Hypersensitivity Diagnosis. Development of an Automatic and Quantitative IgE Concentration Detection Algorithm

Utilized in the Self-designed Allergen Disease Diagnosis System


Master's Thesis, 2014

76 Pages


Excerpt

CONTENTS

CHAPTER-1 Background
1.1 Introduction
1.2 Diagnosis of Allergic Diseases
1.3 Research Status
1.4 In-vitro Allergy Test Products

CHAPTER 2 In-Vitro Allergen Testing Device
2.1 Preparation of Reagent
2.2 Experimental Device
2.3 System Assembly

Chapter 3 Verification of System Stability and Grayscale Image Analysis
3.1 Introduction
3.2 Color calibration of the imaging system by intensity ratio analysis
3.3 Gray Value Analysis of Colored Reaction Product

CHAPTER 4 COLOR MODELS AND IMAGE ANALYSIS
4.1 Introduction
4.2 Color Models

5. Polynomial Regression
5.1 Background
5.2 Null hypothesis
5.3 Experiment Motive
5.4 Procedure Details

6. Summary and Future Work

7. References

Abstract

Allergic diseases are seen to affect a large number of people around the world every year. The proper diagnosis of allergic disease is an important factor to maintain the good health condition of a general population. Although there are several techniques available for the diagnosis of allergic disease, most of the methods fail to cover all of the criteria to make it acceptable.

This study introduces general methods of allergy diagnosis which are in practice in current scenario and different image processing techniques that can be incorporated into it to make the process even easier. In this study ELISA technique is proposed to fulfill all the basic criteria like sensitivity, accuracy and most importantly cost effectiveness. ELISA is an immunoassay that involves the detection of Immunoglobulin type E commonly referred as IgE. An enzyme linked anti-human globulin is made to react with IgE, if present in the patient's serum which will produce the colored product when the substrate of the enzyme is added. Depending upon the concentration of the colored product, the concentration of IgE is determined. The experiment condition like wet condition and plastic container can significantly change the detected color of the allergen card and hence the result value. This proves that the system environment indeed affects calculated IgE concentration. In order to reduce this negative effect, we need to find an effective image feature which is little influenced by system environment. The content of this study mainly focuses on different color models of the color images that can be used in image processing technology which are further utilized to define its relationship with IgE concentration or the severity of the disease. Involving the features of both binary and color image models it gives the better comparison of the different image features in different color models. These image features ware tested and result curves were produced that describes the relationship between IgE concentration and image feature. Based on the experimental findings the new diagnostic image feature was introduced. The experiments were done to test the accuracy of the proposed diagnostic image feature which is cheap, sensitive and can detect the IgE concentration quantitatively.

Keywords: Allergic disease, Automatic Diagnosis, IgE, ELISA, Quantitative Detection

List of Figures

Figure 1.1 Prevalence rate of eczema, rhinitis and asthma in different cities in China

Figure 1.2 Prevalence of allergic diseases asthma and rhino conjunctivitis in European countries

Figure 1.3 Oral Food Challenge Diagrams

Figure 1.4 Diagrammatic Representation of ELISA reaction

Figure 1.5 Immuno-CAP Tests

Figure 1.6 ImmunoCAP

Figure 1.7 Protein Microarray Chip for Allergen Detection

Figure 2.1 Allergen Reagent Strip

Figure 2.2 Paper Reagent Strip after ELISA Reaction

Figure 2.3 LED Light Source

Figure 2.4 Optical Filters

Figure 2.5 Camera and Lens A) Outer Appearance of Camera B) Camera Lens

Figure 2.6 Electric Translation Stage

Figure 2.7 Imaging System Assembly

Figure 3.1 Images of the Allergen Card A) RGB Image B) Gray Scale Image

Figure 3.2 IgE Vs Gray Value Curve at Different Wavelength

Figure 3.3 IgE Concentration Vs Multi Spectral Area Curve

Figure 3.4 IgE Concentration Vs Color Difference

Figure 3.5 IgE Vs Gray Value curve for Different Monochromatic Light

Figure 4.1 RGB Color Model A: Primary colors representation. B: Color cubes

Figure 4.2 One of the image data of group 1 with cat allergen reaction product in third Small Square box from upside down

