IMMUNE ASSAY FOR DETECTING BIOLOGICALLY ACTIVE PROTEINS

Description

TECHNICAL AREA

[0001] The present invention relates to an immunoassay for detecting a biologically active protein, a device in which such an immunoassay is arranged, the use of an auxiliary protein, and a method for detecting a biologically active antigen. STATE OF THE ART

[0002] There are many different immunoassays for the analysis of an individual's body fluids, such as whole blood, serum, saliva, milk, or urine.

[0003] One problem with common immunoassays that use antibodies is that the antibodies are of animal origin. Antibodies are produced either in animals or in animal cell cultures. CHO (Chinese hamster fallopian tube cells) and HEK-293T (human embryonic kidney cells) are primarily used. This cell culture-based production is very expensive, so existing capacities are mainly used for the production of high-priced, therapeutic antibodies. Antibodies used in diagnostics have much lower profit margins and are therefore still frequently produced in animals. Besides the associated animal suffering, such animal-produced antibodies also suffer from low binding reliability, limited availability, and the risk of contamination with human pathogens.Currently, there are no technical solutions that would allow the cost-effective production of purely vegan assays in economically relevant production quantities.

[0004] Alternatives for producing antibodies, as well as other complex proteins, are quite limited. Bacteria such as Escherichia coli They cannot carry out the necessary post-translational modifications to the antibodies, and production rates are extremely low. The same applies to yeast- or Pichia-based production systems. Here, too, the antibodies are rarely folded correctly by the host cells, and production rates are similarly low to those in bacteria. Transgenic plants have been discussed as alternative producers for many years, but here, too, the very low production rates preclude economic viability.

[0005] There are attempts to use higher plants to produce antibodies, as disclosed, for example, in US patent 6,080,560 A. Further approaches are disclosed in publications, for example, by Melnik Stanislav et al. ( DOI: 10.1111 / pbi. 12746 ) and Ayala Marta et al. ( DOI: 10.1007 / 978-1-59745-407-0_7The problem with these plants is that antibody production rates in plants are generally very low, since typically only cells from certain plant tissues have integrated the antibody genes into their genome. This means that only specific tissues produce small amounts of antibodies. The plant is a chimera; it contains many tissues that have not incorporated any antibody genes, further reducing the yield. For an antibody to be produced over several generations, it must also be ensured that the germline cells have integrated the antibody genes into their genome to guarantee continuous production. Finally, continuous production requires self-propagation or grafting of the transgenic plants. With "natural" sexual reproduction, the transgene would no longer be present after a few generations.

[0006] An alternative method uses agroinfiltration with genetically modified plant viruses, which are used to infect plants. A problem with these plants is that they are transiently modified, meaning the antibody genes are not integrated into any of the plant genomes. This means they can only stably express antibodies for one generation. With this approach, virtually every cell in the plant can produce the antibodies as the virus spreads throughout the plant. However, there is no integration into the plant genome, and the plant does not survive this procedure, dying within a few days. This means that consistently high antibody production cannot be guaranteed, and legally, it always involves the generation of new transgenic plants, with all the associated regulatory implications.

[0007] Furthermore, purification is problematic because higher plants produce significant amounts of fibrous material, which makes the separation and isolation of antibodies difficult and is therefore not suitable for providing antibodies of comparable quality and quantity to the animal alternatives.

[0008] Furthermore, several research documents and patent specifications for the expression of antibodies from diatoms are already known. Examples in the literature for secreting antibodies from diatoms include those by Hempel. et al. ( DOI: 10.1186 / 14 75-2859-11-126 ), Samuels et al. ( DOI: 10.1038 / s41598-022-11053-7 ) and Hempel et al. ( DOI: 10.1371 / JOURNAL.PONE.0028424) published works. A common feature of these approaches is that these antibodies are expressed extracellularly, which has the unfortunate disadvantage that these methods are unable to produce homogeneous, uniform antibodies of high quality and in technically feasible quantities that can be used commercially as an alternative to established antibodies from animal sources. This is also the case with the following patent specifications. The production of antibodies in Phaeodactylum tricornutum,a microalga, has been described. For example, EP 2 671 950 A1 discloses the expression and secretion of recombinant, fully assembled protein complexes by microalgae. Here, the microalgae are expressed extracellularly, which theoretically leads to easier separation of the proteins; a regrettable disadvantage of this method is the very low production rates, which unfortunately means that the antibodies can only be purified with considerable effort. Patents EP 2 444 495 A1 and

[0009] EP 2 660 323 A1 describes an approach to producing therapeutic antibodies using a transformed microalga called Phaeodactylum tricornutum.The transformed microalga contains a nucleic acid sequence encoding a therapeutic antibody, a functional fragment, or a derivative thereof, coupled to a heterologous signal peptide, all surgically linked to a promoter. These transformed microalgae express the therapeutic antibodies extracellularly into the medium; these are therefore secreted antibodies. Unfortunately, this approach yields only small amounts of therapeutic antibodies, which is due to the inefficient use of extracellular expression. TASK

[0010] The present invention therefore addresses the technical problem of providing an immunoassay that overcomes the disadvantages known from the prior art, in particular the provision of recombinant antibodies from a diatom or single-celled plant in high quality and technically usable quantities. SOLUTION

[0011] To solve the technical problem from the prior art, a preferred embodiment for detecting a biologically active antigen, in particular a hormone, protein or vaccine, in a biological sample from an individual comprises the use of recombinant antibodies, e.g. a first antibody, a second antibody and / or a further antibody, obtained from a diatom, unicellular plant or Viridiplantae, wherein the recombinant antibody has a heterologous diatom-, plant- or microalga-specific signal peptide and / or a glycosylation pattern different from that of a native antibody obtained from an individual (as defined herein).

[0012] According to a preferred embodiment, at least two antibodies, e.g. the first antibody and the second antibody, and especially preferably all antibodies used, were obtained from a diatom, unicellular plant or Viridiplantae.

[0013] The problem is solved by an immunoassay with the features of claim 1, as well as a device and a method with the features of the dependent claims. Further advantageous embodiments can be found in the dependent claims, the description, and the exemplary embodiments. The advantages of the immunoassay and the other components will become apparent from the following further description.

[0014] According to a preferred embodiment of the present invention, an immunoassay for detecting a biologically active antigen, in particular a hormone, protein or vaccine, in a biological sample of an individual is provided, comprising the following components: a sample application area for applying a biological sample from an individual, wherein the biological sample is preferably urine, whole blood, saliva, milk, or serum from an individual; a capture area, wherein the capture area comprises an immobilized first antibody directed against the biologically active antigen, in particular against the hormone, protein, or vaccine; a conjugate area, wherein the conjugate area comprises a second antibody directed against the biologically active antigen, in particular against the hormone, protein, peptide, or vaccine; and optionally a control area comprising an antibody directed against the detection antibody; wherein the first antibody and the second antibody are recombinant antibodies derived from a diatom or unicellular plant, wherein the immunoassay comprises at least one further antibody and / or an auxiliary protein, wherein the auxiliary protein acts as a blocker to saturate free surfaces of a reaction vessel or membrane, wherein the further antibody and the auxiliary protein are derived from a diatom and / or viridiplanta and / or unicellular plant, and wherein the recombinant antibody is provided in a purity of at least 90%, and wherein the immunoassay is vegan.

[0015] Furthermore, it has been revealed that Immunoassay dissolved for the detection of a biologically active antigen, in particular a hormone, protein or drug, in a biological sample from an individual, exhibiting: a sample application area for applying a biological sample from an individual, wherein the biological sample is preferably urine, whole blood, saliva, milk or serum; a capture area, wherein the capture area comprises an immobilized first antibody directed against the biologically active antigen, in particular against the hormone, protein, peptide or drug; a conjugate area, wherein the conjugate area comprises a second antibody directed against the biologically active antigen, in particular against the hormone, protein, peptide or drug; wherein the first antibody and / or the second antibody is a recombinant antibody obtained from a diatom or single-celled plant, wherein the recombinant antibody is provided in a purity of at least 90%, preferably at least 95%. In an alternatively preferred embodiment, the recombinant antibody is provided in a purity of 80 to 99%, preferably 85 to 99%, particularly preferably 90 to 99%, and most preferably 95 to 99%.

[0016] An individual's biological sample can be whole blood, serum, saliva, urine, or milk. Preferably, the individual's biological sample is urine, whole blood, or serum; urine is particularly preferred.

[0017] According to a preferred embodiment of the present invention, the immunoassay is a lateral flow immunoassay. For example, the lateral flow immunoassay comprises a sample application area, a conjugate area, and a capture area arranged on a membrane, wherein a sample application area and a capture area on the membrane are fluidly connected to each other via a flow path, and wherein a conjugate area is arranged in the flow path. For example, the membrane is a nitrocellulose membrane. In the following, the term "embodiment" or "design" or related terms refer to embodiments in the description and refer to the invention only if they fall within the scope of protection defined by the claims.

[0018] Preferably, the immunoassay provides a nitrocellulose membrane.

[0019] Alternatively, preferably the immunoassay is an enzyme-linked immunosorbent assay (ELISA), e.g. a direct ELISA, an indirect ELISA, a direct sandwich ELISA or an indirect sandwich ELISA, preferably the ELISA is a direct sandwich ELISA or an indirect sandwich ELISA.

[0020] It may be useful for the first antibody and / or the second antibody, in particular the mobilized of the two antibodies, to be labeled with a dye and / or an optically active nanoparticle, in particular a gold nanoparticle.

[0021] However, it may be provided that the first antibody and / or the second antibody, in particular the mobilized of the two antibodies, is coupled to an enzyme designed to induce a dye or a luminescence reaction. GENERAL BENEFITS

[0022] For the provision of the immunoassay or a method for detecting a biologically active antigen in a biological sample from an individual, recombinant antibodies are used which are not of mammalian synthetic origin, so that animals or animal cell cultures, in particular mammalian cell cultures, can be advantageously dispensed with.

[0023] Furthermore, by synthesizing / expressing the recombinant antibodies used herein in a diatom, not only can similarly high expression rates be achieved with much lower energy and resource requirements, but the risk of the antibodies being contaminated with human pathogens, as is the case with synthesis / expression in animals or animal cell cultures, is also eliminated. DETAILED DESCRIPTION OF THE INVENTION

[0024] In one embodiment, an immunoassay for detecting a biologically active antigen, in particular a hormone, protein or drug substance in a biological sample from an individual, comprises: a sample application area for applying a biological sample from an individual, wherein the biological sample is preferably urine, whole blood, saliva, milk or serum; a capture area, wherein the capture area comprises an immobilized first antibody directed against the biologically active antigen, in particular against the hormone, protein or drug; a conjugate area, wherein the conjugate area comprises a second antibody directed against the biologically active antigen, in particular against the hormone, protein or drug; wherein the first antibody and / or the second antibody is a recombinant antibody obtained from a diatom, unicellular plant or Viridiplantae, wherein the recombinant antibody contains a heterologous diatom-, plant- or microalga-specific signal peptide.

[0025] In a particularly preferred embodiment, an immunoassay is provided for the detection of a biologically active antigen, in particular for the detection of a hormone, protein or drug substance in a biological sample of an individual, comprising: a sample application area for applying a biological sample from an individual, wherein the biological sample is preferably urine, whole blood, saliva, milk or serum; a capture area, wherein the capture area comprises an immobilized first antibody directed against the biologically active antigen, in particular against the hormone, protein, peptide or drug; a conjugate area, wherein the conjugate area comprises a second antibody directed against the biologically active antigen, in particular against the hormone, protein, peptide or drug; wherein the first antibody and / or the second antibody is a recombinant antibody obtained from a diatom or a single-celled plant, the recombinant antibody being provided in a purity of at least 90%, preferably at least 95%. In an alternatively preferred embodiment, the recombinant antibody is provided in a purity of 80 to 99%, more preferably 85 to 99%, particularly preferably 90 to 99%, and most preferably 95 to 99%.

[0026] A distinction can be made between two different types of contamination: Contamination by non-proteins, for example pigments; contamination by other proteins that are not antibodies.

[0027] Both groups of impurities can disrupt the purification and function of the antibodies, both by negatively affecting the stability of the antibodies and by reducing the specificity of the immunoassays.

[0028] Non-proteins can generally be residues from the separation process, especially cell fragments, components of the culture medium, and, in higher plants, for example, fibers. In the production of antibodies in diatoms, this group primarily includes pigments, which can be detected and easily separated by spectrophotometry because they have a much lower molecular weight than the antibodies and exhibit different chemical properties. Common methods known to those skilled in the art, such as dialysis, ultrafiltration, size exclusion chromatography, or charge-dependent separation (e.g., ion exchange chromatography), are suitable for this purpose. This separation is complex, and the effort required increases, relative to the amount of antibody isolated, as the proportion of antibodies in the culture decreases. After separation, the amount of non-proteins can preferably be determined gravimetrically, and in the case of pigments, particularly preferably spectroscopically.

[0029] Impurities from non-antibody proteins can be analyzed by denaturing SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis), combined with Coomassie staining on the one hand, and immunostaining to identify the antibodies, preferably the antibody chains produced in diatoms, on the other. For this purpose, the protein mixtures isolated from diatoms are separated according to their size after the addition of a suitable buffer and denaturation (10 min at 80 °C). This standard procedure in molecular biology is well known to those skilled in the art (e.g., Reinard, Molecular Biological Methods 2.0 (UTB, p. 229 ff; ISBN 978-3825287955)). The proteins separated by SDS-PAGE are stained with Coomassie, and the color intensity is measured densitometrically and / or compared with a standard. This allows the determination of the amount of protein present in each band.

