Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/20—Dermatological disorders
  • G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/24—Immunology or allergic disorders

Definitions

• the field of the subject matter disclosed herein is sensitivity testing for food intolerance, and especially as it relates to testing and possible elimination of selected food items as foods that exacerbate or worsen symptoms or foods that, when removed, alleviate symptoms in patients diagnosed with or suspected to have psoriasis.
• Food sensitivity also known as food intolerance
• psoriasis a type of autoimmune disease
• the underlying causes of psoriasis are not well understood in the medical community.
• Psoriasis may be visually diagnosed, along with various tests to exclude various other inflammatory or infectious conditions.
• treatment of psoriasis may often be less than effective and may present new difficulties due to immune suppressive or modulatory effects.
• elimination of other one or more food items has also shown promise in at least reducing incidence and/or severity of the symptoms.
• psoriasis is often quite diverse with respect to dietary items triggering symptoms, whereby no standardized test to help identify trigger food items with a reasonable degree of certainty is known, thus leaving such patients often to trial-and-error.
• the subject matter described herein provides systems and methods for testing food intolerance in patients diagnosed with or suspected to have psoriasis.
• One aspect of the disclosure is a test kit with for testing food intolerance in patients diagnosed with or suspected to have psoriasis.
• the test kit includes a plurality of distinct food preparations coupled to individually addressable respective solid carriers.
• the plurality of distinct food preparations have an average discriminatory p-value of ⁇ 0.07 as determined by raw p-value or an average discriminatory p-value of ⁇ 0.10 as determined by FDR multiplicity adjusted p- value.
• Another aspect of the embodiments described herein includes a method of testing food intolerance in patients diagnosed with or suspected to have psoriasis.
• the method includes a step of contacting a food preparation with a bodily fluid of a patient that is diagnosed with or suspected to have psoriasis.
• the bodily fluid is associated with gender identification.
• the step of contacting is performed under conditions that allow IgG from the bodily fluid to bind to at least one component of the food preparation.
• the method continues with a step of measuring IgG bound to the at least one component of the food preparation to obtain a signal, and then comparing the signal to a gender-stratified reference value for the food preparation using the gender identification to obtain a result.
• the method also includes a step of updating or generating a report using the result.
• Another aspect of the embodiments described herein includes a method of generating a test for food intolerance in patients diagnosed with or suspected to have psoriasis.
• the method includes a step of obtaining test results for a plurality of distinct food preparations.
• the test results are based on bodily fluids of patients diagnosed with or suspected to have psoriasis and bodily fluids of a control group not diagnosed with or not suspected to have psoriasis.
• the method also includes a step of stratifying the test results by gender for each of the distinct food preparations. Then the method continues with a step of assigning for a predetermined percentile rank a different cutoff value for male and female patients for each of the distinct food preparations.
• Still another aspect of the embodiments described herein includes a use of a plurality of distinct food preparations coupled to individually addressable respective solid carriers in a diagnosis of psoriasis.
• the plurality of distinct food preparations are selected based on their average discriminatory p-value of ⁇ 0.07 as determined by raw p-value or an average discriminatory p-value of ⁇ 0.10 as determined by FDR multiplicity adjusted p-value.
• Table 1 shows a list of food items from which food preparations can be prepared.
• Table 2 shows statistical data of foods ranked according to 2-tailed FDR multiplicity- adjusted p-values.
• Table 3 shows statistical data of ELISA score by food and gender.
• Table 4 shows cutpoint values of foods for a predetermined percentile rank.
• Figure 1A illustrates ELISA signal score of male psoriasis patients and control tested with peach.
• Figure IB illustrates a distribution of percentage of male psoriasis subjects exceeding the 90 th and 95 th percentile tested with peach.
• Figure 1C illustrates a signal distribution in women along with the 95 th percentile cutoff as determined from the female control population tested with peach.
• Figure ID illustrates a distribution of percentage of female psoriasis subjects exceeding the 90 th and 95 th percentile tested with peach.
• Figure 2A illustrates ELISA signal score of male psoriasis patients and control tested with cucumber.
• Figure 2B illustrates a distribution of percentage of male psoriasis subjects exceeding the 90 th and 95 th percentile tested with cucumber.
• Figure 2C illustrates a signal distribution in women along with the 95 th percentile cutoff as determined from the female control population tested with cucumber.
