阅读说明：本技术 可溶性和免疫反应性寨卡病毒ns1多肽 (Soluble and immunoreactive Zika virus NS1 polypeptide ) 是由 E.法茨 M.格勒克 P.明希 A.里德尔 C.舒尔茨 G.塔巴雷斯 于 2018-04-23 设计创作，主要内容包括：本发明涉及适合于检测分离的生物样品中的针对寨卡病毒的抗体的多肽,其包含寨卡病毒NS1侧翼结构域特异性氨基酸序列及其变体,其中没有另外的寨卡病毒特异性氨基酸序列存在于所述多肽中。该多肽不与针对来自蜱传脑炎病毒、登革病毒1-4、西尼罗病毒、黄热病毒或日本脑炎病毒的结构相关抗原产生的抗体免疫交叉反应,但与针对全长寨卡病毒NS1抗原产生的抗体免疫反应。还公开了产生可溶性和免疫反应性寨卡病毒NS1多肽的方法以及用于检测分离的样品中的对于寨卡病毒特异性的抗体的方法和试剂盒。(The present invention relates to polypeptides suitable for detecting antibodies to Zika virus in an isolated biological sample comprising Zika virus NS1 flanking domain-specific amino acid sequences and variants thereof, wherein no additional Zika virus-specific amino acid sequences are present in the polypeptides. The polypeptide does not immunologically cross-react with antibodies raised against structurally related antigens from tick-borne encephalitis virus, dengue virus 1-4, west nile virus, yellow fever virus or japanese encephalitis virus, but is immunologically reactive with antibodies raised against the full-length zika virus NS1 antigen. Also disclosed are methods of producing soluble and immunoreactive Zika virus NS1 polypeptides and methods and kits for detecting antibodies specific for Zika virus in an isolated sample.)

1. A polypeptide suitable for detecting antibodies to zika virus in an isolated biological sample comprising amino acid sequences specific for the flanking domains of zika virus NS1, wherein no amino acid sequence from the zika virus NS1 β -ladder domain is present in the polypeptide.

2. The polypeptide of claim 1, wherein the Zika virus NS1 flanking domain-specific amino acid sequence consists essentially of SEQ ID No.1 or 2, and wherein no amino acid sequence from the Zika virus NS1 β -ladder domain is present in the polypeptide.

3. The polypeptide of any one of claims 1 or 2, wherein no additional Zika virus-specific amino acid sequences are present in the polypeptide.

4. The polypeptide of any one of claims 1 to 3, wherein said Zika polypeptide is fused to a chaperone.

5. The polypeptide of any one of claims 1 to 4, wherein the chaperone is selected from SlyD, SlpA, FkpA, and Skp.

6. The polypeptide of claim 5, consisting of SEQ ID NO 21.

7. The polypeptide of any one of claims 1 to 6, wherein the Zika NS1 polypeptide is not immunologically cross-reactive with an antibody raised against a structurally related antigen comprising any of SEQ ID NOs 5 or 6 from tick-borne encephalitis virus and/or comprising any of SEQ ID NOs 7 to 14 from dengue virus 1-4 and/or comprising any of SEQ ID NOs 15 or 16 from West Nile virus and/or comprising any of SEQ ID NOs 17 or 18 from yellow fever virus and/or comprising any of SEQ ID NOs 19 to 20 from Japanese encephalitis virus, but is immunologically cross-reactive with an antibody raised against a full length Zika virus NS1 polypeptide according to SEQ ID NOs 3 in an immunoassay for detecting antibodies to Zika virus.

8. A method of producing soluble and immunoreactive zika virus NS1 flanking domain polypeptides, the method comprising the steps of:

a) culturing a host cell transformed with an expression vector comprising an operably linked recombinant DNA molecule encoding the Zika virus NS1 flanking domain polypeptides according to any one of claims 1 to 7,

b) expressing the Zika virus NS1 polypeptide, and

c) purifying the Zika virus NS1 polypeptide.

9. Method for the detection of antibodies specific for Zika virus in an isolated sample, wherein a Zika virus polypeptide according to any one of claims 1 to 7 or a Zika virus polypeptide obtained by the method according to claim 8 is used as a capture agent for the anti-Zika virus antibody and/or as a binding partner for the anti-Zika virus antibody.

10. A method for detecting antibodies specific to Zika virus in an isolated sample, the method comprising

a) Forming an immunoreaction mixture by mixing a body fluid sample with a Zika virus polypeptide according to any one of claims 1 to 7 or a Zika virus polypeptide obtained by the method of claim 8,

b) maintaining the immunoreaction mixture for a period of time sufficient to immunoreactive antibodies to the Zika virus polypeptide present in a sample of bodily fluid with the Zika virus polypeptide to form an immunoreaction product; and

c) detecting the presence and/or concentration of any of the immunoreaction products.

11. The method for detecting antibodies specific for Zika virus in an isolated sample according to any one of claims 9 or 10, wherein the antibodies detected are IgG or IgM antibodies.

12. Use of the Zika virus polypeptide according to any one of claims 1 to 7 or the Zika virus polypeptide obtained by the method of claim 8 in an in vitro diagnostic test for the detection of anti-Zika virus antibodies.

13. A kit for detecting anti-zika virus antibodies comprising a zika virus polypeptide according to any one of claims 1 to 7 or a zika virus polypeptide obtained by the method of claim 8.

14. Kit according to claim 13, comprising at least microparticles coated with avidin or streptavidin and a polypeptide according to any one of claims 1 to 7 or obtained by the method according to claim 8 covalently coupled to biotin in separate containers or in separate compartments of a single container unit.

15. The kit according to claim 13, comprising at least microparticles coated with avidin or streptavidin and a μ -capture binding partner covalently coupled to biotin in separate containers or in separate compartments of a single container unit.

16. A method of detecting antibodies to Zika virus in an isolated biological sample putatively containing antibodies to at least one other non-Zika flavivirus by using a Zika virus NS1 polypeptide comprising the complete or partial sequence of the beta-ladder domain as a specific binding partner, wherein cross-reactivity to said non-Zika flavivirus is eliminated by: adding a polypeptide comprising the NS1 β -ladder domain of the zika virus in an unlabeled form as a quencher, and in one embodiment, adding the polypeptide comprising the β -ladder domain as a quencher.

Summary of the invention:

the present invention relates to polypeptides comprising an amino acid sequence specific for the NS1 domain of Zika virus and variants thereof, wherein no amino acid sequence from the NS1 β -ladder domain of Zika virus is present in said polypeptides, and their use in immunoassays, suitable for detecting antibodies against Zika virus in an isolated biological sample. In one embodiment, the Zika virus NS1 domain-specific amino acid sequence consists essentially of SEQ ID No.1 or 2. In another embodiment, no additional Zika virus-specific amino acid sequences are present in the polypeptide. The polypeptide does not immunologically cross-react with an antibody raised against a structurally related antigen from tick-borne encephalitis virus comprising the NS1 polypeptide of dengue virus 1-4, West Nile virus, yellow fever virus or Japanese encephalitis virus, but is immunologically reactive with an antibody raised against the full length Zika virus NS1 antigen according to SEQ ID NO: 3.

