WO1995007298A1

WO1995007298A1 - Natural rubber latex antigens associated with allergies

Info

Publication number: WO1995007298A1
Application number: PCT/US1994/010226
Authority: WO
Inventors: Harri Alenius; Kevin J. Kelly; Viswanath P. Kurup; Kristiina Turjanmaa; Jordan N. Fink
Original assignee: Ansell Incorporated
Priority date: 1993-09-08
Filing date: 1994-09-07
Publication date: 1995-03-16
Also published as: AU7832694A

Abstract

The present invention relates to a 27 kD natural rubber latex (NRL) antigenic protein which can cause severe allergic reactions in humans sensitized to natural rubber. The present invention further pertains to antibodies and fragments of such antibodies which specifically bind to the 27 kD NRL antigen. The present invention additionally relates to the use of the 27 kD antigen of the invention to detect antibodies specific for the antigen. Methods of detecting the antigen using antibodies specific for the 27 kD protein are also disclosed in the present invention.

Description

NATURAL RUBBER LATEX ANTIGENS

ASSOCIATED WITH ALLERGIES

REFERENCE TO RELATED APPLICATION

The present application is a continuing application of application Serial No. 08/117,594, filed September 8, 1993. The content of this prior application is incorporated by reference in its entirety. Field of the Invention

The invention relates to the field of natural rubber latex (NRL) antigens, which can cause serious allergic reactions in humans or mammals sensitized to such antigens. The present invention additionally relates to antibodies, both polyclonal and monoclonal, which are capable of recognizing and binding to the NRL antigens of the invention. The present invention further relates to methods of use of the NRL antigens and antibodies of the invention. Background of the Invention

Numerous reports on IgE- ediated hypersensitivity to natural rubber latex (NRL) have been published in the medical literature since the first description of a patient with immediate latex glove allergy [1]. The accumulated data from Europe and the U.S.A. support the view that NRL proteins are the responsible allergens. Furthermore, health care workers and patients with spina bifida are the major risk groups for such reactions (for review, see [2]).

Systemic allergic reactions upon exposure to NRL have been reported to occur during surgical and other invasive procedures both in children and adult patients. Severe allergic reactions seem to be especially frequent in patients with spina bifida [8-10]. We have previously demonstrated different IgE anti-latex reactivity by ELISA in sera from latex allergic patients from the U.S. and Finland [11] and speculated that populations and patients may be exposed to different antigenic materials in their natural surroundings and, consequently, mount different immune responses to NRL antigens. Genetic predisposition, evidenced by the fact that atopic individuals are at increased risk for developing latex hypersensitivity, may also be an important factor determining the outcome of latex allergy in different populations and patient series.

NRL proteins that cause such allergic reactions are found in raw NRL (sap of the rubber tree, Hevea brasxliensis) and may be eluted from a wide variety of manufactured products, such as surgical gloves, catheters and other medical devices, as well as from non-medical rubber products like household gloves, condoms and toy balloons. However, the amount of extractable rubber proteins is not necessarily proportional to their antigenicity [3]. Several allergenic proteins, with molecular weights ranging mostly between 10 to 100 kilodaltons (kD), have recently been detected in NRL, ammoniated NRL and extracts of latex gloves [4-7], but their overall biological significance has not yet been established.

In the present invention we have used immunoblot techniques to compare IgE antibody responses in latex allergic patients from two different geographical locations (U.S.A. and Finland). The patients tested presented symptoms ranging from anaphylaxis to contact urticaria, and having associated clinical conditions, such as spina bifida and other congenital defects. Healthy patients served as controls. Our efforts have lead to the discovery of a previously unrecognized 27 kD NRL antigen. Allergic patients with spina bifida exhibit a strong IgE immune response towards a multitude of NRL antigens, and confirming our preliminary report [12], especially IgE reactivity to the previously unrecognized 27 kD NRL antigen. Summary of the Invention

The present invention relates to an allergenic protein of about 27 kilodalton (kD) present in natural rubber latex products. Methods of obtaining and using the purified protein of about 27 kD are also disclosed in the present invention. In particular, the 27 kD protein of the invention can be used to detect the presence of antibodies specific for the 27 kD protein in a patient. Patients having antibodies to the 27 kD protein of the invention are at risk in developing an allergic reaction upon future exposure to latex products.

The invention additionally pertains to antibodies (polyclonal or monoclonal) or fragments of such antibodies, which specifically bind to the 27 kD protein of the invention. Use of such antibodies allows for the detection of the 27 kD NRL antigen. Use of these antibodies is particularly suited for detecting the presence or absence of the NRL antigen of the invention in a finished latex product such as surgical or exam gloves. Testing latex products in the manufacturing process provides a screen for safe products for use on individuals allergic to particular NRL antigens. Brief Description of the Drawing

This invention will be described in greater detail by reference to the drawing in which:

Figure 1 shows an immunoblot analysis of IgE binding to NRL antigens in sera from 20 latex allergic patients. The notations in Fig. 1 have the following meanings. U.S. patients: 1-7, 12, spina bifida; 13-14, other congenital anomalies. Finnish patients: 1-2, spina bifida; 4-5, other congenital anomalies; 9, 11, children with no histories of multiple operations; 12, 14, 17, 19, adults. C, control subject. Patient numbers equal to those given in Table 1. Molecular weight markers (kD) are indicated on the left side of each of the two geographic patient groups. Description of the Preferred Embodiments

A. Isolation and Purification of NRL Antigens

In accordance with this invention, the NRL antigen of about 27 kD can be isolated from the sap of Hevea brasllieπslε . Alternatively, the antigen of the invention can be prepared by extracting the antigen from latex products such as latex gloves, catheters and other medical devices or instruments. Non-medical rubber products can be used to extract the antigen of the invention. Other potential sources of the NRL antigen of the invention will be readily apparent to those of ordinary skill in the art.

