. Next, three individual charcoal-stripped plasma samples were spiked with 100-1300 pM testosterone and the recovery capability of the assay was analyzed. The results were compared to a standard curve of 0-8000 pM testosterone in assay buffer.

[0208] The imprecision of the assay was evaluated by analyzing one male and one female sample in triplicate on two separate days. The charcoal-stripped samples were spiked with 100-1000 pM testosterone.

[0209] The ability of the TR-F immunoassay to discern between male and female samples was analyzed by directly measuring plasma samples diluted to 20% in assay buffer (Figure 11) .

[0210] 5.2 Results

[0211] The half-maximal effective concentration (EC50) for the TR-F immunoassay was determined using 12 concentration points of testosterone ranging between 0 and 25000 pM. The EC50 for the assay was 600.7 pM (Figure 9A) . The LoD was 14.9 pM and the LoQ was 35.8 pM (figure 9B) . These values indicate the capability of the assay to detect testosterone at low concentrations. Given that physiological free testosterone levels range from 160 to 800 pM in men and 9 to 30 pM in women, the assay is well-suited for measuring male testosterone levels . However, for female samples , where concentrations are significantly lower, the LoD of 14 . 9 pM suggests that the assay may detect testosterone near the lower end of the reference range , but the LoQ of 35 . 8 pM indicates that precise quantification may be challenging, particularly for samples with very low concentrations . This highlights the need for careful interpretation of results when measuring free testosterone in female samples .

[0212] The effect of the matrix on the assay performance was determined using charcoal-stripped plasma and serum samples pooled from different donors . Adding plasma to the immunoassay led to only minimal changes to the signal levels ( Figure 10 ) . Meanwhile , the presence of serum affected the signal levels more signifi cantly, and only plasma samples were therefore used for the subsequent experiments .

[0213] To analyze the recovery capability of the immunoassay, three individual charcoal-stripped plasma samples were spiked with variable amounts of testosterone ( Table 3 ) . Higher recoveries were observed in the male samples , most l ikely due to incomplete removal of endogenous testosterone .

[0214] Table 3. Recovery of testosterone from spiked plasma samples. The recovery capability of the testosterone TR- F immunoassay was assessed using tree charcoal-stripped plasma samples spiked with 50-800 pM testosterone.

[0215] Spiked (pM) Plasma

[0216] Sample 1 Sample 2 Sample female) (male) (male)

[0217] Found CV% Found CV% Found

[0218] 50 4.3 23.6 7.9 23.6

[0219] 100 3.7 49.0 5.5 47.8

[0220] 200 59.7 7.4 157.2 0.9 183.0

[0221] 400 92.1 2.8 378.1 1.6 257.1

[0222] 800 364.3 3.5 917.2 5.4 578.8

[0223] The intraassay, interassay, and total imprecision of the were assessed with immunoassay charcoalstripped male and female samples.

[0224] The immunoassay could clearly differentiate between male and female samples, even when the plasma was diluted to 20% (Figure 11) .

[0225] REFERENCES

[0226] Lehmusvuori et al. (2012) . Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes. BioTechniques, 53(5) , 301- 303.

[0227] Leivo et al. (2019) Development of anti-immunocomplex specific antibodies and non-competitive time- resolved fluorescence immunoassay for the detection of estradiol. Analytical and bioanalytical chemistry, 411, 5633-5639.

[0228] Raiko et al. (2021) . Supersensitive photon up- conversion based immunoassay for detection of cardiac troponin I in human plasma. Clinica Chimica Acta, 523, 380-385.

[0229] Valjakka et al. (2002) . Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-tes- tosterone Fab in Complex with Testosterone. IMPROVED AFFINITY RESULTS FROM SMALL STRUCTURAL CHANGES WITHIN THE VARIABLE DOMAINS. J. Biol. Chem. , Vol. 277, No. 46, pp. 44021-44027.

Claims

CLAIMS1. An anti-immunocomplex (anti-IC) antibody specific for an immunocomplex formed between testosterone and an anti-testosterone primary antibody, wherein the anti-IC antibody enables detection of free testosterone in a sample at a half maximal effective concentration (EC50) of less than 1000 pM.

2. The anti-IC antibody according to claim 1, having no substantial specificity towards free testosterone or to the anti-testosterone antibody as such.

3. The anti-IC antibody according to claim 1 or 2, having a light chain variable region comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO : 2 , and CDR3 of SEQ ID NO: 3; and a heavy chain variable region comprising CDR1 of SE ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6, or a mutant thereof having one or more conservative sequence variations that do not substantially affect binding specificity of the anti-IC antibody .