• Figure 2D illustrates a distribution of percentage of female psoriasis subjects exceeding the 90 th and 95 th percentile tested with cucumber.
• Figure 3A illustrates ELISA signal score of male psoriasis patients and control tested with tea.
• Figure 3B illustrates a distribution of percentage of male psoriasis subjects exceeding the 90 th and 95 th percentile tested with tea.
• Figure 3C illustrates a signal distribution in women along with the 95 percentile cutoff as determined from the female control population tested with tea.
• Figure 3D illustrates a distribution of percentage of female psoriasis subjects exceeding the 90 th and 95 th percentile tested with tea.
• Figure 4A illustrates ELISA signal score of male psoriasis patients and control tested with tomato.
• Figure 4B illustrates a distribution of percentage of male psoriasis subjects exceeding the 90 th and 95 th percentile tested with tomato.
• Figure 4C illustrates a signal distribution in women along with the 95 th percentile cutoff as determined from the female control population tested with tomato.
• Figure 4D illustrates a distribution of percentage of female psoriasis subjects exceeding the 90 th and 95 th percentile tested with tomato.
• Figures 5A illustrates distributions of psoriasis subjects by number of foods that were identified as trigger foods at the 90 th percentile.
• Figures 5B illustrates distributions of psoriasis subjects by number of foods that were identified as trigger foods at the 95 th percentile.
• Table 5A shows raw data of psoriasis patients and control with number of positive results based on the 90 th percentile.
• Table 5B shows raw data of psoriasis patients and control with number of positive results based on the 95 th percentile.
• Table 6A shows statistical data summarizing the raw data of psoriasis patient populations shown in Table 5A.
• Table 6B shows statistical data summarizing the raw data of psoriasis patient populations shown in Table 5B.
• Table 7A shows statistical data summarizing the raw data of control populations shown in Table 5A.
• Table 7B shows statistical data summarizing the raw data of control populations shown in Table 5B.
• Table 8A shows statistical data summarizing the raw data of psoriasis patient populations shown in Table 5 A transformed by logarithmic transformation.
• Table 8B shows statistical data summarizing the raw data of psoriasis patient populations shown in Table 5B transformed by logarithmic transformation.
• Table 9A shows statistical data summarizing the raw data of control populations shown in Table 5 A transformed by logarithmic transformation.
• Table 9B shows statistical data summarizing the raw data of control populations shown in Table 5B transformed by logarithmic transformation.
• Table 10A shows statistical data of an independent T-test to compare the geometric mean number of positive foods between the psoriasis and non-psoriasis samples based on the 90 th percentile.
• Table 10B shows statistical data of an independent T-test to compare the geometric mean number of positive foods between the psoriasis and non-psoriasis samples based on the 95 th percentile.
• Table 11A shows statistical data of a Mann-Whitney test to compare the geometric mean number of positive foods between the psoriasis and non-psoriasis samples based on the 90 th percentile.
• Table 11B shows statistical data of a Mann-Whitney test to compare the geometric mean number of positive foods between the psoriasis and non-psoriasis samples based on the 95 th percentile.
• Figure 6A illustrates a box and whisker plot of data shown in Table 5A.
• Figure 6B illustrates a notched box and whisker plot of data shown in Table 5A.
• Figure 6C illustrates a box and whisker plot of data shown in Table 5B.
• Figure 6D illustrates a notched box and whisker plot of data shown in Table 5B.
• Table 12A shows statistical data of a Receiver Operating Characteristic (ROC) curve analysis of data shown in Tables 5A-11 A.
• ROC Receiver Operating Characteristic
• Table 12B shows statistical data of a Receiver Operating Characteristic (ROC) curve analysis of data shown in Tables 5B-1 IB.
• Figure 7A illustrates the ROC curve corresponding to the statistical data shown in Table 12 A.
• Figure 7B illustrates the ROC curve corresponding to the statistical data shown in Table 12B.
• Table 13A shows a statistical data of performance metrics in predicting psoriasis status among female patients from number of positive foods based on the 90 th percentile.
• Table 13B shows a statistical data of performance metrics in predicting psoriasis status among male patients from number of positive foods based on the 90 th percentile.
• Table 14A shows a statistical data of performance metrics in predicting psoriasis status among female patients from number of positive foods based on the 95 th percentile.
• Table 14B shows a statistical data of performance metrics in predicting psoriasis status among male patients from number of positive foods based on the 95 th percentile
• test kits and methods are now presented with substantially higher predictive power in the choice of food items that could be eliminated for reduction of psoriasis signs and symptoms.