Since the 27 kD antigen is a protein, standard extraction and purification techniques can be used to isolate and purify the antigen of the invention. Such techniques commonly employed include extraction, precipitation, ion exchange chromatography, affinity chromatography, gel filtration and the like. The preferred method to isolate the NRL antigen of the invention is by affinity chromatography using a monoclonal antibody bound to a column matrix.

Using an affinity chromatography purification procedure, the NRL antigen of about 27 kD can be substantially purified from the sources described above. As used herein, the term "substantially pure" or "substantially purified" is meant to describe a protein which is substantially free of any compound normally associated with the protein in its natural state, i.e., substantially free of contaminating proteins and other components. The term is not meant to exclude the presence of minor impurities that do not interfere with the antigenic or binding activity of the protein.

B. Antibodies Specific for NRL Antigens

The present invention concerns antibodies that are capable of binding or directed against the 27 kD protein of the invention. An antibody is said to be "capable of binding" or "directed against" a protein if it is capable of specifically reacting with the protein, thereby forming an antigen-antibody complex.

The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used herein is meant to include intact molecules as well as fragments of such molecules capable of binding the antigen of the invention. Examples of antibody fragments that can be used in accordance with the invention include Fab and F(ab')₂ fragments. Other antibody fragments that may be used in the invention will be readily apparent to those of skill in the art.

The antibodies used in the present invention may be prepared by any of a variety of methods. For example, a protein preparation containing the 27 kD antigen can be administered to an animal to induce the production of sera containing polyclonal antibodies capable of binding the antigen. In the preferred method, a preparation of the antigen of the invention is purified to render it substantially free of natural contaminants. The purified preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

The antibodies of the present invention also include monoclonal antibodies (or fragments thereof). Monoclonal antibodies can be prepared using well-known hybridoma technology. In general, such procedures involve immunizing an animal with substantially pure NRL antigen.

The splenocytes of the immunized animal are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line can be employed in accordance with the present invention. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilutions. The hybridoma cells obtained through such a selection are then assayed to identify clones that secrete antibodies capable of binding to the antigen of the invention.

As noted, Fab and F(ab')₂ and other fragments of antibodies can be used in the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments). Alternatively, hapten-binding fragments can be produced through recombinant DNA technology or through synthetic chemistry.

C. Assays for Detecting NRL Antigens

The antibody or fragments of antibodies of the invention can be used to detect the presence or absence of the 27 kD NRL antigen in a sample. Detection can be accomplished using any of a variety of assays well known in the art. Briefly, the 27 kD NRL antigen of the invention can be detected by contacting a sample with an antibody specific for the 27 kD NRL antigen. The sample and antibody mixture are incubated for a time and under conditions sufficient for the antibody and antigen in the sample to form an antigen-antibody complex. The antigen-antibody complex may then be detected using well known detection techniques.

Examples of materials that may contain the 27 kD NRL antigen of the invention include, but are not limited to, medical latex products such as gloves, catheters, and surgical devices or instruments. Non-medical latex products may also be tested with the antibodies of the invention for the presence of the 27 kD NRL antigen.

Antibodies or fragments thereof can be labeled using any of a variety of labels and methods of labeling. Examples of types of labels that can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, fluorescent labels, toxin labels, and chemiluminescent labels. Examples of suitable enzymic, radioactive, flourescent, toxin and chemiluminescent lables will be readily apparent to one of skill in the art. Those of ordinary skill in the art will know of other suitable labels that can be employed in accordance with the invention.

Binding of labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Examples of coupling techniques include the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimido-benzyl-N- hydroxy-succinimide ester method, and the like.

The detection of the NRL antigens of the invention can be improved through the use of carriers. For example, an antibody specific for the 27 kD antigen can be bound to a carrier to aid in the detection of the antigen. Well-known carriers include glass, polystyrene, polypropylene, -1- polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface can be flat such as a sheet, test strip, etc. Those skilled in the art will note many other suitable carriers or will be able to ascertain the same by use of routine experimentation.

The antibodies of the present invention can also be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody, or fragment of antibody, is bound to a solid support that is insoluble in the fluid being tested (i.e. , blood, lymph, liquified stools, tissue homogenate, etc.), and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing a quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled molecule to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether the antigen is present or may be quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of antigen.

In another type of "sandwich" assay, which may also be useful to detect the 27 kD antigen, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition of a solution of labeled antibody to the fluid sample is followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.

