**3.2 Research development for point-of-care tests**

Although there are some commercial products for the assay of celiac disease in the market, the effort has not been stopped to devise a low-cost kit with high


**Table 1.**

*Performance of QUANTA lite celiac disease tests in a high-risk population.*

accuracy. In 2005, Korponay-Szabó et al. developed a POCT that could rapidly detect the autoantibodies of tTG from blood sample [35]. The test is based on a Nunc-Immunostick (Denmark) principle with a four-wing stick. The two wings of the stick are pre-covered with gelatine to recognize and capture self-tTG/anti-tTG antibody complexes from the hemolyzed patient blood sample. The third wing is fixed with anti-human IgA antibodies, to combine with plasma IgA, which is used as a positive control. The fourth wing has no coating leading to the absence of antibody capture, and it serves as a negative control. In the test process, one drop of blood is inserted into the hemolyzing solution and incubated with the stick for 15 min. Then, the stick is washed with water and immersed for another 15 min in the solution of peroxidase-labeled anti-human IgA. To obtain a visible signal, the stick is washed again and then inserted into tetramethyl benzidine solution, which is used as a color reagent, to observe the color change. If these three wings become blue within 5 min, it means that the result is positive for celiac disease. Negative result can be confirmed when only the IgA-sensitive part turns blue. If no blue color appears, it indicates that the sample was IgA deficient, and the test is invalid. It was demonstrated that the sensitivity and specificity were 97.0 and 96.9%, respectively, after testing 164 human blood samples of untreated celiac patients [35].

In recent years, another design for POCT device to detect celiac disease has been based on electrochemical biosensor. The desirable features of POCT, such as low cost and ease of operation, match well with the utilization of electrochemical biosensor [36]. It makes biosensors suitable for the commercial applications. Moreover, commercial device to detect blood glucose has been widely used in clinics and households, boosting motivation of researchers to devise electrochemical sensors for the rapid test of celiac disease.

In 2012, Adornetto et al*.* developed a novel fast immunosensor to achieve anti-tTG antibody detection based on magneto-electrochemistry from serum samples [37]. In this system, tTG antigen-coated magnetic beads are used to capture and detect antibodies against tTG from positive serum samples. Alkaline phosphatase-labeled anti-human IgA antibodies are used as a control. Magnetized screen-printed electrodes coupled with a portable instrument serving to read out electrochemical signal, which is produced after the addition of α-naphthyl phosphate that is enzymatically converted into the electrochemically active α-naphthol product. The device was used to analyze 107 blood serum samples (46 positive vs. 61 negative samples), and it was able to identify with a clinical sensitivity of 100% and a specificity of 98.36%, while the cutoff was 1.0 AU/ml. This is comparable to spectrophotometric ELISA kits (98.57%, 100%, and 7.00 AU/ml, respectively).

Interestingly, in 2015, Adornetto et al. have engineered another electrochemical immunoassay system to detect the IgA anti-tTG antibodies [38]. The authors described a similar system, but the biggest change was that this device is used for the detection of celiac disease in saliva sample with high sensitivity. This is the first report that overcomes the problem associated with saliva samples, such as low levels of IgA anti-tTG antibodies and high liquid viscosity. In this device, magnetic beads were covered with the tTG antigen to react with antibodies against IgA antitTG, which would typically be present in saliva samples of positive celiac disease patients. The marker in this case was the conjugate of anti-human IgA and alkaline phosphate enzyme. The electrochemical transducer was created with a strip of eight magnetized screen-printed electrodes. This device showed the clinical sensitivity of 95% and specificity of 96% when analyzed in 66 saliva samples. The results show the suitability for this POCT as noninvasive screening for celiac disease.

In another study, a modular electrochemical peptide-based sensor was developed to detect anti-DGP antibody [39]. In this approach, firstly a short helical support peptide (SP) was immobilized on the surface of a gold electrode, followed

**29**

*Challenges with Point-Of-Care Tests (POCT) for Celiac Disease*

validation for the potential in POCT application.