Figure 4.3 G channel Vs IgE curve when the allergen cards were dry

Figure 4.4 IgE Vs G Channel Curve when the allergen cards were wet in plastic container

Figure 4.5 Comparison curves for G channel in wet and dry conditions

Figure 4.6 Comparison diagram for experimental and calculated curve

Figure 4.7 Comparison of IgE Vs Luma Intensity curve in wet and dry Condition

Figure 4.8 RGB geometry in HSL color model

Figure 5.1 Second order polyfit curve

Figure 5.2 Fourth order polyfit curve

Figure 5.3 Fifth order polyfit curve 64

List of Tables

Table 1 Comparison of Oral Food Challenges

Table 2 Different Allergens and their Respective IgE Concentration

Table 3 Gray Value Result of Different Color Bands

Table 4 Concentration of IgE in Different Samples Used in the Experiment

Table 5 Allergen Reagent Strips Corresponding Serum IgE Concentrations

Table 6 Concentration of IgE Used in ELISA for Different Cards

Table 7 Comparison of G channel value in wet and dry conditions

Table 8 Calculation of coefficients and constant of the experimental curve

Table 9 Comparison of different Image Features of YCbCr Color Model

Table 10 Comparison of different Color Features of HSV Color Model

CHAPTER-1 Background

1.1 Introduction

Allergy is a hypersensitivity reaction mediated by immunological mechanisms, so it is also known as hypersensitivity disorder of the immune system. A substance that causes the reaction is called allergen. Allergy is one of the four forms of hypersensitivity and is called type I or immediate hypersensitivity. These reactions are distinctive because of the excessive activation of certain white cells called mast cells and basophils by a type of antibody called immunoglobulin E (IgE). Generally, allergic hypersensitivity is of two types IgE mediated and non-IgE mediated. IgE mediated hypersensitivity is classified again into two types, Atopic and Non-atopic.

World Allergy Organization (WAO) after a survey of 20 long years found nearly a quarter of world's total population is affected by allergic diseases directly or indirectly. It has been estimated by World Health Organization (WHO) that the treatment costs of allergic diseases have exceeded 20 billion US$. So the WHO has laid its focus on the prevention, control and the treatment of the disease [[1],[2]]. The incidence of the allergic diseases has been increasing and the disease is always associated with complications. Allergy is not just a disease; it includes several types of diseases including atopic dermatitis, food allergies, allergic rhinitis, asthma and a series of diseases affecting not only one organ, but a number of organs such as skin, respiratory tract, eye etc. The world's total incidence rate of pollen induced allergic rhinitis and asthma are 5% and 22% respectively. Seasonal allergic rhinitis gradually progresses to asthma in 25-38% of the cases that don't involve desensitization therapy. And the condition kept on getting worse, eventually developing into perennial asthma, emphysema, pulmonary heart diseases etc affecting people's health and lives seriously. Meanwhile, WHO has estimated there are 150 million people suffering from asthma with 18 million deaths per year [[2], [3], [5]].

Survey conducted earlier shows 20% of the world's population suffering from allergy[[6]], 300 million people suffered from asthma and 255,000 died of asthma in the year 2005[[7]]. International Study of Asthma and Allergies in Children (ISAAC) was conducted across different regions of the world, which indicated that China's allergy problems could not be ignored [[8]].

Survey done by Fei Li and colleague[[9]] shows that incidence of allergic diseases like Eczema, Rhinitis etc, are high in China to affect the average health condition of a general population. The crude prevalence was estimated in their work and it was represented by gender across cities. The average prevalence of asthma, allergic rhinitis and eczema across eight cities was 3∙3% (3∙1, 3∙6), 9∙8% (9∙4, 10∙2) and 5∙5% (5∙2, 5∙8), respectively. Below is the graphical representation of this data.