• Food sensitivity also known as food intolerance
• psoriasis a type of autoimmune disease
• the underlying causes of psoriasis are not well understood in the medical community.
• Psoriasis may be visually diagnosed, along with various tests to exclude various other inflammatory or infectious conditions.
• treatment of psoriasis may often be less than effective and may present new difficulties due to immune suppressive or modulatory effects.
• elimination of other one or more food items has also shown promise in at least reducing incidence and/or severity of the symptoms.
• psoriasis is often quite diverse with respect to dietary items triggering symptoms, whereby no standardized test to help identify trigger food items with a reasonable degree of certainty is known, thus leaving such patients often to trial-and-error.
• inventive subject matter provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
• the numbers expressing quantities or ranges, used to describe and claim certain embodiments of the disclosure are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
• test kit or test panel that is suitable for testing food intolerance in a patient that is diagnosed with or suspected to have psoriasis.
• a test kit or panel will include one or more distinct food preparations (e.g., raw or processed extract, which may include an aqueous extract with optional co-solvent, which may or may not be filtered) that are coupled to (e.g., immobilized on) individually addressable respective solid carriers (e.g., in a form of an array or a micro well plate), wherein each distinct food preparation has an average discriminatory p-value of ⁇ 0.07 as determined by raw p-value or an average discriminatory p-value of ⁇ 0.10 as determined by FDR multiplicity adjusted p-value.
• distinct food preparations e.g., raw or processed extract, which may include an aqueous extract with optional co-solvent, which may or may not be filtered
• respective solid carriers e.g., in a form of an array or a micro well plate
• the average discriminatory p- value is determined by comparing assay values of a first patient test cohort that is diagnosed with or suspected of having psoriasis, with assay values of a second patient test cohort that is not diagnosed with or suspected of having psoriasis.
• the assay values can be determined by conducting assays for the first and second patient test cohorts with the distinct food preparation.
• the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the disclosure are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some
• the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that, the numerical ranges and parameters setting forth the broad scope of some embodiments of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some
• exemplary food preparations include at least two, at least four, at least eight, or at least 12 food preparations prepared from foods 1-59 of Table 2.
• the exemplary food preparations can include at least two of peach, cucumber, tea, tomato, broccoli, cauliflower, almond, green pepper, grapefruit, tobacco, eggplant, rye, oat, cantaloupe, cabbage, cane sugar, sweet pot, pineapple, avocado, orange, spinach, honey, swiss cheese, malt, mustard, wheat, apple, chocolate, yogurt, and goat milk. Still further especially contemplated food items and food additives from which food preparations can be prepared are listed in Table 1.
• the methods described herein comprise the one of one or more distinct food preparations having an average discriminatory p-value, wherein the average discriminatory p-value for each distinct food preparation is determined by a process that includes comparing test results of a first patient test cohort that is diagnosed with or suspected of having psoriasis, with test results of a second patient test cohort that is not diagnosed with or suspected of having psoriasis.
• test results e.g., ELISA
• test results are obtained for various distinct food preparations, wherein the test results are based on contacting bodily fluids (e.g., blood saliva, fecal suspension) of the first patient test cohort and the second patient test cohort with each food preparation.
• bodily fluids e.g., blood saliva, fecal suspension
• such identified food preparations will have high
• discriminatory power will have a p-value of ⁇ 0.15, ⁇ 0.10, or even ⁇ 0.05 as determined by raw p-value, and/or a p-value of ⁇ 0.10, ⁇ 0.08, or even ⁇ 0.07 as determined by False Discovery Rate (FDR) multiplicity adjusted p-value.
• FDR False Discovery Rate
• each distinct food preparations will have an average discriminatory p-value of ⁇ 0.05 as determined by raw p-value or an average discriminatory p-value of ⁇ 0.08 as determined by FDR multiplicity adjusted p-value, or even an average discriminatory p-value of ⁇ 0.025 as determined by raw p-value or an average discriminatory p-value of ⁇ 0.07 as determined by FDR multiplicity adjusted p-value.
• the FDR multiplicity adjusted p-value may be adjusted for at least one of age or gender, and in certain embodiments adjusted for both age and gender.