**4. Outlook for the future POCT development**

by functionalization of SP with DGP and methylene blue (MB), which are used as the antigen and electrochemical tag, respectively. When the added anti-DGP IgG monoclonal antibody was recognized and then bound with the DGP, the transfer electrons efficiency between DGP and gold surface was reduced, leading to a signal decrease in a potentiometer. This unique modular style could guarantee the background of high currents when DGP antibody is absent, even at low surface densities of DGP. This means that this system could achieve a low-limit detection of anti-DGP. Although this system presents a great potential, it was not properly assessed on a real human serum or saliva sample from celiac patients. Nevertheless, it represents an alternative direction for the future development of POCT device. A creative electrochemiluminescence immunosensor for the test of tTG was designed based on the detection platform of a membrane-templated gold nanoelectrode ensemble [40, 41]. In this platform, tTG antigen was first immobilized on the surface of polycarbonate to capture the target anti-tTG antibody present in the sample. Then it could react with the biotinylated secondary antibody, which was labeled with ruthenium-based electrochemiluminescence reagent modified with streptavidin. The application of an oxidizing potential could induce the generation of intense and sharp electrochemiluminescence signal, which was used to analyze different concentrations of anti-tTG. The result showed that its linear range was between 1.5 ng/mL and 10 μg/mL, with a detection limit of 0.5 ng/mL. This system was applied to detect human sera samples from five celiac patients and two healthy controls as a proof of concept for screening test of celiac disease with great outcomes. Nevertheless, this test still requires more human samples and more vigorous

Recently, our group has developed a novel one-step test for screening celiac disease [41]. The test is based on a precipitation principle of gliadin peptide-coated gold nanoparticles. In this test, diluted serum is added to the prepared peptidecoated gold colloids in a small tube. If AGA antibodies are present in serum, it causes agglutination of gold colloids and essentially leads to a colloid precipitation. The test was used on 30 human serum samples (26 positive celiac samples and 4 controls) in a blinded assessment. The test demonstrated an overall sensitivity and specificity of over 85%, indicating that this assay has potential to be adopted as screening tool for celiac disease. Furthermore, this test could be a part of an exclusion-based diagnostic strategy in testing high risk of celiac disease populations.

Over the last decade, several POCTs have been introduced to the market, but these tests have not been widely accepted by clinicians [42]. Moreover, the 2009 National Institute for Clinical Excellence (NICE) guideline CG 86 recommended that self-tests and/or POCT for celiac disease should not be used as a substitute for laboratory-based tests [43]. Therefore, even though the current POCT devices can be used to detect celiac disease with a relatively high sensitivity, their specificity somewhat lags behind lab-based tests. However, one of the biggest drawbacks of the

Most of the lab-based tests for celiac disease are performed by trained clinical technicians, but POCTs are generally aimed to be performed by a non-trained person. Multiple steps and components, such as blood drawn from a finger prick, accurate amount of blood requirement, addition of dilatants or other solutions and visual interpretation, would typically introduce user errors leading to a decrease in accuracy of the tests. The usability of POCTs has been assessed on the example of HIV self-testing kits [44]. In this particular study, authors found that almost 50%

current available POCTs is their lack of usability by a non-trained person.

*DOI: http://dx.doi.org/10.5772/intechopen.81874*

### *Challenges with Point-Of-Care Tests (POCT) for Celiac Disease DOI: http://dx.doi.org/10.5772/intechopen.81874*

*Celiac Disease - From the Bench to the Clinic*

for the rapid test of celiac disease.

accuracy. In 2005, Korponay-Szabó et al. developed a POCT that could rapidly detect the autoantibodies of tTG from blood sample [35]. The test is based on a Nunc-Immunostick (Denmark) principle with a four-wing stick. The two wings of the stick are pre-covered with gelatine to recognize and capture self-tTG/anti-tTG antibody complexes from the hemolyzed patient blood sample. The third wing is fixed with anti-human IgA antibodies, to combine with plasma IgA, which is used as a positive control. The fourth wing has no coating leading to the absence of antibody capture, and it serves as a negative control. In the test process, one drop of blood is inserted into the hemolyzing solution and incubated with the stick for 15 min. Then, the stick is washed with water and immersed for another 15 min in the solution of peroxidase-labeled anti-human IgA. To obtain a visible signal, the stick is washed again and then inserted into tetramethyl benzidine solution, which is used as a color reagent, to observe the color change. If these three wings become blue within 5 min, it means that the result is positive for celiac disease. Negative result can be confirmed when only the IgA-sensitive part turns blue. If no blue color appears, it indicates that the sample was IgA deficient, and the test is invalid. It was demonstrated that the sensitivity and specificity were 97.0 and 96.9%, respectively,

after testing 164 human blood samples of untreated celiac patients [35].