In one embodiment, the polypeptide does not cross-react immunologically with antibodies raised against a structurally related antigen from tick-borne encephalitis virus comprising any of SEQ ID NOs 5 or 6 and/or from dengue virus 1-4 comprising any of SEQ ID NOs 7 to 14 and/or from west nile virus comprising any of SEQ ID NOs 15 or 16 and/or from yellow fever virus comprising any of SEQ ID NOs 17 or 18 and/or from japanese encephalitis virus comprising any of SEQ ID NOs 19 to 20, but is immunologically reactive with antibodies raised against the full length zika virus NS1 antigen according to SEQ ID No. 3.

Also disclosed are methods of producing soluble and immunoreactive Zika virus NS1 polypeptides, and methods of detecting antibodies specific for Zika virus in an isolated sample using the Zika virus NS1 polypeptide as a binding partner for anti-Zika virus antibodies as described above. These methods detect IgG or IgM class antibodies or both. Further disclosed is the use of the NS1 Zika virus polypeptide in an in vitro diagnostic test for detecting anti-Zika virus antibodies.

A further aspect is a kit for detecting anti-zika virus antibodies comprising a zika virus polypeptide as previously specified.

Another aspect is a method of detecting antibodies to zika virus in an isolated biological sample putatively containing antibodies to at least one other non-zika flavivirus by using a zika virus NS1 polypeptide comprising the complete or partial sequence of the beta-ladder domain as a specific binding partner, wherein cross-reactivity to the non-zika flavivirus is eliminated by: adding a polypeptide comprising the NS1 β -ladder domain of the zika virus in an unlabeled form as a quencher, and in one embodiment, adding the polypeptide comprising the β -ladder domain as a quencher.

Description of the disclosed amino acid sequences:

the mature NS1 protein contains 352 amino acid residues (NS1, 1-352). Within the NS1 protein, the flanking domain comprises 151 amino acid residues and spans the NS1 amino acid region 30-180. Thus, by adding 29 amino acid positions, the flanking domain positions (1-151) are easily converted to the NS1 numbering. Vice versa, by subtracting up to 29 amino acid positions, the NS1 amino acid position is easily converted to flanking domain numbering; aa 1 (flank) = aa 30 (NS1), aa 2 (flank) = aa 31 (NS1), aa 3 (flank) = aa 32 (NS1), and so on. In a similar manner, the β -ladder domain positions (1-162) were readily converted to the NS1 numbering by adding 190 amino acid positions, since the 191-352 positions of NS1 correspond to the β -ladder domain.

SEQ ID NO:1Zika virus NS1 flanking domain aa 30-180, position 179X= A or S or C

SEQ ID NO:2Zika virus NS1 flanking domain aa 30-180 with C55, C143, C179A

SEQ ID NO:3Zika virus NS1 full length aa 1-352; this sequence is also disclosed as strain MR766, UniProt IDW8Q7Q 3; the corresponding number in the full-length Zika precursor polyprotein is aa 795-

SEQ ID NO:4Zika virus NS1 beta-ladder domain aa 191-352

SEQ ID NO:5Tick-borne encephalitis (FSME) virus NS1 flanking domain aa 30-180 according to UniProt ID P14336; a european subtype strain Neudoerfl;X= A or C or S

SEQ ID NO:6:Tick-borne encephalitis (FSME) virus NS1 full-length aa 777-1128 according to UniProt ID P14336; a european subtype strain Neudoerfl; 352 amino acids

SEQ ID NO:7Aa 30-180 flanking domain of dengue virus type 1 NS 1;X= A or C or S

SEQ ID NO:8Dengue virus type 1 NS1 full length aa 776-1127, 352 amino acids based on UniProt ID W8FUV0

SEQ ID NO:9Aa 30-180 flanking domain of dengue virus type 2 NS 1;X= A or C or S

SEQ ID NO:10According to the dengue virus type 2 NS1 full-length aa 776-one 1127 of the UniProt ID P29990 strain Thailand/16881/1984, 352 amino acids

SEQ ID NO:11Aa 30-180 flanking domain of dengue virus type 3 NS 1;X= A or C or S)

SEQ ID NO:12Dengue virus type 3 NS1 full length aa 774-1125, 352 amino acids based on UniProt ID W8FRG8

SEQ ID NO:13Aa 30-180 flanking domain of dengue virus type 4 NS 1;X= A or C or S)

SEQ ID NO:14According to the dengue virus type 4 NS1 full-length aa 775-1126 of UniProt ID Q58HT7, strain Philippines/H241/1956, 352 amino acids

SEQ ID NO:15West nile virus NS1 flanking domain aa 30-180;X= A or C or S

SEQ ID NO:16West Nile Virus NS1 full-length aa 788-1139, 352 amino acids according to UniProt ID P06935

SEQ ID NO:17Aa 30-180 flanking domain of yellow fever virus NS 1;X= A or C or S

SEQ ID NO:18The full-length aa 779-1130 strain 17D vaccine of NS1 yellow fever virus based on UniProt ID P03314

SEQ ID NO:19Japanese encephalitis virus NS1 flanking domain aa 30-180；X= A or C or S

SEQ ID NO:20Japanese encephalitis virus NS1 full-length aa 795-1146, 352 amino acids according to UniProt ID Q9YJ16

SEQ ID NO:21Fusion protein of tandem escherichia coli SlyD and Zika virus NS1 flanking domain aa 30-180

SEQ ID NO:22Fusion protein of series connected Escherichia coli SlyD and Zika virus NS1 beta-ladder domain (aa 191-352 strain Mr766)

SEQ ID NO:23Fusion protein of Escherichia coli SlyD and Zika virus NS1 beta-ladder domain (aa 191-352 strain Mr766)

Detailed Description

The currently available ELISA format immunoassay for the detection of anti-zika virus antibodies employs the NS1 antigen as the immunoreactive reagent. However, we found that these assays lack specificity, resulting in a considerable number of false positive results. Surprisingly, by limiting the zika NS1 antigen to its flanking domains (as explained further below), the number of erroneously reactive samples can be significantly reduced while maintaining high sensitivity of the assay.

