FI116486B - Homologous or heterologous immunological method for simultaneous detection of at least two latex allergens and in the method useful test package - Google Patents

Description

116486

Homologous or heterologous immunoassay for simultaneous determination of at least two latex allergens and a test kit useful in the method

FIELD OF THE INVENTION The present invention relates to immunochemical assay methods for the detection of residual allergenic proteins or peptides derived from natural rubber latex in a final product made from or containing natural rubber latex, and immunochemical assays useful in such methods.

Background of the Invention

Products containing natural rubber latex (NRL) of rubber tree, Hevea brasiliensis, are widely used due to the low cost of natural rubber and favorable processing properties, although the reactions to the numerous allergenic proteins contained in NRL are well known and documented. For example, 15 NRL-containing products, such as surgical gloves and many other equipment (such as catheters, tubing, masks, etc.) used by healthcare professionals, or NRL-containing products, such as protective gloves, used by kitchen staff cause most of these reactions. In health care, NRL-based medical devices are a potential hazard not only for the person-20 community but also for patients undergoing examination or surgery. Even glove powder: may contain NRL products adsorbed on NRL powder particles. · On the coils and thus cause both sensitization and counter-reactions.

• | * ..; In the rubber industry, workers at different stages of production may be physically exposed to NRL allergenic products and may be exposed ';;; '25 also for NRL proteins in the air. In addition, even the normal population is in daily contact with various NRL-containing products such as household gloves, condoms, and balloons made by dipping. · As well as hoses, tires, erasers and the like.

Although natural rubber latex gloves have been used in 30 medical and industrial applications for over 100 years, the medical and scientific community has not been aware of latex allergy as a single disease until about 10-15 years ago. Currently, latex allergy is considered to be a serious global health problem: a significant proportion (3-15%) of health personnel: ·: * * and about 1% of the total population are exposed to natural rubber latex. When susceptible individuals are exposed to latex allergens, IgE-mediated allergy may develop. Latex gloves are considered the major source and thus the largest single source of exposure and account for most of the immediate Type 1 or IgE-mediated hypersensitivity reactions to latex. In the Western world, about one fifth of people are atopic (prone to producing large amounts of IgE-5 antibodies) and thus generally have an increased risk of developing latex allergy.

Well-defined risk groups include healthcare workers and other workers wearing gloves, and children with spinal cord or other congenital malformations who have undergone many surgical procedures at an early age. Clinical manifestations of latex allergy range from mild contact urticaria to fatal anaphylaxis, and the severity of the condition is emphasized by the fact that the first sign of sensitization may occur as a life-threatening reaction.

Latex allergens are proteins or polypeptides that elute from manufactured products upon contact with the skin, mucous membranes, or other tissues. According to the current nomenclature maintained by the International Union of Immunological Societies (IUIS) of the WHO, nine latex allergens, which are characterized at primary structural level and are included in the official list of allergens, are designated as shown in Table 1.

20 Table 1. Nomenclature of natural rubber allergens; '; Nomenclature Name Molecular

'. : weight, kD

Hevbl Rubber Elongation Factor (REF) 14.5

Hevb2 p-1,3-Glucanase 36 25 Hev b3 Rubber Particle Associated Protein 23 * ;;; * Hev b4 Micro-Helical Protein Complex 50-57 *: ···: 100-110 **

Hev b5 Acid 16 kD Protein 16: Hevb6.01 Prohevin 21! * ,, · * 30 Hevb6.02 Hevin 4.7. * · Hevb6.03 Prohevin C Area 14

Hev b7 Patatin-like protein 46

Hevb8 The 15.7 'reduced form of my profile, ** the non-reduced form

M M I

The complete primary structure of these named latex allergens (except Hev b 4) is currently known, and the genes encoding these proteins have been cloned and corresponding recombinant proteins have been produced. To date, four of these allergens (Hev b1, Hev b3, Hev b5 and Hev b6.02) have been unquestionably identified from manufactured latex products. Nel and Gujuluva (Annals of Allergy, Asthma and Immunology 81 (1998) 388-398) report the percentages of allergic responses to Hev b 1, Hev b 3, Hev b 5, and Hev b 6.02 / Hev b 6.01. (hevein / prohevein) in different patient groups as shown in Table 2.

10 Table 2. Major latex allergens

Antigen Rate of Allergic Response in Different _Patients

Hevein / Prohevein 75-83% LSP

75% HCW

_27% SBP_

Hev b 1 22% LSP

51-100% SBP

_52% HCW_

Hevb 3_76% SBP_

Hev b 5 95% HCW

_152% LSP_. ' LSP = Latex Sensitive People HCW = Healthcare Worker '; · · 'SBP = spinal cord patient; · ·: Other latex allergens are likely to play a role in common allergen cross reactions. Despite recent advances in the identification and characterization of latex allergens, including the production of polytonal and monoclonal antibodies to these allergens, * and there are no suitable commercial tests available to measure the residual allergen content of NRLs. 20 products. In particular, there is no method available for clinically relevant j. for simultaneous measurement of latex allergen.

"". Serious allergic reactions to the latex products and the prevalence of the condition have led international and national health authorities to seek 4,116,486 remedies. The use of alternative synthetic materials would be an easy solution. However, due to its low cost and reasonable price, natural rubber latex is important. for the economy of the country, synthetic materials cannot compete with the NRL as a raw material, and while many of the proteins that have been shown to be allergenic could be removed from the NRL by modern technology, they cannot be completely removed from the NRL because these proteins provide both the manufacturing characteristics of the NRL, that is to say, the frontier properties and the like, and the elasticity and comfort of use of the final NRL products. 10 As a result, the specific objective of the managing authorities, taking and selling only non-allergenic or low-allergen medical or industrial latex products to stop sensitization cannot be easily achieved. A viable approach would be to develop and provide a specific and reliable test for measuring and monitoring the amount of latex allergens in NRL-containing products, which should be easy enough for users and producers of these products.

As obviously required, the FDA in the US and CEN in Europe have recently introduced total protein measurement by a modified Lowry's 20 method as a first means of monitoring putative allergen levels in medical rubber latex medical gloves.

•, however, so far there is no agreement on acceptable protein levels \; in medical gloves. In addition, and significantly, total protein and · '·'; allergen levels do not correlate satisfactorily with each other. However, since no suitable commercial tests for measuring the specific allergen residues in NRL containing products are available, insensitive and unrealistic total protein measurements are currently in use.

The total protein of NRL-containing products is currently measured by the conventional Lowry method [Lowry, O.H., et al., J. Biol. Chem.

193, 265-275 (1951)], or modified by the Lowry method approved by the FDA (ASTM D 5712-95) and CEN, or by the LEAP method (i.e., Latex. · ELISA for Antigenic Protein; Guthrie Research Institute, Sayre, Pa, USA), the only commercially available test kit for measuring latex antigens. The detection limit for total protein in these methods ranges from about 0.06 to 9.3 mg / ml and has several drawbacks. For example, a relatively large amount of sample is required, the presence of miscible agents such as surfactants and non-latex proteins may lead to false results and time consuming to perform the methods. Another disadvantage is that the method is based on the absorbance of certain amino acid residues and these amino acids must be present in the peptide chain in an average fashion. The major disadvantage, however, is the obvious fact that they measure the total protein or antigen content of the sample, rather than the concentration of a particular allergen.

European Patent Application 0 704 457 proposes and claims a method for the qualitative and / or quantitative determination of the allergen content of natural rubber latex in a latex-containing material. The assay is based on the use of three proteins isolated by chromatographic techniques in a substantially purified form from natural rubber latex, namely Hev b 4, Hev b 2 and Hev b 3, and the use of selected monoclonal antibodies raised against said allergenic proteins. Only one of the disclosed proteins, i.e. Hev b 3, has been unquestionably shown to be present in the manufactured latex products. In addition, each allergen is measured separately.

Simultaneous measurement of the residual concentration of clinically relevant latex allergens in the finished NRL product is necessary because the response to the various latex allergens contained in the product differs from individual to individual: in other words, allergic individuals are allergic to different latex allergens.

: '· Thus, even for individuals diagnosed with latex allergy, the natural: * · latex product can cause a fatal reaction in another allergic. * ·. 25 individuals, but may be completely harmless to another allergic individual who, on the other hand, may get a serious reaction from another product containing !!! repeat specific allergen. This means that the conventional method of specifically detecting only one latex allergen is neither reliable nor safe for the user. Simultaneous measurement of at least two clinically relevant latex allergy: ··· · 30 genes, let alone simultaneous measurement of all clinically relevant latex allergens, significantly increases. reliability, safety and applicability of the method.