[0030] In parallel, another SDS-PAGE can be performed with identical sample preparation, but this time the sample is not stained; instead, it is transferred to a nitrocellulose membrane. On this membrane, the bands caused by the two antibody chains are clearly visible. By adding a first, specifically targeted antibody that is labeled (e.g., with biotin, a radioisotope, a reporter enzyme, an oligonucleotide, or a fluorophore), or by adding a second, labeled antibody that is directed against the first antibody, the two chains of the antibody to be detected become clearly visible. Any bands not labeled in this way are contaminating proteins.

[0031] In a preferred embodiment, the ratio of recombinant antibody to total protein is determined by SDS-PAGE after separation and spectroscopic quantification of the non-proteins. This method enables the determination of the absolute amount of antibody, total protein, and non-protein of a recovered recombinant antibody according to the present invention.

[0032] The purity of the purified recombinant antibody provided for an immunoassay according to the present invention is calculated according to the following formula: Reinheit % = Menge des rekombinanten Antikörpers Menge der Gesamtproteine + Menge der Nichtproteine × 100 %

[0033] The purity of the recombinant antibody is determined at the time after purification, before the antibody is applied to an immunoassay in accordance with the present invention or otherwise made available.

[0034] In an alternative embodiment of the present invention, the recombinant antibody is provided in a protein purity of at least 90%, more preferably at least 95%. In an alternatively preferred embodiment, the recombinant antibody is provided in a protein purity of 80 to 99%, more preferably 85 to 99%, more preferably 90 to 99%, and most preferably 95 to 99%.

[0035] The protein purity of the recombinant antibody is calculated using the following formula: Proteinreinheit % = Menge des rekombinanten Antikörpers Menge der Gesamtproteine × 100 %

[0036] In a preferred embodiment, the ratio of recombinant antibody to total protein is determined by SDS-PAGE. The protein purity of the recombinant antibody is determined after purification, before the antibody is applied to an immunoassay according to the present invention or otherwise made available.

[0037] A particularly preferred embodiment is one in which the high production rate of the recombinant antibody according to the invention allows it to be obtained in a high concentration, preferably 20–1000 mg / L culture. Cell debris and diatom-specific proteins can be easily separated because interfering fibers are absent. This provides a recombinant antibody with a purity of at least 90%, preferably at least 95%. Due to the high purity of the antibody, together with the high homogeneity and uniformity of the glycosylation pattern, an antigen can be detected specifically and at a low concentration, i.e., with a low detection limit of the immunoassay, thus achieving an improvement over conventional immunoassays based on animal antibodies.The higher homogeneity of the antibodies reduces the number of non-specific cross-reactions (non-specific signals), making the result of the immunoassay more reliable in accordance with the present invention, while maintaining a consistently high sensitivity of the antibody.

[0038] The higher purity of the antibodies makes them more stable and longer-lasting because antibody-degrading proteases are removed along with the foreign proteins. Furthermore, the purity and homogeneity of the antibodies result in a lower number of non-specific cross-reactions (non-specific signals), leading to more reliable and reproducible results.

[0039] In a particularly preferred embodiment, which in Fig. 13As shown, the additional bands due to non-specific reactivity are clearly visible in the commercially acquired antibody. These bands are absent on the gel pathways of the diatom-derived antibody, demonstrating that it exhibits higher specificity than the commercial, animal-derived antibody. Besides reducing false-positive signals, this also has the advantage of requiring a smaller amount of protein for the immunoassay.

[0040] An immunoassay is a test that uses the binding of antibodies to antigens to identify specific substances and / or quantify the amount of the substance present. Immunoassays can be used to diagnose diseases, but also to analyze the physiological state of an individual, such as a human being. Examples of disease diagnoses include the detection of various cancers and the detection of an infection like COVID-19. Examples of analyzing a physiological state include female conception (ovulation tests) or pregnancy tests. The terms immunoassay and immunoassay are used interchangeably.

[0041] Immunoassays can be used in various technical variations. The best-known variations are the enzyme-linked immunosorbent assay (ELISA or ELISA test) and the lateral flow immunoassay (LFA), in which a membrane, e.g., a paper-based platform, serves for the detection and quantification of analytes in complex mixtures. The biological sample is placed on a test device, and the results are displayed within 5–30 minutes. Well-known examples of antibody-based LFAs are the rapid COVID-19 tests and pregnancy tests.

[0042] For the purposes of this invention, the term "biologically active antigen" (also referred to as "antigen") means any substance, in particular molecules with a molecular weight of approximately 4,000 Daltons or higher, preferably approximately 2,000 Daltons or higher, and most preferably 770 Daltons or higher, that causes the body to initiate an immune response against that substance. Antigens include toxins, chemicals, bacteria, viruses, proteins, peptides, or other substances originating from outside the body. In the humoral immune response of vertebrates, antibodies are produced. These antibodies, produced for the humoral immune response, can also be used independently of an immune response and outside the body to recognize, identify, and / or quantify the antigen. For the purposes of this invention, biologically active antigen and biologically active protein can be used interchangeably.

[0043] From a biological perspective, biologically active antigens are molecules against which an antibody recognizing them exists in an individual (as defined herein). Specifically, this includes molecules that represent proteins and / or hormones from humans and mammals, which function either as markers for a disease in the individual (for example, the diagnosis of breast cancer via the detection of the Herceptin2 receptor) or as markers for the individual's physiological state (for example, pregnancy). Alternatively, and preferably, molecules that represent proteins and / or hormones in animals and / or plants are also included, particularly for food analysis.Biologically active antigens, in particular sequences of hormones, proteins or vaccines or drugs, especially preferably hormones, which either serve as markers for a disease of the individual (for example, diagnosis of breast cancer via the detection of Herceptin2 receptor) or as markers for the physiological state of the individual (for example, pregnancy), are known to those skilled in the art or can be obtained from relevant databases and textbooks.

[0044] Furthermore, proteins from human pathogenic organisms, especially bacteria (e.g., bacterium erythrocytes), are used as biologically active antigens. Streptococcus mutans (these cause tooth decay) or viruses (e.g., the spike protein of Covid 19).

[0045] Preferably, the biologically active antigen is a hormone from an individual, e.g., a human hormone selected from the group comprising cortisol, thyroxine, somatotropin, vasopressin, testosterone, and estrogens. Corresponding native antibodies for the detection of these hormones are known to those skilled in the art or can be found in the relevant literature.

[0046] In a preferred embodiment, the biologically active antigen is a peptide hormone from an individual, e.g., a human peptide hormone selected from the group comprising gonadotropin-releasing hormone (10 amino acids), insulin (A-chain: 21 amino acids, B-chain: 30 amino acids), somatostatin (14 amino acids), and glucagon (29 amino acids). Corresponding native antibodies for the detection of these hormones are known to those skilled in the art or can be found in the relevant literature.

[0047] In a preferred embodiment, the biologically active antigen is a protein hormone of an individual, e.g., a human protein hormone, which can serve, for example, as a physiological marker. Suitable protein hormones are, for example, selected from the group comprising parathyroid hormone (84 amino acids) and hCG (human chorionic gonadotropin: α-subunit: 92 amino acids, β-subunit: 145 amino acids). Corresponding native antibodies for the detection of these hormones are known to those skilled in the art or can be found in the relevant literature.

[0048] Furthermore, the biologically active antigen can be a protein, in particular a human protein, that serves as a disease marker and is selected from the group including Her2 (human epidermal growth receptor 2, which is overexpressed on the surface of cancer cells and represents a cancer marker); IgE antibodies (which are produced by the human body due to an allergic reaction); small signaling proteins, such as interleukin 5 (which is associated with allergic diseases, including allergic rhinitis and asthma); and interferons (which have immunostimulatory, especially antiviral and antitumor, effects and can serve as markers for viral infections). Corresponding native antibodies for the detection of the hormones are known to those skilled in the art or can be found in the relevant literature.

[0049] The biologically active antigen can be a protein of a human pathogenic organism, e.g. the spike protein of a coronavirus, such as SARS-CoV-1 (for the detection of SARS infections) or SARS-CoV-2 (for the detection of a Covid-19 infection) or the HIV-1 nucleocapsid protein (for the detection of an HIV infection).

[0050] In a preferred embodiment, the biologically active antigen is a hormone, a protein, and / or a drug substance. Particularly preferred is a hormone, especially a hormone that serves as a marker for the physiological state of the individual (for example, pregnancy), particularly preferably human chorionic gonadotropin, and most preferably human chorionic gonadotropin.

[0051] A lateral flow immunoassay (hereinafter also referred to as "LFA") typically uses three antibodies: A first immobilized antibody, located in the capture area (also referred to as the test zone) and also called the capture antibody. This antibody, like the second antibody (also referred to as the detection antibody), is directed against the antigen, preferably against a different epitope of the antigen than the detection antibody; a second antibody, preferably located in the conjugate area and also referred to as the detection antibody, which is directed against an epitope of the antigen, e.g., a hormone, a protein, or a peptide such as human chorionic gonadotropin, wherein the second antibody is preferably conjugated to marker particles, e.g., gold, silver, latex, carbon, nanoparticles, fluorescent dyes, or enzymes. A further antibody, preferably located in a control area (control zone), preferably immobilized, and directed against a detection / control antibody.

[0052] Preferably, the first antibody (e.g., the capture antibody), the second antibody (e.g., the detection antibody), and / or the further antibody (e.g., the capture antibody in the control zone), and especially preferably all antibodies, are obtained from a diatom, unicellular plant, or Viridiplantae and are characterized by the characteristics defined herein.

[0053] Particularly preferably, the first antibody (e.g., the capture antibody), the second antibody (e.g., the detection antibody) and / or the further antibody (e.g., the capture antibody in the control zone), and particularly preferably all antibodies, are obtained from a diatom and are characterized by the characteristics defined herein.

[0054] An advantage over the use of animal antibodies lies in the consistent quality and reproducibility of the antibodies used in the test, thus improving the overall quality of the immunoassay. This is because, for example, the antibody-producing animals die after a certain period, and antibodies from a different animal exhibit different properties; that is, the quality of antibodies produced using conventional methods is highly variable. Furthermore, antibodies and auxiliary proteins derived from diatoms, single-celled plants, or viridiplants, unlike polyclonal antibodies from individuals (as defined herein), are precisely defined antibodies, meaning their amino acid sequence is predefined and consistent.

[0055] The term "individual" (here also referred to as "subject"), as used in the present invention, refers to any mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate), in particular rodents, cloven-hoofed animals, ungulates, preferably with odd toes, or primates. In a particularly preferred embodiment, the individual is a primate, in particular a human. Unless otherwise specified, the term "individual" does not denote a specific age and therefore includes adults, elderly individuals, children, and newborns.

[0056] In a preferred embodiment of the immunoassay, the individual is a mammal, preferably a primate, more preferably a human.

[0057] In a preferred embodiment, the recombinant antibodies, e.g., the first antibody and / or the second antibody and / or the further antibody, are obtained by expression from diatoms, green algae (including Chlorobionta), or a seed plant. Particularly preferably, the recombinant antibodies are obtained from diatoms or green algae, especially from diatoms, as e.g., Phaeodactylum tricornutum, obtained. One advantage of this is that antibodies from plants, especially diatoms, cannot contain endogenous pathogens. An example of this is bovine spongiform encephalopathy (BSE), which is why animal antibodies, particularly for therapeutic applications, can pose a risk. Therefore, these must be rigorously tested for potential human pathogens, which is not necessary with the recombinant antibodies according to the invention, especially those obtained from diatoms.

[0058] The term "antibody" here refers to an immunoglobulin (Ig) or an immunoglobulin derivative, such as those produced by the acquired immune system of vertebrates. Examples of naturally occurring antibodies are the M (IgM), G (IgG), A (IgA), and E (IgE) class antibodies, particularly from mammals such as humans, rabbits, mice, rats, camels, llamas, goats, and / or horses. Artificial formats based on such proteins are also included; examples are scFvs and scFv-Fc.

[0059] In some preferred embodiments of the immunoassay, "antibody" herein refers to an immunoglobulin (Ig) or an immunoglobulin derivative such as that produced by the acquired immune system of vertebrates and / or cartilaginous fish. Examples of naturally occurring antibodies are class M (IgM), D (IgD), G (IgG), A (IgA), and E (IgE), NAR (IgNAR), particularly from mammals such as humans, rabbits, mice, rats, camels, llamas, goats, and / or horses, and / or cartilaginous fish such as sharks. Artificial formats based on such proteins are also included; examples include scFvs, scFv-Fc, or single-domain antibodies / nanobodies.

[0060] A "native antibody" is a natural antibody such as is found in an individual as defined herein, in particular a vertebrate, especially preferably a mammal, most especially preferably a primate, in particular a human.

[0061] According to a preferred embodiment, the antibody has at least one variable region and one constant / conserved region.

[0062] Preferably, the variable region is of vertebral, preferably mammalian, origin, particularly preferably human, murine, equine, canine and / or camel-like origin, most preferably human, murine, equine and / or camel-like origin, particularly human, murine and / or equine origin. The variable region may exhibit at least 50% sequence identity in its amino acid sequence to homologous sequence regions of a vertebral, preferably mammalian (as defined above), preferably human, antibody.

[0063] In a preferred embodiment, the constant region is of vertebral, preferably mammalian, and particularly preferably human, canine, murine, equine, goat-like, and / or camel-like origin. The constant region can exhibit at least 50% sequence identity in its amino acid sequence to homologous sequence regions of a vertebral, preferably mammalian (as defined above), and preferably human, antibody.

[0064] The invention discloses the provision of a nucleic acid sequence with an increased expression rate for the production of recombinant proteins, comprising at least one expression cassette for the expression of one or more peptides. According to the invention, the expression cassette has at least one promoter element and at least one first transcription unit encoding a protein, wherein the promoter element consists of the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence with a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, most preferably at least 95%, and further preferably at least 99% to SEQ ID NO: 1.