When samples positive for anti-Zika virus antibodies were tested with two different fragments of the NS1 antigen of Zika (i.e., the so-called "flanking" domain antigen and the so-called "beta ladder" domain antigen), it was evident that both antigens were able to detect anti-Zika antibodies. However, we found that the flanking domain antigen did not cross-react with dengue antibody positive samples, whereas the Zika NS1 β -ladder domain antigen cross-reacts with dengue antibody positive samples, leading to false positive results and false conclusions. Additional blocking experiments with NS1 antigen against zika positive sera and related arboviruses finally showed that these related arbovirus NS1 antigen hardly quenched the zika NS1 flanking domain antigenic signal, whereas the zika NS1 β -ladder domain antigenic signal was significantly quenched. We conclude that competition-related arbovirus NS1 antigen significantly blocks beta-ladder antigen binding to immunoglobulins. Thus, we were able to show that the Zika NS1 flanking domain antigen is less susceptible to immunological cross-reactivity with other arbovirus NS1 homologues. As a result, the zika NS1 flanking domain enables an immunoassay with excellent specificity against zika antibodies that is capable of diagnosing zika virus infection in the presence of other (recent or past) arbovirus infections, in one embodiment, distinguishing zika virus infection from dengue virus infection.

The present invention therefore relates to a polypeptide suitable for detecting antibodies against Zika virus in an isolated biological sample, comprising a Zika antigen having amino acid sequences specific for the flanking domains of Zika virus NS1, wherein no amino acid sequence from the NS1 β -ladder domain of Zika virus is present in the polypeptide. In one embodiment, no additional Zika virus NS 1-specific amino acid sequence is present in the polypeptide, and in one embodiment, no additional Zika virus-specific amino acid sequence is present in the polypeptide. In one embodiment, the Zika virus NS1 domain-specific amino acid sequence consists essentially of SEQ ID No.1 or 2, and in one embodiment, consists of SEQ ID No.1 or 2. In particular, no amino acid sequence from the C-terminal so-called β -ladder domain is present in the polypeptide of the invention. In one embodiment, SEQ ID NO 4 is absent from the polypeptide.

In one embodiment, the invention relates to the use of said polypeptide in an immunoassay method for the detection of antibodies against Zika virus.

The term "Zika virus" is equivalent to the abbreviation "Zika" or "ZIKV"; these acronyms refer to the same virus.

The terms "NS 1", "NS 1 antigen", "NS 1 polypeptide" are used synonymously and refer to the first non-structural antigen within the virus precursor polyprotein and to (unless otherwise specified) the full-length antigen NS 1. The structure of this protein has been described in Akey et al, supra, for West Nile Virus and dengue Virus 2. For Zika NS1, Song et al (supra) have described the structure of the C-terminal domain (amino acid residues 172-352), and Brown et al (supra) have described the complete NS1 three-dimensional structure in further detail. The Zika NS1 sequence comprises 352 amino acids and is shown in SEQ ID NO 3. The term "NS 1-flanking" or "NS 1-flanking domain", "NS 1-flanking variant" or "NS 1-flanking region" refers to a domain within the NS1 polypeptide, and thus is part of the sequence of NS 1. For the Zika NS1 flanking domain, this is exemplified in SEQ ID NO1 and 2. Furthermore, unless further indicated, the terms "polypeptide", "polypeptides", "antigen" and "antigens" are to be understood as synonyms.

The synonymous terms "beta-ladder", "beta-ladder domain" or "ladder-tip antigen" or "ladder tip", "ladder tip domain", "ladder tip polypeptide", "ladder tip antigen" refer to the NS1 domain located adjacent the C-terminus of the flanking domain. For west nile virus and dengue virus 2, this domain has been described in Akey et al, supra. For Zika virus, the NS1 domain has been described by Song et al (supra) and Brown et al (supra). For the Zika NS1 beta-ladder domain, the amino acid sequence is illustrated in SEQ ID NO: 4.

These definitions apply to all arboviruses within this specification. The following arboviruses belonging to the flaviviridae family may be abbreviated as follows: west Nile virus (West Nile, WNV), tick-borne encephalitis virus (TBEV or FSME), dengue virus 1-4 (dengue, four strains of dengue: DENV 1-4), Yellow Fever Virus (YFV), Japanese Encephalitis Virus (JEV).

According to the present invention, the Zika virus NS1 domain-specific amino acid sequence is an amino acid sequence in which no amino acid sequence from the Zika virus NS1 β -ladder domain is present in the polypeptide. In one embodiment, no additional Zika virus-specific amino acid sequences are present in such polypeptides. For example, the Zika NS1 flanking domain polypeptide contains only the flanking domain sequence, and in one embodiment, SEQ ID No.1 or 2. In one embodiment, SEQ ID NO 4 is not present in the polypeptide sequence. In another embodiment, no additional Zika virus-specific amino acid sequence is present in this sequence. The absence of NS1 beta-ladder domain specific sequences, and in one embodiment, the absence of additional zika NS1 specific sequences or additional zika specific sequences, supports the goal of reducing or completely avoiding cross-reactivity with antibodies raised against other arboviruses.

However, variants of Zika NS1 flanking domain polypeptides are also contemplated. These variants can be easily generated by those skilled in the art by conservative or homologous substitution of the disclosed amino acid sequences, such as for example substitution of alanine or serine for cysteine, or valine for isoleucine, or vice versa. The term "variant" in this context also relates to a protein or a protein fragment (i.e., a polypeptide or peptide) that is substantially similar to the protein. For example, modifications such as C-or N-terminal truncations at one or both termini of 1 to 10 amino acids, in one embodiment 1 to 5 amino acids, are within the scope of the claimed flanking domain antigens of zika NS 1. In particular, a variant may be an isoform that shows amino acid exchanges, deletions or insertions compared to the amino acid sequence of the most prevalent isoform of the protein. In one embodiment, such substantially similar proteins have at least 80%, in another embodiment at least 85% or at least 90%, and in yet another embodiment at least 95% sequence similarity to the most prevalent isoform of the protein. The term "variant" also relates to post-translationally modified proteins such as glycosylated or phosphorylated proteins. According to the present invention, the variants are classified as Zika NS1 flanking domain variants as long as the immunoreactivity in the in vitro diagnostic immunoassay is unchanged or largely maintained, i.e., the variants are still able to bind to and detect the anti-Zika antibodies present in the isolated sample, while antibodies raised against other arboviruses are not detected or are detected to a much lesser extent. In addition, the overall three-dimensional structure of the zika polypeptide remains unchanged such that the epitope that was previously present (i.e., in the wild type) and accessible for binding to an antibody is still present and accessible in the variant.

A "variant" is also a protein or antigen that has been modified, for example, by covalent or non-covalent attachment of a label or carrier moiety to the protein or antigen. Possible labels are radioactive, fluorescent, chemiluminescent, electrochemiluminescent, enzymes or others like e.g. digoxigenin (digoxigenin), digoxigenin (digoxigenin) or biotin. Such markers are known to those skilled in the art.

When the provided polypeptide sequence information, designated in the form of SEQ ID NO, is described by the term "consisting essentially of" (i.e. the sequence), this means that the sequence is present as literally set forth, but may also be present as a variant that does not substantially affect the essential characteristics of the polypeptide in terms of immunological binding to an antibody. An example of this is the deletion or addition of only few amino acids at the N-and/or C-terminus of the peptide, and the exchange of similar amino acids, such as, for example, alanine for serine, isoleucine for valine, and vice versa.