Thus, there is an urgent need for a reliable, repeatable, sensitive, and specific test for the simultaneous measurement of residual levels of clinically relevant latex allergens in NRL-containing products. In addition to the test used by skilled laboratory personnel, there is an urgent need for a rapid test that is suitable for latex allergic or at-risk users or for workers in the latex industry.

The present invention provides a solution and methods for overcoming the drawbacks and disadvantages described above.

It is an object of the present invention to provide reliable quantitative analytical methods for the simultaneous measurement of the residual content of clinically relevant natural rubber latex allergens in a final product made from or containing natural rubber latex. Such reliable quantitative analytical methods could, for example, enable health authorities to control and monitor latex-based products. Similarly, such quantitative analysis methods would enable manufacturers of rubber products to specifically and reliably control the concentration of latex allergens in their products and to provide safe products for the user.

Another object of the invention is to provide rapid qualitative / semi-quantitative screening methods for the simultaneous measurement of the residual content of clinically relevant natural coumulate latex allergens from a final product made from or containing natural rubber latex. Such qualitative / semi-quantitative screening methods would enable the latex allergy or atopic or other users to quickly and reliably analyze the product being used and thus be useful to the users of the product and / or, for example, to the patient for surgical or medical examination; f * ': It is yet another object of the invention to provide a means for carrying out the methods of the invention, that is, test kits for use in the quantitative or qualitative / semiquantitative compositions of the invention. in titanium analytical methods.

Brief Description of the Invention It has been unexpectedly found that it is possible to develop new immuno-chemical methods for the simultaneous measurement of clinically relevant latex allergens using well-known immunological principles and methods. Accordingly, the present invention provides the use of immunochemical analysis methods based on recombinant luonnonkumilateksial-allergens for use, and in particular the individual recombinant latex allergens: 35 or with a suitable fusion partner formed in the fusion proteins of the preferred and specific combination use, and with those responsive 7 116 486 antibodies specific for clinically significant luonnonkumilateksiallergeeni at the same time. In this context, the term "clinically relevant or clinically relevant latex allergens" refers to those natural rubber latex allergens which are clinically significant because they retain their allergenic properties in the manufacturing processes and can be found in end products made from and containing natural rubber latex.

The present invention relates to a homologous or heterologous immunological method for the simultaneous detection and quantification and qualitative and / or semi-quantitative detection of at least two clinically significant specific latex-siallergens in a method comprising contacting a sample of said rubber product with and (b) detecting bound allergens with a mixture of appropriately labeled secondary antibodies, wherein the method is characterized in that said primary antibodies or primary and / or secondary antibodies. the compositions comprising at least two (mono) -specific polyclonal antibodies or monoclonal antibodies directed against: Γ: at least two clinically relevant latex allergens; and please keep: '·. * Allergenic properties in production processes, or an appropriate fusion with a party generated against fusion proteins, said intermediate. ···. Twenty-two different clinically significant latex allergens are selected from the group consisting of ····. min extension factor (REF or Hev b1), rubber particle-associated protein (Hev b3), acidic 16 kD protein (Hev b5), and hevin (Hev b6.02).

In the preferred embodiment, monoclonal antibodies are used as primary and / or secondary antibodies. In another preferred embodiment: monoclonal antibodies are used as primary antibodies and polyclonal antibodies are used as secondary antibodies.

In a particularly preferred embodiment of the invention, four clinically important latex allergens found monoclonal antibodies, which are formed .35 corresponding four clinically important latex allergens or their 8 116 486 with a suitable fusion partner formed in the fusion proteins.

The present invention further relates to test kits for performing the method of the invention. In particular, the present invention relates to 5 test kits for determining at least two clinically relevant specific latex alller genes in a product made from or containing natural rubber latex, wherein the test kit comprises (1) a first set of at least two antibodies, wherein the antibodies are (mono) specific polyclonal or monoclonal antibodies, 10 which are directed to the respective at least two different clinically relevant specific latex allergens that retain against allergenic properties in production processes, or with a suitable fusion partner formed in the fusion proteins, wherein the first antibodies to said bound to the solid phase separately or as a mixture of , 15 (2) at least two antibodies of the second series, wherein the antibodies are (mono) specific polyclonal or monoclonal antibodies directed against at least two The various clinically significant lateksial-allergen that retain the allergenic properties in production processes, or with a suitable fusion partner formed in the fusion protein and 20 en against another specific epitope, wherein said antibodies are suitably labeled, _: wherein said at least two different clinically significant lateksialler - the gene is selected from the group consisting of a rubber elongation factor (REF or Hev b1), a rubber particle associated protein (Hev b3), an acidic 16 kD protein (Hev b5) and a hevin (Hev! ·· \ 25 b6.02), · ' ··. (3) the reagent or reagents needed for detection, and »·. (4) the standards required for the quantification of said clinically relevant specific latex allergens.

In addition, the test kit may optionally contain (5) a reagent or reagents to elute clinically relevant latex allergens from a sample suspected of containing such allergens, and / or (6) a reagent or reagents for possible washing steps, and / or ( 7) latex allergen control. * *. reagent or reagents.

In a preferred embodiment of the invention, the test kit contains reagents for detecting four clinically relevant latex allergens. In another preferred embodiment of the invention, the test kit 9 116486 of the invention contains reagents for the detection of all clinically relevant latex allergens, as appropriate.

The present invention enables the simultaneous detection of at least two, preferably four, and most preferably all four clinically relevant latex allergens that are currently known to retain their IgE-binding ability during tough rubber manufacturing processes and which have been identified in the final products. These latex allergens are thus Hev b1, Hev b3, Hev b5 and Hev b6.02, all of which are undeniably demonstrated in finished NRL-containing products.

Although known as the principle of these immunochemical methods, they have not previously been used in the field of latex allergy to simultaneously detect and measure the residual concentrations of clinically relevant latex allergens in a final product containing natural rubber latex. The simultaneous use of selected purified and standardized recombinant latex allergens and their specific antibodies reactive with them in a specifically defined combination renders the methods of the invention highly specific and extremely sensitive and capable of detecting specific latex allergens or their epitopes in the nanogram.

The methods of the invention are suitable for quality assurance and product monitoring of rubber manufacturers and assist companies in the production and marketing of low-allergen or low-allergen products: they provide a means for health authorities to perform monitoring and market analysis, and for health care professionals and other users. : *. *. for testing non-NRL material.

• 1 * 25 Brief Description of Patterns · · ·

* I

Fig. 1 shows a standard curve of a recombinant avidin-Hev b 6.02 fusion protein and a Hev b 6.02 concentration of hand samples on a microtiter plate coated with a monoclonal antibody raised against a recombinant avidin-Hev b 6.02 fusion protein.

Figure 2 shows a standard curve of a recombinant MBP-Hev b5 fusion protein and Hev b 5 in hand samples on a microtiter plate coated with [. * A recombinant MBP-Hev b5 monoclonal antibody. fusion protein.

10 116486

Detailed Description of the Invention

The homologous or heterologous immunochemical methods of the present invention are based on the idea of simultaneous use of (mono) specific polyclonal and / or monoclonal antibodies directed against specific, clinically relevant recombinant latex allergens and recombinant fusion proteins of said allergens. for detection and identification and / or quantification of NRL-containing products in a single assay.

Recombinant latex allergens or their fusion proteins for the production or use of (mono) -specific polyclonal or monoclonal antibodies as standards in the methods of the present invention were prepared by the baculovirus expression system (BAC-TO-BAC, GIBCO BRL, Gaithersburg, MD, USA). use primers. Specifically, recombinant Hev b 6.02 and / or its recombinant fusion protein with avidin or maltose binding protein (MBP), recombinant Hev b 5 and / or its recombinant fusion protein with avidin or MBP, recombinant Hev b 1 and / or fusion protein with avidin or MBP; and recombinant Hev b 3 and / or its recombinant 20 fusion protein with avidin or MBP. For a detailed description of the manufacturing methods, reference is made to Examples 2-6. However, any known conventional expression method for the preparation of recombinant proteins can be used. Thus, prokaryotic expression cysts such as E. coli, Bacillus subtilis and the like, and other eukaryotic expression systems, such as yeast, fungal and mammalian cells, are equally suitable for this purpose.

. * '. Recombinant latex allergens can be prepared with both prokaryotic and eukaryotic expression systems as single proteins or, as fusion proteins with conventionally used fusion partners such as avidin, streptavidin or maltose-binding, S-transferase or other available isolation and purification label. Bacterial expression systems are well characterized and inexpensive and produce large amounts of the desired proteins. Although most of the post-translational modifications are missing from the produced recombinant proteins, they can be used as highly specific immunomodulators for antibody production. Eukaryotic expression systems, on the other hand, produce proteins with post-translational modifications.