Figure 1.1 below shows the prevalence of Asthma, Rhinitis and Eczema in major cities of China. It can be seen clearly that the prevalence of Eczema is high almost in all of the major cities of China affecting at an average of 5 people per 100. The least occurring allergic reaction also accounts for around 1 out of 100. So in general it can be understood that an average of 3 people out of 100 is infected with allergic disease in China.

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Figure 1.1 Prevalence rate of eczema, rhinitis and asthma in different cities in China [9].

The prevalence rate of different kinds of allergic diseases is high in western European countries too. A survey done by the European Environment and Health Information System (ENHIS) [[10]] shows that the prevalence rate of allergic disease is high not only in adult population, but it is affecting the children of different age groups as well. The spread of the allergic disease like Asthma and Rhino conjunctivitis was found to be in between 4 to 28% in different countries of Europe. The graphical representation of the survey data of ENHIS is given above as Figure 1.2.

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Figure 1.2 Prevalence of allergic diseases, [A] asthma and [B] rhino conjunctivitis in European countries [[10]].

Allergic disease is a chronic allergic inflammation associated with the exposure of allergens. Experiments have shown that some people develop allergic symptoms after exposing them to the allergens while the others living in the same environment may not develop such symptoms. There is no any theory that best describes this variation. The disease cannot be diagnosed accurately and early enough so the prevention could not be effective.

1.2 Diagnosis of Allergic Diseases

The effective management of allergic diseases is possible only if the available methods could make an accurate diagnosis. For assessing the presence of allergen-specific IgE antibodies, two different methods can be used, a skin pricks test or an allergy blood test. Both of these methods are recommended and have the similar diagnostic value [[11], [12]]. Diagnosis of allergy generally involves these two tests also known as in vitro test and in vivo test. The skin prick test in which an allergen is introduced into the subject is in vitro test while the other tests that are done outside the patient's body by taking patients sample like serum is called in vivo test.

1.2.1 Skin Prick Test

Skin prick testing or SPT demonstrates an allergic response to a specific allergen. The skin prick test in conjunction with an allergy focused history can be used to detect the presence of an allergy to either a food or inhaled substance (allergen). It is one of the most common allergy tests performed in an allergy clinic by specially trained staff. SPT is a simple, safe and quick test providing results within 15 minutes. The skin prick test introduces a tiny amount of allergen into the skin, causing a small, localized allergic response in the form of wheal (bump) and flare (redness) at the site of testing. These tests can be carried out on all age groups, including children. This test is generally done in the fore arm and the range of the allergens that can be tested is as few as 3 or 4 or up to about 25 allergens. It involves the following general steps.

- The skin is coded with a marker or pen to identify the allergens to be tested.
- A drop of the allergen extract solution is placed on the skin.
- The skin is then pricked through the drop using the tip of lancet.

The skin may become itchy within a few minutes and become red and swollen with a "wheal" in the centre. The wheal has a raised edge which slowly expands to reach its maximum size in 15 minutes. This wheal clears off for most people within an hour. No reaction to SPT may indicate that the patient is not sensitive to that allergen. Negative reactions may occur for other reasons also, for example, if the patient is taking anti-histamines or the medications that block the effect of histamine. So it is necessary the results are interpreted by health care professional experienced in allergy. In some cases, a blood test may be needed to clarify the results.

1.2.2 Oral Food Challenge

A growing number of patients present to the allergist with the laboratory findings of food sensitization and a weak likelihood of reaction. Clinical history, skin prick testing (SPT), and serum immunoglobulin E (sIgE) concentration can provide data which is only suggestive for the likelihood of reaction. Ultimately, the diagnosis should be provided by an oral food challenge (OFC). It is a safe test that can be carried out even in the office of a board-certified allergist.

