JP2011505557A - A novel method to assess symptoms in patients associated with hypersensitivity to specific allergens - Google Patents

Abstract

The present invention relates to the field of allergic diseases, and in particular, to a method for assessing whether a patient's symptoms are associated with hypersensitivity to a particular allergen. The invention further relates to a computer program product comprising means for controlling the steps of the method for use with an apparatus for analyzing binding assays. The invention also relates to such a device.

Description

  The present invention relates to the field of allergic diseases, and in particular, to a method for assessing whether a patient's symptoms are associated with hypersensitivity to a particular allergen.

  It is well known that allergen-specific IgE antibodies are associated with allergic diseases when individuals are exposed to allergens. We have found that absolute specific IgE antibody levels provide more information on the relationship between IgE-mediated sensitization and respiratory symptoms than just the presence of specific IgE. Shown before. The inventors have found that total IgE is a poorer predictor of wheeze than the sum of specific IgE. These data suggested that labeling whether a subject is sensitizing based on an arbitrary cut-off is oversimplifying the undivided property in relation to wheezing. Under certain circumstances, the association is small, especially after a strong stimulation of the immune system, such as after immunotherapy. Furthermore, the association becomes less noticeable after intense exposure to livestock, especially cats. An explanation for this is that severe exposure overloads IgE stimulation and can be directed to another Th2 response with production of more IgG4 and less IgE antibodies. Furthermore, this emphasizes the view that the dichotomous use of IgE antibodies is oversimplified so that the results are right.

In a first aspect, the invention relates to a method for obtaining a parameter for assessing whether a symptom in a subject is associated with hypersensitivity to an allergen or an allergen component,
Obtaining a body fluid sample containing immunoglobulin from the patient;
Determining the amount in the sample of a first type of immunoglobulin that specifically binds to the allergen or allergen component;
Determining the amount in the sample of a second type of immunoglobulin that specifically binds to the allergen or allergen component; and then the first type relative to the amount of the second type of immunoglobulin in the sample. Relating the amount of immunoglobulin of

  In a preferred form of the invention, the first and second types of immunoglobulins are the group consisting of IgA, IgD, IgE, IgG and IgM, such as IgG or IgE, or their subtypes such as IgG4. Selected from.

  In a preferred form of the invention, the step of relating the amount of the first type of immunoglobulin to the amount of the second type of immunoglobulin in the sample comprises the first and second types of immunoglobulins. Each forming a ratio between the quantities.

  In one form of the invention, the amount of the first and / or second type of immunoglobulin is each the sum of serum immunoglobulins.

  The allergen is preferably a respiratory allergen, and many such as animals and plants such as cats, cockroaches, dogs, mice, grass pollen, tree pollen, horses, house dust fleas, mold and / or olive pollen From allergen sources.

  Symptoms can be any allergy-like or allergy-related symptoms such as persistent wheezing, asthma, rhinitis, rhinoconjunctivitis, eczema and others.

In a second aspect, the invention relates to a method for assessing whether a subject's symptoms are related to hypersensitivity to allergens, the method comprising:
Obtaining parameters according to the method according to the first aspect;
-Specifying the probability of hypersensitivity to the allergen for the patient by relating the obtained value of the parameter to a predetermined correlation between the parameter and hypersensitivity to the allergen.

In a third aspect, the invention relates to a method for assessing whether a subject's symptoms are related to hypersensitivity to allergens, the method comprising:
Obtaining parameters according to the method according to the first aspect;
-Comparing the value of the obtained parameter with at least one previously determined value of the parameter, wherein the previously determined value is then combined with a probability score for the symptom to be associated with hypersensitivity to the allergen. Then-based on a comparison between the value obtained and the previously determined value for the parameter, the probability of a symptom to be associated with hypersensitivity is higher or lower than the probability score associated with the previously determined value A process of evaluating the above.

  The subject in the embodiment described above can be either a human or an animal.

  Quantification of immunoglobulins in a sample from a subject can be performed by conventional methods used in the art, for example, by immunoassays such as ImmunoCAP (Phadia AB, Uppsala, Sweden), ELISA, microarrays, etc.