In addition, the materials for use in the assays of the invention are ideally suited for preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, test tubes, and the like. Each of said container means comprises one of the separate elements to be used in the method. For example, one of said container means can comprise an antibody specific for the 27 kD antigen. Such antibody can be bound directly to the inner walls of a container. A second container can comprise detectably labeled antibody in lyophilized form or in solution. The carrier can contain, in addition, a plurality of containers each of which comprises different, predetermined and known amounts of antigen. These latter containers can then be used to prepare a standard curve from which can be interpolated the results obtained from the sample containing the unknown amount of antigen.

D. Diagnostic Uses for NRL Antigens Diagnostic uses of the 27 kD NRL antigen of the invention include the detection of antibodies specific for the 27 kD antigen in a sample obtained from a patient. Assays to detect the antibody specific for the NRL antigen can be accomplished by any of a variety of assay methods. Briefly, the method to detect an antibody in a sample comprises contacting a sample with the 27 kD NRL antigen of the invention. The sample-antigen mixture is incubated for a time and under conditions sufficient for the antigen and the antibody in the sample to form an antigen-antibody complex. The formation of the antigen-antibody complex can then be determined using conventional techniques.

Examples of assay techniques are disclosed in Section C in the Description of the Preferred Embodiment. However, the assays described in Section C are modified to the extent needed to use an antigen rather than an antibody as the detecting component of the assay. Thus, for example, in the "sandwich" assay described in Section C, a quantity of unlabeled 27 kD antigen is bound to a solid support. The solid phase is contacted with a sample suspected of containing antibodies to the 27 kD NRL antigen to form a binary solid phase antigen-antibody complex. A detectably labeled antibody can then be added to permit detection of the ternary complex formed between the solid phase antigen, antibody, and labeled antibody.

In accordance with assays using antigen to detect antibodies in a sample, the antigen can be labeled using any of a variety of labels and methods of labeling. Examples of types of labels that can be used in the present invention include enzyme labels, radioactive labels, fluorescent labels, toxin labels and chemiluminescent labels. Coupling these labels to an antigen can be accomplished using standard techniques known to those of ordinary skill in the art.

To detect antibodies specific for the 27 kD antigen, various samples can be assayed. Of particular interest are biological samples obtained from a patient. Such biological samples can be body tissue, body fluids (such as blood, urine, tear drops, saliva, serum, and cerebrospinal fluid) , feces, cellular extracts, and the like. In particular, biological samples obtained from patients suffering from spina bifida or other congenital defects can be used in accordance with the present invention.

This invention is described in greater detail in the following examples. Example 1: Preparation of NRL Antigen

The latex from Hevea brasillensis was collected in Malaysia directly into containers, deep-frozen immediately, and stored at -70°C. For the extraction of soluble proteins, NRL was centrifuged at 100,000 g for 90 minutes, following which the supernatant was collected and stored at -70°C. Alternatively, antigen was prepared by diluting the sap of the latex tree in saline or by extracting latex gloves in buffered saline for 24 hours.

Example 2 : Detection of NRL Antibodies in Patients USA Subjects

Sera from 12 patients with spina bifida, (4 females and 8 males) aged 4 to 17 years, 2 male patients with other congenital anomalies, aged 5 and 25 years, and 5 female health care workers, aged 31 to 39 years, were studied. All patients had immediate wheal and flare skin prick test reactions to NRL extracts prepared as previously described [11] and had histories of anaphylaxis on NRL contact. Control sera from non-allergic subjects were obtained from 3 spina bifida patients, aged 4 to 7 years, with no clinical evidence of anaphylaxis and negative skin prick tests to NRL, and from 8 skin test negative health care workers who had no histories compatible with latex allergy. Finland Subjects

Sera were obtained from 11 latex allergic children (5 females and 6 males) aged 1 to 15 years, 3 of whom had spina bifida, 4 had other congenital anomalies and histories of multiple operations and 4 had no associated anomalies or histories of surgical operations. Sera were also obtained from 9 adult latex allergic patients (7 females and 2 males, 6 of whom were health care workers) aged 32 to 66 years, all presenting with cutaneous symptoms. Serum samples from 10 non-allergic adult subjects served as controls. All 20 patients were positive in skin prick testing with two surgical latex glove extracts (Exona , Semperit, Vienna, Austria and Triflex , Baxter, Lessines, Belgium) prepared as previously described [13] and all controls were negative. Rubber Extract

The sap from Hevea brasiliensis (SAP) was collected in Malaysia directly to special containers, deep-frozen immediately, and stored at -70°C. For the extraction of soluble proteins, SAP was dissolved 1:2 in phosphate-buffered saline and centrifuged at 100,000 g for 90 minutes, following which the supernatant was collected and stored at -70°C. Protein assays

The total protein concentration of the SAP was determined using the Bradford's or Lowry's method with bovine gamma globulin as the standard. Total protein staining of the extract was performed with a commercial kit (Amersham, UK) as previously described [6]. Immunoblotting