The Zika NS1 flanking domain antigens of the present invention were soluble, stable and immunoreactive, i.e., they were suitable as antigens for use in immunological assays. This means that the antigen according to the invention is soluble in physiological buffer conditions, e.g. in phosphate buffered systems at ambient temperature without the addition of detergents. The antigen is also capable of binding to or being recognized and bound by an antibody specific for the Zika NS1 flanking domain (such as, for example, an anti-Zika antibody present in an isolated sample such as human serum).

In one embodiment, the addition of non-zika specific linker or peptide fusion amino acid sequences to the zika NS1 flanking domain polypeptides is possible because these sequences are not specific for anti-zika virus antibodies and do not interfere with in vitro diagnostic immunoassays.

In one embodiment, the Zika NS1 flanking domain antigen may be fused to a chaperone. The terms "fusion protein," "fusion polypeptide," or "fusion antigen" refer to a protein comprising the Zika NS1 flanking domain polypeptide and at least one chaperone-derived protein portion that provides the role of the fusion partner.

Chaperones are well known folding accessory proteins that assist in the folding and maintenance of structural integrity of other proteins. Examples of folding aids are described in detail in WO 03/000877. In accordance with the present invention, a peptidyl-prolyl isomerase class of chaperones such as those of the FKBP family can be used to fuse with the Zika NS1 flanking domain antigen variants. Examples of FKBP chaperones suitable as fusion partners are FkpA, SlyD and SlpA. Other chaperones suitable as fusion partners for the flanking antigens of Zika NS1 are Skp, a trimeric chaperone from the periplasm of E.coli, which does not belong to the FKBP family. It is not always necessary to use the complete sequence of the chaperone. Functional fragments of chaperones (so-called modules or polypeptide binding motifs capable of binding) which still have the desired ability and function can also be used (cf. WO 98/13496).

In a further embodiment of the invention, at least one or at least two modules of the FKBP chaperone (such as e.g. e.coli SlyD, SlpA or FkpA) are used as fusion moiety for expressing the zika NS1 flanking domain antigen. The chaperone Skp can likewise be used as fusion partner. The fusion of the two FKBP-chaperone domains results in increased solubility of the resulting fusion polypeptide. The fusion moiety may be located at the N-terminus or C-terminus or at both termini of the flanking domain antigens of Zika NS1 (sandwich-like).

In one embodiment, the zika NS1 flanking domain antigen is fused to an oligomeric chaperone. Oligomeric chaperones are chaperones that naturally form dimers, trimers or higher order multimers, allowing the assembly of multiple monomeric subunits into well-defined functional quaternary structures through specific non-covalent interactions. Thus, covalently fused antigens are also forced to a higher epitope density. Preferred oligomeric chaperones are FkpA and Skp. The multimeric antigens are particularly useful for detecting IgM antibodies and thus early immune responses immediately following infection.

In one embodiment, the Zika NS1 flanking domain polypeptide is fused to one, two or more chaperone molecules of the bacterium SlyD, SlpA, FkpA or Skp, in one embodiment E.coli SlyD, SlpA, FkpA or Skp. In a further embodiment, the Zika NS1 flanking domain polypeptide consists of SEQ ID NO: 21.

Another embodiment of the invention is Zika NS1 antigen that is not immunologically cross-reactive with an antibody raised against a structurally related antigen from tick-borne encephalitis virus comprising any of SEQ ID NO. 5 or 6 and/or from dengue viruses 1-4 comprising any of SEQ ID NO. 7 to 14 and/or from West Nile virus comprising any of SEQ ID NO. 15 or 16 and/or from yellow fever virus comprising any of SEQ ID NO. 17 or 18 and/or from Japanese encephalitis virus comprising any of SEQ ID NO. 19 to 20, but is immunologically reactive with an antibody raised against the full length Zika NS1 antigen according to SEQ ID NO. 3. In a further embodiment, the Zika NS1 antigen is a Zika NS1 flanking domain antigen, wherein the Zika specific sequence consists essentially of SEQ ID NO:1 or 2, and in one embodiment consists of SEQ ID NO:1 or 2.

The term "non-immune cross-reactivity" means an undesired immunoreactivity that is strongly reduced or completely eliminated. The term "immunological cross-reactivity" has been created to illustrate unwanted binding of immunoglobulins due to the similarity in sequence or structure of the antigen and the immunogen against which the antibody has originally been raised. In one embodiment, the Zika virus NS1 flanking domain polypeptides exhibit complete abolition or strong reduction in immune activity toward an antibody or subset of antibodies raised against the homologous or related corresponding arbovirus NS1 antigen named above, as compared to the full-length Zika virus NS1 polypeptide. In yet another embodiment, the zika virus NS1 flanking domain polypeptides show strongly reduced immunological cross-reactivity towards antibodies or antibody subsets raised against dengue virus, in one embodiment towards antibodies raised against dengue virus types 1, 2, 3, 4. In a further embodiment, the strongly reduced immunological cross-reactivity of the polypeptide of the flanking domain of Zika virus NS1 is also applicable to antibodies or subsets of antibodies raised against yellow fever virus.

The expression "immunogenecity cross-reaction" also refers to the case where in an immunoassay in the form of a double antigen sandwich for the detection of antibodies, the sample antibody (i.e. the analyte antibody) is bound by two specific antigens: one capable of binding to a solid phase and the other carrying a label, the sample antibody being sandwiched between two antigens. In the presence of analyte antibodies, labeled antigen-within the resulting ternary immune complex-is recruited to the solid phase and generates a signal. In the present case, the zika NS1 flanking domain polypeptides were labeled and the measured signal was set to 100%. In a parallel or subsequent experiment, the same assay is performed with another aliquot of the same (positive) sample, and in addition, a non-labeled antigen having an amino acid sequence suspected of competing with the labeled antigen is added to the mixture. In the present case, full-length NS1 polypeptide of TBEV, DENV1-4, WNV, YFV or JEV was added. In another embodiment, an NS1 polypeptide consisting of only the NS1 flanking domain of TBEV, DENV1-4, WNV, YFV or JEV or comprising only the NS1 flanking domain of TBEV, DENV1-4, WNV, YFV or JEV is added. The flavivirus (in this example: Zika) NS1 polypeptide is not susceptible to signal quenching when the signal obtained after measurement is maintained at about at least 70% signal recovery, in one embodiment at least 80% signal recovery, in one embodiment at least 85% signal recovery, and in one embodiment at least 90% signal recovery of the original signal. It is not outweighed by the added antigen and is therefore resistant to potentially cross-reactive species. For illustration, such blocking experiments are described in example 2 (table 2).