  In a fourth aspect, the present invention provides an interior of a processing means for coupling with a device for analyzing a binding assay, comprising software code means adapted to control the steps of the method according to the first or second aspect. It relates to computer program products that can be loaded directly into memory.

In a fifth aspect, the invention relates to an apparatus for analyzing a binding assay, said apparatus being adapted to perform a method according to the first or second aspect,
-Means (10) for receiving a value representative of the amount of the first type of immunoglobulin that specifically binds to the allergen;
-Means (11) for receiving a value representative of the amount of a second type of immunoglobulin that specifically binds to said allergen; and-the relationship between said first and second type of immunoglobulin by the user Means (30) for communicating to

  A schematic diagram of such an apparatus is shown in FIG. The aforementioned means should typically be regarded as software defined functional means, i.e. not necessarily a physical entity, in the digital processing part of the device. The devices can be in one physical device, such as a desktop computer, but may be distributed across multiple physical devices, such as servers located at different locations.

FIG. 6 is an approximate expected probability curve for persistent wheeze of Fel d 1-specific IgG values derived from a given cat-specific IgE value and multivariate logistic regression analysis. Higher IgE antibody levels for cats are associated with a higher probability of allergic disease, wheezing or asthma where lower IgG antibody levels exist. The higher the IgG antibody level, the less this relationship. Approximate expected probability curve for persistent wheezing of Fed d 1-specific IgG4 values derived from a given cat-specific IgE value and multivariate logistic regression analysis. Higher IgE antibody levels for cats are associated with a higher probability of allergic disease, wheezing or asthma. This relationship does not change with high IgG4 antibody levels. Approximate expected probability curve between Fed d 1-specific IgG values derived from 5-year cumulative Fel d 1 exposure, cat-specific IgE values and multivariate logistic regression analysis. Higher exposure to cats is associated with higher levels of IgE and IgG antibodies. Approximate expected probability curves between Fed d 1-specific IgG4 values derived from 5-year cumulative Fel d 1 exposure, cat-specific IgE values and multivariate logistic regression analysis. Higher exposure to cats is associated with higher levels of IgE and IgG4 antibodies. It is the schematic of the apparatus which concerns on the 5th aspect of this invention.

Definitions An allergen source is to be interpreted herein as an organism such as a cat, dog, birch, etc. from which the allergen is derived.

  Allergen is to be interpreted herein as a desensitizing environmental factor derived from an allergen source, such as grass pollen or an extract from dog dander etc.

  An allergen component is to be construed herein as an isolated or recombinantly produced molecule that is a component of an allergen such as Fel d 1 from cats, Phl p 1 etc. from Pseudomonas aeruginosa.

Further described the present invention in the detailed description below of embodiments of the invention, wherein the allergen is derived from cats, immunoglobulins IgG, an IgG ₄ and IgE, symptoms associated with hypersensitivity is wheezing. This example should not be considered as limiting the scope of the invention, which is defined by the appended claims.

Example Example 1
Method Experimental population
The Manchester asthma and allergy study is a birth cohort study based on randomized populations, described in detail elsewhere (12, 13). Subjects were recruited from a prenatal clinic where all pregnant women (first 3 months of pregnancy) were screened for eligibility. Parents were interviewed and had a skin prick test. The children were tracked prospectively and attended laboratory examinations at the age of 3 and 5 years (± 4 weeks). The study was approved by the Local Research Ethics Committee. Informed consent was obtained in writing from the subject's family and consent was obtained from the child.

Although retention in the observed birth cohort based on this population was excellent, we analyzed data from nearly half of the subjects (primarily because the children refused venipuncture). We emphasize that the analysis does not eliminate or include any differences between children in any relevant parameters in the analysis. In addition, the prevalence of allergic sensitization among the parents of children is similar to that of teenagers in UK ²¹ , suggesting that the subject is representative of the general public.