A method described previously in detail [6] was used to perform immunoblots. Briefly, NRL (total protein concentration adjusted to 450 μg/ml) was diluted 1:3 into electrophoresis buffer containing 2% sodium dodecyl sulphate (SDS) and 5% 2^-mercaptoethanol and kept in boiling water for 2 minutes. The dissolved samples, 45 l/cm of gel, were applied to 12.5% acrylamide gels containing 0.1% SDS, using a 4% stacking gel. Electrophoresis was carried out for 45 minutes at a constant voltage of 200 V (Mini Protean II Cell; Bio-Rad, Richmond, Calif., USA). After electrophoresis, the proteins were transferred from the gel to 0.45-μm pore size nitrocellulose filters (Trans-Blot; Bio-Rad) in an electroblotting buffer containing 25 mmol of Tris-HCl, 150 mmol of glycine and 20% methanol, pH 8.3, for 1 hour at 100 V (Mini Trans-blot cell, Bio-Rad). After blotting, the membrane was stained with Ponceau-S red to demonstrate successful transfer of proteins. The membrane was then cut into 3-mm wide strips, washed 3 times for 5 minutes in 0.1% Tween-tris-buffered saline (TBS) and incubated for 1 hour in a blocking buffer containing 5% non-fat milk powder in TBS.

Sera diluted 1:5 in the blocking buffer were added, and the strips incubated overnight at room temperature with continuous shaking. The strips were then washed 4 times for 5 minutes. Then biotinylated goat antihuman IgE antibody (Vector, Burlingame, Calif.) diluted 1:2000 in blocking buffer was added and the strips were incubated for 30 minutes at room temperature. After washing 4 times, streptavidin-conjugated alkaline phosphatase (Bio-Rad) diluted 1:5000 in TBS was added and the strips were incubated for 30 minutes at room temperature. The strips were again washed and then placed in a color development solution (Bio-Rad) . The reaction was stopped after 6 minutes by rinsing in tap water. Latex-RAST and total IgE determinations

For the US patients, latex RAST determinations were kindly done by Jay Slater, M.D., of the Children's National Medical Center, Washington, D.C. Values exceeding 1.0 kU/L were considered positive. When only one of two parallel test recordings exceeded 1.0 kU/L, the result was considered indeterminate. Thirteen (72%) of the 18 U.S. patients tested showed a positive RAST result and 2 (patients No. 7 and 15, Table 1) an indeterminate result. Three patients (all health care workers) had negative RAST results.

A commercial latex RAST using the CAP method (Pharmacia, Uppsala, Sweden) was used for the Finnish patients. Values exceeding 0.4 kU/L were regarded positive. Eighteen (90%) of the 20 Finnish patients had positive RAST results. Standard laboratory methods were used for total IgE measurements. Three (17%) of the 18 U.S. patients and 13 (65%) of the 20 Finnish patients had elevated total IgE levels (Table 1). Results

IgE binding patterns from the 39 sera to 6 major NRL antigens, chosen for comparative analysis, are shown in Table 1 and results from immunoblot analyses for 20 sera (10 U.S. and 10 Finnish patients) are compiled in Fig. 1. All but one of the 12 latex allergic spina bifida patients from the U.S. had IgE antibodies binding to at least 2 of the 6 antigens; 5 patient sera recognized all 6 antigens and 9 patient sera reacted with at least 3 antigens (Table 1).

Ten of the 12 (83%) U.S. and the 2 of the 3 Finnish spina bifida patients had serum IgE binding to a previously undescribed 27 kD antigen. One of the 2 U.S. and one of the 4 Finnish patients with congenital anomalies had also similar IgE antibodies to the 27 kD antigen, but these antibodies were not detected in adult sera from either country.

The most frequently occurring co-existing IgE antibody was against a 14 kD antigen; 13/14 U.S. and 5/7 Finnish patients with spina bifida or other congenital anomalies showed positive immunoblots (Table 1). None of the 5 U.S. adults, but 2/9 of the Finnish adult latex allergic patients showed IgE binding to the 14 kD protein.

A third major antigen in the two patient groups was a 20 kD protein: IgE antibodies against it were recorded in 9/12 U.S. spina bifida patients and in 5/11 Finnish children. Only one of the 5 U.S. health care workers but 8/9 of the Finnish adult latex allergic patients had IgE to the 20 kD antigen. Reactivity patterns with 30, 45 and 75 kD allergens were variable (Table 1) . Three health care worker sera from the U.S. and one Finnish patient with spina bifida showed no anti-latex IgE antibodies in immunoblotting and also all the 21 control sera, including samples from the 3 U.S. spina bifida patients with no findings compatible with latex allergy, gave negative results.

Table 1

IgE immunoblot reactivity against 6 major natural rubber allergens in sera from latex allergic patients from the U.S. and from Finland.

PRESENCE OF IgE

PATIENT SYMPTOMS LATEX ' TOTAL ANTIBODY TO LATEX

No. and OF LATEX ASSOCIATED RAST IgE ANTIGENS (kD)

SEX/AGE ALLERGY CONDITION (kU/L) (kU/L) 14 20 27 30 45 75

U.S.