[0065] The term "promoter" as used in the present invention refers to a polynucleotide sequence located upstream of a gene that regulates the transcription of a functional gene. The promoter forms a recognition and binding site for an RNA polymerase, which initiates the transcription of the gene.

[0066] For the purposes of this invention, the term "homology" refers to the similarity between nucleotide sequences of DNA or RNA and / or between amino acid sequences of proteins.

[0067] In a preferred embodiment, the SEQ ID NO:1 represents a promoter, hereinafter referred to as HASP1 ​​mod<, which has proven to be particularly suitable for regulated protein production. The HASP1 ​​mod< promoter is a promoter element derived from the natural HASP1 ​​promoter, wherein a partial sequence of the natural HASP1 ​​promoter has been duplicated.

[0068] The SEQ ID NO:1 is as follows, where the underlined part represents the duplication:

[0069] The provision of nucleic acid sequences whose promoter element repetitively displays individual HASP1 ​​sequence segments, for example, with respect to the start codon ATG between -100 and -1, between -200 and -101, between -300 and -201, between -400 and -301, and / or between -500 and -401, has been revealed. These segments can be combined in any configuration and copy number. This results in a new sequence that exhibits less than 85% homology to the native HASP1 ​​promoter.

[0070] According to a preferred embodiment, at least one transcription unit comprises a polynucleotide encoding an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence with a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, most preferably at least 95%, further preferably at least 99% to SEQ ID NO:2.

[0071] The SEQ ID NO:2 represents an amino acid sequence of the hinge region of an antibody, derived from equine immunoglobulin sequences and proven to be particularly protease-resistant. The use of this protease-resistant hinge region significantly reduces the proteolysis of manufactured antibodies of various formats and from different species, both in vivo as well as in vitro

[0072] The SEQ ID NO:2 is as follows: VIKEPCCCPKCP

[0073] In a preferred embodiment, a vector or an isolated nucleic acid can be provided, which comprises a nucleic acid according to the invention in simple or repetitive form, as well as the provision of a cell comprising a vector or an isolated nucleic acid or a nucleic acid sequence or an amino acid sequence according to the invention.

[0074] Furthermore, an amino acid comprising SEQ ID NO: 2 or an amino acid sequence with a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, most preferably at least 95%, and more preferably at least 99% to SEQ ID NO: 2 is provided.

[0075] In the present document, the cell is a photosynthetically active cell, in particular a unicellular plant, preferably a diatom.

[0076] Preferably, the recombinant antibody in the joint region has a cysteine-rich amino acid sequence of at least 20 amino acids, preferably at least 15 amino acids, and particularly preferably at least 12 amino acids, wherein at least 33% of the amino acids comprise cysteine. According to a particularly preferred embodiment, the amino acid sequence in the joint region comprises or consists of (a) the sequence VIKEPCCCPKCP, or (b) a sequence identity that differs from this amino acid sequence by a maximum of 30%, particularly by a maximum of 20%, and particularly preferably by a maximum of 15%, or (c) an amino acid sequence that, compared to the variant according to (a), has only one amino acid substitution.

[0077] In a particularly preferred embodiment, the production rate is 20 mg / L to 1000 mg / L, and particularly preferably 30 mg / L to 1000 mg / L of antibodies per liter of culture. This high production rate is essential for the technical application of the antibodies, as separation and purification at values ​​below 20 mg / L is not technically feasible and therefore also essential for economic viability with a reasonable purification effort. These values ​​are within the range for commercially used animal CHO cell cultures ( Chinese Hamster Ovary typical range and thus far exceeds the capacity previously achieved for diatoms, microalgae, single-celled plants and / or viridiplantae. The realization that diatoms or other microalgae, with their production capacities, could become true competitors of CHO cells was not foreseeable until now.

[0078] A particularly preferred embodiment is one in which recombinant antibodies are obtained from a diatom or unicellular plant culture at a concentration of 20 mg / L to 1000 mg / L, preferably 30 mg / L to 800 mg / L, or alternatively at least 30 mg / L to 160 mg / L. This high production rate is the result of the inventors' discovery that various modifications to a diatom can lead to an unexpectedly high increase, for example, by a factor of at least more than 100 compared to the prior art. This enables the technical feasibility of obtaining and purifying antibodies from diatoms that exhibit high purity and homogeneity. The production rate in the diatom could be increased from a conventional maximum of 3 mg of antibody per liter of culture to, for example, at least 160 mg of antibody per liter of culture.Particularly preferably, the production rate in diatoms can be increased from a conventional maximum of 3 mg antibody per liter of culture to 300 mg antibody per liter of culture, representing a factor of 100. In some highly preferred embodiments, the production rate can be increased to up to 1000 mg / L of culture, representing a factor of 333.

[0079] A culture includes the diatoms, culture medium and all other additives necessary for the provision of the recombinant antibodies.

[0080] A culture medium comprises a liquid, preferably an aqueous, saline liquid, which provides suitable nutrients, temperatures, pH values ​​and other conditions that promote the growth and reproduction of cells, particularly preferably of microalgae, even more preferably of diatoms.

[0081] In some formulations, the recombinant antibody is provided at a concentration of at least 100 mg / L culture, preferably at least 250 mg / L; most preferably at least 500 mg / L. This exceeds previously reported values ​​by a factor of at least 33, preferably by at least 83, and most preferably by a factor of 166.

[0082] According to a preferred embodiment of the immunoassay, the glycosylation of the first antibody and / or the second antibody has a modified glycosylation pattern compared to the corresponding native antibody; preferably, the glycosylation has a more homogeneous pattern with a homogeneity factor in the range of 1 to 3; particularly preferably, the glycosylation has a homogeneous, mannose-rich N-glycan pattern with a homogeneity factor preferably in the range of 1 to 3.

[0083] The homogeneity factor is defined as the ratio of the number of baseline-separated, defined peaks in the chromatogram, determined by HPLC (High-Performance Liquid Chromatography) and / or UPLC (Ultra-Performance Liquid Chromatography) coupled with MS and / or HRMS and / or UV / Vis and / or diode array, between an antibody as defined in the present compound, preferably an antibody expressed from a diatom, and the corresponding native antibody and / or an animal antibody. A homogeneity factor of 1 means that there is 1 fewer peak, a factor of 2 means that there are 2 fewer peaks, and so on. In a particularly preferred embodiment ( Figs. 5-7 ) a homogeneity factor of 3 is achieved, the comparison antibodies which originate from a human cell culture ( Fig. 5 and 6 ) each have 6 peaks, while the recombinant antibody according to the present invention has only 3 peaks.

[0084] In a preferred embodiment of the immunoassay, the glycosylation of the first antibody and / or the second antibody exhibits increased homogeneity of the glycosylation pattern compared to the corresponding native antibody, preferably a homogeneous, mannose-rich N-glycan pattern, without requiring additional addition of mannose to the culture medium.

[0085] The differences in antibody glycosylation patterns compared to mammalian systems arise from the presence of various "mannose-rich" N-glycans (from mannose-5 to mannose-9). In contrast to natural mammalian cells, which have a relatively low mannose content, commercially used cell cultures such as CHO cells exhibit a higher mannose content due to the intentional addition of mannose. CHO-based antibodies often display a markedly heterogeneous distribution of glycosylation. Figs. 5-7A clear differentiation can be observed. The consistency of glycosylation is often not present in CHO, Expi, and other animal or human cells or cell cultures (recognizable by the numerous peaks in the outlined area of Figure 5 ).

[0086] This inconsistency affects the specificity and stability of the antibodies. Therefore, mannose is often added to the medium of animal or human cell cultures to achieve greater uniformity (despite the associated disadvantages). Diatoms naturally have a high mannose content. Furthermore, our antibodies exhibit remarkable homogeneity; that is, antibodies derived from different cells in a culture show uniform glycosylation patterns. Figure 5 This significantly reduces the likelihood of undesirable properties such as the recognition of foreign proteins and fluctuations in antibody stability.

[0087] Recombinant antibodies are currently produced either in animals or using human or animal-based expression systems, i.e., eukaryotic cell lines. Mammalian cell systems are preferred, especially when dealing with complex proteins, such as antibodies, where elaborate post-translational modifications are essential for analytical or therapeutic efficacy. These post-translational modifications include the glycosylation patterns of antibodies. A major problem with currently produced recombinant proteins is their inconsistent glycosylation patterns. Recombinant antibodies from currently used expression systems, particularly those derived from individuals, are frequently hyperglycosylated, meaning, for example, that increased amounts of mannose residues are incorporated, often with unusual branching patterns.These can "break down" (degrade) and / or this can lead to the proteins being ineffective, or to unwanted side reactions by the immune system.

[0088] In a preferred embodiment, the glycosylation (also referred to as the glycosylation pattern) of the antibody (as defined herein), e.g., of the first antibody and / or the second antibody and / or each subsequent antibody, differs from that of the corresponding native antibody as it occurs in the individual. Particularly preferred is the glycosylation pattern of the recombinant antibodies (as defined herein) being more homogeneous than the glycosylation pattern of the native antibody as expressed in an individual (as defined herein) (cf. e.g., Fig. 5 and 6 opposite Fig. 7The glycosylation pattern of recombinant antibodies (as defined herein) exhibits, for example, fewer branches than the native antibody, which, as mentioned above, can "terminate" (degrade) and / or (in combination) lead to the antibodies being ineffective or to undesirable side reactions by the immune system. A recombinant antibody disclosed herein (i.e., an antibody derived from diatoms, single-celled plants, or viridiplants) is thus a so-called biosimilar. Such antibodies, whose glycosylation pattern differs from that of the corresponding native antibody, are preferably preferred as markers for an individual's disease or as markers for the individual's physiological state.

[0089] In a preferred embodiment of the immunoassay, the recombinant antibody is a mosaic antibody, wherein the mosaic antibody comprises at least a first sequence selected from at least one first organism and at least a second sequence selected from at least one second organism. The first and second organisms are different organisms. The first sequence can be the heavy chain or a portion thereof, and the second sequence can be the light chain or a portion thereof. The hinge region, as a defined segment of the heavy chain sequence, can be derived either from the same organism as the remainder of the heavy chain sequence or, preferably, from a different organism.

[0090] In an alternative embodiment, the first sequence can be selected from a first and a second organism. In a further alternative embodiment, the second sequence can be selected from a first and a second organism.

[0091] The production of mosaic antibodies results in novel and non-naturally occurring antibodies. These antibodies are meticulously designed using in silico processes, leading to sequences not found in nature. Process optimizations have resulted in the creation of antibody regions that exhibit sequence matches with database sequences from a range of animal and human sources. Consequently, the produced antibodies are mosaic antibodies, or mosaic proteins, containing genetic sequences from various species.

[0092] The disclosed methods represent a departure from conventional methods for producing chimeric antibodies. While chimeric antibodies have already been produced in animal cell cultures, this approach requires the use of transgenic animals. In contrast, the aforementioned method enables the production of chimeric antibodies in diatoms, thus eliminating the need for transgenic organisms, particularly transgenic animals.

[0093] For example, the antibodies synthesized using this method have a basic structure that corresponds to the sequence of human immunoglobulin IgG4.

[0094] In a preferred embodiment, this mosaic protein can have the following outlined structure, consisting of a heavy chain, light chain and hinge region: a) Heavy chain (exemplary structure): CH1 - CH3 - without hinge region at least 80% homologous to human IgG4; for example, CH1 without hinge comprises 116-118 amino acids, CH2 and CH3 together 215-220 amino acids, the hinge region typically comprising 10-14 amino acids. The 80% homology refers to the minimum 331 and maximum 338 amino acids of CH1, CH2, and CH3, which at 80% homology results in a total of 264-270 amino acids. The hinge region corresponds to the protected sequence at least 80%. Further templates for the hinge region, which are particularly resistant to proteolytic degradation, and preferably from the order of Perissodactyla especially preferred the Equidaeoriginates. The variable chain is homologous to the IgG sequences of very different organisms, preferably selected from the list consisting of human, mouse, and rabbit. b) Light chain (Exemplary structure): The constant region of the light chain is homologous to those from the order of Perissodactyla especially preferred the Equidae. Variable chain is homologous to the light chain IgG sequences of very different organisms, preferably selected from the list consisting of humans, mice, and rabbits, but especially preferentially to variable chains of human kappa-type light chains. Here, too, it was found that these have an influence on production levels and protease resistance.

[0095] Preferably, the different combinations lead to an increased or decreased production rate of the target protein or antibody, depending on the sequence used.

[0096] An important aspect is the variability of domain combinations within the sequences. Depending on the chosen sequence arrangements, different production rates of the target protein are achieved. This flexibility makes it possible to adjust protein production to the desired level.

[0097] In summary, this disclosure presents an approach to antibody production using diatoms, generating non-naturally occurring mosaic antibodies and / or mosaic proteins with sequences from multiple species. This innovation avoids reliance on transgenic animals and offers advantages such as customizable protein designs, improved production control, and potential applications in various fields, including diagnostics and therapeutics. In particular, properties such as stability, selectivity, and production rate can be specifically optimized and controlled through the targeted design of these antibodies.

[0098] In a preferred embodiment, the immunoassay comprises at least one further antibody and / or an auxiliary protein, wherein the further antibody and / or the auxiliary protein is preferably obtained from a diatom and / or unicellular plant and / or Viridiplantae and is particularly preferably vegan.

[0099] Vegan within the meaning of the present invention, specifically a vegan immunoassay within the meaning of the present invention, is an assay that is produced using biotechnological processes and methods in accordance with the principles of veganism. This means that these products are developed without the use of animal materials and / or animal cell cultures or by-products, and that no animal testing is carried out in their production.

