Protein Microarray Chip Research Articles

Automatic detection of multi-antibiotic residues simultaneously in aquatic products by visual protein microarray chips

A 96-well microplate-based protein microarray chip which can simultaneously detect three different families of antibiotics using a multiplexed approach has been developed for the first time. All steps in the microarray chip can be completed through an automated biochip analyzer, enabling high throughput analysis of 96 samples within 60 min according to the set program. The microarray chip can be visually evaluated by the color depth of array points, and can also be quantified through a scanner. Under the optimal conditions, the limits of detection (LODs) of nitrofuran metabolites (NFMs), chloramphenicol (CAP), and fluoroquinolones (FQs) were 0.11–0.15 μg/kg, 0.07 μg/kg and 6.42 μg/kg, respectively. This microarray chip can be applied to different aquatic products, including crucian carp, shrimp, and scallops, with the recovery ranged from 89.4 % to 111.2 %. These results demonstrate new strategies for the high-throughput detection of multi-antibiotic residues and present potential of protein chip technologies for daily supervision.

Preparation of substrates for microarray protein chips with different ending functional groups

Protein microarray chips are composed of three components, these are pre-treatment substrates, surface chemical modification, and immobilizing protein on substrate surfaces. In this study, self-assembly monolayers are used for surface chemical modification. Using this method, silanization on a glass and silicon chip is achieved, forming the terminal group substrates. Modification of the substrate surface to provide COOH and NH2 terminal functional groups provides a mechanism to proteins to immobilize on the substrate surface. To observe immobilized proteins on the substrate surface, they are first labeled with Cy5 fluorescent dye before analysis using a GenePix 4000B Microarray Scanner. The scanner induces fluorescence in the labelling dye and the resulting light is analyzed to provide information concerning both the quantity of immobilized protein, and the orientation of attachment. The antigen of the HSV-1 virus, a common human virus, was used in this study, performing an antigen-antibody analysis to determine the efficacy of the method under test for clinical diagnosis.

Uptake and tumor-suppressive pathways of exosome-associated GKN1 protein in gastric epithelial cells.

Gastrokine 1 (GKN1) is a stomach-specific tumor suppressor that is secreted into extracellular space as an exosomal cargo protein. The objective of this study was to investigate the uptake and tumor-suppressive pathways of exosome-associated GKN1 protein in gastric epithelial cells. Immunofluorescent and Western blot analysis were used to investigate gastric-specific uptake of HFE-145-derived exosomes. Binding affinity of HFE-145 derived exosomes with integrin proteins was examined using protein microarray chip. Tumor suppressor activities of exosome-carrying GKN1 protein were analyzed using transwell co-culture, MTT assay, BrdU incorporation, immunoprecipitation, and Western blot analysis. HFE-145-derived exosomes were internalized only into HFE-145 gastric epithelial cells and gastric cancer cells. Gastric-specific uptake of stomach-derived exosomes required integrin α6 and αX proteins. Clathrin and macropinocytosis increased the uptake of exosomes into gastric epithelial cells, whereas caveolin inhibited the uptake of exosomes. Transwell co-culture of AGS cells with HFE-145 cells markedly inhibited viability and proliferation of AGS cells. Following uptake of HFE-145-derived exosomes in recipient cells, GKN1 protein bound to HRas and inhibited the binding of HRas to b-Raf and c-Raf which subsequently downregulated HRas/Raf/MEK/ERK signaling pathways in AGS, MKN1 cells, and MKN1-derived xenograft tumor tissues. In addition, exosomal GKN1 protein suppressed both migration and invasion of gastric cancer cells by inhibiting epithelial-mesenchymal transition. Gastric-specific uptake of exosomes derived from gastric epithelial cells requires integrin α6 and αX proteins in both gastric epithelial cells and exosomes. Exosomal GKN1 protein inhibits gastric carcinogenesis by downregulating HRas/Raf/MEK/ERK signaling pathways.