Food challenge is done by letting the patient to consume an increasing amount of suspected food at fixed intervals under observation. Generally, the patient is feed increasing doses of the suspected food at 10-30 minutes until a reaction occurs or a normal amount of food is eaten without producing symptoms. The negative blind challenges end with an open challenge. Oral food challenges are important for the diagnosis of food allergy.

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Figure 1.3 Oral Food Challenge Diagrams [[13]].

Table 1 Comparison of Oral Food Challenges

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Three types of oral food challenges as shown in the above diagram are open oral food challenge (OFC), Double-blind placebo-controlled food challenge (DBPCFC) and Single-blind placebo-controlled food challenge (SBPCFC). Comparison of these three food challenge tests is shown in the table below.

1.2.3 Biochemical Analysis of Allergic Reaction

In the early stage of allergy, a type I hypersensitivity reaction occurs that involves the encounter of the allergen exposed for the first time and its reposition to the TH2 cells by Antigen Presenting Cells (APC). TH2 lymphocyte is a subset of T lymphocyte that produces a cytokine, Interleukin-4 (IL-4). The TH2 cells when comes in contact with the antigen brought by APC, it gets activated and interact with other lymphocytes called B cells, whose role is to produce antibodies. Coupled with signals provided by IL-4 this interaction stimulates the B cell to begin production of a large amount of a particular type of antibody known as IgE. Analysis of cytokines and other biochemical compounds in the blood is a useful aid in the diagnosis of allergy.

1.2.4 Serum Specific Immunoglobulin E (sIgE)

An allergy blood test involves the quantification of specific IgE antibodies in the blood. This test is quick and simple and unlike the SPT, a blood test could be performed irrespective of age, skin condition, medication, symptoms and pregnancy. This test measures the concentration of specific IgE antibodies in the blood. Quantitative IgE test results also facilitate the differentiation and ranking of different allergic substances according to onset of the symptoms. A general rule of IgE concentration interpretation is that the higher the IgE antibody value, the greater the likelihood of symptoms. Allergens at low levels that are not producing symptoms today can't predict the future symptom development. The quantitative allergy blood result can help determine what a patient is allergic to and it also helps to predict and follow the disease development, estimate the risk of a severe reaction and explain cross-reactivity. [[14]]

1.3 Research Status

1.3.1 In-Vitro Allergen Detection Technology

There is a wide variety of in vitro allergen detection tests including enzyme linked immunosorbent assay (ELISA). These are western blot test or immunoblotting (IBT), enzyme linked immunofluorescence assay (IFA), chemiluminescence immunoassay (CLIA), Radio-immunosorbent assay (RIA) and radiation allergen assay (RAST).

(1) Radioactive Allergen Assay

Radioactive allergen assay also called radio-immunoassay is a very sensitive in-vitro immunoassay technique used to quantify the concentration of antigen by the use of antibodies. Although the RIA technique is most sensitive and extremely specific requiring special equipments, it remains one among the least expensive immunoassay methods.

(2) Enzyme-linked Immuno-Sorbent Assay

ELISA is the abbreviated form of the Enzyme Linked Immuno-Sorbent Assays. This technique is based on attaching an enzyme labeled anti-human globulin to the test IgE attached to the allergen. The enzyme upon the addition of its substrate gives the colored product.

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Figure 1.4 Diagrammatic Representation of ELISA Reaction [[38]]

The indirect type of ELISA reaction was used in this experiment. The basic principle of Indirect ELISA reaction lies in the fact that the antigen (allergen in this research case) is coated on the stable solid phase. It is then followed by the addition of the suspected serum, the IgE against the specific allergen if present in the suspected serum will bind with the allergen and forms allergen-IgE complex. Now, to produce a visible colored reaction product secondary antibody linked with an enzyme is added. The secondary antibody is generally manufactured in equine serum by introducing the human IgE. This secondary antibody is also called as anti-human globulin antibody. The enzyme linked secondary anti-human globulin antibody now will bind with the primary antibody. The unbound components in the reaction complex can be washed away by pouring distilled water on it. Finally, substrate of the enzyme linked to the secondary antibody is added which will react with the enzyme linked to the secondary antibody and produces the colored product. The intensity of the colored product is directly proportional to the IgE concentration. Below is the diagrammatic representation of ELISA reaction.