In one embodiment, the full length NS1 or NS1 flanking domain peptides from tick-borne encephalitis virus comprising any of SEQ ID NOs 5 or 6 and/or from dengue virus 1-4 comprising any of SEQ ID NOs 7 to 14 and/or from west nile virus comprising any of SEQ ID NOs 15 or 16 and/or from yellow fever virus comprising any of SEQ ID NOs 17 or 18 and/or from japanese encephalitis virus comprising any of SEQ ID NOs 19 to 20 are added in the blocking experiments described above.

In another embodiment, the Zika NS1 flanking domain polypeptides are immunoreactive with an antibody or subset of antibodies raised against the full-length NS1 antigen as set forth in SEQ ID NO. 3. This means that the signal of the flanking domain polypeptide of zika NS1 should be completely quenched (100%) after addition of the full-length zika NS1 polypeptide in the assay setup disclosed above.

The method of how to determine immunological cross-reactivity to Zika-associated virus is further described in example 2.

The zika NS1 polypeptide (flanking and β -ladder domains) and the polypeptides used in the blocking experiments of example 2 can be generated and prepared by recombinant DNA techniques and protein purification techniques known in the art. Another aspect of the invention is therefore a recombinant DNA molecule encoding the Zika NS1 flanking domain antigen, in one embodiment the antigen according to SEQ ID NO1, 2 or 21 and variants thereof as further defined above.

The term "recombinant DNA molecule" refers to a molecule made by combining two otherwise isolated segments of DNA sequences by the manual manipulation of the isolated segments of a polynucleotide by genetic engineering techniques or by chemical synthesis. In doing so, polynucleotide segments having the desired functions can be joined together to generate the desired combination of functions. Recombinant DNA techniques for expressing proteins in prokaryotic or lower or higher eukaryotic host cells are well known in the art. They have been described, for example, by Sambrook et al, (1989, Molecular Cloning: A laboratory Manual).

The recombinant DNA molecule according to the present invention may also contain a sequence encoding a linker peptide of 5 to 100 amino acid residues between the flanking domain antigen of zika virus NS1 and the fusion moiety and also between several fusion moieties. Such linker sequences may, for example, carry proteolytic cleavage sites.

A further aspect of the invention is an expression vector comprising an operably linked recombinant DNA molecule according to the invention, i.e. a recombinant DNA molecule encoding an antigen of the flanking domain of zika virus NS1 and optionally a peptidyl-prolyl isomerase chaperone such as the FKBP-chaperone, wherein the FKBP-chaperone is selected from the group consisting of FkpA, SlyD and SlpA. In an alternative embodiment, the recombinant DNA molecule encodes a fusion protein comprising the flanking domain antigens of zika virus NS1 and Skp. Expression vectors comprising recombinant DNA according to the present invention can be used to express the zika virus NS1 flanking domain antigen in a cell-free translation system or can be used to transform host cells for expression of the zika virus NS1 flanking domain antigen according to methods well known in the art. Another aspect of the invention therefore relates to a host cell transformed with an expression vector according to the invention. In one embodiment of the invention, the recombinant Zika virus NS1 flanking domain antigen was produced in E.coli cells.

An additional aspect is a method for generating soluble, stable and immunoreactive antigens of the flanking domain of Zika virus NS 1. The Zika virus NS1 flanking domain antigen can be produced as a fusion protein comprising the Zika virus NS1 flanking domain antigen and a chaperone. Preferably, chaperones are used, such as Skp or peptidyl prolyl isomerase class chaperones like FKBP chaperones. In a further embodiment of the invention, the FKBP chaperone is selected from SlyD, FkpA and SlpA.

The method comprises the following steps:

a) culturing a host cell transformed with the above expression vector containing a gene encoding an antigen of the NS1 flanking domain of Zika virus

b) Expressing the genes encoding the antigens of the NS1 flanking domains of Zika virus

c) Purifying the Zika virus NS1 flanking domain antigen.

Optionally, as an additional step d), it is necessary to perform a functional solubilization to bring the antigen of the flanking domain of Zika virus NS1 into a soluble and immunoreactive conformation by means of refolding techniques known in the art.

Yet another embodiment is a method for producing soluble, stable and immunoreactive antigens of the flanking domain of Zika virus NS1 in a cell-free in vitro translation system.

An additional aspect of the present invention relates to a method for detecting anti-Zika antibodies in an isolated human sample, wherein Zika virus NS1 flanking domain antigens according to the present invention are used as binding partners for the antibodies. The invention therefore encompasses a method for detecting antibodies specific for zika virus in an isolated sample, the method comprising a) forming an immunoreaction mixture by mixing a sample of bodily fluid with zika virus NS1 flanking domain antigens according to the invention, b) maintaining the immunoreaction mixture for a period of time sufficient to allow antibodies present in the sample of bodily fluid against the zika virus NS1 flanking domain antigens to immunoreactive with the zika virus NS1 flanking domain antigens to form an immunoreaction product; and c) detecting the presence and/or concentration of any of the immunoreaction products.

In a further aspect, the method is suitable for detecting antibodies to Zika virus of the IgG and IgM subclasses or both classes in the same immunoassay.

Immunoassays for the detection of antibodies are well known in the art, and methods and practical applications and procedures for carrying out such assays are also well known in the art. The zika NS1 antigen according to the present invention can be used to improve assays for detecting anti-zika antibodies independent of the label used and independent of the mode of detection (e.g., radioisotope assay, enzyme immunoassay, electrochemiluminescence assay, etc.) or assay principle (e.g., test strip assay, sandwich assay, indirect test concept or homogeneous assay, etc.).

In one embodiment of the present invention, the immunoassay is a microparticle-based immunoassay using microparticles as a solid phase. "particle" as used herein means a small, localized object that can be attributed to a physical property such as volume, mass, or average size. The microparticles may thus be of symmetrical, spherical, substantially spherical or spherical shape, or of irregular, asymmetrical shape or form. The size of the particles contemplated by the present invention may vary. In one embodiment, the particles used are in the form of spheres, for example particles having diameters in the nanometer and micrometer range. In one embodiment, the microparticles used in the method according to the present disclosure have a diameter of 50 nanometers to 20 micrometers. In a further embodiment, the microparticles have a diameter of 100nm to 10 μm. In one embodiment, the microparticles used in the method according to the present disclosure have a diameter of 200nm to 5 μm or 750nm to 5 μm.

The microparticles as defined above may comprise or consist of any suitable material known to the person skilled in the art, for example they may comprise or consist essentially of an inorganic or organic material. Generally, they may comprise, consist essentially of, or consist of a metal or an alloy of metals or organic materials, or comprise, consist essentially of, or consist of carbohydrate components. Examples of contemplated particulate materials include agarose, polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals, alloys or composites. In one embodiment, the microparticle is a magnetic or ferromagnetic metal, alloy or composition. In further embodiments, the material may have specific properties, and for example be hydrophobic or hydrophilic. Such particles are typically dispersed in aqueous solutions and retain a small negative surface charge, keeping the particles separated and avoiding non-specific aggregation.