In recent years, another design for POCT device to detect celiac disease has been based on electrochemical biosensor. The desirable features of POCT, such as low cost and ease of operation, match well with the utilization of electrochemical biosensor [36]. It makes biosensors suitable for the commercial applications. Moreover, commercial device to detect blood glucose has been widely used in clinics and households, boosting motivation of researchers to devise electrochemical sensors

In 2012, Adornetto et al*.* developed a novel fast immunosensor to achieve anti-tTG antibody detection based on magneto-electrochemistry from serum samples [37]. In this system, tTG antigen-coated magnetic beads are used to capture and detect antibodies against tTG from positive serum samples. Alkaline phosphatase-labeled anti-human IgA antibodies are used as a control. Magnetized screen-printed electrodes coupled with a portable instrument serving to read out electrochemical signal, which is produced after the addition of α-naphthyl phosphate that is enzymatically converted into the electrochemically active α-naphthol product. The device was used to analyze 107 blood serum samples (46 positive vs. 61 negative samples), and it was able to identify with a clinical sensitivity of 100% and a specificity of 98.36%, while the cutoff was 1.0 AU/ml. This is comparable to spectrophotometric ELISA kits (98.57%, 100%, and 7.00 AU/ml, respectively). Interestingly, in 2015, Adornetto et al. have engineered another electrochemical immunoassay system to detect the IgA anti-tTG antibodies [38]. The authors described a similar system, but the biggest change was that this device is used for the detection of celiac disease in saliva sample with high sensitivity. This is the first report that overcomes the problem associated with saliva samples, such as low levels of IgA anti-tTG antibodies and high liquid viscosity. In this device, magnetic beads were covered with the tTG antigen to react with antibodies against IgA antitTG, which would typically be present in saliva samples of positive celiac disease patients. The marker in this case was the conjugate of anti-human IgA and alkaline phosphate enzyme. The electrochemical transducer was created with a strip of eight magnetized screen-printed electrodes. This device showed the clinical sensitivity of 95% and specificity of 96% when analyzed in 66 saliva samples. The results show

the suitability for this POCT as noninvasive screening for celiac disease.

In another study, a modular electrochemical peptide-based sensor was developed to detect anti-DGP antibody [39]. In this approach, firstly a short helical support peptide (SP) was immobilized on the surface of a gold electrode, followed

**28**

by functionalization of SP with DGP and methylene blue (MB), which are used as the antigen and electrochemical tag, respectively. When the added anti-DGP IgG monoclonal antibody was recognized and then bound with the DGP, the transfer electrons efficiency between DGP and gold surface was reduced, leading to a signal decrease in a potentiometer. This unique modular style could guarantee the background of high currents when DGP antibody is absent, even at low surface densities of DGP. This means that this system could achieve a low-limit detection of anti-DGP. Although this system presents a great potential, it was not properly assessed on a real human serum or saliva sample from celiac patients. Nevertheless, it represents an alternative direction for the future development of POCT device.

A creative electrochemiluminescence immunosensor for the test of tTG was designed based on the detection platform of a membrane-templated gold nanoelectrode ensemble [40, 41]. In this platform, tTG antigen was first immobilized on the surface of polycarbonate to capture the target anti-tTG antibody present in the sample. Then it could react with the biotinylated secondary antibody, which was labeled with ruthenium-based electrochemiluminescence reagent modified with streptavidin. The application of an oxidizing potential could induce the generation of intense and sharp electrochemiluminescence signal, which was used to analyze different concentrations of anti-tTG. The result showed that its linear range was between 1.5 ng/mL and 10 μg/mL, with a detection limit of 0.5 ng/mL. This system was applied to detect human sera samples from five celiac patients and two healthy controls as a proof of concept for screening test of celiac disease with great outcomes. Nevertheless, this test still requires more human samples and more vigorous validation for the potential in POCT application.

Recently, our group has developed a novel one-step test for screening celiac disease [41]. The test is based on a precipitation principle of gliadin peptide-coated gold nanoparticles. In this test, diluted serum is added to the prepared peptidecoated gold colloids in a small tube. If AGA antibodies are present in serum, it causes agglutination of gold colloids and essentially leads to a colloid precipitation. The test was used on 30 human serum samples (26 positive celiac samples and 4 controls) in a blinded assessment. The test demonstrated an overall sensitivity and specificity of over 85%, indicating that this assay has potential to be adopted as screening tool for celiac disease. Furthermore, this test could be a part of an exclusion-based diagnostic strategy in testing high risk of celiac disease populations.