Results Symptoms: Confirmed questions (14) gathered information about the symptoms reported by the interviewer as reported by the parent, the disease diagnosed by the doctor and the treatment received. A child was defined as having recently had wheezing if he had had wheezing in the last 12 months at age 5 years. We also assigned children to different wheezing phenotypes according to two follow-up (15) wheezing histories:
No wheezing-no wheezing in the first 3 years of birth and no wheezing by age 5;
Transient early wheezing-wheezing in the first 3 years and no wheezing in the 12 months after 5 years;
Late-onset wheezing-no wheezing in the first 3 years and wheezing in the 12 months after 5 years;
Persistent wheezing-wheezing in the first 3 years and wheezing in the 12 months after 5 years.

Antibody Measurement Allergen specific antibody levels were determined using the ImmunoCAP ™ assay (Phadia AB, Uppsala, Sweden) according to the manufacturer. For the determination of IgE antibodies, commercially available reagents for flea and cat allergen extracts were used. Consistently, the reproducibility and accuracy of the assay has been reported to be very good (18).

  Pure rFed d 1 Allergen ImmunoCAP was used for specific IgG antibody measurements. rFel d 1 was manufactured by Phadia AB and subsequently bound to a solid phase of a flexible and hydrophilic cellulose sponge polymer matrix activated by the CNBr method. Purified components were covalently attached to the activated solid phase via the amino group of the protein according to a standard simplified procedure previously described (18).

Environmental exposure We visited home immediately after birth and at 3 and 5 years of age. Children of the bed, the floor of the children bedroom, the dust samples from the parents of bed and lounge of the floor, was collected in each opportunity by sucking the area of 1m ^{2 2} minutes in a standardized manner. Fel d 1 was assayed using the previously described (21) enzyme-linked immunoassay. Results are expressed as allergen concentration (μg / g). We estimated cumulative allergen exposure over the first 5 years as the sum of allergen levels at 4 locations at 3 time points (ie, 12 separate measurements of allergen exposure).

Statistical methods The main outcome measure was “persistent wheezing” at age 5. The relationship between antibody response and outcome measure was analyzed using logistic regression. The odds ratio (OR) was estimated using a regression model, and a 95% confidence interval (95% CI) was generated according to Wald using a p-value of 0.05 as significance. The approximate expected probability curve was plotted using the results from logistic regression. Specific antibody levels are subjected to a logarithmic transformation prior to analysis; different antibody levels representing increased or decreased risk correlating with antibody levels increasing OR are shown. Since logarithmic transformation was used, all calculations assumed a log scale, i.e., OR was exp (r * b) and r, and the distance between a certain antibody level and a level indicating the absence of antibody was r = ln It was defined as (x2) -ln (x1). The relationship between Fel d 1 exposure and antibody levels was confirmed using linear regression analysis. Calculated statistical analysis was performed using SAS System V8.01.

Results Of the 1211 couples who agreed to participate, 1085 had a successful moon-filled pregnancy (> 36 weeks gestation; children did not need intensive care) and agreed to follow-up. Of these, 128 were randomized to environmental control before birth (19, 20) (excluded from this analysis) and 957 children were followed in an observation cohort. Of the children in the observation cohort, 840 (87.8%) attended a 5-year follow-up. All of these children had interrogation data and 540 provided blood samples for antibody measurements (62%).

  Excluded children were no different from those included with respect to family history, parental smoking, mother's age, socioeconomic status, gestational age, birth weight, wheezing history, and skin test results. A recent wheeze at age 5 was reported by parents of 114 children (21.9%); 257 children did not experience wheezing, 107 had transient early wheezing, 26 started late And 70 had persistent wheezing (61 children could not be classified into 1 of 4 wheezing phenotypes).

Multivariate However, this effect was markedly improved by Fel d 1-specific IgG antibodies. The degree of specific IgE effect decreased as Fel d 1-specific IgG increased. For example, the expected probability of persistent wheezing with cat IgE of 50 kUA / L is 0.82, low IgG for Fel d 1, corresponding to an OR of 7.4 (95% CI 5.9-9.1) at 2 mg / L This decreased to an OR of 2.5 (95% CI 1.5-4.4) when IgG antibody levels to Fel d 1 increased to 6 mg / L. Fel d 1-specific IgG4 did not alter the relationship between cat-specific IgE levels and persistent wheezing (FIG. 4).