1 F/9 AR SB 21.2 453 + + + + + +

2 F/15 AR SB 26.9 950 + + + + +

3 M/8 AR SB 5.2 ND + + + + + +

4 F/9 AR SB 3.1 13 + + + -

5 M/10 AR SB 2.6 133 + + + + + +

6 F/6 AR SB 3.41 40 + - + +

7 M/17 AR SB 1.04 121 - + +ι/- -

8 M/12 AR SB 2.01 <10 + - + -

9 M/10 AR SB 15.0 224 + + + + + +

10 M/10 AR SB 18.7 320 + + + + + +

11 M/8 AR SB 9.8 85 + + + -

12 M/4 AR SB 2.6 13 -+1/-

13 M/5 AR COA 6.3 103 + - +ι/- - -

14 M/25 AR COA 15.0 403 +

15 F/31 AR _ 0.9 138 _ + _ _ +

16 F/38 AR - 0.66 54 - - - +

17 F/35 AR - ND <10

18 F/39 AR - 0.4 <10

19 F/34 AR - 0.86 70

FINLAND

1 M/13 SR SB 2.2 110 + - + + -

2 F/12 LR SB 2.2 98 + - + -

3 F/10 LR SB 1.6 39

1/ weak reaction 4 F/12 LR COA 0 193 + + - - - -

5 F/13 AR COA 32.2 15010 + — + + -

6 F/10 LR COA 11.2 139 + - - + -

7 M/13 LR COA 0.5 934 - - - - + -

8 M/9 LR 0.9 282 _ + __. _ __. _

9 M/2 LR 3.5 4100 + + — + + +

10 M/l AR 0.8 200 — + — — — —

11 M/15 LR 2.3 891 - + - + +

12 F/39 SR 14.4 396 + + _ + + _

13 F/66 LR 13.2 2292 - + — — — -

14 F/46 LR 24.0 3400 - + — +1/+ +

15 M/40 LR 0 256 — — — + + +

16 F/48 LR 0.8 64 _ + _ _ _ _

17 M/32 LR 1.9 15000 +1/+ — ll/+ +

18 F/38 AR 0.5 641 - + - + +

19 F/34 LR 21.9 7075 - + - - - +

20 F/32 LR 4.6 259 — + — — — +

AR = anaphylactic reaction

SR = systemic, non-anaphylactic reaction (incl. generalized urticaria asthma) LR = local cutaneous reaction (incl. contact urticaria, eczema, facial oedema) COA = congenital anomalies and multiple operations SB = spina bifida

Discussion

The present immunoblot results showed that the majority (13/15) of latex allergic patients with spina bifida have IgE class antibodies binding to a previously undescribed NRL 27 kD antigen. Also two children (one from the U.S. and one from Finland) with congenital anomalies and histories of multiple operations had anti-27 kD IgE antibodies. Similar IgE antibodies were not detected in adult latex allergic patients from either country or among pediatric patients not having congenital anomalies and histories of multiple operations.

Previously, a 14 kD protein was suggested by Slater and Chhabra [7] to be a major allergen among U.S. spina bifida patients since all their 14 spina bifida patients had IgE antibodies against it. In the present study we also frequently (92%) found IgE antibodies to the 14 kD protein in the U.S. spina bifida patients. This anti-14 kD IgE antibody was also found in 2/3 Finnish latex allergic patients with spina bifida and in 5/6 patients (2 from the U.S. and 4 from Finland) with other congenital anomalies. In the present study, and also in our previous study [6], antibodies to the 14 kD antigen were detected also in a few of the Finnish adult latex allergic patients, but these patients did not show anti-27 kD antibodies. On the basis of these observations it seems that IgE responses to the 14 kD and especially 27 kD NRL antigens are typical for children with multiple operations, i.e., they develop in conditions under which repeated and/or prolonged exposures to rubber proteins take place.

The reason why IgE antibodies against the 27 kD NRL antigen have not been described in patients with spina bifida in previous works is unclear. It is possible that the same or a similar protein with an apparent molecular weight close to 27 kD was encountered by Slater and Chhabra [7] who found in total protein staining a doublet at 27 and 28 kD in reduced non-ammoniated latex. However, the data given do not allow one to evaluate whether IgE antibodies to these peptides were found in sera of their spina bifida patients. In the present study, the occurrence of anti-27 kD antibodies was closely associated with anti-14 kD antibodies and the possibility that the 27 kD protein represents a dimer form of the earlier described 14 kD peptide may not be excluded. Our results, however, do not support this hypothesis since both 14 and 27 kD peptides were found by total protein staining after SDS-polyacrylamide electrophoresis carried out under reducing and non-reducing conditions.

The numbers of individuals in the different patient groups of the present study are small and the clinical significance of the IgE antibody binding to the 27 kD antigen should be interpreted with caution. The most plausible explanation for the finding is that pediatric patients with spina bifida and/or other congenital anomalies are exposed and sensitized in a different way from the other latex allergic children and adults. Patients with congenital anomalies have, as a rule, undergone several surgical operations and have been frequently in contact with a variety of latex-containing medical devices, some of which could contain the 27 kD peptide. We could not find this protein by total protein staining in extracts of several brands of surgical gloves, but to our knowledge, there are no studies characterizing the occurrence of various NRL antigens in e.g. catheters and other medical and surgical NRL devices.