In one embodiment of the invention, the microparticles are paramagnetic microparticles and the separation of such particles is aided by magnetic forces in the measurement method according to the present disclosure. A magnetic force is applied to pull the paramagnetic or magnetic particles out of the solution/suspension and retain them as desired, while the liquid of the solution/suspension may be removed and the particles may be washed, for example.

All biological fluids known to the expert can be used as isolated samples for the detection of anti-Zika antibodies. The sample generally used is a bodily fluid such as whole blood, serum, plasma, urine or saliva, in one embodiment serum or plasma.

A further embodiment of the invention is an immunoassay for the detection of anti-zika antibodies in an isolated sample, performed according to the so-called double antigen sandwich concept (DAGS). Sometimes, this assay concept is also referred to as a double antigen bridge concept, since the two antigens are bridged by the antibody analyte. In such assays, the ability of an antibody to bind at least two different molecules of a given antigen with its two (IgG, IgE), four (IgA), or ten (IgM) paratopes is required and utilized.

In more detail, an immunoassay for the determination of anti-zika antibodies according to the double antigen bridge format was performed by incubating a sample containing anti-zika antibody with two different zika virus NS1 flanking domain antigens, namely a first ("solid phase" or "capture") zika virus NS1 flanking domain antigen and a second zika virus NS1 flanking domain antigen ("detection" or "reporter") antigen, wherein each of the antigens specifically binds to the-zika antibody. The first antigen may be bound directly or indirectly to a solid phase and typically carries an effector group which is part of a bioaffinity (bioaffine) binding pair.

One type of bioaffinity binding pair suitable for the method according to the invention is a hapten and anti-hapten antibody binding pair. Haptens are organic molecules having a molecular weight of 100-. Such small molecules may be rendered immunogenic by coupling them to a carrier molecule and anti-hapten antibodies may be produced according to standard procedures. The hapten may be selected from sterols, bile acids, sex hormones, corticosteroids, cardiac glycosides, cardiac glycoside-glycosides, bufadienolides, steroid-sapogenines and steroid alkaloids, cardiac glycosides and cardiac glycoside-glycosides. Representative of these substance classes are digoxigenin (digoxigenin), digoxigenin (digitoxin), digoxigenin (gitoxigenin), strophanthin (strophanthin), digoxigenin (digoxin), digoxigenin (digitoxin), ditoxin and strophanthin (strophanthin). Another suitable hapten is, for example, fluorescein. In one embodiment, the bioaffinity binding pair comprises biotin and avidin/streptavidin or digoxigenin (digoxin) and anti-digoxigenin.

In yet another embodiment, the first antigen is conjugated to biotin and the complementary solid phase is coated with avidin or streptavidin. The second antigen carries a label which alone or in complex with other molecules confers a specific detection capability on the antigenic molecule. Thus, an immunoreaction mixture is formed comprising the first antigen, the sample antibody and the second antigen. This ternary complex, consisting of an analyte antibody sandwiched between two antigenic molecules, is called an immune complex or immune reaction product. The solid phase to which the first antigen can bind is added before the sample is added to the antigen, or after the immunoreaction mixture is formed. Maintaining the immunoreaction mixture for a period of time sufficient to allow an anti-Zika antibody in the body fluid sample directed against the Zika virus NS1 flanking domain antigen to immunoreactive with the Zika virus NS1 flanking domain antigen to form an immunoreaction product. The next step is a separation step, in which the liquid phase is separated from the solid phase. Finally, the presence of any of the immunoreaction products is detected in either the solid phase or the liquid phase or both.

In the DAGS immunoassay, the basic structures of the "solid phase antigen" and the "detection antigen" are essentially identical. In the double antigen bridge assay, similar, but different Zika virus NS1 flanking domain antigens, which are immunologically cross-reactive, may also be used. The essential requirement for performing such assays is that the relevant epitope or epitopes are present on both antigens. According to the present invention, the same or different fusion portions (e.g., SlyD fused to the zika virus NS1 flanking domain antigen on the solid phase side and, e.g., FkpA fused to the zika virus NS1 flanking domain antigen on the detection side) can be used for each zika virus NS1 flanking domain antigen, because such variations significantly alleviate the problem of non-specific binding, thus reducing the risk of false positive results.

A further embodiment is a method for detecting class M anti-Zika virus antibodies (i.e., immunoglobulins) (IgM detection). In one embodiment of this method, the zika NS1 flanking domain polypeptide as further disclosed above is applied in a manner such that multivalent IgM antibodies present in the sample specifically bind to the zika NS1 flanking domain antigen. In one embodiment, the zika NS1 flanking domain antigen is provided in multimeric form by chemically cross-linking the antigen or by fusing the antigen to an oligomeric molecule such as an oligomeric chaperone, in one embodiment, FkpA or Skp. In another embodiment, the Zika flanking domain antigens are present in multiple forms by serially linking the individual antigens adjacent to each other. These individual antigen portions may also be separated by linker molecules that are not specific for Zika. In a further embodiment, multiple zika antigens linked in tandem may additionally be multimerized by an oligomerizing molecule such as an oligomeric chaperone, such as, for example, FkpA or Skp. In yet another embodiment, the Zika NS1 flanking domain polypeptides are used in multimeric form, wherein each polypeptide is present in at least repetitive form, and in one embodiment, it is present three to ten times.

In yet another embodiment of the IgM detection method of Zika-antibodies, the IgM class antibodies present in the sample are bound to a solid phase by a so-called mu-capture component, which is typically a binding partner or an antibody fragment that specifically binds to the Fc portion of human IgM molecules, irrespective of the specificity of the IgM molecules. The μ -capture component carries an effector group (such as biotin), which is a moiety with a bioaffinity pair of avidin or streptavidin. In one embodiment, other bioaffinity pairs may also be used, such as, for example, digoxigenin (digoxin) and anti-digoxigenin or additional haptens and anti-haptens as described further above. In one embodiment, the solid phase coated with avidin or streptavidin is then attracted to and binds the μ -capture component. For specific detection of Zika-specific antibodies, the Zika NS1 flanking domain polypeptides described above were used in labeled form for detection of IgM anti-Zika antibodies.

Another embodiment is the use of a Zika NS1 flanking domain polypeptide as detailed above in an in vitro diagnostic test, in one embodiment an immunoassay as defined above, for the detection of anti-Zika virus antibodies.

As a further embodiment, the maximum total duration of the immunoassay method for the detection of zika virus antibodies is less than 1 hour, i.e. less than 60 minutes, in one embodiment less than 30 minutes, in one further embodiment less than 20 minutes, in one embodiment between 15 and 30 minutes, in one embodiment between 15 and 20 minutes. The duration includes aspiration of the sample and reagents required to perform the assay and incubation time, optional washing steps, detection steps and final output.