[0100] In addition to the first and / or second antibody, the immunoassay may also contain a further antibody and / or an additional auxiliary protein. Auxiliary proteins serve, for example, as blockers to saturate the free surfaces (i.e., areas where antibodies are not immobilized and non-specific binding can occur) of a reaction vessel or membrane. Examples of auxiliary proteins that act as blockers include bovine serum albumin (BSA), casein, a modified BSA, or a modified casein, which, for example, saturates free surfaces.

[0101] Auxiliary proteins (as defined herein) can perform several functions: Membrane saturation: The membrane (e.g., the nitrocellulose membrane) binds proteins of any origin with high efficiency. Therefore, antibodies in an immunoassay, such as an LFA, can bind tightly to the membrane. However, between the areas where antibodies saturate the membrane, there are still membrane regions where no protein is bound. Proteins from the sample being tested would bind to these regions and thus lead to false results. If the antigen is immobilized (e.g., in an ELISA), the testing antibodies would bind specifically to the antigen but also non-specifically to the surface. Evaluation would be impossible. These proteins therefore block the protein-binding surfaces, so that antibodies can only bind specifically to their antigens and thus play a central role in immunoassays. These auxiliary proteins are also helpful during the incubation of antibodies and antigen. The excess of auxiliary protein, e.g.,BSA and / or casein from milk ensures that proteases present in the sample, which could degrade antigen or antibody, also have access to BSA or casein as a substrate for proteolysis. Since casein / BSA is present in excess in the solution, the risk of an antibody being accidentally damaged proteolytically is (statistically) lower the more BSA is in the solution.

[0102] According to a particularly preferred embodiment, the immunoassay may, in addition to the recombinant antibody, also include another antibody and / or one or more additional auxiliary proteins. In general, auxiliary proteins serve, for example, to saturate (block) free binding sites on the matrix (i.e., areas where no proteins from the sample to be analyzed are yet immobilized). Since matrices generally bind proteins and antibodies are proteins, antibodies would bind non-specifically to such free sites and would no longer be able to bind to their immobilized antigens. Therefore, in all immunoassays, such areas of a reaction vessel, microtiter plate, or membrane are saturated with one or more auxiliary proteins. Often, particularly in the prior art, such auxiliary proteins are of animal origin, such as bovine serum albumin (BSA) or caseins, which act as blocking agents to saturate free surfaces on the matrix.Casein(e) and Casein(e) are used interchangeably.

[0103] In a particularly preferred embodiment, auxiliary proteins (as defined herein) can perform several functions: Membrane saturation: The membrane (e.g., the nitrocellulose membrane) binds proteins of any origin with high efficiency. Therefore, proteins to be analyzed in an immunoassay, such as antibodies in an LFA, can bind tightly to the membrane. However, between the areas where proteins have bound to the membrane, there are still membrane regions where no protein is bound. In an immunoassay such as ELISA or immunoblot, the antibodies would bind to these unbound regions and would no longer be able to detect their antigens. In LFAs, proteins from the sample being tested would bind to these unbound membrane regions, leading to false results. These auxiliary proteins thus block the protein-binding surfaces, ensuring that antibodies can only bind specifically to their antigens, and therefore play a central role in immunoassays. These auxiliary proteins are also helpful during the incubation of antibodies and antigens.The excess of auxiliary protein, e.g., BSA and / or casein from the milk, ensures that proteases present in the sample, which could degrade antigen or antibody, also have BSA or casein available as a substrate for proteolysis. Since casein / BSA is present in excess in the solution, the risk of an antibody being accidentally damaged proteolytically is (statistically) lower the more BSA is present in the solution.

[0104] Conventionally produced BSA and casein are of animal origin. While initially inexpensive, its purification requires considerable technical effort. In particular, the removal of (human) pathogenic viruses and prions (e.g., BSE, "mad cow disease") is very complex. Other sources, such as bacteria or yeast, cannot produce BSA heterologously because it is not only glycosylated but also contains several post-translationally modified amino acids. The production of vegan BSA from diatoms, unicellular plants, or Viridiplantae, especially diatoms, disclosed herein, therefore has the advantage that the purification and removal of (human) pathogenic viruses and prions are unnecessary.

[0105] The other antibodies or excipients are preferably recombinant antibodies or recombinant proteins which (also as defined herein) are obtained from a diatom, unicellular plant or viridiplantae and therefore have a heterologous diatom-, plant- or microalga-specific signal peptide and / or a glycosylation pattern different from that of a native antibody obtained from an individual (as defined herein).

[0106] In a particularly preferred embodiment, further antibodies or excipients are recombinant antibodies or recombinant proteins which (also as defined herein) are obtained from a diatom or unicellular plant and therefore exhibit a heterologous diatom-, plant- or microalga-specific signal peptide and / or a glycosylation pattern different from that of a native antibody obtained from an individual (as defined herein).

[0107] In a preferred embodiment of the immunoassay, the amino acid sequence of the first antibody and / or the second antibody is that of a vertebral, preferably a mammalian, and particularly preferably a human antibody; or it has an amino acid sequence or consists of an amino acid sequence that exhibits at least 80%, preferably at least 85%, particularly preferably at least 90%, most preferably at least 95%, and in particular at least 97% sequence identity with homologous sequence regions of a vertebral and / or mammalian and / or human antibody. This ensures compatibility with a range of targeted antigens and results in high antibody quality.

[0108] In a preferred embodiment, the nucleotide sequence encoding the first antibody and / or the second antibody is codon-optimized for the host organism from which the first antibody and / or the second antibody is derived, preferably for Phaeodactylum tricornutum codon optimized.

[0109] During codon optimization, the base sequence is also adjusted simultaneously to, for example, facilitate the cloning of the recombinant DNA. One aspect of this is the removal of unnecessary or undesired restriction enzyme recognition sites in the recombinant DNA.

[0110] Also disclosed but not claimed is a nucleic acid that codes for a first antibody, a second antibody, a further antibody and / or an auxiliary protein, wherein the sequence of the nucleic acid is codon-optimized for expression in a diatom, unicellular plant or Viridiplantae, in particular in a diatom.

[0111] A nucleic acid that encodes a first antibody, a second antibody, a further antibody and / or an auxiliary protein is disclosed but not claimed, wherein the sequence of the nucleic acid is codon-optimized for expression in a diatom or unicellular plant, in particular in a diatom.

[0112] The inventors have also developed a method for codon optimization that, unlike conventional methods, does not consider each codon individually. Instead, a position-specific matrix is ​​used to create a codon frequency profile across the entire original sequence. This profile is then used to transfer the codon frequency at each position to the sequence optimized for diatoms. This approach optimizes the folding of antibody chains immediately following translation, as regions that are difficult to fold are translated somewhat more slowly than those that are easily foldable. This directly affects the amount of antibody produced and is also relevant to the high homogeneity of the antibodies produced in diatoms.

[0113] Furthermore, a codon-optimized sequence of nucleic acid for expression in a host organism (as defined herein) has the advantage that the folding of the antibody is improved to match the native counterpart of the antibody, leading to increased stability of the antibody and increased biological activity of the antibody expressed in the host organism (as defined herein).

[0114] Furthermore, a codon-optimized nucleic acid sequence has the advantage that the expression rate in the host organism is increased by at least a factor of 10, preferably at least a factor of 20, particularly preferably at least a factor of 30, and most preferably at least a factor of 40 compared to a non-codon-optimized nucleic acid sequence. For example, this can increase the production rate in diatoms from a conventional maximum of 3 mg antibody per liter of culture to 160 mg antibody per liter of culture. It is particularly preferred that this can increase the production rate in diatoms from a conventional maximum of 3 mg antibody per liter of culture to 100–1000 mg antibody per liter of culture. It is particularly preferred that the production rate in diatoms can be increased from a conventional maximum of 3 mg antibody per liter of culture to 300 mg antibody per liter of culture, which corresponds to a factor of 100.In some highly preferred embodiments, the production rate can be increased to up to 1000 mg / L culture, which corresponds to a factor of 333.

[0115] Preferably, the recombinant antibody is modified in the region of a joint such that it exhibits increased stability against diatom-, plant-, or microalgae-specific proteases, preferably with a stability factor of 1.1 to 5, compared to the native antibody. This increases the antibody yield during purification (also known as the downstream process) and produces fewer interfering degradation products that reduce the purity of the obtained antibodies, resulting in a more homogeneous recombinant antibody from cultivation that provides more specific signals and fewer cross-reactions.

[0116] The increased stability relates to the stability of diatom-derived enzymes, specifically proteases, which can attack the region of the antibody known as the hinge region and enzymatically cleave it. A stability factor associated with the increased time the antibody with the modified hinge region remains stable against proteases under culture conditions, thus increasing production volumes, particularly the yield of functional recombinant antibodies after separation. Since fewer proteolytically cleaved antibody fragments are present, the antibody solution is more homogeneous and allows for more specific detection with significantly fewer cross-reactions.

[0117] In a preferred embodiment, the stability factor between an antibody with a native hinge and a recombinant antibody with a modified hinge, as described in the present invention, is preferably determined by SDS-PAGE. The stability factor can be determined via degradation products of the recombinant antibody that lead to additional bands in the Coomassie stain. By adding specific proteases, preferably diatom-, animal-, human-, plant-, or microalgae-specific proteases, to the recombinant antibody with a modified hinge and the antibody with a native hinge, and performing SDS-PAGE at specific time intervals, it is possible to determine how much intact antibody remains after this time and how many proteolytically generated fragments are detected in the SDS-PAGE.The absolute proportions can be determined densitometrically, and the quotient of the concentration of the recombinant antigen and the native antibody yields the stability factor. In a particularly preferred embodiment, the stability factor is between 1.1 and 10, more preferably between 1.1 and 5, and most preferably between 1.1 and 3.

[0118] According to a preferred embodiment, the amino acid sequence of the antibody, e.g., of the first antibody and / or the second antibody and / or any further antibody, is modified in such a way that it exhibits increased stability against diatom-, animal-, human-, plant- or microalgae-specific proteases.

[0119] For example, the amino acid sequence of the antibody, e.g., the first antibody and / or the second antibody and / or each subsequent antibody, is modified in the hinge region such that it exhibits increased stability against host-specific proteases, particularly those specific to diatoms, plants, or microalgae. This can increase the stability of the antibody in the host organism in which it is expressed, and thus also the yield of intact antibody from the culture.

[0120] A particularly preferred embodiment of the immunoassay is one in which the recombinant antibody is expressed from a stably transformed diatom or unicellular plant, preferably from a stably transformed diatom, over several generations, preferably for at least 60 generations, more preferably for at least 80 generations, and most preferably for at least 100 generations. This ensures high yields with simultaneously high antibody quality, thereby guaranteeing the quality of assays across different batches.

[0121] A generation concludes with a cell division and denotes a unit of cellular replication. For example, 60 generations correspond to 60 successive cell divisions originating from a single parent cell. Similarly, the "generation time" refers to the time interval between two successive generations of organisms in a population. It is the time a single cell or organism needs to divide and produce two new cells or organisms. The generation time is a fundamental parameter that characterizes the growth rate of single-celled organisms. It indicates how quickly a population of genetically identical cells can multiply under optimal conditions (mitosis).Shorter generation times indicate faster growth rates and higher reproductive capacities, while longer generation times indicate slower growth rates and potentially more complex cellular processes.

[0122] In a preferred embodiment, the generation time is between 6 and 48 hours, preferably between 12 and 24 hours. This generation time is significantly shorter than that of higher viridiplantae known from the prior art, such as... N. tabacum. The short generation time allows for faster growth of the culture and thus a significantly increased production capacity for antibodies than is possible with higher plants.

[0123] Transiently modified higher plants are genetically modified plants in which foreign genetic material, such as genes encoding specific proteins or traits, is introduced into the plant cells for a short period. This modification is temporary and does not lead to the integration of the foreign genes into the plant's genome. Instead, the foreign genes are expressed, and the desired traits are produced only for a limited time. This information is thus lost after one generation, resulting in significant effort in cultivating cell cultures and controlling product quality. In contrast, the antibodies according to the present invention can be stably expressed over numerous generations, enabling high quality and controllable conditions.

[0124] A stably transformed culture enables the expression of the antibody for at least 60 generations, preferably for at least 80 and particularly preferably for at least 100 generations.

[0125] In a preferred embodiment, the antibody is stably expressed in a culture for at least 30 days, more preferably for at least 40 days, and most preferably for at least 60 days after inoculation of the culture.

[0126] In addition to the other advantages over higher plants that produce transient or stable antibodies, diatoms do not contain fibers, which greatly facilitates antibody purification.

[0127] An antibody is preferably expressed intracellularly in connection with the immunoassay, ideally in a stably transformed diatom. Previously, extracellular secretion was used in the prior art for obtaining antibodies from diatoms, as this allows the antibodies to be released directly into the culture medium. Theoretically, this would facilitate isolation, but the authors made the unexpected observation that intracellular secretion leads to significantly higher production volumes.

[0128] This outstanding achievement of the inventors, together with the other properties of the antibodies described here, led to a dramatic increase in antibody production, preferably by a factor of 100 or more.

[0129] In a preferred embodiment, the first and / or second antibody can be an antibody directed against human chorionic gonadotropin (hCG), preferably an hCG antibody. hCG is a glycoprotein hormone that is produced even before implantation of the embryo and indicates pregnancy very early on. During pregnancy, it is primarily produced by the placenta. It plays a crucial role in maintaining the corpus luteum, which in turn produces progesterone to support the early stages of pregnancy. Anti-hCG antibodies can be used in various applications, including diagnostic pregnancy tests. Pregnancy tests detect the presence of hCG in urine or blood, indicating pregnancy.These antibodies are used as recognition elements to bind to hCG molecules and generate a measurable signal that confirms pregnancy. This enables the early detection of pregnancy, particularly as point-of-care testing (PoCT). PoC, as used in the present invention, means point-of-care; analogously, PoCT means point-of-care testing. Patient-centered self-diagnosis. This concept refers to medical diagnostic tests that are performed close to the patient, usually outside the traditional laboratory environment.