Although IgE antibodies to the 14 and 27 kD NRL peptides were frequently observed in connection with spina bifida, such antibodies (or other IgE anti-latex antibodies) were not found in the sera from the 3 non-latex allergic U.S. spina bifida patients tested as controls. It is thus likely that spina bifida per se does not imply this type of IgE reactivity to NRL. All the 10 U.S., but only 2/20 Finnish latex allergic patients had experienced anaphylactic reactions on exposure to NRL, and among the U.S. patients with spina bifida, there was a close association between anaphylaxis and IgE anti-14 and anti-27 kD reactivity. One U.S. and one Finnish patient with other congenital anomalies, both with anaphylactic reactions, also had anti-14 and anti-27 kD IgE antibodies. However, clinical histories of the 2 Finnish spina bifida patients with antibodies against the 14 and 27 kD proteins did not reveal anaphylactic reactions, and anti-14 kD or anti-27 kD IgE antibodies were not seen in the 5 U.S. health care workers all of whom had had anaphylactic reactions. Hence, the present results do not support a clear association between the pattern of immunoblot anti-latex IgE reactivity and particular symptoms of latex allergy.

In summary, we examined by immunoblotting sera from 39 latex allergic patients, 19 from U.S.A. and 20 from Finland, including 15 children with spina bifida and 5 children with other congenital anomalies, for IgE antibodies to natural rubber latex (NRL) antigens. IgE antibodies in 10 of the 12 (83%) U.S. spina bifida patients and in 2 of the 3 Finnish spina bifida patients recognized a previously undescribed 27 kD antigen which together with a 14 kD and a 20 kD peptide appeared to be a major NRL allergens. Two patients with other congenital anomalies, one from the U.S. and one from Finland, also demonstrated anti-27 kD bands. IgE antibodies to the 27 kD antigen were not detected in other latex allergic patients from the U.S. or Finland although most of them showed IgE binding to 14 and/or 20 kD NRL antigens. All 21 controls, including 3 spina bifida patients with no evidence of latex allergy, gave negative immunoblot results. This observation suggests that patients with spina bifida or other congenital anomalies who have been subject to multiple operations and other invasive therapeutic procedures may have been exposed to different antigenic source materials than other latex allergic patients.

In conclusion, our findings demonstrate that the majority of latex allergic spina bifida patients show a vigorous immune response to several latex allergens, the most significant of which appear to be 14 kD, 20 kD and 27 kD proteins. IgE antibody response to a previously undescribed 27 kD protein was noted only in latex allergic pediatric patients with spina bifida or other congenital anomalies and histories of multiple surgical operations. Further studies are needed to show if immune response to the 27 kD antigen is involved in the pathogenesis of clinical manifestations of latex allergy.

Example 3: Preparation and Characterization of Monoclonal Antibodies Against the 27 kD NRL Antigen

Standard techniques for production of monoclonal antibodies were used to produce this monoclonal antibody. Antigen

The antigen was prepared by diluting the sap of the latex tree of Malaysia in saline or by extracting latex gloves in buffered saline for 24 hours. Immunization of Mice

About 100 μl (1 mg/ml protein) antigen in sterile physiological saline was mixed with an equal volume of complete Freund's adjuvant and injected subcutaneously at two sites of the gluteal area of 6- to 8-week-old BALB/c mice. The subcutaneous injection was repeated once more a week after the first injection. The animals were then injected intraperitoneally every week for four more weeks. All intraperitoneal injections were made with antigen in sterile physiological saline without complete Freund's adjuvant. Animals were bled through retro-orbital plexes under anesthesia or through the tail vein, and antibody production was evaluated by biotin-avidin linked immunosorbent assay (BALISA) . Animals showing high titers of antibodies against latex antigens were used for hybridoma production. Those BALB/c mice showing low titers of antibodies were further boosted with antigens and tested as before. Fusions of Lymphocytes with Myeloma Cells

The spleen was removed aseptically and the cells were mechanically dissociated in RPMI 1640 medium (GIBCO) containing streptomycin (100 μg/ml) and penicillin (100 units/ml). The red blood cells were lysed with ammonium chloride and the resulting cells were filtered through a nylon membrane. The viability of the cells was determined by trypan blue dye exclusion staining. Spleen cells were then mixed with NS-1 myeloma cells (P3-NSI/Ag4-l) growing at logarithmic phase at a ratio of 4:1. The cells were centrifuged at 500 g for 5 minutes and again washed with RPMI 1640 medium. Approximately 1 ml of polyethylene glycol (PEG-1500, Sigma) was added to the cell pellet over a 2-minute period while the cells were maintained in a 37°C water bath. The cell suspension was gently stirred using the tip of pipet during the addition of PEG. About 20 ml of medium was gradually added (over a 10-minute period) to the cells in order to dilute the PEG. The cells were centrifuged at 500 g for 5 minutes and resuspended in complete RPMI 1640 medium containing glutamine (2 mmol), fetal calf serum (10% v/v), sodium pyruvate (1 mmol), penicillin (100 units/ml) and streptomycin (10 g/ml). The cells were plated in 92-well tissue culture plates in 100 μl quantities at a concentration of 2.5 x 10 cells/well and incubated in a humidified CO, incubator (5% C0₂ in air) at 37°C. After 24 hours, 100 l of complete medium containing hypoxanthine, aminopterin and thymidine (HAT) was added to each well. Thereafter HAT medium was added to the wells by splitting the medium 1:1 for four consecutive days. Two more HAT changes were made at three-day intervals. After that the cells were grown in hypoxanthine and thymidine medium for the next two weeks with frequent changes of the same medium. The culture fluids were then tested for antibody-producing hybridomas by BALISA using latex antigen-coated polyvinyl microtiter plates. Wells showing antibody-secreting hybridomas were cloned at least two times by limiting dilution using thymocytes as feeder cells. The purified clones were injected intraperitoneally into pristine-primed syngenic mice for ascitic fluid production. Isotyping