An additional subject of the invention is a kit for detecting antibodies against Zika virus comprising the Zika NS1 flanking domain polypeptides disclosed above. In one embodiment, the kit comprises at least microparticles coated with avidin or streptavidin, and the zika NS1 flanking domain polypeptide as previously detailed, in separate containers or in separate compartments of a single container unit. In another embodiment, the microparticles are coated with one partner of other bioaffinity pairs as further described above, such as, for example, digoxigenin (digoxin) and anti-digoxigenin, hapten and anti-hapten. In one embodiment, the Zika NS1 flanking domain polypeptide is covalently coupled to biotin. In one embodiment, the zika NS1 flanking domain is covalently coupled to a second partner of another bioaffinity pair, such as, for example, digoxin and anti-digoxin, hapten and anti-hapten. In another embodiment, the Zika NS1 flanking domain polypeptide is covalently coupled to a detectable label, in one embodiment to an electrochemiluminescent complex. In a further embodiment, chemiluminescent labels, such as, for example, acridinium esters or radioactive or fluorescent compounds or enzymes may be used as labels. In yet another embodiment, the kit comprises in separate containers or in separate compartments of a single container unit at least a microparticle coated with avidin or streptavidin, a first zika NS1 flanking domain polypeptide covalently coupled to biotin and a second zika NS1 flanking domain polypeptide covalently coupled to a detectable label, e.g., an electrochemiluminescent ruthenium complex or an electrochemiluminescent iridium complex.

A further embodiment is a kit for the detection of anti-tacha antibodies of the IgM class comprising in separate containers or in separate compartments of a single container unit at least microparticles coated with avidin or streptavidin and a μ -capture binding partner covalently coupled to biotin. In a further embodiment, the IgM detection kit additionally comprises a zika NS1 flanking domain polypeptide covalently coupled to a detectable label, in one embodiment to an electrochemiluminescent complex.

The term single container unit relates to the fact that: for many automated analyzers, such as the Elecsys Analyzer series from Rochediagnostics, the reagents required to measure a certain analyte are provided in the form of "reagent packs," i.e., as one container unit mounted on the analyzer and containing all the key reagents required to measure the target analyte in different compartments.

In addition, the kits defined above contain control and standard solutions and one or more reagents in solution with usual additives, buffers, salts, detergents, etc. as used by one of ordinary skill in the art, along with instructions for use.

In yet another aspect, the invention relates to a method for detecting antibodies to Zika virus in an isolated biological sample putatively containing antibodies to at least one other non-Zika flavivirus, such as, for example, dengue virus. In this method, a Zika virus NS1 polypeptide comprising the complete or partial sequence of the β -ladder domain is used as a specific binding partner, i.e., not only the NS1 flanking domain. In this setup, cross-reactivity against other non-zika flaviviruses can be expected due to the presence of beta-ladder domain peptide sequences in the specific binding partners. To eliminate this interference, a polypeptide comprising only the NS1 β -ladder domain of the zika virus was added in an unlabeled form, such that cross-reactive antibodies that are not of the zika origin bind and quench. In one embodiment, a β -ladder domain is added as a quencher, and in a further embodiment said β -ladder domain polypeptide consists essentially of SEQ ID No. 4, and in one embodiment of SEQ ID No. 4.

The following embodiments are also part of the present invention:

1. a polypeptide suitable for detecting antibodies to zika virus in an isolated biological sample comprising amino acid sequences specific for the flanking domains of zika virus NS1, wherein no amino acid sequence from the zika virus NS1 β -ladder domain is present in the polypeptide.

2. The polypeptide of embodiment 1, wherein no additional Zika virus-specific amino acid sequences are present in the polypeptide.

3. The polypeptide of any of embodiments 1 or 2, wherein the Zika virus NS1 flanking domain-specific amino acid sequence consists essentially of SEQ ID No.1 or 2.

4. The polypeptide of any one of embodiments 1 to 3, wherein the Zika virus NS1 flanking domain specific amino acid sequence consists of SEQ ID No.1 or 2.

5. The polypeptide of embodiments 1-4, wherein the Zika virus NS1 flanking domain specific amino acid sequence may be truncated by 1 to 5 amino acids at its N-terminus or C-terminus or both.

6. The polypeptide of embodiments 1-5, wherein the Zika virus NS1 flanking domain-specific amino acid sequences may be modified by conservative amino acid substitutions such that the immunoreactivity of the Zika polypeptide remains unchanged or largely maintained, and in one embodiment, such that the three-dimensional structure of the Zika polypeptide remains unchanged.

7. The polypeptide of any one of embodiments 1 to 6, wherein said Zika polypeptide is fused to a chaperone.

8. The polypeptide according to any one of embodiments 1 to 7, wherein the chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp.

9. The polypeptide according to embodiment 8, consisting essentially of, and in one embodiment consisting of SEQ ID NO 21.

10. The polypeptide of any one of embodiments 1-9, wherein the zika NS1 polypeptide is not immunologically cross-reactive with an antibody raised against a structurally related antigen comprising any of SEQ ID NOs 5 or 6 from tick-borne encephalitis virus and/or comprising any of SEQ ID NOs 7 to 14 from dengue virus 1-4 and/or comprising any of SEQ ID NOs 15 or 16 from west nile virus and/or comprising any of SEQ ID NOs 17 or 18 from yellow fever virus and/or comprising any of SEQ ID NOs 19 to 20 from japanese encephalitis virus, but is immunologically cross-reactive with an antibody raised against a full length zika virus NS1 polypeptide according to SEQ ID No. 3 in an immunoassay for detecting antibodies to zika virus.

11. A method of producing soluble and immunoreactive zika virus NS1 flanking domain polypeptides, the method comprising the steps of:

a) culturing a host cell transformed with an expression vector comprising an operably linked recombinant DNA molecule encoding the Zika virus NS1 polypeptide according to any one of embodiments 1 to 10,

b) expressing the Zika virus NS1 polypeptide, and

c) purifying the Zika virus NS1 polypeptide.

12. Method for the detection of antibodies specific for Zika virus in an isolated sample, wherein a Zika virus polypeptide according to any one of embodiments 1 to 10 or a Zika virus polypeptide obtained by a method according to embodiment 11 is used as a capture agent for the anti-Zika virus antibody and/or as a binding partner for the anti-Zika virus antibody.

13. A method for detecting antibodies specific to Zika virus in an isolated sample, the method comprising

a) Forming an immunoreaction mixture by mixing a body fluid sample with a Zika virus polypeptide according to any one of embodiments 1 to 10 or a Zika virus polypeptide obtained by the method of embodiment 11,

b) maintaining the immunoreaction mixture for a period of time sufficient to immunoreactive antibodies to the Zika virus polypeptide present in a sample of bodily fluid with the Zika virus polypeptide to form an immunoreaction product; and

c) detecting the presence and/or concentration of any of the immunoreaction products.