The fusion protein approach provides a means for easy chromatographic isolation and purification of the fusion proteins. For example, 5 MBP fusion proteins can be purified by affinity chromatography on an amylose column and avidin fusion proteins on a biotin-agarose column, a 2-iminobiotin-agarose column, or other biotin derivatives coupled to agarose. The recombinant latex allergens can then be cleaved from the fusion protein by a suitable enzyme or chemically without impairing the immunological or other biological properties of the allergen. For example, MBP fusion proteins can be cleaved by factor Xa protease and avidin fusion proteins by enterokinase alpha or thrombin.

For accurate identification, all recombinant latex allergens used for antibody production were carefully analyzed by standard methods such as SDS electrophoresis, immunoblot analysis, N-terminal sequencing and (MALDI-TOF) mass spectrometry. In addition to these structural analyzes, the immunological properties of the recombinant allergenic NRL-derived proteins were analyzed by standard methods, such as the inhibition ELISA assay, using characterized sera from patients with NRL allergy. Immunological properties were also compared with the corresponding purified native rubber tree proteins. Functional properties such as interactions with other molecules are studied when appropriate. . ; dirty, optical biosensor technology using devices such as BiaCore or laSys.

: \. In all cases, the structural and functional properties of the latex allergens produced by recombinant DNA technology were very similar or! identical to the corresponding native proteins.

The recombinant latex allergens that are clinically significant and "· *> 'can maintain their allergenicity of the manufacturing process, the fusion portion with the side with or without a fusion partner, form a basis to provide specificity of I *: 30 sifisiä panels of monoclonal antibodies and (mono) specific for polyclonal antibodies. For present purposes, polyclonal and monoclonal antibodies were prepared against the various recombinant latex alleles, genes, or fusion proteins thereof, and their isotypes were determined using

1 I

standard procedures well known in the art. In this regard, ♦ ... T 35 refers to Example 8.

'' * I

12 116486

Generally, the antibodies useful in the methods of the present invention may be either mono- or polyspecific polyclonal antibodies, monoclonal antibodies, or recombinant single-chain monoclonal antibodies or antigen-binding fragments thereof that are specific for clinically relevant latex allergens. When used in the homologous immunochemical method of the invention (the allergen binding antibody, i.e., the primary antibody is derived from the same animal species), the monoclonal antibodies must be specific for different epitopes. When used in the heterologous immunochemical method of the invention (the allergen-binding antibody is derived from different animal species), the monoclonal antibodies (provided that anti-mouse subclass-specific labeled monoclonal or polyclonal antibodies are used) must belong to different mouse isotypes. In cases where the binding antibody is a monoclonal antibody and the detection antibody or secondary antibody is a labeled polyclonal antibody, the mouse isotype is not critical.

Preferably, the antibodies useful for binding latex allergens in the present invention are monoclonal antibodies or mono-specific polyclonal antibodies or functional fragments thereof that are specific for certain molecular epitopes on clinically relevant latex allergens. The most preferred antibodies for binding of latex allergens are monoclonal antibodies. Any type of monoclonal antibody can be used, but the antibodies that are included. . Mouse IgG subclasses (isotypes) are preferred because they can be efficiently purified by immunoaffinity chromatography.

Preferably, in the present invention, the detection of latex allergens •;:; useful antibodies are their monoclonal antibodies or monospecific polyclonal antibodies or functional fragments thereof.

> I

pathways that are specific to other particular molecular epitopes in clinically relevant latex allergy genes. In a preferred embodiment of the invention; In one embodiment, polyclonal antibodies raised against a mixture of clinically relevant recombinant latex allergens are used for simplicity of use.

I ,. For the simultaneous administration of clinically relevant latex allergens; capture would be possible, either as primary or binding antibodies; The specific antibodies used in the immunochemical methods according to the invention as 35 DAR or detection antibodies were pre-mixed in predetermined amounts. These specific amounts were determined on the basis of separate analyzes of each individual recombinant Nanten allergen or its fusion protein, for example by ELISA. Thus, the primary and secondary antibody mixtures useful in the invention are recombinant Hev b 6.02 and Hev b 5 5 or recombinant Hev b 6.02 and Hev b 3 or recombinant Hev b 6.02 and recombinant Hev b 1 or any suitable mixture of two clinically relevant latex allergy or fusion protein monoclonal antibodies thereof, wherein the primary and secondary antibodies are directed against different epitopes of the respective latex allergens. Alternatively, mixtures of primary and secondary antibodies useful in the invention are monoclonal antibodies against all clinically relevant recombinant latex allergens, recombinant forms of Hev b 6.02, Hev b 5, Hev b 3 and Hev b 1 or their fusion proteins. mixtures of primary and secondary antibodies directed against different epitopes of the respective latex allergens. Alternatively, binding antibodies may be used separately, i.e., they may be attached to different portions of the solid phase.

Detecting antibodies can be labeled with any conventional label. Thus, a label useful in the methods of the present invention is an enzyme label, a biotin-avidin system, a fluorescence, phosphorescence or luminescence label, a radioactive label or a chromophore, a gold label, or any other conventionally used label. Inexpensive labels. *. ; are enzyme labels such as horseradish peroxide.

Suitable materials for the solid phase are synthetic materials, '..! Such as polystyrene, polyvinyl chloride, polyamide and other conventionally used synthetic polymers as well as natural polymers such as cellulose, derivatives of natural polymers such as cellulose acetate and nitrocellulose, paper and glass.

The solid phase may be in the form of a microtiter plate, tube, stick or bead, as is conventionally used in the art. Paper strips, sheets or films also form a suitable solid phase useful in the invention. Preferred solid phases are microtiter plates, tubes, sticks and strips of paper or nitrocellulose. The most preferred materials are microtiter plates and, in a rapid quaternary or semi-quantitative method, a synthetic material; · * 35 molded stick and also nitrocellulose film strip.

'* · T> »14 116486

Primary antibodies are fixed to the solid phase by any conventional means. Typically, incubation of the antibody solution in a suitable buffer, such as PBS, phosphate buffer and the like, with a solid phase for a predetermined time will produce repeatable attachment. The nitrocellulose 5 can be coated by simply dispensing or spraying the antibody solution in a suitable buffer as a line or pattern on the membrane. The paper strips may be coated by covalent chemical coupling using glutaraldehyde or cyanogen bromide as the coupling agent by methods known in the art.

The solid phase may contain discrete regions, e.g., wells in a microtiter plate or bands on a polystyrene stick or nitrocellulose membrane containing a suitable antibody mixture. Alternatively, specific antibodies to the individual recombinant latex allergens or their fusion proteins may be coupled to different wells or zones with a solid phase. For example, microtiter plates may be formed containing separate rows of wells coated with specific binding antibodies to each of the clinically relevant latex allergens.

The detectable latex allergens are relatively easily water soluble. Thus, generally, the solvent used to prepare a sample of the product suspected of containing latex allergens may be any suitable aqueous buffer such as phosphate buffer, phosphate buffered saline (PBS), acetate buffer, TRIS buffer and the like about. , 10 to 200 mM and a pH of about 6-9. A preferred buffer is 100 mM PBS. You know •] '; For these purposes, for example, to stabilize the antibody or to increase the viscosity of the solution, additives such as sugars may be added.

* »25 The test sample may be any product suspected of containing latex allergens, such as medical or protective gloves, tubing, catheter, '··· * mask and the like. It may also be a solution or a sample taken from a particular manufacturing step.

The present immunochemical methods may utilize: any known immunochemical principle provided that the requirements of the claims are met, such as Enzyme Linked ImmunoSorbent Test, Turbidometry, Nephelometry, Competitive Assays, Time-Resolved Fluorometry (see Immunoassay, Diamandis, EP and Christopoulus, TK, Ed. (1997); i * 35 AACC Press, USA).

'C 1 1 6486 15

The test kit of the present invention contains the reagents necessary to carry out the immunochemical methods of the present invention. Thus, the test kit contains a first set, preferably a mixture, of (mono) specific polyclonal or monoclonal antibodies, preferably monoclonal antibodies, fixed in a solid phase, such as one of the aforementioned solid phases. The first series antibodies are directed to each of the corresponding clinically relevant with the specific latex allergens or a suitable fusion partner formed in the one-fusion protein is a fusion of the epitope.

In a presently preferred embodiment, the kit comprises a solid phase coupled with a mixture of monoclonal antibodies against at least two clinically relevant latex allergens, for example, recombinant Hev b 5 and recombinant Hev b 6.02 or their recombinant fusion proteins, recombinant Hev b 6.02 15 and recombinant Hev b 1 or suitable recombinant fusion proteins thereof, recombinant Hev b 6.02 and recombinant Hev b 3 or suitable recombinant fusion proteins thereof, or any of two clinically relevant latex allergens or against a mixture of fusion proteins. In a particularly preferred embodiment, the kit contains 20 solid phases coupled with a mixture of monoclonal antibodies against all latex allergens known to be of clinical relevance, i.e., recombinant Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02 or suitable recombinant fusion proteins thereof, or with two mixtures each containing a different pair of monoclonal antibodies: '*; 25 two clinically relevant latex allergens.