Chip architecture-enabled sensitivity enhancement of oblique-incidence reflectivity difference for label-free protein microarray detection

Oblique-incidence reflectivity difference (OIRD) is a promising optical technique for detection of protein microarray chip with the compelling features of being label-free, real-time, high-throughput and nondestructive, but its sensitivity requires essential improvement for practical applications. In this work we report a simple yet highly efficient strategy to boost the sensitivity of OIRD by rationally engineering the biochip architecture via introducing a polymer film (polydopamine, PDA) to the glass surface. With anti-Zearalenone (ZEN) monoclonal antibody as a model target, up to 2 orders of magnitude sensitivity enhancement was demonstrated on this engineered chip compared to conventional silanized glass chip. Experimental investigation indicates that the OIRD sensitivity is positively correlated to the polymer thickness until reaching the maximal sensitivity at 90.2 nm thickness; a detection limit of as low as 5.0 ng mL−1 is achieved on this optimal chip. Theoretical calculation unveils that the polymer film serves as an interference layer to enhance the light-matter interaction for improved sensitivity, and the OIRD sensitivity possesses a periodic dependence on the film thickness. This work reports for the first time an interference-based versatile strategy to boost OIRD sensitivity and reveals the underlying mechanism, and thus paves a new way toward sensitive and label-free OIRD detection of protein microarrays.

Development of a Protein Microarray Chip with Enhanced Fluorescence for Identification of Semen and Vaginal Fluid.

The detection of body fluids has been used to identify a suspect and build a criminal case. As the amount of evidence collected at a crime site is limited, a multiplex identification system for body fluids using a small amount of sample is required. In this study, we proposed a multiplex detection platform using an Ag vertical nanorod metal enhanced fluorescence (MEF) substrate for semen and vaginal fluid (VF), which are important evidence in cases of sexual crime. The Ag nanorod MEF substrate with a length of 500 nm was fabricated by glancing angle deposition, and amino functionalization was conducted to improve binding ability. The effect of incubation time was analyzed, and an incubation time of 60 min was selected, at which the fluorescence signal was saturated. To assess the performance of the developed identification chip, the identification of semen and VF was carried out. The developed sensor could selectively identify semen and VF without any cross-reactivity. The limit of detection of the fabricated microarray chip was 10 times better than the commercially available rapid stain identification (RSID) Semen kit.

Antigen Analysis of Pre-Eclamptic Plasma Antibodies Using Escherichia Coli Proteome Chips

Pre-eclampsia is one of the main causes of perinatal mortality and morbidity. Many biomarkers for diagnosing pre-eclampsia have been found but most have low accuracy. Therefore, a potential marker that can detect pre-eclampsia with high accuracy is required. Infection has been reported as a cause of pre-eclampsia. In recent years, protein microarray chips have been recognized as a strong and robust tool for profiling antibodies for infection diagnoses. The purpose of the present study was to profile antibodies in the human plasma of healthy and pre-eclamptic pregnancies to identify suitable biomarkers. In this study, an Escherichia coli chip was probed with samples from 29 individuals (16 pre-eclamptic women and 13 healthy pregnant women) to profile plasma antibodies. Bioinformatics tools were used to analyze the results, discover conserved motifs, compare against the entire human proteome, and perform protein functional analysis. An antibody classifier was identified using k-top scoring pairs and additional samples for a blinded test were collected. The findings indicated that compared with the healthy women, the pre-eclamptic women exhibited 108 and 130 differentially immunogenic proteins against human immunoglobulins G and M, respectively. In addition, pre-eclamptic women developed more immunoglobulin G but less immunoglobulin M against bacterial surface proteins compared with healthy women. The k-top scoring pairs identified five pairs of immunogenic proteins as classifiers with a high accuracy of 90% in the blind test. [AG] [ISV] GV [AE] L [LF] and [IV] [IV] RI [AG] [AD] E were the consensus motifs observed in immunogenic proteins in the immunoglobulin G and immunoglobulin M of pre-eclamptic women, respectively, whereas GA [AG] [AL] L [LF] and [SRY] [IQML] [ILV] [ILV] [ACG] GI [GH] [AEF] [AK] [ATY] [RG] N [IV] were observed in the immunoglobulins G and immunoglobulin M of healthy women, respectively.