[0130] In a preferred embodiment, the immunoassay with a recombinant antibody directed against the hCG protein is provided as a lateral flow assay, particularly preferably as a kit. This allows the immunoassay to be made available close to the patient, enabling simple and rapid on-site diagnostics.

[0131] In a preferred embodiment, the biologically active antigen is a part of the epitope of a virus, preferably a pathogenic virus, such as influenza, SARS-CoV-2, RSV, adenovirus, Strep A, norovirus, rotavirus, or HIV. Preferably, the immunoassay utilizes highly specific antibodies that recognize and bind to the selected part of the epitope, enabling rapid and accurate identification of viral infections. This approach facilitates early diagnosis, which allows for timely and targeted treatment, as well as accurate monitoring of the course of infection and the ability to quickly detect outbreaks, ultimately contributing to timely public health responses and effective containment strategies.

[0132] In a preferred embodiment, the biologically active antigen is a tumor-associated sequence, preferably an HLA complex and / or a sequenced part of a tumor epitope and / or a tumor marker, for example, IFN-γ, IL-8, PSA, CEA, AFP, DCP, CA 125, HER2 / neu. This enables early detection and treatment of malignant transformations and can thus drastically increase a patient's chances of recovery. In a preferred embodiment as a lateral flow assay, this can serve as a point-of-care test (POCT) for early diagnosis. In a particularly preferred embodiment as an LFA or ELISA, the immunoassay serves as a diagnostic tool performed by medical personnel in laboratories or medical facilities.

[0133] IFN-γ (interferon-gamma) is a cytokine produced by immune cells in response to infections and plays a key role in immune responses against pathogens. An antibody-based immunoassay enables the precise detection of IFN-γ in patient samples, thus aiding in the diagnosis of immune system disorders, the monitoring of autoimmune diseases, and the assessment of response to immunotherapy.

[0134] IL-8 (interleukin-8) is a chemokine involved in inflammatory responses and the recruitment of immune cells. Detecting IL-8 using antibody-based immunoassays helps to understand inflammatory conditions such as autoimmune diseases, allergies, and infections, and allows for precise monitoring and evaluation of treatment.

[0135] PSA (prostate-specific antigen) is a protein produced by the prostate gland. Elevated levels can indicate prostate problems, including cancer. Antibody-based immunoassays allow for accurate PSA measurement and aid in the early detection of prostate cancer, risk assessment, and monitoring of treatment effectiveness.

[0136] CEA (carcinoembryonic antigen) is a glycoprotein found in elevated concentrations in certain cancers, particularly colorectal cancer. Antibody-based immunoassays allow for the sensitive detection of CEA, which is helpful in cancer diagnosis, monitoring treatment progress, and detecting potential relapses.

[0137] AFP (alpha-fetoprotein) is a protein produced during fetal development. Elevated levels in adults can indicate liver disease or certain cancers, such as liver cancer. An antibody-based immunoassay allows for the accurate detection of AFP, thus supporting early diagnosis and monitoring of treatment success.

[0138] DCP (des-gamma-carboxyprothrombin) is a protein produced by liver cells. Elevated DCP levels are associated with liver cancer. Antibody-based immunoassays allow for accurate measurement of DCP, thus aiding in the early detection of hepatocellular carcinoma and the monitoring of treatment success.

[0139] CA 125 (Cancer Antigen 125) is a protein that is elevated in some cancers, particularly ovarian cancer. Antibody-based immunoassays offer a reliable method for measuring CA 125 levels, which is helpful in diagnosing ovarian cancer, monitoring disease progression, and assessing treatment success.

[0140] HER2 / neu (human epidermal growth factor receptor 2) is a protein involved in regulating cell growth. Elevated levels are associated with certain aggressive types of breast cancer. Detecting HER2 / neu using antibody-based immunoassays helps identify appropriate treatment strategies, predict disease progression, and monitor treatment success in breast cancer patients.

[0141] In a preferred embodiment of the immunoassay, the biologically active antigen is a characteristic sequence for identifying a protein, preferably an enzyme tag, most preferably selected from the list consisting of the 6xHis tag, Strep tag, c-Myk tag, Flag tag, and GST tag. This enables an immunoassay to detect these protein tags and thus provide the specificity and sensitivity to accurately quantify, purify, and characterize proteins in various research contexts.

[0142] The 6xHis tag is a short peptide sequence with six histidine residues, often genetically fused to proteins. This fusion tagging enables efficient purification of the labeled protein using immobilized metal affinity chromatography (IMAC) due to the strong binding affinity between histidine and divalent metal ions, such as nickel. In an antibody-based immunoassay, specific antibodies can recognize and bind to the 6xHis tag, facilitating the detection and quantification of the protein. The technical advantage of the immunoassay lies in its high specificity and sensitivity, allowing for precise measurement of the labeled protein even in complex biological samples.

[0143] The Strep-Tag is a peptide tag characterized by an eight-amino acid sequence (WSHPQFEK) that exhibits a high binding affinity for the streptavidin protein. When fused to a target protein, the Strep-Tag allows for straightforward purification through interaction with streptavidin-coated surfaces. In an antibody-based immunoassay, antibodies that recognize the Strep-Tag can selectively bind to the tag of the desired protein. This approach enables efficient and specific detection of the target protein, making it valuable for various research and diagnostic applications.

[0144] The c-Myc tag is derived from the c-Myc protein and consists of ten amino acids (EQKLISEEDL). It is commonly used as a fusion tag to facilitate the detection and purification of proteins. In antibody-based immunoassays, antibodies against the c-Myc tag can bind specifically to the tag, enabling sensitive detection and quantification of the labeled protein. The technical advantage of this technique lies in its versatility, as it can be used for a wide range of protein studies and assays.

[0145] The flag tag is a peptide sequence (DYKDDDDK) typically attached to the N- or C-terminus of a protein to facilitate its identification and isolation. It is recognized by commercially available anti-flag antibodies, enabling straightforward protein detection and purification. The technical advantage of an antibody-based immunoassay for the flag tag lies in its robustness and widespread availability, making it a popular choice for researchers working with recombinant proteins.

[0146] The GST tag, derived from the enzyme glutathione S-transferase, is frequently used for protein expression, purification, and interaction studies. Linking the tag to glutathione-conjugated matrices enables efficient, single-step purification. In antibody-based immunoassays, antibodies specific for the GST tag can detect and quantify the tagged protein. The technical advantage of this immunoassay lies in its simplicity and the ability to achieve high protein yield and purity through the affinity purification step.

[0147] In a preferred embodiment of the immunoassay, the biologically active antigen is a sequence associated with nutritional parameters, such as transcobalamin II, ferritin, homocysteine, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or calcitriol. This allows for the monitoring of several important parameters, enabling an individual or subject, preferably a human, to monitor and improve their health. In particular, individuals with certain dietary habits may exhibit deficiencies in specific vitamins and trace elements, and an immunoassay according to the present invention, preferably configured as a low-fat immunoassay (LFA), and more preferably as a point-of-care test (POCT), can help to identify and correct these deficiencies. In an alternative embodiment, the result of the immunoassay is merely a recommendation and not a medical indication.

[0148] Transcobalamin II (TCII) is a transport protein that plays a crucial role in the transport of vitamin B12 (cobalamin) in the body. It binds to vitamin B12 and facilitates its transport to cells for various biochemical processes. The presence of TCII can indicate a vitamin B12 deficiency or certain diseases. An antibody-based immunoassay targeting TCII allows for the accurate detection and quantification of this protein in clinical samples, thus aiding in the diagnosis and monitoring of disorders related to vitamin B12 metabolism.

[0149] Ferritin is a protein that stores and releases iron in a controlled manner, thus contributing to iron homeostasis in the body. Measuring ferritin levels is crucial for assessing iron status and diagnosing conditions such as iron deficiency anemia or iron overload. An antibody-based immunoassay, preferably an LFA assay performed as a point-of-care test (POCT), targeting ferritin, allows for the precise quantification of this protein in blood or tissue samples, providing valuable information about an individual's iron levels and overall health.

[0150] Homocysteine ​​is an amino acid produced from the metabolism of methionine. Elevated homocysteine ​​levels in the blood are associated with an increased risk of cardiovascular disease and other health problems. Detection of homocysteine ​​using an immunoassay, preferably an LFA as a point-of-care test (POCT), provides a reliable method for assessing an individual's cardiovascular risk and monitoring the effectiveness of measures to lower homocysteine ​​levels.

[0151] Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are omega-3 fatty acids found in certain fish oils and known for their potential health benefits, including supporting cardiovascular health and cognitive function. Measuring blood levels of EPA and DHA provides information about an individual's omega-3 fatty acid status and aids in the development of dietary recommendations. An antibody-based immunoassay, preferably an LFA (low-fat antibody) performed as a point-of-care test (POCT), targeting EPA and DHA, allows for accurate quantification of these fatty acids and assists in personalized dietary assessment.

[0152] Calcitriol is the active form of vitamin D and plays a crucial role in maintaining calcium and phosphorus balance, bone health, and various physiological processes. Monitoring calcitriol levels is important for assessing vitamin D status and diagnosing conditions such as rickets and osteoporosis. An antibody-based immunoassay, preferably an LFA assay performed as a point-of-care test (POCT) specific for calcitriol, allows for the precise measurement of this hormone in blood samples, thus aiding in the evaluation of an individual's vitamin D status and the determination of appropriate interventions.

[0153] In a preferred embodiment, the recombinant antibody is obtained from a diatomaceous algae. In a particularly preferred embodiment, the diatomaceous algae Phaeodactylum tricornutum.This enables the provision of an antibody with the properties disclosed herein, in particular high homogeneity and purity, which makes it possible to provide an immunoassay according to the invention.

[0154] Preferably, the immunoassay is a lateral flow immunoassay which provides at least one fluid-connected sample application area, one conjugate area and one capture area arranged on a membrane.

[0155] A lateral flow immunoassay (hereinafter also referred to as "LFA") typically uses three antibodies: A first immobilized antibody, located in the capture area (also referred to as the test zone), is also called the capture antibody. Like the second antibody (also referred to as the detection antibody), this antibody is directed against the antigen, preferably against a different epitope of the antigen than the detection antibody. A second antibody, preferably located in the conjugate area and also referred to as the detection antibody, is directed against an epitope of the antigen, e.g., a hormone, a protein, or a peptide such as human chorionic gonadotropin. The second antibody is preferably conjugated to marker particles, e.g., gold, silver, latex, carbon, nanoparticles, or enzymes. A further antibody, preferably located in a control area (control zone), preferably immobilized, is directed against a detection / control antibody.

[0156] Preferably, the first antibody (e.g., the capture antibody), the second antibody (e.g., the detection antibody), and / or the further antibody (e.g., the capture antibody in the control zone), and especially preferably all antibodies, are obtained from a diatom, unicellular plant, or Viridiplantae and are characterized by the features defined herein.

[0157] The first antibody (e.g., the capture antibody), the second antibody (e.g., the detection antibody), and / or the further antibody (e.g., the capture antibody in the control zone), and especially all antibodies, obtained from a diatom or single-celled plant and characterized by the features defined herein are particularly preferred.

[0158] In a preferred embodiment, the LFA is provided as a point-of-care test (POCT). In a particularly preferred embodiment, the LFA is provided as a kit comprising at least the LFA and instructions. This allows for easy use by the patient themselves and can thus enable faster diagnostics outside of healthcare system infrastructure.

[0159] Preferably, in an embodiment relating to LFA and sandwich ELISA, the first antibody is directed against a first domain of the biologically active antigen (as defined herein). Preferably, in an embodiment relating to LFA and sandwich ELISA, the second antibody is directed against a second domain of the biologically active antigen (as defined herein).

[0160] Preferably, in an embodiment referred to in ELISA, the first antibody is directed against a domain of the biologically active antigen (as defined herein) and the second antibody is preferably directed against a domain of the first antibody.

[0161] In a particularly preferred embodiment, an enzyme-linked immunosorbent assay (ELISA) immunoassay is performed, wherein at least a. a sample application area is provided for the application of a biological sample, which b. is bounded by a collection area designed as a vessel boundary and / or part of a vessel boundary, preferably as a microtiter plate, and c. a conjugate area which is spatially arranged in the sample application area.

[0162] ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used laboratory technique for detecting and quantifying the presence of specific proteins or antibodies in a sample. In some forms of ELISA, a target antigen or antibody is immobilized on a solid surface, and then specific enzyme-bound antibodies are used to detect and quantify the amount of antigen, which correlates with the amount of bound antibody. In a sandwich ELISA, two different antibodies are used. The first antibody is immobilized on the ELISA plate and binds specifically to the antigen in the sample. The second (detection antibody) is labeled, for example, with an enzyme, and binds to a different epitope on the same antigen. Among the technical advantages of ELISA is its high sensitivity, which allows for the detection of low concentrations of target molecules.It is versatile and suitable for a wide range of sample types and molecules, including proteins, peptides, and small molecules. An ELISA can provide quantitative information about the amount of the target molecule present in a sample. It is one of the most important tools in molecular biology and is indispensable in analytical and diagnostic procedures. In addition to the antibodies and the sample to be analyzed, a microtiter plate as a support material and a reader, known as an ELISA reader, are required. There are various ways to perform the procedure; in the simplest form, the antigen to be analyzed is pipetted into the wells of the microtiter plates, where it binds firmly to the polystyrene of the plates. Remaining free binding sites on the polystyrene are blocked with a blocking reagent (e.g., acetylcysteine).vegan BSA) saturated, so that the subsequently added antibodies can only bind to their antigens and not to free binding sites of the polystyrene.