The test kit also contains a second series, preferably a mixture. * ··. (mono) specific polyclonal or monoclonal antibodies, preferably

* I

monoclonal antibodies, each of which is directed against the respective clinic. , Cally significant specific latex allergens or a suitable fusion • 'i, / 30 established with the second party of the fusion protein specific to an epitope of the • *. In a presently preferred embodiment, the test kit contains antibodies to at least two clinically significant latex allergens, for example, recombinant Hev b 5 and recombinant Hev b 6.02 or both. den suitable anti-recombinant fusion proteins, recombinant Hev b 6.02 and recombinant Hev b 1 or their appropriate recombinant fusion proteins, recombinant Hev b 6.02 and recombinant Hev b 3 16 116486 or their against suitable recombinant fusion proteins, or any mixture of two clinically relevant latex allergens or their appropriate fusion proteins. In a particularly preferred embodiment, the solid phase is coupled with a mixture of monoclonal antibodies against all latexial-5 allergens known to be of clinical relevance, i.e., recombinant Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02 or suitable recombinant fusion proteins thereof. These antibodies are optionally appropriately labeled with a suitable label, such as one of the aforementioned labels, and / or optionally bound to the solid phase described above to allow detection.

Alternatively, the test kit contains labeled polyclonal antibodies raised against a mixture of the corresponding specific clinically relevant latex allergens or fusion proteins thereof with the appropriate partners to enable detection of latex allergens. In a presently preferred embodiment, the kit comprises antibodies against at least two clinically relevant latex allergens, for example, recombinant Hev b 5 and recombinant Hev b 6.02, or their appropriate recombinant fusion protein, recombinant Hev b 6.02, and recombinant Hev b 1. or against their appropriate recombinant fusion protein 20, recombinant Hev b 6.02 and recombinant Hev b 3 or their suitable recombinant fusion protein, or against any combination of two clinically relevant latex allergens or their appropriate fusion proteins. In a particularly preferred embodiment, the kit contains polyclonal antibodies formed at least from recombinant 25 Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02, or a suitable recombinant thereof. * * '. nant fusion protein mixture.

. Alternatively, specific antibodies to individual recombinant latex allergens or their fusion proteins may be coupled to a solid phase. . to different pits or zones. For example, microtiter plates containing separate wells of wells coupled with specific binding antibodies to each of the clinically relevant latex allergens can be constructed.

The kit also contains the reagent or reagents needed; '; such as the label enzyme substrate, the stopping reagent, the development reagent, the pH indicator when the reaction changes pH, the reagent 35 to change the ionic strength, the detergent and the like. In addition, the test kit according to the invention may contain a positive and / or a negative control sample. In an embodiment using an unlabeled secondary antibody, a labeled tertiary antibody, such as an HRP-labeled anti-IgG antibody, is included in the test kit of the invention.

The standards required for the quantification / semiquantification / qualification of specific clinically relevant latex-5 allergens are also included in the test kits of the invention. In the presently preferred embodiment, the standards are two recombinant clinically relevant allergens, such as recombinant Hev b 5 and recombinant Hev b 6.02 or their respective recombinant fusion proteins, recombinant Hev b 6.02 and recombinant Hev b 1 or 10 their appropriate recombinant fusion proteins H, and recombinant Hev b 3 or their suitable recombinant fusion proteins or any two clinically relevant latex allergens or their suitable fusion proteins. In a particularly preferred embodiment, the test kit of the present invention comprises at least recombinant Hev b 1, Hev b 3, Hev b 5 15 and Hev b 6.02, or appropriate recombinant fusion proteins thereof, preferably all in one flask.

In addition, the test kit may optionally contain a reagent or reagents for eluting clinically relevant latex allergens from a sample suspected of containing such allergens. Such reagents include e.g. PBS, phosphate buffer, acetate buffer or any of the above buffers. Similarly, washing solutions or concentrates thereof such as PBS-: 0.05% Tween may be present.

As will be apparent to a person skilled in the art, the idea of the invention can be implemented in many different ways now and in the future. Thus, the following examples are in no way intended to limit the invention and its embodiment. * * *. but are given by way of illustration only.

»· ** <· Example 1

Production of Native Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02 • · *

Native Hev b1 was substantially purified by the method of Alenius, H. et al. Arch. *; · * 30 Allergy Immunol. 109 (1996) 362-368]. Native Hev b 3 iT was substantially purified following the principles described for Hev b1 by Alenius, H. et al. (Supra). Native Hev b 5 was substantially purified by Akasawa, A. et al., [J. Biol. Chem. 271: 25389-25393 (1996)]. Native Hev ** ** b 6.02 was substantially purified by Alenius, H. et al. Immunol. 156: 351618-1625 (1996)].

116486 18

Example 2

Preparation of recombinant Hev b 6.02

For the preparation of recombinant Hev b 6.02 or hevein (SEQ ID NO: 11), the hevein coding fragment was amplified by PCR from p1Prohev plasmid essentially according to Airienne, K.J. et al. Express. Purif. 9 (1997) 100-108]. PCR-Prohev6 [(5 '- TA TGT GGA TCC GAC GAC GAC GAC AAA GAG CAA TGT GGT CGG CAA GCA-3' (SEQ ID NO: 3); δHI site underlined; enterokinase site thickened; heme sequence italicized) was used and Prohev2 [5 '- AAC ACA AGC 10 TT C TTA GTC TTT GCA ATT GCT TTG GC-3' (SEQ ID NO: 4); Hind Sweat underlined; stop codon thickened; hevein sequence in italics) as an inverse primer. PCR was performed as described by Airenne, K.J., and Kulomaa, M.S. [Gene 167 (1995) 63-68]. After digestion with Sg / II + H / ndlll (Promega, Madison, WI, USA), the PCR product was applied to a 1.5% preparative agarose gel. A fragment of the expected size was recovered from the gel as described by Heery et al., TIG 6 (1990) 173 and further purified by Magic ™ DNA Clean-up (Promega). The purified fragment was subcloned into the pFastBACI donor plasmid (GIBCO BRL, Gaithersburg, MD, USA), and recombinant viruses containing the Hev b 6.02 sequence were generated using the Bac-TO-Bac Baculovirus expression system (GIBCO BRL, Gaithersburg, USA). by.

* ·; Alternatively, recombinant Hev b 6.02 may be obtained by cleavage of a * ♦ · ”club enzymatically from a protein fusion protein (cf. Example 3).

* # * »

Example 3 Preparation of recombinant avidin-Hev b 6.02 fusion protein

To generate viruses containing both Hev b 6.02 and avidin coding regions, the amplified fragment containing the Hev b 6.02 sequence obtained as described in Example 2 was subcloned into the pFastBACI-] ··· [pbacAVs + C based vector which contains a form of secretion of recombinant avidin, essentially Airenne, KJ et al. [Prot. Express. Purif. v: 17 (1999) 39-145].

Subsequently, for the preparation of the avidin-Hev b 6.02 fusion protein, ~ 5 X 10 7 Sf9 cells (ATCC CRL 1711) in SF-900 II SFM serum-free medium containing biotin and Pluronic F68 (GIBCO BRL, Gaithersburg, 35 MD, USA; 041 -94100A) had been removed, inoculated to a final volume of 25 ml with 250 ml 19 116486

Erlenmeyer flask. Recombinant viruses were added to produce many 2 Pfu / cell infections. After incubation for three days at 27 ° C in a shaker at 125 rpm, cells were pelleted by centrifugation (1000 X g, 22 ° C, 5 min) and the button was frozen in liquid nitrogen and stored at -70 ° C.

The crude avidin-Hev b 6.02 fusion protein was purified and characterized as described by Airenne, K.J. et al. (1999) [supra]. The purified fusion protein was shown to have the same equivalents of biotin-binding properties as native egg white avidin. The hevein moiety was also found to have the same or identical functional and structural properties 10 as the native hewey.