Some of the notable advantages of ELISA are

- The method is quick and convenient.
- Antigens of very low concentration can be detected since the capture antibody binds only with the specific antigen.
- It is safe to perform as there is no need of applying any bio-hazardous or radioactive substances. Reagents used are generally un-hazardous.
- The technique can be applied to a wide variety of allergens.

Disadvantages

- Immuno-reactivity of the antibody might be adversely affected by labeling with enzymes.
- Minimal Signal amplification.

(3) Immuno-CAP for IgE Detection

The ImmunoCAP IgE testing has been a standard test which can replace all of the traditional practices for the allergic diagnosis in which the coefficient of variation is very high. The ImmunoCAP technique being not only sensitive and accurate also gives the quantitative results. It’s why the test ImmunoCAP should replace other allergy test for allergic diagnosis. Immuno-CAP is a highly sensitive and fully automated assay and are designed in such a way that they are able to produce highly reliable and reproducible results. These assays can be performed to detect a wide variety of the allergens including cats, insects, venom, mites, food allergens etc. This device are used not only in a large commercial laboratory but also in a small clinical laboratory.

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Figure 1.5 Immuno-CAP Tests [15].

Test Principle

The allergen of interest, covalently coupled to solid phase cellulose ImmunoCAP is made to react with the sample containing IgE. The unbound IgE is washed away and the complex is then allowed to react with enzyme labeled anti-human IgE. This step is again followed by washing and then the substrate for the enzyme used to label anti IgE is added and is incubated. After stopping the reaction fluorescence of the final complex is measured. The response value is directly proportional to the concentration of the antibodies in the serum. To calculate the result the response value is transformed into the IgE concentration by using a calibration curve based on the World Health Organization (WHO) reference.

Instrument Set up

The UniCAP (Pharmacia Diagnostic) provides an automated system for all of the experiment steps like specimen incorporating, reagent holding, washing, reading, calculation etc. all of the procedures is carried out in controlled environment condition at constant temperature. Data are store and integrated daily. In this system calibration is carried out automatically and compared with stored values. The linearity of the test procedure results in more precise result and also supports the fast processing of the sample. Various models of UniCAP are available for laboratories of different sizes. The instrument with highest capacity has the input of 240 tests per hour with a total capacity of 1200 tests/day.

Immunocap test procedure requires only one blood sample to detect many allergens and the test result being extremely accurate has drawn the attraction of medical personnel and researchers to use this technique as a standard test for quantitative IgE detection.

(4) Sensitive Colorimetric Assay

This technique involves the coupling of allergy ELISA assays with several advantageous concept of microarray technology for IgE responsive to allergen. This method uses three dimensional highly porous substrate of thickness 0.1 µm that binds a large number of allergen protein and uses the same concept of sandwich ELISA as in current in-vitro IgE testing. This method would increase the sensitivity of the test to detect IgE concentration in blood as low as 0.1 IU. Studies and the research work did previously have shown the Zeta-grip protein microarrays to be cost effective, highly sensitive and miniaturized way to conduct ELISA-like assays.

Detection of allergen is done by applying one of the two different techniques in this system. The first one is silver staining and another one is to produce color precipitate by using Alkaline Phosphate (ALP).

The multiplex microarray system is superior to other ELISA for the diagnosis of allergic disorders because of its sensitivity. The WHO standard with cutoff of 0.35 IU has been reduced even less by this technique and provided newer sensitive assay for immunological studies in allergic condition. This has made the diagnosis of allergic disease more accurate. The ImmunoCAP method employed more often for allergic diagnosis and the research purpose has now been surpassed by the development of this colorimetric micro assay.