• test kit or panel is stratified for use with a single gender
• at least 50% (or 70% or all) of the plurality of distinct food preparations when adjusted for a single gender, have an average discriminatory p-value of ⁇ 0.07 as determined by raw p-value or an average discriminatory p-value of ⁇ 0.10 as determined by FDR multiplicity adjusted p- value.
• stratifications e.g., dietary preference, ethnicity, place of residence, genetic predisposition or family history, etc.
• stratifications e.g., dietary preference, ethnicity, place of residence, genetic predisposition or family history, etc.
• test kit or panel may vary considerably, and contemplated formats include micro well plates, dip sticks, membrane-bound arrays, etc. Consequently, the solid carrier to which the food preparations are coupled may include wells of a multiwall plate, a bead (e.g., color-coded or magnetic), an adsorptive film (e.g., nitrocellulose or micro/nanoporous polymeric film), or an electrical sensor (e.g. a printed copper sensor or microchip). [0077] Consequently, the inventors also contemplate a method of testing food intolerance in patients that are diagnosed with or suspected to have psoriasis.
• a bead e.g., color-coded or magnetic
• adsorptive film e.g., nitrocellulose or micro/nanoporous polymeric film
• electrical sensor e.g. a printed copper sensor or microchip
• such methods will include a step of contacting a food preparation with a bodily fluid (e.g., whole blood, plasma, serum, saliva, or a fecal suspension) of a patient that is diagnosed with or suspected to have psoriasis, and wherein the bodily fluid is associated with a gender identification.
• a bodily fluid e.g., whole blood, plasma, serum, saliva, or a fecal suspension
• the step of contacting can be performed under conditions that allow an immunoglobulin such as IgG (or IgE or IgA or IgM) from the bodily fluid to bind to at least one component of the food preparation, and the IgG bound to the component(s) of the food preparation are then quantified/measured to obtain a signal.
• the signal is then compared against a gender-stratified reference value (e.g., at least a 90th percentile value) for the food preparation using the gender identification to obtain a result, which is then used to update or generate a report (e.g., written medical report; oral report of results from doctor to patient; written or oral directive from physician based on results).
• a gender-stratified reference value e.g., at least a 90th percentile value
• a report e.g., written medical report; oral report of results from doctor to patient; written or oral directive from physician based on results.
• At least some, or all of the different food preparations have an average discriminatory p-value of ⁇ 0.07 (or ⁇ 0.05, or ⁇ 0.025) as determined by raw p- value, and/or or an average discriminatory p-value of ⁇ 0.10 (or ⁇ 0.08, or ⁇ 0.07) as determined by FDR multiplicity adjusted p-value.
• food preparations are prepared from single food items as crude extracts, or crude filtered extracts
• food preparations can be prepared from mixtures of a plurality of food items (e.g., a mixture of citrus comprising lemon, orange, and a grapefruit, a mixture of yeast comprising baker' s yeast and brewer' s yeast, a mixture of rice comprising a brown rice and white rice, a mixture of sugars comprising honey, malt, and cane sugar.
• a plurality of food items e.g., a mixture of citrus comprising lemon, orange, and a grapefruit, a mixture of yeast comprising baker' s yeast and brewer' s yeast, a mixture of rice comprising a brown rice and white rice, a mixture of sugars comprising honey, malt, and cane sugar.
• food preparations can be prepared from purified food antigens or recombinant food antigens.
• Each food preparation is immobilized on a solid surface (typically in an addressable manner, such that each food preparation is isolated), it is contemplated that the step of measuring the IgG or other type of antibody bound to the component of the food preparation is performed via an ELISA (enzyme-linked immunosorbent assay) test.
• exemplary solid surfaces include, but are not limited to, wells in a multiwell plate, such that each food preparation may be isolated to a separate microwell.
• the food preparation will be coupled to, or immobilized on, the solid surface.
• the food preparation(s) will be coupled to a molecular tag that allows for binding to human immunoglobulins (e.g., IgG, etc.) in solution.
• the inventors also contemplate a method of generating a test for food intolerance in patients diagnosed with or suspected to have psoriasis. Such a test is applied to patients already diagnosed with or suspected to have psoriasis, in certain embodiments, the authors do not contemplate that the method has a diagnostic purpose. Instead, the method is for identifying triggering food items among already diagnosed or suspected psoriasis patients.