Example 4

Preparation of recombinant MBP-Hev b 1 fusion protein and recombinant Hev b 1

To produce recombinant Hev b 1, i.e., REF or the rubber extension factor (SEQ ID NO: 5), the Hev b 1 coding fragment was amplified by PCR from REF-pMALc-2. The PCR forward primer was (5- ATGGCTGAA-GACGAAGACAACCAA-3 '; Hev b 1 sequence italicized; SEQ ID NO: 6), and the reverse primer (5' - GATATCAAGCTTTCAATTCTCTCCATAAAACACCTTA-3 ',

H / ndlll site underlined; Hev b 1 sequence in italics; SEQ ID NO: 7). PCR

20 was performed in a staple fashion by Airennen, K.J. and Kulomaa, M.S. [Gene 167 (1995) 63-68]. After digestion of Hindi (Promega, Madison, WI, USA):: The PCR product was applied to a 1.5% preparative agarose gel. The fragment of expected size was recovered from the gel as described by Heery et al., TIG 6: 173 (1990) and further purified by ethanol precipitation. Puh-, ···. The 25 isolated fragments were subcloned into pMAL-c2 plasmid digested * »

Xmn \ + Hind \\\ - Enzymes \\\ s. The sequence of the recombinant plasmid was checked by an automatic DNA sequencer.

When this recombinant plasmid was introduced into E coli JM109 host cells according to the manufacturer's instructions (New England BioLabs Inc., Hitchim, Herfordshire, 30 UK), an MBP-Hev b1 fusion protein with a Factor Xa cleavage site was produced. between the tag and the allergen. Purification of MBP-Hev b 1 Fusion Protein ···. The assay was performed according to method 1 designed to purify soluble MBP fusion protein expressed in the cytoplasm from pMAL-c2 vector (New England BioLabs Inc., Hitchim, Herfordshire, UK). The isolation was based on ': 35 affinity chromatography on an amylose column.

Cleavage of the 116486 MBP tag from Hev b 1 can be performed by Factor Xa protease to obtain recombinant Hev b 1.

Example 5

Preparation of Recombinant MBP-Hev b 3 Fusion Protein and Recombinant Hev b 3 5

To produce recombinant Hev b 3, a rubber particle-associated protein (SEQ ID NO: 8), the Hev b 3 coding fragment was amplified by PCR from pBS + SRPP. The PCR primer was (5 '- ATGGCTGAAGAGG-TGGAGGA - 3'; Hev b 3 sequence italicized; SEQ ID NO: 9) and the reverse transcript (5 '- GATAT CAAGCTT TT A TGA TGCCTCA TCTCCAA - 3', H / ndlll-). underlined; Hev b 3 sequence in italics; SEQ ID NO: 10). PCR was performed as described by Airenne, K.J. and Kulomaa, M.S. (Gene 167 (1995) 63-68). After digestion with / ndllIIII (Promega, Madison, WI, USA), the PCR product was applied to a 1.5% preparative agarose gel. The fragment of expected size was recovered from the gel as described by Heery et al. (TIG 6: 173, 1990) and further purified by ethanol precipitation. The purified fragment was subcloned into pMAL-c2 plasmid digested with Xmn + Hind III. The recombinant plasmid was sequenced by automated DNA sequencing.

When this recombinant plasmid was introduced into E coli JM 109 host cells according to the manufacturer's instructions (New England BioLabs Inc., Hitchim, Herford: shire, UK), an MBP-Hev b 3 fusion protein with the Xa cleavage site MBP was obtained. between the tag and the allergen. Purification of MBP-Hev b 3 fusion protein was carried out according to method 1 designed for soluble MBP-fu, · * ·. 25 sioprotein expressed in the cytoplasm from the pMAL-c2 vector (New England BioLabs Inc., Hitchim, Herfordshire, UK). The isolation was based on affinity chromatography on an amylose column.

. . Cleavage of the MBP tag from Hev b 3 can be performed with the Fac: j: Tor Xa protease to yield recombinant Hev b 3.

30 Example 6

Preparation of Recombinant BP-Hev b 5 Fusion Protein (To produce recombinant Hev b 5, an acidic 16 kD protein (SEQ ID NO: 12), the Hev b 5 coding fragment was amplified by PCR from the Hevb5-pMALc-2 plasmid The PCR primer was (5 '-ATGGC-21 116486 CAGTGTTGAGGTTGA-3'; Hev b 5 sequence italicized, SEQ ID NO: 1), and the reverse primer (5 '-GATATCAAGCTT7TATTCCTCTGI ITI TTC-3', H / ndll1-koh-1). underlined, Hev 3 sequence in italics; SEQ ID NO: 2) PCR was performed as described for Airennen, KJ and Kulomaa, MS [Gene 167 (1995) 63-68] Hindi (Promega, Madison) , WI, USA) after digestion, the PCR product was applied to a 1.5% preparative agarose gel. The fragment of the expected size was recovered from the gel as described by Heery et al., TIG 6 (173) (1990) and further purified The purified fragment was subcloned into pMAL-c2 plasmid digested with Xmn1 + Hind III. the sequence of the ecombinant plasmid was checked by an automated DNA sequencer.

When this recombinant plasmid was introduced into Eco / JM109 host cells according to the manufacturer's instructions (New England BioLabs Inc., Hitchim, Herfordshire, UK), an MBP-Hev b 5 fusion protein with factor Xa cleavage site 15 MBP was obtained. tag and allergen. Purification of MBP-Hev b 5 fusion protein was performed according to method 1 designed for purification of soluble MBP fusion protein expressed in cytoplasmic vector pMAL-c2 (New England BioLabs Inc., Hitchim, Herfordshire, UK). The isolation was based on affinity chromatography on an amylose column.

Cleavage of the 20 MBP tag from Hev b 5 can be performed by Fac Tor Xa protease to obtain recombinant Hev b 5.

> r ·

Example 7 · · ·

Preparation of polyclonal antibodies using recombinant avidin • ·

Against Hev b 6.02 Fusion Protein • ·. · · ·, 25 Polyclonal antibodies recombinant avidin-Hev b 6.02 fusion protein were prepared using standard procedures as follows. Rabbits were immunized intramuscularly with 0.05-0.1 mg of recombinant avidin-Hev b 6.02 fusion protein prepared as described in Example 3. In the En-: · first immunizations, the antigen was suspended in 0.2 ml of physiological saline and Freund's complete adjuvant (1: 1) and injected into four rabbit body parts (0.05 ml each). The following effects were used in Freund's. incomplete adjuvant (1: 1). The effects were given 3-4 times every 3-4 weeks · *. The last boost was given intravenously. Blood was collected after 7-9 days.

22 116486

Similar methods are used to make polyclonal antibodies against recombinant Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02, or their appropriate recombinant fusion proteins, or both, or mixtures thereof.

Example 8

Preparation of Monoclonal Antibodies Against Recombinant Hev b6.02 Protein or Recombinant Hev b6.02-Avidin Fusion Protein and Recombinant Hev b5-MBP Fusion Protein Galfren and Millstein [ G. and Milstein, C., Meth. Enzymol. 73: 1981, pp. 1-46] and Godingin, GW, [Monoclonal Antibodies: Principles and Practice (Production and Application of Monoclonal Antibodies, 1986), Cell Biology, Biochemistry and Immunology, Academic Press, Harcourt 15 Brace Jovanovich Publishers] Recombinant Hev b 6.02 or its recombinant fusion protein with avidin (avidin-Hev b6.02) and recombinant Hev b 5 or its recombinant fusion protein maltose binding protein (MBev) MBP-Hev b5) were used as antigens in BALB / c mice An antigen solution containing 15 µg of the antigen-responding antigen in 100 µl of 0.1 M phosphate buffered saline (PBS), pH 7.0 - 7.4 was mixed with 150 pl of Freund's complete adjuvant and injected intraperitoneally into mice, every three weeks, an intramuscular boost of the same dose (15 µg) of antigen emulsified.

Freund's incomplete adjuvant in equal amounts at four sites. 25 (60 µl / well). After four weeks, the antigen was injected intravenously as an ···· inhalation of 20 µg in 150 µl of 0.1 M PBS. Two weeks later, three days before the fusion, the same booster dose was administered.

,, The standard technique for the production of hybridomas was applied to these studies (see Galfre, G., Milstein, C., supra). Briefly, pancreatic cells from immunized mice were prepared and fused to the murine myeloma line Sp2 / 0-Ag-14 (abbreviated as Sp2), a total non-producer · ·. variant selected from a hybridoma fusion between MOPC-21 and BALB / c pancreatic cells. Polyethylene glycol (PEG), MW 4000, was used as the fusion agent and the hybridomas were cultured in RPMI-1640 medium buffered with HCO 3 '/: 35 CO 2 containing 10% fetal calf serum (FCS). Hypoxanthine, amine 23 116486 nopterin and thymidine (HAT) were used to select the hybridoma. Thymocytes from BALB / c mice were used in the early stages of hybridoma cultivation as well as in recloning.

Hyperidomas were first screened for the production of specific monoclonal antibodies using a conventional indirect ELISA and a homologous antigen. Thereafter, or in parallel, the hybridomas were screened on a fusion protein partner, whereby the fusion protein was used as an immunogen with recombinant purified homologous antigen and natural rubber latex as well as other glove extracts (Tables 2 and 3).