Discussion It is well known that exposure to allergens can lead to the production of IgE, IgG and IgG4 antibodies. In susceptible individuals, IgE antibody levels are largely correlated with clinical illness triggered by exposure to allergens. However, it is also well known that large exposures can lead to IgG and IgG4 antibody formation but not disease development. The present invention quantitatively establishes the negative effect of IgG antibody on the correlation between IgE antibody and clinical disease, but not IgG4 antibody.

Claims (13)

A method of obtaining a parameter for assessing whether a symptom in a subject is related to hypersensitivity to an allergen or allergen component
Obtaining a body fluid sample containing immunoglobulin from the patient;
Determining the amount in the sample of a first type of immunoglobulin that specifically binds to the allergen or allergen component;
Determining the amount in the sample of a second type of immunoglobulin that specifically binds to the allergen or allergen component; and then the first type relative to the amount of the second type of immunoglobulin in the sample The method comprising the step of relating the amount of immunoglobulin of   2. The method of claim 1, wherein the first and second types of immunoglobulins are selected from the group consisting of IgA, IgD, IgE, IgG and IgM and their subclasses.   3. The method of claim 2, wherein the first and second types of immunoglobulins are selected from the group consisting of IgG and IgE. The method according to any one of claims 1 to 3 one of the first and second types of immunoglobulin subtype IgG _4.   Correlating the amount of the first type of immunoglobulin in the sample with the amount of the second type of immunoglobulin, each forming a ratio between the amount of the first and second immunoglobulins The method according to claim 1, comprising:   The method according to any one of claims 1 to 5, wherein the amounts of the first type immunoglobulin and / or the second type immunoglobulin are each the sum of serum immunoglobulins.   The method according to any one of claims 1 to 6, wherein the allergen or the allergen component is a respiratory allergen or an allergen component.   An allergen source wherein the allergen or allergen component is selected from the group consisting of animals and plants such as cats, cockroaches, dogs, horses, mice, grass pollen, tree pollen, olive pollen, house dust fleas and molds The method according to claim 1, which is derived from   9. The method according to any one of claims 1 to 8, wherein the symptom is selected from the group consisting of persistent wheezing, asthma, rhinitis, rhinoconjunctivitis, eczema or other allergy related or allergic symptoms. A method for assessing whether a subject's symptoms are related to allergen hypersensitivity, comprising:
Obtaining the value of the parameter according to the method according to any one of claims 1 to 9;
-Specifying the probability of hypersensitivity to the allergen for the patient by relating the obtained value of the parameter to a predetermined correlation between the parameter and hypersensitivity to the allergen. A method for assessing whether a subject's symptoms are related to allergen hypersensitivity, comprising:
Obtaining parameters according to the method according to any one of claims 1 to 9;
-Comparing the value of the obtained parameter with at least one previously determined value of the parameter, wherein the previously determined value is then combined with a probability score for the symptom to be associated with hypersensitivity to the allergen. Then-based on a comparison between the value obtained and the previously determined value for the parameter, the probability of a symptom to be associated with hypersensitivity is higher or lower than the probability score associated with the previously determined value The method comprising the step of assessing.   Loadable directly into the internal memory of a processing means coupled with a device for analyzing binding assays, including software code means adapted to control the steps of the method according to any one of claims 1-11 Computer program product. A device for analyzing a binding assay comprising:
-Means (10) for receiving a value representative of the amount of the first type of immunoglobulin that specifically binds to the allergen;
Means (11) for receiving a value representative of the amount of a second type of immunoglobulin that specifically binds to the allergen; and-the amount of the second type of immunoglobulin in the sample to the first Means (20) for relating the amount of immunoglobulins of the type; and-the apparatus comprising means (30) for communicating the relationship between the first and second types of immunoglobulins to the user .

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