14. The method for detecting antibodies specific for Zika virus in an isolated sample according to any one of embodiments 12 or 13, wherein the antibodies detected are IgG antibodies.

15. The method for detecting antibodies specific to Zika virus in an isolated sample according to embodiment 13 or 14, wherein the immune reaction is carried out in a double antigen sandwich format, the method comprising

a) Adding to the sample a first Zika virus polypeptide according to any one of embodiments 1 to 10 or a polypeptide produced by the method of embodiment 11 and a second Zika virus polypeptide according to any one of embodiments 1 to 10 or produced by the method of embodiment 11, which first Zika virus polypeptide can be bound directly or indirectly to a solid phase and which first Zika virus polypeptide carries an effector group that is part of a bioaffinity binding pair and which second Zika virus polypeptide carries a detectable label, wherein the first and second Zika virus polypeptides specifically bind to the anti-Zika virus antibody,

b) forming an immunoreaction mixture comprising the first Zika virus polypeptide, the sample antibody, and the second Zika virus polypeptide, wherein a solid phase carrying the corresponding effector group of the bioaffinity binding pair is added before, during, or after forming the immunoreaction mixture,

c) maintaining the immunoreaction mixture for a period of time sufficient to immunoreactive Zika virus antibodies directed against the first and second Zika virus polypeptides in a sample of bodily fluid with the first and second Zika virus polypeptides to form an immunoreaction product,

d) separating the liquid phase from the solid phase

e) Detecting the presence of any of the immunoreaction products in the solid phase or the liquid phase or both.

16. The method for detecting an antibody specific for Zika virus according to embodiment 15, wherein said first Zika virus polypeptide carries a biotin moiety and said second Zika virus polypeptide is labeled with an electrochemiluminescent moiety, in one embodiment with a ruthenium or iridium complex.

17. The method for detecting antibodies specific for zika virus in an isolated sample according to any one of embodiments 12 or 13, wherein the antibodies detected are IgM antibodies.

18. The method for detecting antibodies of the IgM class specific for zika virus of embodiment 17, wherein the zika NS1 flanking domain polypeptides are used in multimeric form, in one embodiment wherein the polypeptides are present in at least repetitive form, in one embodiment three to ten times.

19. The method according to any one of embodiments 12 or 13, wherein the detected antibodies are IgM antibodies, and wherein the IgM antibodies are captured on a solid phase by a μ -capture binding partner.

20. The method for detecting IgM antibodies specific for zika virus in an isolated sample according to any one of embodiments 17 to 19, wherein the immune reaction is carried out in a μ -capture format, the method comprising:

a) adding to the sample a μ -capture binding partner that can bind directly or indirectly to a solid phase and a Zika virus polypeptide according to any one of embodiments 1 to 10 or produced by the method according to embodiment 11, and the μ -capture binding partner carries an effector group that is part of a bioaffinity binding pair, and the Zika virus polypeptide carries a detectable label,

wherein the mu-capture binding partner specifically binds to the Fc portion of human IgM antibody and the Zika virus polypeptide specifically binds to the anti-Zika virus antibody,

b) forming an immunoreaction mixture comprising the mu-capture binding partner, the sample antibody and the Zika virus polypeptide, wherein a solid phase carrying the corresponding effector group of the bioaffinity binding pair is added before, during or after forming the immunoreaction mixture,

c) maintaining the immunoreaction mixture for a period of time sufficient to immunoreactive IgM antibodies directed to the Zika virus polypeptide in a sample of bodily fluid with the Zika virus polypeptide to form an immunoreaction product,

d) separating the liquid phase from the solid phase,

e) detecting the presence of any of the immunoreaction products in the solid phase or the liquid phase or both.

21. The method according to any one of embodiments 12 to 20, wherein the method does not use Zika NS1 antigen from the β -ladder domain, in one embodiment does not use a polypeptide comprising an amino acid sequence according to SEQ ID NO. 4.

22. Use of a Zika virus polypeptide according to any one of embodiments 1 to 10 or a Zika virus polypeptide obtained by the method of embodiment 11 in an in vitro diagnostic test for the detection of anti-Zika virus antibodies.

23. Use of the Zika virus polypeptide according to any one of embodiments 1 to 10 or the Zika virus polypeptide obtained by the method of embodiment 11 in an in vitro diagnostic test for the detection of anti-Zika virus antibodies according to any one of the methods of embodiments 12 to 21.

24. A kit for detecting anti-zika virus antibodies comprising a zika virus polypeptide according to any one of embodiments 1 to 10 or a zika virus polypeptide obtained by the method of embodiment 11.

25. Kit according to embodiment 24, comprising at least in separate containers or in separate compartments of a single container unit microparticles coated with avidin or streptavidin and a polypeptide according to any one of embodiments 1 to 10 or obtained by a method according to embodiment 11 covalently coupled to biotin.

26. The kit according to embodiment 24, comprising at least in separate containers or in separate compartments of a single container unit microparticles coated with one partner, such as a hapten/anti-hapten, and a polypeptide according to any one of embodiments 1 to 10 or obtained by the method according to embodiment 11, wherein the bioaffinity pair is a hapten/anti-hapten, in one embodiment digoxigenin (digoxin)/anti-digoxigenin.

27. The kit according to embodiment 24, further comprising a second polypeptide according to any one of embodiments 1 to 10 or obtained by the method according to embodiment 11, said second polypeptide carrying a detectable label.

28. The kit according to embodiment 24, comprising at least microparticles coated with avidin or streptavidin and a μ -capture binding partner covalently coupled to biotin in separate containers or in separate compartments of a single container unit.

29. The kit of embodiment 24, wherein said Zika virus polypeptide carries a detectable label.

30. A method of detecting antibodies to Zika virus in an isolated biological sample putatively containing antibodies to at least one other non-Zika flavivirus by using a Zika virus NS1 polypeptide comprising the complete or partial sequence of the beta-ladder domain as a specific binding partner, wherein cross-reactivity to said non-Zika flavivirus is eliminated by: adding a polypeptide comprising the NS1 β -ladder domain of the zika virus in an unlabeled form as a quencher, and in one embodiment, adding the polypeptide comprising the β -ladder domain as a quencher.

31. The method of embodiment 30, wherein the added polypeptide comprising Zika virus NS1 beta-ladder domain consists essentially of SEQ ID NO. 4, and in one embodiment consists of SEQ ID NO. 4.

32. Use of the NS1 beta-ladder domain of zika virus, in one embodiment consisting essentially of SEQ ID NO:4, in one embodiment consisting of SEQ ID NO:4, as an agent for reducing interference in an immunoassay for detecting anti-zika virus antibodies.

The invention is further illustrated by the examples.