(5) Automated Micro fluidic Based Immunoassay Cartridge

In vitro testing of allergen specific immunoglobulin E (IgE) has been used along with traditional skin prick testing for more than thirty years. Such immunoassay is a useful tool in monitoring immunotherapy and the diagnosis of children, infants and adult with atopic allergy. Although ImmunoCAP allergy test is the classical test for the diagnosis of allergy that is used frequently there are still many other tests which are being widely used for this purpose. For example CLA allergy test, IMMULITE, AlaSTAT etc. With these several tests available for the quantitative measurement of specific IgE by using a non-specific calibration curve, the level of serum IgE is expressed as international unit per milliliter (IU/ml, where 1 IU is equivalent to 2.4 ng). Because there is no specific IgE standard available currently the specific IgE level measured by this immunoassay may vary test to test due to the difference in the source of the allergen material.

Recently there has been an increased interest of the use of microarray for the specific IgE quantification. The underlying main reason for researches to be focused on such microarray is the ability of the system to detect target allergen in a wide range (50+) with minimal serum (25-100 µl). This section summarizes the review of the development of a micro fluidic cartridge for the automated analysis of specific IgE using allergen extracts. These automated micro fluidic cartridge devices are entirely injection molded and the protein extract is bound to an inexpensive nitrocellulose coat. The cost of this micro fluidic cartridge device is less than one-tenth of the activated slide typically employed in microarray experiments. Additionally, the cartridge is driven by low cost analyzer composed of a solenoid actuator array and a low resolution cooled charge-coupled device (CCD) camera for chemiluminescence detection. The time required for the cartridge allergen screening is less than 30 min. The system has decentralized allergen screening function and near-patient testing. Other advantages that this allergen screening system has includes ease of use, short analysis time, low instrumentation cost etc.

This technique allows production of neat channels thinner than hair which is impossible in traditional transparent plastic technology. It is very important in the field of cell separation. For allergic reaction this technology may help in preparing allergen suspension. It may help to separate raw materials from allergen extract obtained from the natural sources. The net allergen obtained then after contains pure allergen with high concentration. This allergen when used with patients sera produces intense reaction product.

1.4 In-vitro Allergy Test Products

(1) UniCAP with ImmunoCAP Allergen Detection System

In-vitro allergen detecting test products have been produced rapidly in recent decades. Current allergen detection technology is fully automated. UNICAP 100 RAST-FEIA and ImmunoCAP are widely used at home and abroad for the detection of biological system allergic diseases. The system consists of the three parts; reagent and sample handling zone, result detection place and software processing of the information [[16]]. These parts with the whole system can be seen clearly in the figure 1.6.

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Figure 1.6 ImmunoCAP[[39]]

(2) Protein Microarray

Protein microarray or microarray chip is the combination of protein microarray technology and the immunoblotting and immunologic techniques. Due to the development of high sensitivity and high specificity allergic disease sIgE protein chip, the system is capable of screening a large number of allergic diseases [[17]].

Abbildung in dieser Leseprobe nicht enthalten

Figure 1.7 Protein Microarray Chip for Allergen Detection [[17]]

In the protein microarray experiment, human serum IgE can be added to the allergens like dust mite, pollen etc. Meanwhile, positive and negative controls can also be added for the better comparison of the reaction result. The optimal reaction parameter can be maintained effectively in this system. Protein microarray test is simple and cost effective but the method is susceptible by the interference of man-made signal [[18]].

(3) Allergen Detection Reagent Strip

The detection of allergy involves the use of paper reagent strips as one of the most prompt method in allergic diagnosis. Typical image analysis process usually begins with the image acquisition, the image is worth to obtain as gray scale or color images separated by the threshold setting, and then the image processing is carried out. The processed image is then analyzed to obtain statistics [19]. Existing technologies such as Germany Mediwiss detector's Rapid and U.S. ASI's IVT detector uses a full spectrum white light on the reagent strip. The reflected signal is then analyzed to obtain RGB three color components. However, there will be some different color components overlap which will cause the metamerism. This will inevitably lead to some limitations when the original information is analyzed, thus limiting the ability to improve the quantitative detection of allergens [20, 21].