[0163] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used laboratory technique for separating proteins according to their molecular weight. In SDS-PAGE, proteins are denatured and coated with the anionic detergent sodium dodecyl sulfate (SDS) to impart a uniform negative charge. They are then loaded into a porous polyacrylamide gel and exposed to an electric field, causing the proteins to migrate according to their size. Among the technical advantages of SDS-PAGE is its high resolution, which allows for the precise separation of proteins in complex mixtures. It provides quantitative and qualitative information about protein samples, thus aiding in protein identification and characterization. SDS-PAGE is compatible with various sample types and can process both denatured and, with some methodological modifications, native proteins.

[0164] Proteins separated in this way by SD-PAGE can be transferred to a protein-binding membrane, such as nitrocellulose, using Western blot transfer. This makes the previously separated proteins accessible for the binding of antibodies, which are added after saturation of the membrane regions remaining free after Western blotting. As is typical for immunoassays, a cascade of at least two different antibodies is often used. The preceding cascade results in signal amplification because the second antibody binds to the first. The second antibody is often coupled to an enzyme such as alkaline phosphatase (ALP) or horseradish peroxidase (HRP) to visualize the binding. Furthermore, signal amplification often occurs because the second antibody can bind to the first antibody multiple times.After addition, depending on the amount of bound antigen or antibody, an insoluble product is formed by the enzyme reaction, which precipitates directly at the point to which the antigen migrated in the SDS-PAGE, thus clearly identifying the antigen.

[0165] Alternatively, preferably the immunoassay is an enzyme-linked immunosorbent assay (ELISA), e.g. a direct ELISA, an indirect ELISA, a direct sandwich ELISA or an indirect sandwich ELISA, preferably the ELISA is a direct sandwich ELISA or an indirect sandwich ELISA.

[0166] According to a preferred embodiment, the conjugate region, the sample deposition region, and the capture region can be located in the same environment, for example, in a vessel, preferably a well of a microtiter plate or test tube, and separated from one another by temporally separated additions and washing steps. In an embodiment associated with an ELISA, an antigen corresponding to a biological sample from an individual is immobilized on the wall of the vessel, thus forming the sample deposition region. Subsequently, a first antibody directed against the biologically active antigen is added, which binds to the antigen and is thus immobilized on the wall of the vessel, forming the capture region. After an optional washing step, a second antibody directed against the first antibody is added, forming the conjugate region.The regions are fluidically connected but temporally separated. In one embodiment associated with a sandwich ELISA, the first antibody is initially immobilized on the vessel wall, thus forming the capture region. Subsequently, a biological sample from an individual is added, forming the sample deposition region. After an optional washing step, a second antibody directed against the biologically active antigen is added, forming the conjugate region. The regions are fluidly connected but temporally separated.

[0167] In a particularly preferred embodiment, the second antibody is provided as an antibody-enzyme conjugate. This allows the detection of a first antibody and quantification of the signal by the enzyme in an ELISA when a soluble product is formed, or in an immunoblot when the product of the enzyme reaction precipitates at the site of its formation.

[0168] In a preferred embodiment, a first antibody, preferably derived from a diatom, is directed against an antigen, and a second antibody, also preferably derived from a diatom and coupled to an enzyme, preferably alkaline phosphatase (AP) or horseradish peroxidase (HRP), is directed either against the first antigen (LFA or sandwich ELISA) or against the first antibody (ELISA or immunoblot). Preferably, the second antibody can bind to the first antibody multiple times, which can lead to signal amplification. Particularly preferably, the second antibody can bind to the first antibody between 1 and 10 times, most preferably between 1 and 5 times. The resulting signal amplification thus preferably has a factor of 1 to 10, more preferably 1 to 5.This strong amplification allows the ELISA to detect low concentrations of antigen, or requires only a small amount of first and / or second antibody to allow detection above the detection limit. Due to the low concentration, the relevant antigens can be detected earlier in the course of the disease, thus contributing to rapid identification and treatment, which can, for example, break chains of infection.

[0169] In one embodiment associated with an LFA, the invention preferably provides a device comprising: a container, in particular a housing, and an immunoassay according to the invention, in particular a lateral flow immunoassay, arranged therein.

[0170] Also disclosed is a device comprising a housing (8), e.g. a container, in particular a housing, and an immunoassay arranged therein (as defined herein), in particular a lateral flow immunoassay.

[0171] In a preferred embodiment, the housing of the device is essentially, and in particular exclusively, made of a cellulose material, preferably paper or cardboard. This eliminates the need for plastics.

[0172] According to a particularly preferred embodiment, the housing of the device is essentially, and especially more than 90%, made of bioplastics, preferably polylactic acid (PLA), polyhydroxyalkanoates (PHA), or starch-based plastics. In this context, bioplastics are defined as plastics that are biodegradable, i.e., that can break down naturally into environmentally compatible substances, for example, in composting facilities or in nature.

[0173] Preferably, the housing of the device is formed from at least two layers of cellulose material that at least partially enclose the membrane. The ends of the layers of cellulose material can be overlapped in such a way that they are flush with each other.

[0174] In a preferred embodiment, the membrane is surrounded by a housing according to the present invention, which consists predominantly, preferably 90 to 100%, of cellulose fibers that are water-resistant, preferably at least for transport and the service life, and are made from sustainable, preferably recovered plant fibers and are biodegradable, e.g. by microbiological degradation in the environment or by composting.

[0175] Biodegradable, as used in the present invention, means that at least 90-100% of the components, preferably 95-100% of the components, and most preferably 99-100% of the components can be broken down biologically, for example by microbiological degradation in the environment and / or preferably by industrial composting according to EN 13432.

[0176] In a preferred embodiment, markings (symbols, signs, geometric shapes) are arranged on the housing at the membrane, allowing for easy interpretation of the immunoassay result. Particularly in the embodiment of a kit where instructions are provided alongside the assay, ease of use is ensured even for non-medical personnel. This allows the kit to be used as a point-of-care test (POCT).

[0177] For example, the ends of at least two layers of the cellulose material, which preferably form the housing of the device, are embossed and / or punched at the side areas where they contact each other, thereby firmly joining them together. For example, the ends of the layers are joined together by an embossed seam (9).

[0178] In a preferred embodiment, the sample application area is only partially located within the housing. The sample application area can be designed as a pad (cushion). This pad can consist of a fluffy, porous, or fibrous material capable of rapidly absorbing liquid.

[0179] According to a preferred embodiment, the membrane has a fixing area (7) in the distal region (viewed in the direction of flow from the application area at the proximal end of the membrane), preferably distal to the capture area, by which the membrane is fixed in the housing of the device.

[0180] In the distal region of the membrane, a fracture point (10) can be provided on the housing of the device as an example of a separation area, through which the distal end of the housing, preferably with the distal end of the membrane, can be separated.

[0181] Particularly preferably, the device is provided such that the container, preferably a housing, is made of sustainable and water-resistant materials, preferably paper and / or molded fiber. This allows for the provision of an environmentally friendly immunoassay, in particular an environmentally friendly LFA immunoassay. Pregnancy tests alone produce approximately 900 tons of plastic annually, which is why providing a housing that does not use plastic but is made of paper or molded fiber is desirable.

[0182] Disclosed is a kit comprising a device and instructions for performing the immunoassay. Particularly preferably, the kit includes further components, e.g., cotton swabs or other sampling utensils, buffer solution as a mobile phase mixture, further solutions, desiccants for drying, and other aids known to those skilled in the art for performing an immunoassay as a point-of-care test (POCT) or test kit for medically trained personnel.

[0183] Furthermore, a kit is disclosed which includes a device as defined herein and instructions for carrying out the immunoassay arranged in the device.

[0184] The use of an immunoassay (as defined herein) for the detection of a biologically active protein in a biological sample from an individual is disclosed but not claimed.

[0185] Also disclosed is a method for detecting a biologically active protein in a biological sample from an individual, the method comprising the following steps: a) Obtaining a biological sample from an individual; b) Analyzing the biological sample using an immunoassay designed to detect the biologically active protein.

[0186] Providing instructions along with the immunoassay ensures easy use of the kit as a point-of-care test (POCT). This offers several technical advantages, including rapid results due to shorter transport times, immediate clinical decisions, and improved patient management. PoCT minimizes potential pre-analytical errors, increases efficiency in emergency situations, and supports timely treatment. The decentralized nature of PoCT facilitates the monitoring of chronic diseases and infectious diseases.

[0187] In one embodiment, the use of the immunoassay as a lifestyle product, particularly for the detection of nutrition-related parameters, is disclosed. This enables the monitoring of parameters associated with deficiencies or undernutrition and is not a medical recommendation, but rather allows a person to identify potential health risks. This can be used, for example, for independent dietary monitoring.

[0188] A lifestyle product is an item or service provided because of its alignment with a particular lifestyle, set of values, or personal identity. These products often transcend their functional purpose and are chosen by consumers to reflect and enhance their desired lifestyle, interests, and self-expression, without necessarily implying a medical recommendation. In this context, the immunoassay is not a medical product but a tool that allows a subject to review and optimize their lifestyle.

[0189] The present document further discloses a method for detecting a biologically active antigen in a biological sample, preferably urine, whole blood, saliva, milk or serum, comprising the following steps: a) Obtaining a biological sample from an individual; b) Analyzing the biological sample using an immunoassay according to any one of claims 1 to 15, which is configured to detect the biologically active antigen.

[0190] This method enables the application of the immunoassay, allowing for the rapid and precise analysis of biologically active antigens. In a particularly preferred embodiment, the biological sample is provided by the individual performing the immunoassay; in an alternative embodiment, the sample is obtained by a medically trained professional. Preferably, no further treatment of the biological sample is required after collection. Particularly preferably, the biological sample can be applied directly to the immunoassay. This allows for the rapid and direct determination of a biologically active protein.

[0191] In one embodiment, the use of the immunoassay as a point-of-care (PoC) diagnostic tool is disclosed, particularly for the detection of antigens that are part of the epitope of a virus, preferably a pathogenic virus, e.g., influenza, SARS-CoV-2, RSV, adenovirus, Strep A, norovirus, rotavirus, HIV, and / or a recombinant antibody directed against human chorionic gonadotropin, preferably an hCG antibody. This offers several technical advantages, including rapid results due to shorter transport times, immediate clinical decision-making, and improved patient management. PoCT minimizes potential pre-analytical errors, increases efficiency in emergency scenarios, and supports timely treatment. The decentralized nature of PoCT facilitates the monitoring of chronic diseases and infectious diseases.

[0192] In a preferred embodiment, at least one auxiliary protein for the cover, mask and / or support protein in an immunoassay is further disclosed, wherein the auxiliary protein is obtained recombinantly from a stably transformed diatom or unicellular plant.

[0193] Immunoassays, such as ELISAs, dot blots, immunoblots, and LFAs, require auxiliary proteins in addition to antibodies. These proteins stabilize the antibody solutions and block the protein-binding surfaces. For immunoassays, LFAs, and dot blots, the surface is preferably nitrocellulose, while for ELISAs, polystyrene is preferred. In the prior art, predominantly animal proteins, such as bovine or calf serum, skimmed milk powder, or casein, are used. To provide a vegan immunoassay, preferably a vegan LFA and / or vegan ELISA, these proteins must be replaced with animal-free alternatives. Synthetically produced alternatives are known, such as ROTI®<Block; however, these are not suitable for all applications and are very expensive and not economically competitive.

[0194] This document discloses, analogous to recombinant antibodies, the provision of auxiliary proteins derived from a stably transformed diatom, unicellular plant, or higher plant. For this purpose, analogous to antibodies, the sequence of bovine serum albumin (BSA) is codon-optimized and inserted as a gene in Phaeodactylum tricornutum introduced and expressed there.

[0195] After digestion of the diatoms, the vegan BSA can be purified, preferably by affinity chromatography, or, in a preferred embodiment, used directly as a crude extract in various immunobiochemical procedures without further purification.

[0196] The auxiliary protein according to the present invention is particularly preferred, preferably a vegan protein expressed from a diatom, selected from the list including BSA, casein, or gelatin. Bovine serum albumin (BSA) is frequently used as a blocking agent to prevent the non-specific binding of antibodies to the test surfaces and to reduce background noise. Casein is another blocking agent that helps to prevent non-specific interactions and improve the signal-to-noise ratio in assays. Gelatin is a collagen-derived protein that can also be used to block non-specific binding in immunoassays.

[0197] In a preferred embodiment, the purification of the antibodies follows a method familiar to those skilled in the art; that is, after lysis and centrifugation and / or ultrafiltration, purification is carried out either via protein A, protein G, or via the tag sequences used, for example, the 6xHis tag. This represents a further advantage of the method according to the invention, because, unlike higher plants that produce transient or stable antibodies, diatoms do not contain fibers that would make antibody purification extremely difficult. This allows for technically efficient and economically viable use of the recovered antibodies. The purification process therefore largely corresponds to the method used, for example, for animal cell cultures such as CHO cells, and is familiar to those skilled in the art and is described below.

[0198] Preferably, the cells of the production clone, in this example a diatom, are destroyed by a so-called gentle disruption process, whereby the product, the antibody according to the present invention, is protected. Gentle methods include, for example, high pressure, electrical voltage, ultrasound, or disruption by collision in so-called vibrating mills. In a particularly preferred embodiment, the separation is achieved by a combination of a Manton-Gaulin homogenizer and subsequent ultrasonic treatment with a Covaris E220 Focused Ultrasonicator.

[0199] After digestion, the so-called lysate is present, which contains the entire cell contents.

[0200] To obtain a functional antibody, further purification steps are preferably carried out. Insoluble components are separated from the soluble components, e.g., by centrifugation or filtration, particularly preferably by a centrifugation sequence. Pigment-containing components are separated, e.g., by filtration, cooling, chemical precipitation, ion-exchange chromatography, or size-exchange chromatography. Preferably, a sequence of filtration, cooling, chemical precipitation, ion-exchange chromatography, or size-exchange chromatography is used to separate components sequentially and efficiently.