Analysis of PM2.5-induced cytotoxicity in human HaCaT cells based on a microfluidic system

Human exposure to PM2.5 causes several adverse health effects. Skin is the first barrier against harmful environmental substances and can directly contact with PM2.5, but there is no study about PM2.5-induced cytotoxicity in human skin cells on the molecular level partially due to the shortcomings of traditional research methods. In present study, we established a microfluidic system including a cell culture chip integrated with a high-throughput protein microarray chip to investigate the mechanism of PM2.5-mediated cytotoxicity in human HaCaT cells. We found that PM2.5 was lodged inside the cytoplasm, mitochondria and nucleus of HaCaT cells by TEM. Flow cytometry analysis indicated that the cell apoptosis rate increased from 0.49% to 53.4%. The results of protein microarray showed that NF-κB and NALP3 signal transductions were activated in HaCaT cells after PM2.5 stimulations, up-regulating the expression of IL-1β and IL-6, which resulted in inflammatory response in HaCaT cells. Our findings provide a molecular insight into PM2.5-induced skin injury.

Distinct antibody responses of patients with mild and severe leptospirosis determined by whole proteome microarray analysis.

BackgroundLeptospirosis is an important zoonotic disease worldwide. Humans usually present a mild non-specific febrile illness, but a proportion of them develop more severe outcomes, such as multi-organ failure, lung hemorrhage and death. Such complications are thought to depend on several factors, including the host immunity. Protective immunity is associated with humoral immune response, but little is known about the immune response mounted during naturally-acquired Leptospira infection.Methods and principal findingsHere, we used protein microarray chip to profile the antibody responses of patients with severe and mild leptospirosis against the complete Leptospira interrogans serovar Copenhageni predicted ORFeome. We discovered a limited number of immunodominant antigens, with 36 antigens specific to patients, of which 11 were potential serodiagnostic antigens, identified at acute phase, and 33 were potential subunit vaccine targets, detected after recovery. Moreover, we found distinct antibody profiles in patients with different clinical outcomes: in the severe group, overall IgM responses do not change and IgG responses increase over time, while both IgM and IgG responses remain stable in the mild patient group. Analyses of individual patients’ responses showed that >74% of patients in the severe group had significant IgG increases over time compared to 29% of patients in the mild group. Additionally, 90% of IgM responses did not change over time in the mild group, compared to ~51% in the severe group.ConclusionsIn the present study, we detected antibody profiles associated with disease severity and speculate that patients with mild disease were protected from severe outcomes due to pre-existing antibodies, while patients with severe leptospirosis demonstrated an antibody profile typical of first exposure. Our findings represent a significant advance in the understanding of the humoral immune response to Leptospira infection, and we have identified new targets for the development of subunit vaccines and diagnostic tests.

Development of a highly specific HER2 monoclonal antibody for immunohistochemistry using protein microarray chips

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.

Abstract 1706: Evaluation of LGR5 protein expression in colon cancer stem cells

Abstract Cancer stem cells are a subpopulation of cancer cells responsible for cancer initiation, development and metastasis. A number of studies demonstrated that Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5) can drive cancer development through triggering canonical Wnt signaling and its downstream gene expression. LGR5 is an important biomarker specifically expressed on colon cancer stem cells. In this study, we have successfully developed an LGR5 antibody with high specificity to detect endogenous LGR5 expression in different immunoassay application. By screening multiple anti-LGR5 hybridoma clones, antibody generated by clone UMAB212 was proven to be highly specific on our high density protein microarray chip assay. Here our experimental data demonstrated that clone UMAB212 recognizes not only human LGR5 protein but also mouse LGR5 protein in both western blot and flow cytometry applications. No cross-reactivity was observed with the other two LGR family members, LGR4 and LGR6. Furthermore, this antibody also works great on immunohistochemistry application on FFPE tissue blocks. In summary, UMAB212 is great tool for us to study LGR5 protein in different immunoassay setting and it could also be a potential cancer diagnostic reagent. Citation Format: Wei Fu, Rachel Gonzalez, Kehu Yuan, Caiwei Chen, Haitao Wei, Hsiangmin Lu, Qi Ren, Evelin Logis, Sean Kelly, Wei-Wu He, Donghui Ma. Evaluation of LGR5 protein expression in colon cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1706.