The culture fluid and ascites were precipitated with ammonium sulfate (50% saturation). The precipitate was dissolved in a minimal amount of phosphate-buffered saline (PBS) and dialyzed against distilled water at 4°C. Goat anti-human immunoglobulin classes and subclasses were obtained from commercial sources for isotyping (Bio-Rad, Miles, and Sigma). Microtiter plates were coated with 1:100 to 1:1,000 dilution of goat anti-mouse antibodies in carbonate buffer. Ammonium sulfate-precipitated culture fluid and ascites to be tested were added to the wells and the binding of the monoclonals to the specific antisera was demonstrated by peroxidase-labelled anti-mouse polyclonal antibody and substrate as described below. Agar gel double diffusion was also used to isotype the monoclonal antibodies. Reactivity of the Monoclonal Antibody to Latex Materials

Reactivity of the monoclonal antibodies to various latex materials was studied by dot immunobinding assay. Antigens prepared from several latex materials strains were applied to nitrocellulose sheets using a vacuum blotting device

(Bio-Rad). After blocking the nitrocellulose sheets with PBS containing 0.3% Tween 20, they are allowed to react with the monoclonal antibody. The reaction was visualized through immuno-gold strain and silver enhancement according to the method of Daneels, et al.

Crossed Immunoelectrophoresis and Rocket Electrophoresis

Crossed immunoelectrophoresis of the crude latex antigens and monoclonal antibodies was carried out according to the method described before (see Example 2). Antigen purified through an affinity column (monoclonal antibody bound to Sepharose) was tested by rocket electrophoresis and rocket electrophoresis with intermediate gels containing concanavalin A to determine the sugar residues in the antigen.

Western Blot Analysis

The crude latex antigen extract was electrophoresed in sodium dodecyl sulfate (SDS) polyacrylamine gel as described before. A 7.5- to 15% gradient gel was used. About 1 mg of protein was loaded onto a 16 x 16 cm gel and electrophoresed at 30 mA/gel for 2-2.5 hours at 10°C. The proteins from the gels were transferred to nitrocellulose sheets (Bio-Rad) using a semi-dry blotter (Poly Blot, American Bionetics,

Inc., Calif.) according to the specifications and buffer

2 systems recommended by the manufacturer. We used 0.5 mA/cm for 43 minutes to transfer two gels at a time. A piece of the gel, before transfer, was stained by Coomasie blue and after transfer with silver stain to determine the band pattern and efficiency of transfer. A piece of the nitrocellulose membrane was stained with colloidal gold stain and compared with the protein bands transferred to the membrane. The nitrocellulose sheets were cut into small strips and analyzed using monoclonal antibodies according to the methods described before. The glycoprotein bands on the membranes were demonstrated using biotinylated Con-A and Ricinus 120 lectins (Sigma). Example 4: Affinity Purification of NRL Antigen

Ascites collected from mice injected with hybridoma were purified by 50% ammonium sulfate precipitation. The precipitate was redissolved in PBS and dialyzed extensively against PBS. This antibody was then coupled to cynogen bromide-activated sepharose-4B beads (Pharmacia) and packed into a column as reported before. The crude antigens, showing reactivity to the monoclonal antibody in BALISA and rocket electrophoresis, were passed through this column. After washing to remove the non-binding antigen, the Sepharose bound components were eluted with urea as described before. The urea fraction was dialyzed extensively and used in BALISA to detect circulating antibodies in the sera of patients. The protein content was estimated by the method of Lowry et al. This fraction was also used in rocket and affinity rocket electrophoresis to determine its chemical and immunochemical characteristics. Example 5: Biotin-Avidin Linked Immunosorbent Assay

A sensitive enzyme immunoassay using the biotin and avidin system was used to detect antibodies in the sera. This method has been evaluated extensively in our laboratory for the past several years and has been reported before in detail. The purified antigen was used at a concentration of 25 ng protein/ml for coating the plates. Serum dilutions of 1:100 and 1:1,000 were used to detect specific IgE and IgG. The optical density (OD) values at 490 of crude latex antigens and purified fraction adjusted to the same protein concentration were compared.

Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in immunology, hybridoma technology, pharmacology and/or related fields are intended to be within the scope of the following claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for the purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

REFERENCES ^~24~

1. Nutter AF. Contact urticartia to rubber. Br J Dermatol 1979;101:597-598.

2. Levy DA, Charpin D, Pecquet C, Leynadier F, Vervloet D. Allergy to latex. Allergy 1992;47:579-587.

3. Tomazic VJ, Withrow TJ, Fisher BR, Dillard SF. Latex- associated allergies and anaphylactic reactions. Clin Immunol Immunopathol 1992;64:89-97.

4. Carrillo T, Cuevas M, Munoz T, Hinojosa M, Moneo I. Contact urticaria and rhinitis from latex surgical gloves. Contact Dermatitis 1986;15:69-72.

5. Morales C, Basomba A, Carreira J, Sastre A. Anaphylaxis produced by rubber glove contact: case reports and immunological identification of the antigens involved. Clin Exp Allergy 1989;19:425-430.

6. Alenius H, Turjanmaa K, Palosuo T, Makinen-Kiljunen S, Reunala T. Surgical latex glove allergy: characterization of rubber protein allergens by immunoblotting. Int Arch Allergy Appl Immunol 1991;96:376-380.

7. Slater JE, Chhabra SK. Latex antigens. J. Allergy Clin Immunol 1992;89:673-678.

8. Axelsson JGK, Johansson SGO, Wrangsjδ K. IgE-mediated anaphylactoid reactions to rubber. Allergy 1987;42:46-50.

9. Turjanmaa K, Reunala T, Tuimala R, Karkkainen T. Allergy to latex gloves: unusual complication of delivery. Br Med J 1988;297:1029.

10. Slater JE. Rubber anaphylaxis. N Engl J Med 1989;320:1126-1130.

11. Kurup VP, Kelly KJ, Turjanmaa K, Alenius H, Reunala T, Palosuo T, Fink JN. IgE reactivity to latex antigens in the sera of patients from Finland and the United States. J Allergy Clin Immunol (in press).

12. Alenius H, Kelly KJ, Kurup VP, Turjanmaa K, Makinen- Kiljunen S, Reunala T, Palosuo T, Fink JN. Differences in IgE reactivity to latex antigens in sera from patients from the U.S. and Finland. J Allergy Clin Immunol 1993;91:240 (abstract). 13. Turjanmaa K, Reunala T, Rasanen L. Comparison of diagnostic methods in latex surgical glove contact urticaria. Contact Dermatitis 1988;19:241-247.

Claims

WHAT IS CLAIMED IS:

1. A substantially pure natural rubber latex antigen having a molecular weight of about 27 kilodaltons.

2. The antigen of claim 1, wherein said antigen is obtained from Hevea brasiliensis.

3. The antigen of claim 1, wherein said antigen is obtained from a latex product.

4. The antigen of claim 1, wherein said antigen is detectably labeled.

5. The antigen of claim 4, wherein said label is selected from the group consisting of a radioactive, enzymatic, fluorescent, and chemiluminescent label.

6. An antibody which is capable of binding to the antigen of claim 1.

7. The antibody of claim 6, wherein said antibody is a polyclonal antibody.

8. The antibody of claim 6, wherein said antibody is a monoclonal antibody.

9. The antibody of claim 6, wherein said antibody is an antibody fragment.

10. The antibody of claim 6, wherein said antibody is detectably labeled.

11. The antibody of claim 10, wherein said label is selected from the group consisting of a radioactive, enzymatic, fluorescent, and chemiluminescent label.

12. A hybridoma cell which produces the monoclonal antibody of claim 8.

13. A method of detecting the presence or absence of a natural latex rubber antigen having a molecular weight of about 27 kilodaltons in a sample comprising:

(a) contacting said sample with the antibody of claim 6; and

(b) detecting the formation of an immune complex formed between said antibody and said antigen.

14. The method of claim 13, wherein said antibody is detectably labeled.

15. The method of claim 14, wherein said detectable label is selected from the group consisting of a radioactive, enzymatic, fluorescent, and chemiluminescent label.

16. The method of claim 13, wherein said assay is an enzyme linked immunosorbent assay (ELISA).

17. The method of claim 13, wherein said sample is a latex product.

18. The method of claim 17, wherein said latex product is selected from the group consisting of gloves, catheters and surgical instruments.

19. A method of detecting the presence or absence of an antibody in a sample which binds to a natural rubber latex antigen having a molecular weight of about 27 kilodaltons comprising:

(a) contacting said sample with the antigen of claim 1; and

(b) detecting the formation of an immune complex formed between said antigen and said antibody.

20. The method of claim 19, wherein said antigen is detectably labeled.

21. The method of claim 20, wherein said detectable label is selected from the group consisting of a radioactive, enzymatic, fluorescent, and chemiluminescent label.

22. The method of claim 19, wherein said sample is a biological sample selected from the group consisting of blood, urine, tear drops, saliva and serum.

23. The method of claim 19, wherein said sample is a biological sample obtained from a patient suffering from spina bifida.

24. The method of claim 23, wherein said patient is a child.

25. The method of claim 19, wherein said assay is an enzyme linked immunosorbent assay (ELISA) .