The present invention is directed to a method and composition for detecting or quantifying the antigen-specific immunoglobulin E (IgE) in the biological samples by a reverse lateral flow immunoassay using a procedure of coupling the antigens, to which the IgE are specific for and reactive to, to the colored nanoparticles. The formed antigen specific IgE-antigen-nanoparticle complex is then captured and detected by an anti-IgE antibody immobilized on the test line or area of the test strip in the lateral flow immunoassay device.
Over the past decades, numerous patents have been issued involving immuno-chromatographic devices. The standard features of these devices comprise the following:
Two types of chromatographic immunoassays are commonly described, of which in one type, the analytes (proteins, haptens, or small molecules) contained in bodily fluids (urine, whole blood, plasma, serum, and saliva) are detected. The analytes include hCG, FSH, TSH, troponins, myoglobulin, serum proteins, viral or bacterial proteins, haptens, therapeutic drugs, and drugs of abuse.
In the other type of the chromatographic immunoassay, the analyte being detected is (are) human antibody (antibodies) of various classes specifically reactive with agents such as viral or bacterial proteins (HIV, Hepatitis A and C, H. pylori, EBV, Rubella, CMV, HSV, Dengue fever, Lyme, Chagas, TB, Toxoplasma, autoimmune antigens, etc.) or allergens (pollens, molds, dust/mites, foods, animal epithelia, etc.). When it comes to detecting antibody, three formats are typically used:
The U.S. Pat. No. 6,528,325 (Hubscher et al) and its later supplementary version U.S. Pat. No. 6,528,325 B1 (Hubscher et al) claimed a lateral flow-based method for detection of antigen-specific IgM, IgG and IgA using the approach of coupling the antigens to the chromatographic particles, the IgM-, IgG- and IgA-containing immunocomplex were then captured by the anti-IgM, anti-IgG and anti-IgA antibodies dispensed in the test line of the nitrocellulose membrane. In another embodiment of the same patent, the inventors also claimed a method to detect allergen-specific IgE using anti-IgE antibody-coupled chromatographic particles. In this IgE detecting method, the allergen (s) were immobilized in the test line of the bibulous nitrocellulose membrane to detect the IgE-containing complex. Such an anti-IgE antibody conjugated particle-based allergen-specific IgE detecting method is thus referred as “conventional lateral flow Immunoassay” hereafter.
U.S. Pat. No. 7,629,127 (Hubscher) disclosed an improved version of a lateral flow immunoassay described in the U.S. Pat. No. 6,528,325 (Hubscher et al) to enhance the reading and detecting sensitivity for allergen specific IgE detection using a “two-step” approaching by designing a buffer port upstream the sample port, and applying a chase buffer in this buffer port following the sample application in the sample port. In this method, the anti-IgE antibody labelled conjugates were dried in the position between buffer port and sample port. In this two-step” alternative form of the conventional LFIA, the allergens were immobilized in the test area of the strip.
An alternative form of the “conventional lateral flow immunoassay” for IgE detection is the European patent EP1891447A1 (Rundstrom et al), wherein a two-step approach was applied for improved detection of allergen-specific IgE. In this two-step” assay, the IgE-containing sample was first applied to the sample port and let it flow towards to the wicking pad, followed by applying the running buffer in the buffer port that was positioned upstream of the sample port. The dried anti-IgE antibody conjugates were deposited in a position between the buffer port and sample port. In this two-step” alternative form of the conventional LFIA, the allergens were immobilized in the test area of the strip.
Due to sensitivity and/or specificity limit, the prior invention of “conventional lateral flow immunoassay” for allergen-specific IgE detection could not be widely applied for allergy diagnoses, particularly in food allergy diagnosis. It would be desirable to having a highly sensitive and specific lateral flow-based assay that can overcome the low sensitivity problem in the prior art for detecting and quantifying the allergen-specific IgE in biological samples as a rapid screening and diagnostic tool to diagnose IgE-mediated allergies, including food allergy.
This invention is about a novel reverse lateral flow immunoassay (R-LFIA) platform capable of highly sensitively and specifically detecting allergen- and antigen-specific IgE in a rapid manner and a point-of-care setting as a diagnostic tool to diagnose IgE-mediated diseases including allergic and autoimmune diseases.
It is, therefore, one of the objects of the present invention is a novel R-LFIA platform capable of detecting soluble IgE, particularly human IgE with specificity to various antigens and allergens, including but not limit to, food allergens, autoantigens, environmental allergens, insect antigens, parasite antigens, bacterial and virus antigens and synthetic drug antigens or heptens that are responsible for IgE-mediated allergic diseases, autoimmune diseases and other IgE-mediated diseases.
It is, therefore, another one of the objects of the present invention to enable the diagnosis of food allergy such as peanut, milk, egg, shellfish, fish, tree nuts, red meat, wheat, soys, sesame seeds; and IgE-mediated diseases against the autoantigens in a form of soluble proteins, membrane proteins, intracellular proteins, protein-DNA complexes, protein-RNA complexes that are served as the autoantigens responsible for the autoimmune based mast cells and basophil activation such as chronical spontaneous urticaria and other idiopathic acute and chronical urticaria; environmental allergens, such as dust mite, cockroach, pollen, pet dander, mold; IgE against insect allergens such as venom of bees, wasps, hornets, yellow-jackets and fire ants; IgE against parasitic antigens such as helminths, hookworms and other parasitic worms; and IgE against the synthetic drugs, antibiotics and anti-tumor drugs.