10 Positive clones were further tested for their reactivity

Western immunoblotting using recombinant and natural latex proteins. Once specific reactivity was determined, monoclonal antibody isotypes were determined using double immunodiffusion on an agarose gel or ELISA assays for murine immunoglobulin class and subclass specific for non-labeled and appropriately labeled antibodies (Goding, G of Monoclonal antibodies in Cell Biology, Biochemistry and Immunology, 1986, pp.104-107, Academic Press, Harcourt Brace Jovanovich Publishers] The viability and potential of hybridomas to produce monoclonal antibodies to tut-20 kit by cloning and re-cloning selected cell lines. was also to reduce the risk of overgrowth of non-producer cells and to ensure that the antibodies are indeed monoclonal (Tables 3 and 4).

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Table 3. Monoclonal antibodies to Hev b 6.02

Mab Isotype Mab Product Reactivity in Antigen ELISA (= Cell Growth Starter) Name___Bilicity,% ______

Av-Hev b Hev b NRL * GE ** Specific ____ 6.02__6.02 ____ epitope

Hbe.02-1 igG2a 100 __ + __ + __ + + NK ***

Hbe.02-2 lgG1 100 _ [* _ + \ + | ♦ NK_

Table 4. Monoclonal antibodies to Hev b 5

Mab Isotype Mab Product Reactivity in Antigen ELISA (= Cell Growth Stator) Name___bilicity,% ______ MBP-Hev Hev b 5 NRL * GE ** Specific peptide ________dj_

HbS-1 lgG1__85 __ + __ + __ + ♦ 101-114

Hb5-2 lgG1 100 __ + __ + __ + + NK ***

Hb5-3 lgG1__75 __ + __ + _ + ♦ NK_

Hb5-4 lgG1__90 __ + __ + __ ♦ * 101-114

Hb5-5 lgG1 100 __ + __ + __ + * NK_; Hb5-7 lgG1__80 __ + __ + __ + * 101-114!: Hb5-9 lgG1__90 __ + __ + __ + + NK_

Hb5-10 lgG1 100 __ + __ + __ + + 101-114

Hb5-11 IgG2b 100 _ [+ _ + + + 38-51 * NRL - Natural rubber latex.,. · ** GE - Glove extract 5 *** NK - Not known i * * • V Example 9

Determination of the assay conditions for Hev b 6.02 Layer ELISA using latex glove extracts · · To determine and confirm the appropriate assay conditions · 10 was designed for the Layer ELISA for Hev b 6.02. Protective gloves from different manufacturers were used as samples.

25 116486

Monoclonal antibody against recombinant avidin-Hev b 6.02 fusion protein (clone Hb6-2) prepared in Example 8 (10 pg / ml in 50 mM sodium phosphate buffer, pH 6.0) was added to 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) (150 μΙ / well) and incubated for 3 hours or overnight at room temperature. The wells were washed with 0.05% PBS-Tween-20 and blocked with 300 µl of 0.5% bovine serum albumin (BSA) in 50 mM phosphate buffer, pH 6.5. The plates were used after removal of the blocking solution. Samples (glove extracts) or standards (recombinant Hev b 6.02 prepared as described in Example 2) in 10 volumes were added to appropriate wells followed by addition of 100 μΙ assay buffer (50 mM sodium phosphate, 50 mM sodium chloride, 10 mM EDTA). , 0.3% BSA, 0.03% Tween-20, pH 7.4). After incubation for 60 minutes on a horizontal shaker, the plates were washed four times with PBS-0.05% Tween-20. One hundred microliters of polyclonal anti-Hev b 6.02 antibody prepared as described in Example 7 was added at a 1: 2000 dilution and the plates were incubated and washed as in the previous step. One hundred microliters of goat anti-rabbit antibody labeled with HRP at 1: 2000 dilution (Dako P0448) was added, the plates incubated for 15 min and washed as above. Substrate (ABTS, Kirkegaard-Perry) was added (100 μΙ) to each well. After fifteen incubations for 20 minutes, the reaction was stopped with 50 µl of 1% SDS and absorbances were read at 414 nm (Multiscan, Labsystems). Glove extracts were prepared by cutting the gloves into pieces of about 1 cm 2 and extracting them with PBS (1 g / 5 mL PBS) for two hours at room temperature with occasional stirring. After the extracts were centrifuged and filtered through a 0.2 µm filter / * '; 25 samples were determined immediately or frozen for later analysis / * ·. for. Concentrations of glove extract Hev b 6.02 were read from a standard curve. The results for latex glove extracts and a standard curve of 0.5 to 200 pg / L for quantifying Heb b 6.02 are shown in Figure 1.

The concentration of Hev b 6.02 in the glove extracts tested varied significantly: * · 30: the correlation between protein concentration and Hev b 6.02 was poor, indicating that the total protein does not indicate allergenicity or non-allergenicity. Similar results have been obtained using total protein and skin. randomized or RAST inhibition tests.

26 116486

Example 10

Determination of the assay conditions to be used in the Hev b 5 sandwich ELISA using latex glove extracts

A sandwich ELISA for Hev b 5 was designed to determine and confirm the appropriate assay conditions. Protective gloves from different manufacturers were used as samples.

Monoclonal antibody against recombinant avidin-Hev b 5 fusion protein (clone Hb5-3) prepared in Example 8 (10 µg / ml in 50 mM sodium phosphate buffer, pH 6.0, 0.1 M sodium phosphate, pH 6). 0), li-10 were plated in 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) (150 μΙ / well) and incubated overnight at room temperature. The wells were washed and blocked with 300 silicon 0.5% bovine serum albumin (BSA) in 50 mM phosphate buffer, pH 6.5. The plates were used after removal of the blocking solution.

Samples (glove extracts prepared as described in Example 9) or standards (recombinant Hev b 5 prepared as described in Example 4) in a volume of 50 µl were added to appropriate wells followed by addition of 100 µl of assay buffer (50 mM sodium phosphate, 50 mM sodium chloride, 10 mM). EDTA, 0.3% BSA, 0.03% Tween-20, pH 7.4). After incubation for 60 minutes on a horizontal shaker, the plates were washed four times with PBS-0.05% Tween-20. One hundred microliters of anti-Hev b5 monoclonal antibody (clone Hb5-11) conjugated to HRP at 1: 200 dilution was added, the plates incubated for 30 minutes and washed as in the previous step. Substrate (ABTS, Kirkegaard-Perry) was added (100 µl) to each well. Fifteen minutes in-; After V incubation, the reaction was quenched with 50 µl of 1% SDS in distilled water and absorbances were read at 414 nm (Multiscan, Labsystems).

: The Hev b5 concentrations of the glove extract were read from a standard curve. The results for latex glove extracts and a standard curve ranging from 1 to 200 pg / L for quantifying Heb b 5 are shown in Figure 2.

:, ·. The concentration of Hev b 5 in tested glove extracts varied significantly.

! ···, '30 The same amount of Hev b5 can be detected in two glove extracts but the protein concentration was five times higher in the second glove extract (# 1 vs. # v: 5). The total protein does not indicate allergenicity or non-allergenicity.

I I

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27 116486

Example 11

Concurrent determination of Hev b5 and Hev b6.02 in hand samples

For the preparation of plates, monoclonal antibodies against recombinant avidin-Hev b 6.02 fusion protein and recombinant MBP-Hev b5 fusion-5 protein at predetermined concentrations of 1-100 pg / ml in 50 mM phosphate buffer, pH 6.0, (150 µl) were added to 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) (150 μΙ / well), each monoclonal antibody separately for the respective half of the plates, and the plates were incubated for three hours or overnight at room temperature. The wells were washed with 0.1 M PBS-10 Tween-20 (0.05%) and blocked with 300 µl of 0.5% bovine serum albumin (BSA) in 50 mM phosphate buffer, pH 6.5. The plates were used after removal of the blocking solution.

Samples (glove extracts) or standards (predetermined amounts of recombinant Hev b 6.02 and recombinant Hev b5), in a mixture of 50 silicon 15 were added to the correct wells followed by addition of 100 µl of assay buffer (50 mM sodium phosphate, 50 mM sodium chloride, 10 mM EDTA, 0.3% BSA, 0.03% Tween-20, pH 7.4). After incubation for 30 minutes on a horizontal shaker, the plate was washed four times with PBS-0.05% Tween-20 and 100 µl of antibody containing a mixture of recombinant avi-20 din-Hev b 6.02 fusion protein and recombinant Against MBP-Hev b5 fusion protein, in a predetermined ratio and both labeled; horseradish peroxidase at predetermined dilutions of 1: 100 to 1: 100,000 * ·:; was added. The plates were incubated for 30 minutes and washed as above. Alternatively, 1 L of polyclonal antibodies raised against a mixture of said fusion proteins may be used. Substrate (ABTS, Kirkegaard-Perry) was added (100 µl) to each well. After fifteen minutes of incubation *, the reaction was stopped with 50 µl of 1% SDS in distilled water and the absorbances read at 414 nm (Multiscan, Labsystems). Hev; · (Concentrations of b6.02 and Hev b5 in hand samples were read as standard

I · I

30 from the sheds.