[0201] The antibodies are purified by affinity chromatography. This can be done using typical antibody purification methods such as those for protein A or G, or via affinity tags. Additionally, impurities are removed by tangential flow filtration or dead-end filtration and / or dialysis. Finally, the antibody is transferred to a suitable buffer solution.

[0202] To achieve optimal performance, the manufactured antibodies must have a certain degree of purity. The purity of the manufactured antibodies can be verified by a method called polyacrylamide gel electrophoresis. Fig. 10 shows an example of two different anti-hCG antibodies according to the invention, here designated AK_1788 and AK_1882, which have a high degree of purity.

[0203] Gel electrophoresis is a laboratory technique used to separate and analyze molecules such as DNA, RNA, and proteins based on their size and charge. The molecules are placed in a gel matrix, and an electric field is applied, causing them to migrate through the gel. Smaller molecules move faster and migrate further, resulting in distinct bands or patterns that can be visualized. Among the technical advantages of gel electrophoresis is its ability to separate complex mixtures of molecules with high resolution. It is versatile, can accommodate various types of molecules, and provides qualitative and semi-quantitative information about their properties.

[0204] A dot blot is a laboratory technique used in molecular biology and immunology to detect, analyze, and quantify specific biomolecules, such as proteins or nucleic acids (DNA or RNA), in a sample. In a dot blot, a small amount of the target biomolecule is immobilized or "splattered" onto a solid support, usually a membrane. This immobilization can be achieved by directly dabbing the sample onto the membrane. REFERENCE MARK LIST

[0205] 1 Immunoassay / Device 2 Sample application area 3 Capture area 4 Conjugate area 5 Control zone 6 Membrane 7 Fixation area 8 Housing 9 Embosser seam 10 Break point 11 Nucleic acid sequence optimization 12 Insertion of the nucleic acid sequence into a vector 13 Transformation 14 Screening procedure 15 Procedure for the production of recombinant proteins 16 Vector 17 Cells 18 Cell culture Examples of implementation

[0206] The present invention is explained in more detail with reference to the following figures and embodiments, without limiting the invention to these.

[0207] This shows Fig. 1: an immunoassay, in particular a lateral flow immunoassay (test strips) in a schematic view from above; Fig. 2: an immunoassay, in particular a lateral flow immunoassay (test strips) in a schematic view from below; Fig. 3: the schematic view of a device in which an immunoassay, in particular a lateral flow immunoassay (test strip), is arranged within a housing; Fig. 4: the schematic view of a device in which the housing is formed from two layers of paper or cardboard arranged one above the other; Fig. 5: Glycan analysis to determine the composition of the glycosylation of an IgG (antibody) in a hamster cell culture; Fig. 6:Glycan analysis to determine the composition of the glycosylation of IgG (antibody) from Fig. 5 in Expi cells (human cell culture); Fig. 7: Glycan analysis to determine the composition of the glycosylation of IgG (antibody) from Fig. 5 and 6 in Phaeodactylum. Fig. 8: A schematic overview of the provision of antibodies according to the invention. Fig. 9: the repetition of two promoters Fig. 10: Polyacrylamide gel electrophoresis with anti-hCG antibody; GS= size standard, AK_1788 and AK_1882: anti-hCG antibody, stained with Coomassie. Fig. 11: A diagram comparing the binding affinity of two diatom antibody (formats) with sequence-matched antibodies from human cell culture (Expi 293F< ) Fig. 12: ELISA for the detection of human beta-chorionic gonadotropin (hCG) by antibodies produced in diatoms Fig. 13:Use of antibodies according to the invention from diatoms as horseradish peroxidase-labeled secondary antibodies for the detection of primary antibodies in an immunoblot (SDS-Page). mlgG: monoclonal IgG against human interleukin 15 from mouse; mlgG_1: an affinity-purified polyclonal antibody (Goat-a-mouse-IgG-HrRP) produced in goats, directed against the light and heavy chains of mouse IgG (reference antibody); mlgG_2: antibody according to the invention from P. tricornutum, directed against the heavy chain of mouse IgG (antibody according to the invention for immunoassay) Fig. 14: ELISA against the Herceptin 2 receptor (Her2) performed with a rabbit trastuzumab biosimilar (Trastuzumab AC) and with a diatom biosimilar P. tricornutum (hlgG4_D-F). Fig. 15: Dot blot with anti-hCG antibodies AK_1788 and AK_1882

[0208] The Figure 8Figure 1 shows a schematic overview of the relationship between providing a diatom and the production of recombinant antibodies for the immunoassay. All depicted steps (individually or in combination) lead to an improvement / optimization of the heterologous production of proteins, especially antibodies, particularly in the diatom. Phaeodactylum tricornutum.

[0209] First, a sequence optimization (11) of a nucleic acid sequence is performed. This may include, for example, codon optimization and / or the use of particularly protease-resistant genetic elements (as described herein), such as hinge regions derived from equine IgGs (immunoglobulin G).

[0210] The nucleic acid sequence is then inserted into a vector (16) (or an isolated nucleic acid), using individual genetic elements and / or complete expression cassettes repetitively. Furthermore, special inducible promoters are used, in particular a promoter element from the nucleic acid sequence of SEQ ID NO: 1.

[0211] By using specific signal sequences (as described herein), the proteins to be produced heterologously are expressed into the endoplasmic reticulum of the cells, which leads to protection of the proteins from proteases and to successful glycosylation, thereby increasing the yield and maintaining the functionality of the proteins.

[0212] In a next step, the vector (or nucleic acid) is transformed (13) into target cells, preferably photosynthetically active cells, in particular cells of a unicellular plant or viridiplantae, preferably cells of Phaeodactylum tricornutum. The transformation can be carried out ballistically or by means of electroporation in a suitable medium.

[0213] Subsequently, a screening procedure (14) is performed to select cells with an increased expression rate of a nucleic acid sequence. This can involve screening for high-performance producers, i.e., cells with increased expression rates, using reporter genes. Furthermore, the correlation between the expression of the reporter genes and the expression level of the proteins to be produced can be determined. Preferably, multiwell plates are used for both the screening procedure and the culture monitoring.

[0214] Following the screening procedure, a process for the production of recombinant proteins, preferably recombinant antibodies, can be carried out based on the cells identified in the screening procedure with an increased expression rate.

[0215] The production of recombinant proteins (15) takes place in a culture medium that is tailored to the genetic elements used, in particular the promoters used. The cultivation conditions, such as minimum light intensities and aeration levels, are also adapted.

[0216] Both in terms of the nucleic acid sequence and in terms of the vector or the isolated nucleic acid, the use of repetitive genetic elements leads to an enormous increase in expression rates in P . tricornutum. Figure 9A shows the repetition of two HASP1 ​​mod< promoters. In Figure 9BAn exemplary embodiment of a vector according to the invention is shown in which an expression cassette was used repeatedly, here in the form of a triple cassette for the production of antibodies in the scFv-Fc format. In particular, this repetitive use of the expression cassette leads to a significant increase in the yield as well as the number of clones or the proportion of clones that show detectable production, thereby minimizing the overall process effort. In the illustrated embodiment, the expression cassette comprises a promoter element (9.1), a first transcription unit encoding a recombinantly produced protein, and a second transcription unit encoding the reporter gene. gfp encoded, with the individual genetic elements being shown as an example on one expression cassette, but also present in the other expression cassettes.

[0217] Fig. 12Figure 1 shows an ELISA for the detection of human beta-chorionic gonadotropin (hCG) using antibodies produced in diatoms. Fig. 12 The ELISA was performed according to the following procedure: After coupling human chorionic gonadotropin (hCG) to the ELISA plate, the first antibody directed against hCG, which was produced in diatoms and purified by affinity chromatography, was added after saturation with an animal-product-free blocking reagent.

[0218] After the washing step, a second diatom-based antibody was used for amplification and an antibody coupled to the enzyme HRP was used for signal generation in order to start the detection reaction after the substrate was added.

[0219] In this ELISA, a total of 13 test series were performed, of which two samples were intended to show a strong signal and eleven served as controls to obtain a meaningful result. Table 1 lists the corresponding procedures, with the order from left to right corresponding to the steps involved. The number of steps can vary. In this case, five steps were performed. First, the antigen, here hCG, was bound to the plate. Then, free binding sites were occupied using a blocking solution. From the third step onward, the additives differed, as shown in Table 1. These different additives allow for the characterization of the antibodies and serve, among other things, to exclude non-specific binding.

[0220] A total of three different antibodies were found, which were in Phaeodactylum tricornutumThe following antibodies were used in this exemplary ELISA: Firstly, two different antibodies directed against the beta subunit of hCG and corresponding to human IgG4 (AK_1788 and AK_1892) were used. Secondly, a secondary antibody derived from diatoms, corresponding to a mouse antibody directed against human IgG4 (AK_2073), was used. In this example, detection was performed with a polyclonal antibody directed against mouse IgG and conjugated to horseradish peroxidase (HRP) (G-α-mIgG-HRP).

[0221] Table 1 lists the antibody additives associated with the image. A signal is only expected in samples 9 and 13; all other samples serve as controls. Table 1: Groups for ELISA with diatom-derived antigens. anti-mIgG-HRP = HRP-labeled antibody directed against antibody 2073 (AK_2073). AK_1788 and AK_1882 are antibodies directed against hCG; AK_2073 is an antibody directed against antibodies 1788 and 1882. group Coating 1. Encore 2nd encore 3rd encore; 4th Encore 1 Block solution Block solution Block solution Block solution 2 hCG Block solution Block solution Block solution 3 hCG Block solution Block solution Block solution anti-mlgG-HRP 4 hCG Block solution Block solution AK_2073 5 hCG Block solution Block solution AK_2073 anti-mlgG-HRP 6 hCG Block solution AK_1788 Block solution 7 hCG Block solution AK_1788 Block solution anti-mlgG-HRP 8 hCG Block solution AK_1788 AK_2073 9 hCG Block solution AK_1788 AK_2073 anti-mIgG-HRP 10 hCG Block solution AK_1882 Block solution 11 hCG Block solution AK_1882 Block solution anti-mlgG-HRP 12 hCG Block solution AK_1882 AK_2073 13 hCG Block solution AK_1882 AK_2073 anti-mlgG-HRP

[0222] Out of Fig. 12 It is evident that only groups 9 and 13, highlighted in Table 1, exhibit a strong signal with intensities of 0.57 and 0.54, respectively. This indicates that the first and second antibodies have a high affinity for the antigen and the first antibody, respectively. The addition of G-α-mIgG-HRP as a detection antibody demonstrated that these antibodies are selective for the detection of human beta-chorionic gonadotropin (hCG). All other control groups showed no value above 0.25. This suggests that both antibodies AK_1788 and AK_1892 selectively bind the antigen, in this case hCG.

[0223] For the so-called control antibody 2073 required in the LFA, which is placed on the control line, it can be shown that it can bind the antibodies AK_1788 and AK_1892 as desired.

[0224] Thus, the antibodies shown here are complete and functional for the construction of an LFA, in this case a pregnancy test, and enable the detection of pregnancy as well as the staining of the control line of an LFA.

[0225] Fig. 13 This shows an exemplary use of an antibody in an immunoassay (immunoblot) using the SDS-PAGE variant. Here, diatom antibodies are used as secondary antibodies, labeled with horseradish peroxidase for the detection of primary antibodies. Fig. 13 mlgG stands for a monoclonal IgG against human interleukin 15 from mouse, and Pt is the abbreviation for a protein extract from diatoms. Phaeodactylum tricornutum(Pt), which is used as a negative control to show that the antibodies do not recognize proteins from the diatoms. Both mlgG and Pt were separated by SDS-PAGE, the separated proteins from the gel were then transferred to a membrane by Western blot, and subsequently mlgG was detected with a commercial reference antibody (mlgG_1) as well as with two different concentrations of an antibody.

[0226] The comparator antibody used is mlgG_1, a commercial, polyclonal, affinity-purified antibody (goat-α-mouse-IgG-HRP) produced in a goat, directed against the light and heavy chains of mouse IgG, and coupled to horseradish peroxidase (HRP) (comparator antibody). The antibody mlgG_2 (Pt-α-mouse-IgG-HRP) is used in two concentrations (0.75 µg / mL and 0.15 µg / mL). This antibody was produced from diatoms. Phaeodactylum tricornutumThe antibody is derived from the mouse IgG heavy chain and is coupled to horseradish peroxidase (HRP). BlueStar from Nippon Genetics was used as the length standard (M).

[0227] The goat-derived antibody Goat-α-mouse-IgG-HRP recognizes both the light and heavy chains of mouse mlgG, resulting in numerous non-specific and two strong specific signals (arrows). The diatom-derived antibody (mlgG_2) is directed only against the heavy chain (HC, upper arrow only), which is why the light chain, which produces the lower specific band in the comparator antibody, is not detected. The diatom-derived antibody (mlgG_2) is directed only against the heavy chain (HC, upper arrow only), which is why the lower band is not detected. Even in small amounts, the diatom-derived antibody produces similarly strong and, more importantly, more specific signals. With the commercial animal antibody (mlgG_1), several non-specific bands (*) appear.The uppermost bands are still complete IgGs (▼), where the denaturation characteristic of SDS-PAGE has not led to the separation of the IgG into the light and heavy chains. Thus, the antibodies not only show strong signal enhancement at lower concentrations, but also higher specificity compared to the target antibodies, making them a more specific alternative to animal antibodies. This increased sensitivity allows for a smaller amount of antibody to be used, while the higher specificity enables more reliable determination and the avoidance of false-positive results. It was also shown that the antigens have no affinity for the diatom-specific proteins, as evidenced by the absence of bands in the separated protein extracts from P. tricornutum (Pt) is demonstrated.