1.5 Summary

Allergic diseases are always associated with biological hazards which require the effective precautions and the control measures to eradicate it or to keep this hazard to the level minimum. A huge amount of money is spent every year to prevent these risks. These risks involve occupational risk to the medical staffs executing the detection tests and sometimes the allergen used for the detection may cause allergic inflammation in the patient at the region where it was used.

Diagnosis of the allergic disease comprises detection of the allergen or allergen related product in two different conditions i.e. in vivo and in vitro conditions. In vitro detection of allergens is safe, effective, and can achieve high-volume testing, has good prospects. The current in-vitro method of detecting is a qualitative detection for initial stage of the disease, semi-quantitative detection for the development stage and the quantitative detection at severe stage [4]. Qualitative detection rely mainly on visual observation of laboratory personnel, the outcome of the judgment is not accurate enough, and more time-consuming. Semi-quantitative test results can provide only the classification of the test results. Therefore, automated quantitative methods are efficient and quick, accurate and they have become the backbone in the development of the current in vitro allergy testing. This research paper describes the various image processing features that can be used to design an automatic and quantitative methods for the diagnosis of allergic diseases.

CHAPTER 2 In-Vitro Allergen Testing Device

The principal objective of this research is to develop a method for quantitative detection of IgE based on the paper reagent strip technology. These paper reagent strips containing previously determined allergens are used to carry out the Enzyme-linked Immuno-Sorbent Assay (ELISA) reaction along with the standard serum sample. The allergen-IgE complex forms the colored product after the ELISA reaction. These colored products are then photographed with suitable camera and the images are then processed in the image processing lab. Several image features can be tested with varying IgE concentration. The plots between IgE concentrations vs. image feature value were then used to analyze the relationship between IgE concentration and image feature value. This thesis provides a quantitative allergen detection scheme based on the comparison of the various image feature curves and the multi-spectral imaging of the reagent strips.

2.1 Preparation of Reagent

Preparation of reagent is an important step in ELISA reaction so proper care should be given while preparing the reagent and using the same while conducting the ELISA reaction.

2.1.1 Reagent Strips

Figure 2.1 shows the original ELISA reagent strip manufactured by Beijing Hai Ruixiang Tian Biotechnology Limited (北京海瑞祥天生物科技有限公司生产). Each color on the test strip package is specific for individual allergens.

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Figure 2.1 Allergen Reagent Strip

Notes: 1- Positive Control, 2-Dog Epithelium (Dog), 3-Cat Fur, 4-Ragweed, 5-Alternaria Mold, 6-Dust mites, 7-Negative Control, 8-Mugwort, 9-Soybeans, 10-Egg, 11- Peanuts, 12-Milk.

Positive control is the specific binding of the allergen with the IgE. If there is no color production in the positive control, the reagent strip is no longer valid and shouldn’t be used for further analysis. Similarly, negative control is the non-specific binding of IgE-allergen. In theory, IgE don’t bind with negative control reagent strip block and don’t produce any color.

These colored reagent strips when allowed to undergo ELISA reaction starts producing colors of different shades. The intensity of the color is directly proportional to the IgE concentration. The color ranges from white in the negative control to navy blue as shown in the figure 2.2.

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Figure 2.2 Paper Reagent Strip after ELISA Reaction

The standard serum sample was used with specific IgE concentration in order to carry out the ELISA reaction. The research experiment was done by taking the cat fur allergen and dust mite allergen into the account. These cat fur allergens and dust mite allergens are present in the 3rd and 6th allergen blocks respectively. Other allergens like dust mite, rag weed, alternaria etc are

Table 2 Different Allergens and their Respective IgE Concentration

Abbildung in dieser Leseprobe nicht enthalten

2.1.2 Production of the Reagent Strip

The following are the specific steps for each reagent strip test.