♦ I

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Example 12 * »·

Simultaneous determination of Hev b1, Hev b3, Hev b5 and Hev b6.02 in hand samples • #

For the preparation of the plates, monoclonal antibodies to recombinant 35 nant avidin-Hev b 6.02 fusion protein, recombinant MBP-Hev b5 fusion protein 1 6486 28, recombinant MBP-Hev b3 fusion protein, and recombinant MBP-Hev b1 at concentrations of 1-100 pg / ml in 50 mM phosphate buffer, pH 6.0 were mixed and 150 µl was added to 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) (150 μΙ / 5 well) and incubated for 3 hours or overnight at room temperature. The wells were washed with 0.1 M PBS-Tween-20 (0.05%) and blocked with 300 silicon 0.5% bovine serum albumin (BSA) in 50 mM phosphate buffer, pH 6.5. The plates were used after removal of the blocking solution.

Samples (glove extracts) or standards (predetermined amounts of 10 recombinant Hev b 6.02, recombinant Hev b5, recombinant Hev b3 and recombinant Hev b1) were added to the appropriate wells in a volume of 50 μΙ, followed by the addition of 100 μΙ assay buffer ( 50 mM sodium phosphate, 50 mM sodium chloride, 10 mM EDTA, 0.3% BSA, 0.03% Tween-20, pH 7.4). After incubation for 30 minutes on a horizontal shaker, plates were washed four times with 15 PBS-0.05% Tween-20, 100 μΙ of each specific antibody containing recombinant avidin-Hev b polyclonal or monoclonal antibody 6.02 fusion protein, recombinant MBP-Hev b5 fusion protein, recombinant MBP-Hev b3 fusion protein, and recombinant MBP-Hev b1 fusion protein, labeled with horseradish peroxidase (HRP) at a predetermined value of 100: 1 in 100: dilutions. The plates were incubated for 30 min and washed as above. Alternatively, polyclonal antibodies raised against a mixture of said fusion proteins can be used.

• Substrate (ABTS, Kirkegaard-Perry) was added (100 μΙ) to each well.

After incubation for fifteen minutes, the reaction was stopped with 50 µl of 1%: 25 SDS in distilled water and the absorbances read at 414 nm; "(Multiscan, Labsystems). Concentrations of Hev b6.02,. b5, Hev b3 and Hev b1 were read from appropriate standard plots.

Example 13

Establishment of a Qualitative / Semi-Quantitative Rapid Flow Test for the Screening of Latex Allergens

A solution containing the anti-Hev b 6.02 monoclonal antibody (clone Hb6-2) in 50 mM phosphate buffer, pH 7.0 is applied as a thin line to the nitrocellulose membrane. The membrane is dried at + 45 ° C for one hour and stored dry before use. The antibody concentration is determined to give a positive result (blue line) if the concentration of Hev b 6.02 in sample 29 116486 is greater than, for example, 10 pg / l. The sample pad is moistened with a solution containing 1-3% Tween-20, 0-2% sucrose and buffer salts. The absorbent pad is normal chromatography paper (e.g. Whatman 3MM).

The anti-Hev b 6.02 monoclonal antibody raised against an epitope other than Hb6-2 is coated on blue polystyrene particles (e.g., Seradyn particles, 0.1-0.3 µm) and applied to the conjugate pad and dried .

The sample head includes a sample pad. The blue polystyrene particles are dried at one end of this pad, which is in contact with a nitrocellulose film, to allow the fluid to flow along the membrane by capillary forces. The membrane contacts the absorbent pad at one end. The absorbent pad draws fluid from the membrane until it is no longer available.

When a sample suspected of containing latex allergens is brought into contact with the test device, it is absorbed by the sample pad and the liquid releases the dried blue polystyrene particles from the conjugate pad and flows along the membrane. If the sample contains Hev b 6.02, a blue line is formed on the membrane, indicating a concentration of Hev b 6.02 above a given threshold (for example, 10 pg / l).

Example 14 Designing a Qualitative / Semi-Quantitative Rapid Flow Test for Latex Allergens Screening * », '· Four separate lines of monoclonal antibodies against Hev b6.02, Γ *': Hev b1, Hev b3 and Hev b5, respectively is applied to a nitrocellulose membrane. The film is dried at + 45 ° C for one hour and kept dry «· *; · * ·. 25 before use. The concentration of each antibody is determined in such a way that a positive result is obtained (each line having a different color) if the concentration of each of the latex allergens in the sample exceeds a quantified limit. Blue polystyrene. . the nipple particles are coated with a monoclonal antibody against a different epitope than the nitrocellulose membrane (anti-Hev b 6.02, anti-Hev b1, anti-Hev b1, anti-Hev b5 and anti-Hev b5) used in the dashed lines, each with a different color - blue anti-

Hev b for 6.02, red for anti-Hev b1, green for anti-Hev b3 and black for anti-Hev b5 (e.g., Seradyn particles, 0.1-0.3 µm) - and applied to the conjugate pad and dried .

The sample head of the instrument includes a sample pad. The colored polystyrene par-35 particles are dried at one end of this pad, which is in contact with a nitro-116486 cellulose film, to allow fluid to flow along the membrane by capillary forces. The membrane contacts the absorbent pad at one end. The Ab sorbent pad pulls liquid from the membrane until it is no longer available.

When a sample suspected of containing latex allergens is brought into contact with the test device, it is absorbed by the sample pad and the liquid releases the dried colored polystyrene particles from the conjugate pad and flows along the membrane. If the sample contains Hev b 6.02, a blue line is formed on the membrane indicating a concentration of Hev b 6.02 above a given threshold (e.g. 10 pg / l). If the sample contains Hev b1, a red line is formed, if 10 samples contain Hev b3, a green line is formed, and finally, if the sample contains Hev b5, a black line is formed, each line having a specific threshold value. If no lines are formed, the sample does not contain any of these allergens at concentrations above a certain threshold. If all lines are formed, the sample contains all latex allergens at a concentration above a specified threshold.

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SEQUENCE LIST

<110> Finnish Immunotechology Ltd.

<120> <130> <160> 12 <170> Patent Ver. 2.1

<210> 1 <211> 20 <212> DNA

<213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 1 atggccagtg ttgaggttga 20

<210> 2 <211> 30 <212> DNA

, <213> Artificial Sequence> · <220> <223> Description of Artificial Sequence: primer · ... '<400> 2 I; gatatcaagc ttttattcct ctgttttttc 30:.: <2io> 3 <211> 47

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<220> _ 'j' <223> Description of Artificial Sequence: primer <t <400> 3 tatgtggatc cgacgacgac gacaaagagc aatgtggtcg gcaagca 47 32 116486

<210> 4 <211> 35 <212> DNA

<213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 aacacaagct tcttagtctt tgcaattgct ttggc 35

<210> 5 <211> 681 <212> DNA

<213> Hevea brasiliensis <400> 5 ttttgatctt ccatttttgc aaaaggaaat cttcgattat ggctgaagac gaagacaacc 60 aacaagggca gggggagggg ttaaaatatt tgggttttgt gcaagacgcg gcaacttatg 120 ctgtgactac cttctcaaac gtctatcttt ttgccaaaga caaatctggt ccactgcagc 180 ctggtgtcga tatcattgag ggtccggtga agaacgtggc tgtacctctc tataataggt 240 tcagttatat tcccaatgga gctctcaagt ttgtagacag cacggttgtt gcatctgtca 300 ctattataga tcgctctctt cccccaattg tcaaggacgc atctatccaa gttgtttcag 360 caattcgagc tgccccagaa gctgctcgtt ctctggcttc ttctttgcct gggcagacca 420 agatacttgc taaggtgttt tatggagaga attgagcccc aatttgcacc aattgcttcc 480 aactaagcaa gttaatgata tgctcaagaa tatatatcta ttgtgagctt tttttatgtt 540;. · ctcatcctga gtgttgagac tatgttttcg tttgaatatt atactgtgtt ttattatgtg 600 ·, · ttttgaatat tcataatgag aataaagggc caattgaatt attggccaat atgtaatgat 660 • acataaattt cgtgattgag t 681 * · < 210> 6 <211> 24 • *