4. The anti-IC antibody according to any one of claims 1-3, comprising a light chain variable region comprising SEQ ID NO: 7, and a heavy chain variable region comprising SEQ ID NO: 8, or a mutant thereof having one or more conservative sequence variations that do not substantially affect binding specificity of the anti-IC antibody .

5. The anti-IC antibody according to any one of claims 1-4, comprising a light chain polypeptide comprising SEQ ID NO: 9, and a heavy chain polypeptide comprising SEQ ID NO: 10, or a mutant thereof having one or more conservative sequence variations which do not substantially affect binding specificity of the anti-IC antibody .

6. The anti-IC antibody according to any one of claims 1-5, which is in the form of a recombinant IgG or a fragment thereof, preferably a Fab, Fab', Fv, dsFv, scFv, or ds-scFv fragment.

7. The acti-IC antibody according to any one of claims 1-6, comprising a detection tag.

8. The anti-IC antibody according to any one of claims 1-7, wherein the anti-testosterone antibody is : a recombinant full-length IgG antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, an scFv fragment, or a ds-scFv fragment having CDR-L1 of SEQ ID NO: 14, CDR-L2 of SEQ ID NO: 15, CDR-L3 of SEQ ID NO: 16, CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18 and CDR-H3 of SEQ ID NO: 19; or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity; a recombinant full-length IgG antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, an scFv fragment, or a ds-scFv fragment having a light chain variable region of SEQ ID NO: 20 and a heavy chain variable region of SEQ ID NO:21; or a mutant thereof having one or more conservative sequence variations as compared to said sequences, which variations do not substantially affect binding specificity.

9. A method for the preparation of the anti-IC antibody according to any one of claims 1-8, comprising: a) providing an anti-testosterone primary antibody immobilized on a solid support or a carrier, such as a microtiter well or a bead; b) performing a negative selection by reacting the immobilized primary antibody with a recombinant expression library, such as a phage display library; c) collecting a first non-bound fraction of the library;d) contacting the immobili zed primary antibody with testosterone to allow formation of an immunocomplex between said testosterone and the primary antibody; e ) reacting the immunocomplex obtained in step d) with the non-bound fraction of the library obtained in step c) ; f ) removing a second non-bound library, optionally by washing; g) separating and collecting one or more anti- IC antibodies bound to the immunocomplex ; and h) expressing said one or more anti- IC antibodies in any suitable expression system .10 . The method according to claim 9 , wherein the anti- IC antibodies obtained in step g) are subj ected to repeated rounds of steps b) to g) , or to repeated rounds of steps d) to g) prior to carrying out step h) , wherein the number of the repeated rounds is preferably three to five .11 . The method according to claim 9 or 10 , wherein the negative selection step b) is omitted .12 . A non-competitive immunoassay for detecting testosterone in a sample , comprising :- contacting a sample suspected of containing testosterone with an anti-testosterone primary antibody;- allowing the anti-testosterone antibody to form a first immunocomplex with testosterone present in the sample , if any, through specific binding to the testosterone ;- contacting the first immunocomplex with an anti- IC antibody disclosed herein, labelled with a detection tag capable of generating a detectable signal ;- allowing the anti- IC antibody to form a second immunocomplex with the first immunocomplex through specific binding to the first immunocomplex ;- removing any non-bound anti- IC antibody; and- determining that testosterone is present in the sample , if a signal generated by the detection tag can be detected .13 . The immunoassay according to claim 12 , wherein the detection of testosterone includes quantification of testosterone in the sample , the method further comprising :- measuring the intensity of the detectable signal ; and- correlating the measured intensity of the detectable signal to the quantity of testosterone in the sample14 . The immunoassay according to claim 13 , wherein the correlating step is accomplished by plotting a calibration curve based on known testosterone concentrations on the x-axis and corresponding signal intensities on the y-axis , wherein the quantity of testosterone in the sample is determined using the calibration curve and the measured signal intensity .15 . The immunoassay according to any one of claims 12 - 14 , wherein the anti-testosterone primary antibody is immobili zed on a solid surface .16 . An immunoassay kit for detecting or quantifying testosterone in a sample , comprising :- an anti- IC antibody according to any one of claims 1 - 8 ; and- an anti-testosterone antibody, wherein at least one of the anti- IC antibody and the anti-testosterone antibody is labelled with a detection tag .17 . The immunoassay kit according to claim 16, further comprising a calibrator, one or more controls , a washing solution and / or a buffer solution .18 . The immunoassay kit according to claim 16 or 17 , wherein the anti-testosterone primary antibody has any feature disclosed in claim 8 .

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