• test kits that can be used for this method may comprise one or more distinct food preparations having an average discriminatory p-value, wherein the average discriminatory p- value for each distinct food preparation is determined by a process that includes comparing test results of a first patient test cohort that is diagnosed with or suspected of having psoriasis, with test results of a second patient test cohort that is not diagnosed with or suspected of having psoriasis.
• test results for the first and second patient test cohorts are obtained for various distinct food preparations, wherein the test results are based on contacting bodily fluids (e.g., blood saliva, fecal suspension, etc.) of the first patient test cohort and the second patient test cohort with each food preparation.
• bodily fluids e.g., blood saliva, fecal suspension, etc.
• the test results are then stratified by gender for each of the distinct food preparations, a different cutoff value for male and female patients for each of the distinct food preparations (e.g., cutoff value for male and female patients has a difference of at least 10% (abs), etc.) is assigned for a predetermined percentile rank (e.g., 90th or 95th percentile, etc.).
• the distinct food preparations include at least two (or six, or ten, or fifteen) food preparations prepared from food items selected from the group consisting of foods 1-59 of Table 2, and/or items of Table 1.
• the distinct food preparations include a food preparation prepared from a food items other than foods 1-59 of Table 2.
• each distinct food preparation will have an average discriminatory p-value of ⁇ 0.07 (or ⁇ 0.05, or ⁇ 0.025) as determined by raw p-value or an average discriminatory p- value of ⁇ 0.10 (or ⁇ 0.08, or ⁇ 0.07) as determined by FDR multiplicity adjusted p-value.
• ⁇ 0.07 or ⁇ 0.05, or ⁇ 0.025
• ⁇ 0.10 or ⁇ 0.08, or ⁇ 0.07
• a three-step procedure of generating food extracts may provide more accurate results.
• the first step is a defatting step.
• lipids from grains and nuts are extracted by contacting the flour of grains and nuts with a non-polar solvent and collecting residue.
• the defatted grain or nut flour are extracted by contacting the flour with elevated pH to obtain a mixture and removing the solid from the mixture to obtain the liquid extract.
• the liquid extract is stabilized by adding an aqueous formulation.
• the aqueous formulation includes a sugar alcohol, a metal chelating agent, protease inhibitor, mineral salt, and buffer component 20-50 mM of buffer from 4-9 pH. This formulation allowed for long term storage at -70 °C and multiple freeze-thaws without a loss of activity.
• a two-step procedure of generating food extract may provide more accurate results.
• the first step is an extraction step.
• extracts from raw, uncooked meats or fish are generated by emulsifying the raw, uncooked meats or fish in an aqueous buffer formulation in a high impact pressure processor.
• solid materials are removed to obtain liquid extract.
• the liquid extract is stabilized by adding an aqueous formulation.
• the aqueous formulation includes a sugar alcohol, a metal chelating agent, protease inhibitor, mineral salt, and buffer component 20-50 mM of buffer from 4-9 pH. This formulation allowed for long term storage at -70 °C and multiple freeze-thaws without a loss of activity.
• a two-step procedure of generating food extract is may provide more accurate results.
• the first step is an extraction step.
• liquid extracts from fruits or vegetables are generated using an extractor (e.g., masticating juicer, etc) to pulverize foods and extract juice.
• solid materials are removed to obtain liquid extract.
• the liquid extract is stabilized by adding an aqueous formulation.
• the aqueous formulation includes a sugar alcohol, a metal chelating agent, protease inhibitor, mineral salt, and buffer component 20-50 mM of buffer from 4-9 pH. This formulation allowed for long term storage at -70 °C and multiple freeze-thaws without a loss of activity.
• the blocking buffer includes 20-50 mM of buffer from 4-9 pH, a protein of animal origin (e.g., beef, chicken) and a short chain alcohol (e.g., glycerin, etc.).
• a protein of animal origin e.g., beef, chicken
• a short chain alcohol e.g., glycerin, etc.
• Other blocking buffers including several commercial preparations, can be attempted but may not provide adequate signal to noise and low assay variability required.
• ELISA preparation and sample testing Food antigen preparations were immobilized onto respective microtiter wells following the manufacturer's instructions.
• the food antigens were allowed to react with antibodies present in the patients' serum, and excess serum proteins were removed by a wash step.
• enzyme labeled anti-IgG antibody conjugate was allowed to react with antigen-antibody complex.
• a color was developed by the addition of a substrate that reacts with the coupled enzyme. The color intensity was measured and is directly proportional to the concentration of IgG antibody specific to a particular food antigen.