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<213> Artificial Sequence 33 1 1 6486 <220> <22 3> Description of Artificial Sequence: primer <400> 7 gatatcaagc tttcaattct ctccataaaa cacctta 37

<210> 8 <211> 922 <212> DNA

<213> Hevea brasiliensis <400> 8 ataatcagtt gatagcttcc acagtgtttt ccgaaaggca aatctttttt caaacttcag 60 cgactgcgtt ttgaatttgt gatttttaaa ggaaattttc aattatggct gaagaggtgg 120 aggaagagag gctaaagtat ttggattttg tgcgagcggc tggagtttat gctgtagatt 180 ctttctcaac tctctacctt tatgccaagg acatatctgg tccattaaaa cctggtgtcg 240 atactattga gaatgtggtg aagaccgtgg ttactcctgt ttattatatt ccccttgagg 300 ctgtcaagtt tgtagacaaa acggtggatg tatcggtcac tagcctagat ggcgttgttc 360 ccccagttat caagcaggtg tctgcccaaa cttactcggt agetcaagat gctccaagaa 420 ttgttcttga tgtggcttct tcagttttca acactggtgt gcaggaaggc gcaaaagctc 480 tgtacgctaa tcttgaacca aaagctgagc aatatgcggt cattacctgg cgtgccctca 540 ataagctgcc actagttcct caagtggcaa atgtagttgt gccaaccgct gtttatttct 600 ctgaaaagta caacgatgtt gttcgtggca ctactgagca gggatataga gtgtcctctt 660 atttgccttt gttgcccact gagaaaatta ctaaggtgtt tggagatgag gcatcataat 720 ctgcactgga tttggttatt tatctattgt gagctttttt atatgtactt attcagtgtt 780 tagaataagt ctttggtggt gtgttttgga tgtggaataa agggccaatt gca ttgttgg 840. tcaatatata attatgtata acatttcgtg atttgagttg gaatctaaag gttttacaaa 900 • aaaaaaaaaa aaaaaaaaaa aa 922 * ·, * ··. <210> 9 • · ;;; <2ii> 20

· ... · <212> DNA

; <213> Artificial Sequence. # <220> I .:: <223> Description of Artificial Sequence.-Primer * · ·; <400> 9 V · atggctgaag aggtggagga 20

) I

,, 'j' <210> 10 .: <211> 32

<212> DNA

<213> Artificial Sequence <220> 34 116486 <223> Description of Artificial Sequence: primer <400> 10 gatatcaagc ttttatgatg cctcatctcc aa 32

<210> 11 <211> 630 <212> DNA

<213> Hevea brasiliensis <400> 11 ggaagagtta tgaatatatt tatagttgtt ttattatgtt taacaggtgt tgcaattgct 60 gagcaatgtg gtcggcaagc aggtggcaag ctctgcccca ataacctatg ttgtagccag 120 tggggatg ^ T gtggctccac tgatgaatat tgttcacctg atcataactg ccaaagcaat 180 tgcaaagaca gcggcgaagg tgttggtggt ggaagtgctt ccaacgttct tgcgacgtac 240 catttgtata attcacagga tcatggatgg gacttgaatg ccgcaagtgc atattgctct 300 acatgggatg ctaacaagcc atattcatgg cggagcaagt atggctggac tgcattctgc 360 ggtcccgtcg gagcacacgg ccaatcctcc tgtggaaagt gcttgagtgt gacaaataca 420 gggactggag ctaaaacgac agtgaggatt gtggatcagt gtagtaatgg aggactagat 480 ttggacgtga atgttttccg tcaactggac acagatggga aaggatatga acgaggtcat 540 attacagtga actaccaatt tgttgattgt ggagattcct tcaatcctct attctccgtt 600 atgaaatcat cagtaattaa ttaataacat 630 <210> I, 12 <211> 735> ·

. * <212> DNA

<213> Hevea brasiliensis • »♦» · '· <400> 12 ||| gcacgagttc ctctccttaa aaccatctct atatattctt tcaagcttac acacatcctt 60 '... * catttttgct ttccaaatgg ccagtgttga ggttgaatca gctgcaacag cattgccaaa 120 gaatgagaca cctgaggtga ccaaggctga ggagga acctgcctct gagcaagaaa ccgccgatgc tacacctgaa aaagaggaac ctacagctgc 240 '· *' * II tcccgcagaa ccggaagctc cagctcctga aaccgagaag gctgaagaag tagaaaagat 300 tgagaagacc gaggagcctg caccagaagc agatcaaacg actccagagg aaaagccagc 360 tgagcctgag cctgttgcag aggaggagcc caaacatgag accaaagaga ctgaaacaga 420 '·' ggcccctgca gctcctgcag agggagagaa gccagctgaa gaagagaagc caattactga 480 · ,,, 'agcagcagag acagccacca cagaagttcc ggtggaaaaa acagaggaat aaataggtct 540 attatgcata tcaaagaaag ggggggatcg agttcctgtt atttattata ttttctttat 600 S «. taatttatga ttatgattat taaggatcga tttggatggt tttggagata gaacctagag 660 .'Ί tcagtctttg agcaaaaact gggatccatc tatgtcgatc agggcacttg ccagagatga 720 gttactgtga ggtct 735

Claims (11)

116486 A homologous or heterologous immunoassay for the detection and quantification of at least two clinically relevant specific latex allergens in rubber products prepared by simultaneous contacting of a sample of said rubber product with a pr or with a mixture of said antibodies, and (b) detecting bound allergens with a mixture of appropriately labeled secondary antibodies, characterized in that said primary antibodies or mixtures of primary and / or secondary antibodies comprise at least two (mono ) specific polyclonal antibody or monoclonal antibody directed against at least two different clinically relevant latex allergens that retain their allergenic properties when sprosesseissa, or a suitable fusion partner with the generated fusion proteins, said at least two different clinically important latex allergens selected from the group consisting of rubber elongation factor (REF or Hev b 1), associated with a rubber particle protein (Hev b 3) 20 acid 16 kDa protein (Hev b 5 ) and hevein (Hev b6.02). A method according to claim 1, characterized in that said at least two clinically significant latex allergens or their:. formed with a suitable fusion partner fusion protein are Hev b5 and, · · ·, Hev b6.02 or fusion proteins. . ·· !, 25 1 3. A method according to claim, characterized in that, * », characterized in that said at least two clinically important latex allergens or their · * · with a suitable fusion partner formed in the fusion protein are Hev b 1, Hev b 3, Hev b 5 and Hev b6.02 or fusion proteins. The method according to claim 1, characterized in that said secondary antibodies are labeled by coupling them to solid supports of different colors. A test kit for the determination of at least two clinically relevant specific Lax 'allergens in a product made from or containing natural latex rubber, characterized in that the test kit'; ' '; 35 comprises 116486 (1) a first series of at least two antibodies, wherein the antibodies are (mono) specific polyclonal or monoclonal antibodies directed against at least two different clinically relevant specific latex allergens that retain their allergenic properties in the manufacturing processes. , or a suitable fusion partner with the generated fusion proteins, wherein the first antibodies to said bound to the solid phase separately or as a mixture, (2) a second set of at least two antibodies, wherein the antibodies are (mono) specific polyclonal or monoclonal antibodies -aineita which 10 is directed to the respective at least two different clinically significant allergen-lateksial which retain the allergenic properties in production processes, or with a suitable fusion partner formed in the fusion protein do not receive a second specific epitope, wherein mai said antibodies are suitably labeled, wherein said at least two different clinically relevant latex alller genes are selected from the group consisting of a rubber extension factor (REF or Hev b1), a rubber particle associated protein (Hev b3), an acidic 16 kD protein (Hev b5), and hevin (Hev b6.02), (3) the reagent or reagents necessary for detection, and the standards necessary for the quantification of the clinically significant specific latex allergens mentioned (20). Test kit according to claim 5, characterized in that: '. and that it further comprises '·': (5) a reagent or reagents for eluting said clinically significant latex allergens from a sample suspected of containing such. · ·, Allergens. , · 'T 7. claimed in claim 5 or 6 test kit, characterized in that said at least two clinically important latex allergens, or, with the appropriate fusion partner formed in the fusion protein are: • ;:; 30 Hev b5 and Hev b6.02 or their fusion proteins. A test kit according to claim 5 or 6, characterized by T; in that said at least two clinically important latex allergens or with a suitable fusion partner are generated fusion protein Hev b 1, Hev b 3, Hev b 5 and Hev b6.02 or fusion proteins. Test kit according to claim 5 or 6, characterized in that said solid support is a microtiter plate or a nitrocellulose membrane. »37 1 1 6 4 8 6 A test kit according to claim 5 or 6, characterized in that the secondary antibodies used in step 2) are labeled by attaching them to solid supports of different colors. • (»• ·» · * »1 1 6486