It is another one of the objects of the present invention to provide a novel reverse lateral flow immunoassay assay, including a test strip and/or a cassette for holding the test strip, that can detect one or more allergen- or antigen-specific IgE in a sample qualitatively, semi-quantitatively, and quantitatively, in a point-of-care setting, and in a rapid manner.
The invention may be understood by referring to the following description, claims, and accompanying drawings. This description of an embodiment, set out below to enable one to practice an implementation of the invention, is not intended to limit the preferred embodiment, but to serve as a particular example thereof. Those skilled in the art should appreciate that they may readily use the conception and specific embodiments disclosed as a basis for modifying or designing other methods and systems for carrying out the same purposes of the present invention. Those skilled in the art should also realize that such equivalent assemblies do not depart from the spirit and scope of the invention in its broadest form.
More than 32 million Americans suffer from food allergies, for example, peanut allergy. Some of them unnecessarily given the high false positive rate of current diagnostics. Thus, the oral food challenge (OFC) remains the gold standard for a definitive diagnosis of food allergy. However, because OFC is a high-risk resource-intensive procedure needing close supervision in well-equipped medical facility, is not routinely accessible to a large portion of food allergy patients. Many are left without a confirmed diagnosis until the allergic reaction accidently occurs. It is unequivocally agreed that accurate food allergy diagnostics are urgent medical needs.
Flow Immunoassay has been widely used as an inexpensive rapid diagnostic suitable for Point-Of-Care (POC) settings for various diseases, including environmental allergies. However, the current C-LFIA format cannot achieve sufficiently high sensitivity to serve as a meaningful diagnostic for many allergies. Given the limitations of the high false positivity of all available commercial allergen specific-IgE tests and the low sensitivity of C-LFIA, this invention of a R-LFIA for allergen specific IgE detection is designed to mitigate these problems.
The R-LFIA format for peanut allergic IgE test not only displays high specificity by filtering out low affinity, cross-reactive IgE but also significantly enhances the IgE detection sensitivity by ˜30 fold compared to that of the C-LFIA format, thus reaching the threshold for clinically application for peanut allergy diagnosis.
The R-LFIA format also is applicable for other food allergic IgE and other environmental allergen and autoantigen specific IgE test with the same or similar high sensitivity and specificity.
The said R-LFIA is a test strip comprised the following components, with following characteristics:
The said test procedures for the said reverse lateral flow immunoassay comprise the following steps:
The antigen (hereafter using peanut allergen as an example) specific-IgE in testing samples binds to the allergens pre-conjugated to the Gold Nano-Particle (GNP) that was dried down to the conjugate pad, leaving other IgE remaining unbound (
In R-LFIA format, the allergen-specific IgE, as well as other Ig isotypes, would directly compete to bind to the allergen coupled to GNP (
Comparison with conventional lateral flow immunoassay (C-LFIA). For C-LFIA (
Example 1. Detection sensitivity comparison between R-LFIA and C-LFIA using the same peanut allergic sample (PL14231). The asterisk represents the ending dilutions to be detected with the respective methods (
Example 2. The ending dilutions detected by R-LFIA from three (PA2, PA3 and PA6) peanut allergic plasma samples. The corresponding IU level measured with ImmunoCAP method is used for comparison to determine the R-LFIA sensitivity limit (
Example 3. R-LFIA detection results using various volume of a peanut allergic plasma. As low as 0.5 uL of PA3 sample sufficiently resulted in visible positive signal measured with R-LFIA (
Example 4. The peanut specific IgE level in random normal population without known peanut allergy. While 90% random normal population did not show any detectable peanut specific IgE, about 10% did display weak positivity with AU<6.0 (For example, the samples NS45,
Example 5. Specificity of the R-LFIA for peanut specific IgE detection. The peanut IgE R-LFIA was only reactive with the recombinant IgE specific for Ara h1 and Ara h2, but not cross-reactive with 100 IU/ml allergen specific IgE to milk (Bos d5 & Bos d8), shrimp (Pen a1), egg (Gal d1), fish (Gad m1), birch pollen (Bet v1), and house mite (Der p1). As a positive control, the PA sample PL 26259 displayed strongly positive reactivity (
Example 6. Correlation of R-LFIA IgE test results with that of the Basophil Activation Test (BAT). All 11 BAT-confirmed peanut allergic samples exhibited high AU levels using peanut IgE R-LFIA (
Example 7. Diagnostic cutoff value of the peanut IgE R-LFIA. With the data available from this pilot study, if a cutoff value is set at AU=6, then the R-LFIA displays 100% (11/11) positive predictive value, and 100% (251/251) negative predictive value for peanut allergy diagnosis (
Example 8. R-LFIA IgE test results for the plasma samples with peanut specific IgE level>17.5 IU/mL (Class IV and above of ImmunoCAP classification) (
Example 9. The ending dilutions detected by R-LFIA from three shrimp allergic plasma samples. The corresponding IU level measured with ImmunoCAP method is used for comparison to determine the R-LFIA sensitivity limit (
Example 10. R-LFIA IgE test results for the plasma samples with shrimp specific IgE level>17.5 IU/mL, which are Class IV and above of ImmunoCAP classification (
This application is based upon and claims the benefit of U.S. Provisional Patent Application No. 62/926,528 filed with the U.S. Patent and Trademark Office on 27 Oct. 2019 and titled “A reverse lateral flow immunoassay method for measurement of antigen-specific IgE”, which application is incorporated herein by reference in its entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/054027 | 10/2/2020 | WO |
Number | Date | Country | |
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62926528 | Oct 2019 | US |