[0228] Fig. 14shows an ELISA of animal antibodies and antibodies against the Herceptin 2 receptor (Her2), performed with a rabbit trastuzumab biosimilar (Trastuzumab AC) and with a diatom biosimilar. Phaeodactylum tricornutum (hlgG4_D-F).

[0229] The OD value refers to the optical density. It is a measure of the absorption of light by a sample in a microplate well. The OD value is used to quantify the presence or concentration of a specific molecule, in this case an antigen, in the sample being tested. Higher OD values ​​generally indicate a higher concentration of the target molecule in the sample, while lower values ​​indicate lower concentrations. This measurement is a crucial component in evaluating the results of ELISA experiments and determining the strength of the reaction between antigen and antibody.

[0230] The OD values ​​in this example show that across all concentration ranges, the antibodies from the diatom Phaeodactylum tricornutum The antibodies obtained (hlgG4_D-F) show higher specific activity against the Herceptin 2 receptor antigen than the animal analogues (trastuzumab AC). This is likely due to higher purity of the antibodies, which are also present in, among other things, Figs. 5-7 (Glycosylation pattern), Fig. 13 (SDS-Page immunoblot) demonstrates this. The higher activity allows for more sensitive detection of the antigens and thus a reduction in the detection limit. Due to the low concentration, corresponding antigens can be detected earlier in the course of the disease, thus contributing to rapid identification and treatment, which can, for example, break chains of infection.

[0231] Fig. 15This shows a dot blot test. In the dot blot assay, one antibody (AK_1788, directed against hCG) is conjugated with colloidal gold, and another antibody (AK_1882, directed against a different epitope on hCG) is applied in a circular pattern to a suitable membrane. The membrane and the colloidal gold-labeled antibodies are then incubated together with an hCG-containing solution. The anti-hCG antibodies used are from the ELISA. ( Fig. 13 ) They are therefore tested in a setup corresponding to an LFA model. Both the membrane-bound and the colloidal gold-labeled antibodies bind the hCG from the solution simultaneously. If both antibodies are able to bind the hCG, a distinct color change occurs at the location of the membrane-bound antibody compared to the surrounding area. Fig. 15This discoloration is shown with the two anti-hCG antibodies. This demonstrates that both antibodies bind to the hCG molecule simultaneously and can therefore detect hCG using a typical LFA structure. Example 1: Production of antibodies from diatoms and purification for immunoassay

[0232] The exemplary embodiment, the course of which is described in Fig. 8 The diagram illustrates the production of an antibody using this method. This antibody, in IgG format, is directed against equine interleukin 31 and was produced using more than 160 mg of purified eqlgG * L -1< cell culture within a 14-day culture period.

[0233] As a first step, codon usage will be... P. tricornutumThe starting sequence was adapted. In this case, it originates from a human scFv library and was codon-optimized before use in diatoms. The required genetic elements, typically the variable region of the light chain (VL) and the variable region of the heavy chain (VH), were synthesized by IDT-DNA (Coralville, Lowa) to ensure optimal fit in the generated vectors. ( Fig. 9B ). In this process, interfering restriction sites were also removed or modified. One variant of the vectors for antibody production in the scFv-Fc format is described in Fig. 9BThis is illustrated. Here, the variable region of the light chain (kappa or lambda format (V Lκ or V Lλ)) as well as the variable region of the heavy chain (VH) can be used. In this example, the constant regions of both chains are derived from horses. Constructs containing constant antibody regions from other host organisms (e.g., mouse or human) have also been generated. Further variants of the vectors have been produced and successfully used for the heterologous production of other formats such as Fab or scFv-Fc. Fig. 9B The example shown contains the finished vector construct and, in addition to the ones in P . tricornutum The elements to be introduced also include bacterial and genetic elements that facilitate cloning. E. coli (colE1 origin and gentamicin resistance gene). In the construct, the gene of interest was inserted in three copies. After ballistic or electroporation-based transformation of P . tricornutumThe resulting clones must not only be checked for the uptake of the antibody genes, but also for the rapid identification of clones that express the introduced antibody gene particularly strongly. The underlying screening method according to the invention enables the correlation of the gfp (Green Fluorescent Protein) caused fluorescence, which is measured in a special microtiter plate reader, with the later expected amount of antibody formed.

[0234] In comparison to production rates of a maximum of 3 mg antibody per L of culture described in the prior art, 160 mg of purified antibody could be obtained from 1 L of culture in this system. To achieve this, new media compositions, aeration with more than 3 L per min of compressed air, and illumination with a luminous intensity of 100–1000 W per m² were used in a reactor column. An example media composition is given in Table 2: Table 2: Media composition substance concentration Tris-HCl pH 8 10 mM NaNO 3 30 mM Vitamins for P. tricornutum Over 10x (f / 2 Medium) Trace elements for P. tricornutum Over 10x (f / 2 Medium) Glycerin At least 100 mM NH₂PO₄ At least 360 µM

[0235] After typically fourteen to twenty days of fed-batch culture, the diatoms can be harvested and processed.

[0236] Purification follows the classical procedure; that is, after lysis and centrifugation and / or ultrafiltration, purification is carried out either via protein A, protein G, or via the employed tag sequences, for example, the 6xHis tag. This is another advantage of the immunoassay, because unlike higher plants that produce transient or stable antibodies, diatoms do not contain fibers that would significantly complicate antibody purification. The purification process therefore largely corresponds to the method used, for example, for animal cell cultures such as CHO cells, and is familiar to those skilled in the art and is described below. The cells of the production clone, in this example a diatom, are destroyed by a so-called gentle digestion, whereby the product, the antibody as defined in the present invention, is preserved. Gentle methods include, for example,High pressure, electrical voltage, ultrasound, or disruption by collision in so-called vibrating mills are used; in this case, a combination of a Manton-Gaulin homogenizer followed by ultrasonic treatment in an ultrasonicator was employed. After disruption, the lysate, containing the entire cell contents, is obtained. To obtain a functional antibody, purification steps are performed. Insoluble components are separated from the soluble components, for example, by centrifugation or filtration—in this case, a centrifugation sequence. Pigment-containing components are separated by a sequence of filtration, cooling, chemical precipitation, ion-exchange chromatography, or size-exchange chromatography.

[0237] The antibodies are purified by affinity chromatography. This is performed either using a typical antibody purification method, such as protein A or G, or via affinity tags. Impurities are then removed by tangential flow filtration or dead-end filtration and / or dialysis. Finally, the antibody is transferred to a suitable buffer solution.

[0238] To achieve good performance, the produced antibodies must have a certain degree of purity. In this example, the purity of the produced antibodies is checked by a method called polyacrylamide gel electrophoresis. Fig. 10 shows an example of two different anti-hCG antibodies, here labeled A-hCG-1 and A-hCG-12, which have a high degree of purity. Example 2: Provision of a pregnancy test

[0239] The lateral flow pregnancy test uses intracellularly produced antibodies from diatoms. Phaeodactylum tricornutum. These antibodies have a concentration of approximately 160–360 mg / L per liter of culture during production. These antibodies are specifically targeted to human chorionic gonadotropin (hCG), which consists of an α-subunit with 92 amino acids and a β-subunit with 145 amino acids. The test includes a first antibody, a capture antibody; a second antibody, a detection antibody; and a third antibody, a control antibody.

[0240] The detection antibodies are characterized by being labeled with colloidal gold nanoparticles. The first immobilized antibody, located in the capture zone, functions as the capture antibody. The second antibody, located in the conjugation zone and referred to as the detection antibody, is also directed against the antigen, preferably against a different epitope of the antigen than the detection antibody. This second antibody is conjugated to a marker particle, in this case, colloidal gold nanoparticles.

[0241] In a preferred embodiment of the immunoassay, the first antibody and / or the second antibody, in particular the mobilized second antibody, is labelled with a dye and / or an optically active nanoparticle, in particular a gold nanoparticle.

[0242] Another antibody, which is preferably located in the control region (control zone) and is immobilized, is directed against a detection antibody.

[0243] The purified antibodies, including the first, second, and subsequent antibodies, are combined with a plant-based coating protein (diatom-expressed BSA) and applied to a nitrocellulose membrane. This configuration creates a lateral flow immunoassay featuring a sample application area, a conjugate area, and a capture area integrated on the membrane. The sample application area and capture area are fluidically connected on the membrane via a flow path, with the conjugate area located within this flow path. This design facilitates assay execution and result interpretation.

[0244] This membrane is surrounded by a housing according to the present invention, which consists predominantly (at least 90%) of cellulose fibers that are water-resistant (at least for transport and service life) and are made from sustainable, preferably recovered plant fibers and are biodegradable (e.g. by composting).

[0245] For ease of use as a point-of-care test (POCT), markings (symbols, signs, geometric shapes) are applied to the housing on the membrane, allowing for easy interpretation of the immunoassay result. Particularly in the embodiment of a kit in which instructions are provided alongside the assay according to the invention, ease of use is also ensured for non-medical personnel.

Claims

1. Immunoassay for detecting a biologically active antigen, particularly a hormone, protein or pharmaceutical substance in a biological sample of an individual, provided with: - a sample application area for applying a biological sample of an individual, wherein the biological sample is preferably urine, whole blood, saliva, milk or serum; - a capture area, wherein the capture area is provided with an immobilized first antibody that is directed against the biologically active antigen, in particular against the hormone, the protein, the peptide or the pharmaceutical substance; - a conjugate area, wherein the conjugate area is provided with a second antibody directed against the biologically active antigen, particularly against the hormone, protein, peptide or pharmaceutical substance; characterized in that the first antibody and the second antibody is a recombinant antibody obtained from a diatom or unicellular plant, wherein the immunoassay comprises at least a further antibody and / or an auxiliary protein, wherein the auxiliary protein saturates free surfaces of a reaction vessel or a membrane as a blocker, wherein the further antibody and the auxiliary protein were obtained from a diatom and / or Viridiplantae and / or unicellular plant, and wherein the recombinant antibody is provided in a purity of at least 90%, and wherein the immunoassay is vegan.

2. Immunoassay according to claim 1, wherein the recombinant antibody is obtained in a concentration of 20-1000 mg / L, preferably 30-800 mg / L, alternatively at least 30-160 mg / L of the culture of a diatom or unicellular plant.

3. Immunoassay according to one of claims 1 or 2, wherein the glycosylation of the first antibody and / or the second antibody has a modified glycosylation pattern compared to the corresponding native antibody, preferably the glycosylation has a more homogeneous pattern with a homogeneity factor in the range from 1 to 3, particularly preferred the glycosylation has a homogeneous, mannose-rich N-glycan pattern with a homogeneity factor in the range preferably in the range from 1 to 3.

4. Immunoassay according to any one of claims 1 to 3, wherein the amino acid sequence of the first antibody and / or the second antibody a) comprises a vertebral, preferably mammalian, particularly preferred a human antibody; or b) comprises or consists of an amino acid sequence which has at least 80 %, preferably at least 85 %, particularly preferably at least 90 %, most preferably at least 95 %, in particular at least 97 % sequence identity to homologous sequence regions of a vertebral and / or mammalian and / or human antibody.

5. Immunoassay according to any one of claims 1 to 4, wherein the first antibody and / or the second antibody is an antibody directed against human chorionic gonadotropin (hCG).

6. Immunoassay according to any one of claims 1 to 5, wherein the biologically active antigen is a part of the epitope of a virus, preferably a pathogenic virus, for example influenza, SARS-CoV-2, RSV, adenovirus, Strep A, norovirus, rotavirus, HIV.

7. Immunoassay according to any one of claims 1 to 6, wherein the biologically active antigen is a tumor-associated sequence, preferably an HLA complex and / or a sequenced part of a tumor epitope and / or a tumor marker, for example IFN-γ, IL-8, PSA, CEA, AFP, DCP, CA 125, HER2 / neu.

8. Immunoassay according to any one of claims 1 to 7, wherein the biologically active antigen is a characteristic sequence for identifying a protein, preferably an enzyme tag, particularly preferred selected from the list consisting of His6-tag, Strep-tag, c-Myk-tag, Flag-tag and GST-tag.

9. Immunoassay according to any one of claims 1 to 8, wherein the biologically active antigen is a sequence associated with nutritional parameters, for example transcobalamin II, ferritin, homocysteine, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), or calcitriol.

10. Immunoassay according to any one of claims 1 to 9, wherein the immunoassay is a lateral flow immunoassay providing fluid-connected at least a sample application area, a conjugate area and a capture area fluid-connected on a membrane.

11. Immunoassay according to any one of claims 1 to 9, wherein the immunoassay is carried out as an enzyme-linked immunosorbent assay (ELISA) immunoassay, wherein at least a. a sample application area for applying a biological sample is provided, which is confined by b. a capture area which is designed as a vessel boundary and / or part of a vessel boundary, preferably as a microtiter plate, and c. a conjugate area, which is spatially arranged in the sample application area.

12. Device, provided with: - a container, particularly a housing, and - an immunoassay according to any one of claims 1 to 11 arranged therein, particularly a lateral flow immunoassay.

13. Device according to claim 12, wherein the container, preferably a housing, is configured from sustainable and water-resistant materials, preferably paper and / or fiber cast.

14. Method for detecting a biologically active protein in a biological sample, preferably urine, whole blood, saliva, milk or serum, comprising the following steps: a. Obtaining a biological sample from an individual; b. Analyzing the biological sample with the use of an immunoassay according to any one of claims 1 to 12, which is adapted to detect the biologically active protein.

15. Use of an auxiliary protein as covering, masking and / or support protein in an immunoassay according to claim 1 to 12, characterized in that the auxiliary protein is obtained from a diatom and is vegan.