• the final list foods will be shorter than 50 food items, or equal or less than of 40 food items.
• signal scores will be compared between psoriasis and controls using a permutation test on a two-sample t-test with a relative high number of resamplings (e.g., >1,000, or >10,000, or even >50,000).
• the Satterthwaite approximation can then be used for the denominator degrees of freedom to account for lack of homogeneity of variances, and the 2-tailed permuted p-value will represent the raw p-value for each food.
• False Discovery Rates (FDR) among the comparisons will be adjusted by any acceptable statistical procedures (e.g., Benjamini-Hochberg, Family-wise Error Rate (FWER), Per Comparison Error Rate (PCER), etc.).
• Foods were then ranked according to their 2-tailed FDR multiplicity-adjusted p- values. Foods with adjusted p-values equal to or lower than the desired FDR threshold are deemed to have significantly higher signal scores among psoriasis than control subjects and therefore deemed candidates for inclusion into a food intolerance panel.
• a typical result that is representative of the outcome of the statistical procedure is provided in Table 2.
• the ranking of foods is according to 2-tailed permutation T-test p-values with FDR adjustment.
• the final 90th and 95th percentile-based cutpoints for each food and gender will be computed as the average 90th and 95th percentiles across the 1000 samples.
• the number of foods for which each psoriasis subject will be rated as "positive” was computed by pooling data across foods. Using such method, the inventors will be now able to identify cutoff values for a predetermined percentile rank that in most cases was substantially different as can be taken from Table 4.
• Figures 1A-1D Typical examples for the gender difference in IgG response in blood with respect to peach is shown in Figures 1A-1D, where Figure 1A shows the signal distribution in men along with the 95 th percentile cutoff as determined from the male control population.
• Figure IB shows the distribution of percentage of male psoriasis subjects exceeding the 90 th and 95 th percentile
• Figure 1C shows the signal distribution in women along with the 95 th percentile cutoff as determined from the female control population.
• Figure ID shows the distribution of percentage of female psoriasis subjects exceeding the 90 th and 95 th percentile.
• Figures 2A-2D exemplarily depict the differential response to cucumber
• Figures 3A-3D exemplarily depict the differential response to tea
• Figures 4A-4D exemplarily depict the differential response to tomato.
• Figures 5A-5B show the distribution of psoriasis subjects by number of foods that were identified as trigger foods at the 90 th percentile (5 A) and 95 th percentile (5B). Inventors contemplate that regardless of the particular food items, male and female responses were notably distinct.
• IgG response results can be use to compare strength of response among given foods
• the IgG response results of a patient are normalized and indexed to generate unit-less numbers for comparison of relative strength of response to a given food.
• one or more of a patient' s food specific IgG results e.g., IgG specific to tomato and IgG specific to cucumber
• the normalized value of the patient' s IgG specific to tomato can be 0.1 and the normalized value of the patient' s IgG specific to cucumber can be 0.3.
• the relative strength of the patient' s response to cucumber is three times higher compared to tomato. Then, the patient' s sensitivity to cucumber and tomato can be indexed as such.
• one or more of a patient' s food specific IgG results can be normalized to the global mean of that patient' s food specific IgG results.
• the global means of the patient' s food specific IgG can be measured by total amount of the patient' s food specific IgG.
• the patient' s specific IgG to shrimp can be normalized to the mean of patient' s total food specific IgG
• the global means of the patient's food specific IgG can be measured by the patient' s IgG levels to a specific type of food via multiple tests. If the patient has been tested for his sensitivity to shrimp five times and to pork seven times previously, the patient' s new IgG values to shrimp or to pork are normalized to the mean of five-times test results to shrimp or the mean of seven-times test results to pork.
• the normalized value of the patient's IgG specific to shrimp can be 6.0 and the normalized value of the patient's IgG specific to pork can be 1.0. In this scenario, the patient has six times higher sensitivity to shrimp at this time compared to his average sensitivity to shrimp, but substantially similar sensitivity to pork. Then, the patient' s sensitivity to shrimp and pork can be indexed based on such comparison.
• Table 5A and Table 5B provide exemplary raw data.
• the data indicate number of positive results out of 90 sample foods based on 90 th percentile value (Table 5A) or 95 th percentile value (Table 5B).
• Average and median number of positive foods was computed for psoriasis and non-psoriasis patients. From the raw data shown in Table 5A and Table 5B, average and standard deviation of the number of positive foods was computed for psoriasis and non-psoriasis patients.