(1) Dilutions of human serum were done in accordance with the table in a ratio of 1ml of the prepared solution. The prepared solution was then added to the reagent strip and left on the shaker for 1 hour which ensures the proper reaction between allergen and IgE.
(2) The liquid remaining after step 1 was discarded and 2 ml buffer solution was added. It was then placed on the shaker for 5 minutes. The liquid was then drained and the reagent strips were washed three times by adding diluents on it.
(3) Liquid remaining in plastic container was discarded and 1 ml bovine serum, HRP secondary antibody solution was added. The plastic container containing reagent strip and this secondary antibody was placed on the shaker for 20 minutes.
(4) Liquid in step 3 was discarded, 2 ml buffer was added 2ml and the plastic container is placed on the shaker for 5 minutes. The liquid was drained and washed with diluents for three times.
(5) Liquid remaining after step 4 was discarded, 1 ml substrate solution was added and the plastic container containing the reagent strips was placed on the shaker for 10 minutes.
(6) Liquid remaining after step 5 was discarded, the test strip was removed from the kit and the reagent strips were allowed to dry by leaving it in the room temperature for 10 minutes.

The resulting color can be used in the following experiments. The two allergens (cat and dust mite) compared to IgE concentrations can be analyzed with experimental results.

2.2 Experimental Device

The images of the reagent strips required for the further analysis is obtained by the machine vision system. The system was installed in the laboratory by bringing together various essential components of the system including camera, light source, sliding stage etc. The system after installation was checked for the reliable performance by carrying out a number of calibration tests. The details of calibration test is described in chapter 3.2. A typical machine vision system includes light source, cameras, lenses, frame grabbers etc. The machine visions are of two types (CMOS or CCD). This system takes an image of the target reagent strip and the image signal is converted into the digital signal. This study describes the utilization of this system for image data acquisition and the analysis of the reagent strips. The principal constituents of the machine vision system are described below.

2.2.1 Light Source Circuit

It comprises of three identical LED surface light source, soft diffusion plate, cooling fan, LED drive power supply etc. LED surface light source uses CL-L251-MC4 planar type, 4 watt LED white light source (Citizen Electronics Co., Ltd. Japan). It is shown in Figure 10.

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Figure 2.3 LED Light Source

2.2.2 Multispectral Light Filter Module

Multispectral white light contains light rays at various wavelengths. Optical filters when used allow the light of specific wavelength to pass through and strike the object i.e. reagent strips. Multispectral light filter module comprises of filter, wheel and bracket. These filters used in this research experiment are able to filter the light beam and produce seven different light wavelengths (430, 450, 530, 550, 570, 590, and 610). The module is assembled by placing the filters into the wheel which is then placed on the camera in such a way that the proper contact between the camera lens and the filter is ensured. Optical filters manufactured by Huizhong Xinda Technology Co., Ltd. were used to conduct the research experiments which are shown in figure 11.

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Figure 2.4 Optical Filters

2.2.3 Image Acquisition Module

Image acquisition module is composed of camera, lenses, black sleeve to use as protective covering which prevents the additional light from entering into the experiment atmosphere. The camera used in the experiment was a product of Fuzhou Tucson, TCC-1.4IICHICE. The camera was a CCD type. The imaging system of CCD camera uses charge couple device.

[...]

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Details

Title
Hypersensitivity Diagnosis. Development of an Automatic and Quantitative IgE Concentration Detection Algorithm
Subtitle
Utilized in the Self-designed Allergen Disease Diagnosis System
College
Peking University
Author
Year
2014
Pages
76
Catalog Number
V506858
ISBN (eBook)
9783346121387
Language
English
Tags
hypersensitivity, disease, allergen, self-designed, utilized, algorithm, detection, concentration, quantitative, automatic, development, diagnosis, system
Quote paper
Satish Thapaliya (Author), 2014, Hypersensitivity Diagnosis. Development of an Automatic and Quantitative IgE Concentration Detection Algorithm, Munich, GRIN Verlag, https://www.grin.com/document/506858

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