• Table 6A and Table 7A show exemplary statistical data summarizing the raw data of two patient populations shown in Table 5A.
• the statistical data includes normality, arithmetic mean, median, percentiles and 95% confidence interval (CI) for the mean and median representing number of positive foods in the psoriasis population and the non- psoriasis population.
• Table 6B and Table 7B show exemplary statistical data summarizing the raw data of two patient populations shown in Table 5B.
• the statistical data includes normality, arithmetic mean, median, percentiles and 95% confidence interval (CI) for the mean and median representing number of positive foods in the psoriasis population and the non-psoriasis population.
• Table 8A and Table 9A show exemplary statistical data summarizing the raw data of two patient populations shown in Table 5 A.
• the raw data was transformed by logarithmic transformation to improve the data interpretation.
• Table 8B and Table 9B show another exemplary statistical data summarizing the raw data of two patient populations shown in Table 5B.
• the raw data was transformed by logarithmic transformation to improve the data interpretation.
• Table 10A and Table 11A show exemplary statistical data of an independent T- test (Table 10A, logarithmically transformed data) and a Mann-Whitney test (Table 1 1 A) to compare the geometric mean number of positive foods between the psoriasis and non- psoriasis samples.
• Table 10A and Table 1 1 A indicate statistically significant differences in the geometric mean of positive number of foods between the psoriasis population and the non-psoriasis population. In both statistical tests, it is shown that the number of positive responses with 90 food samples is significantly higher in the psoriasis population than in the non-psoriasis population with an average discriminatory p-value of ⁇ 0.0001.
• Table 10B and Table 11B show exemplary statistical data of an independent T- test (Table 10A, logarithmically transformed data) and a Mann-Whitney test (Table 1 IB) to compare the geometric mean number of positive foods between the psoriasis and non- psoriasis samples.
• Table 10B and Table 11B indicate statistically significant differences in the geometric mean of positive number of foods between the psoriasis population and the non-psoriasis population.
• Table 12A shows exemplary statistical data of a Receiver Operating
• ROC Characteristic
• the above test can be used as another 'rule in' test to add to currently available clinical criteria for diagnosis for psoriasis.
• the absolute number and percentage of subjects with 0 positive foods is also very different in psoriasis vs. non-psoriasis subjects, with a far lower percentage of psoriasis subjects (8.3%)) having 0 positive foods than non-psoriasis subjects (15.4%).
• the data suggests a subset of psoriasis patients may have psoriasis due to other factors than diet, and may not benefit from dietary restriction.
• Table 12B shows exemplary statistical data of a Receiver Operating
• ROC Characteristic
• the above test can be used as another 'rule in' test to add to currently available clinical criteria for diagnosis for psoriasis.
• the absolute number and percentage of subjects with 0 positive foods is also very different in psoriasis vs. non-psoriasis subjects, with a far lower percentage of psoriasis subjects (16.5%)) having 0 positive foods than non- psoriasis subjects (35%>).
• the data suggests a subset of psoriasis patients may have psoriasis due to other factors than diet, and may not benefit from dietary restriction.
• Method for determining distribution of per-person number of foods declared "positive” To determine the distribution of number of "positive” foods per person and measure the diagnostic performance, the analysis was performed with 90 food items from the Table 1, which shows most positive responses to psoriasis patients.
• the 90 food items includes chocolate, grapefruit, honey, malt, rye, baker's yeast, brewer' s yeast, broccoli, cola nut, tobacco, mustard, green pepper, buck wheat, avocado, cane sugar, cantaloupe, garlic, cucumber, cauliflower, sunflower seed, lemon, strawberry, eggplant, wheat, olive, halibut, cabbage, orange, rice, safflower, tomato, almond, oat, barley, peach, grape, potato, spinach, sole, and butter.
• each food- specific and gender-specific dataset was bootstrap resampled 1000 times. Then, for each food item in the bootstrap sample, sex-specific cutpoint was determined using the 90th and 95th percentiles of the control population. Once the sex-specific cutpoints were determined, the sex-specific cutpoints was compared with the observed ELISA signal scores for both control and psoriasis subjects. In this comparison, if the observed signal is equal or more than the cutpoint value, then it is determined "positive" food, and if the observed signal is less than the cutpoint value, then it is determined "negative" food.

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