Abstract 3393: Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology

Abstract Programmed death-1 (PD-1) expresses in many tumors in response to inflammation. It is up-regulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands PD-L1 and PDL2 and serves as a key checkpoint of the immune system. Pembrolizumab is the first anti-PD-1 therapy approved in the United States and received FDA's Breakthrough Therapy designation for advanced melanoma. Thus, to evaluate PD1 protein levels in formalin-fixed paraffin-embedded tissue samples, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is needed. To develop the best monoclonal antibody for PD-1 IHC analysis, 41 monoclonal antibodies were generated. 8 of them work for IHC application on FFPE tissue sections. To identify clones that are mono-specific on target, only two clones passed our high density protein microarray chip tests. Other immunoassays and species cross-reactivity tests were also explored. In summary, two newly generated monoclonal antibodies demonstrated ultra-specificity against PD-1 protein and superior performance for IHC analyses. These two clones could be utilized as companion diagnostic reagents to stratify cancer patients before pembrolizumab prescription. Citation Format: Caiwei Chen, Yanlin Tang, Haitao Wei, Kehu Yuan, Guiyin Wu, Jian Chen, Boyang Chu, Guangli Wang, Youmin Shu, Wei-Wu He, Donghui Ma. Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2015-3393

Abstract 561: Development of a highly specific Met antibody for screening Met overexpression in NSCLC

Abstract Approximately 5%-22% of non-small cell lung cancer (NSCLC) patients with secondary resistance to EGFR TKIs had evidence of amplification of the Met oncogene. This dysregulation of Met signaling was implicated in the proliferation and eventual metastasis of NSCLC, and therefore represents an attractive target for anticancer drug development. Currently, there are a number of clinical trials for Met targeted small molecule and monoclonal antibody therapies. Immunohistochemistry (IHC) analysis of the Met protein will be very important for these Met targeted therapies and requires a highly specific antibody to prevent false positives. To develop a highly specific monoclonal antibody against Met for IHC analysis in NSCLC, we generated 97 monoclonal antibodies. IHC evaluation showed that 8 of the antibodies had suitable performance in Met positive NSCLC patient tissues. The 8 Met monoclonal antibodies were further tested on a high density protein microarray chip containing 17,000 human proteins. The microarray analysis showed that clone 3G5 was highly specific as evidenced with it only reacting with Met on the microarray. This new 3G5 Met monoclonal antibody will be a promising candidate as a diagnostic reagent for detecting Met protein in NSCLC with minimal false positives. Citation Format: Caiwei Chen, Hsiangmin Emily Lu, Haitao Wei, Kehu Yuan, Donghui Ma, Wei-Wu He. Development of a highly specific Met antibody for screening Met overexpression in NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 561. doi:10.1158/1538-7445.AM2015-561

Using Protein Microarray Technology to Screen Anti‐PD‐1 Monoclonal Antibodies for Specificity and Applications in Anatomic Pathology

Programmed death‐1 (PD‐1) expresses in many tumors in response to inflammation. It is up‐regulated in activated T lymphocytes and inhibits T‐cell function upon binding to its ligands PD‐L1 and PDL2 and serves as a key checkpoint of the immune system. KEYTRUDA (pembrolizumab) is the first anti‐PD‐1 therapy approved in the United States and received FDA's Breakthrough Therapy designation for advanced melanoma. Thus, to evaluate PD1 protein levels in formalin‐fixed paraffin‐embedded tissue samples, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is needed.To develop the best monoclonal antibody for PD‐1 IHC analysis, 41 monoclonal antibodies were generated. 8 of them work for IHC application on FFPE tissue sections. To identify clones that are mono‐specific on target, only two clones passed our high density protein microarray chip tests. Other immunoassays and species cross‐reactivity tests were also explored.In summary, two newly generated monoclonal antibodies demonstrated ultra‐specificity against PD‐1 protein and superior performance for IHC analyses. These two clones could be utilized as companion diagnostic reagents to stratify cancer patients before KEYTRUDA prescription.This research is partly supported by Chinese 863 research grant number 2014AA020909.

Development and Evaluation of Highly Specific Monoclonal Antibodies against Human LGR5 for the Identification of Colon Cancer Stem Cells

The cancer stem cells is a subpopulation of cancer cells which is responsible for cancer initiation, development and metastasis. Detection and identification of cancer stem cells have been considered as one of the most important objectives for clinical diagnostic and therapeutic purposes. Research evidences indicated that Leucine‐rich repeat containing G‐protein‐coupled receptor 5 (LGR5) is one of the biomarkers specifically expressed on colon cancer stem cells, which is activated by extracellular Wnt molecules to trigger canonical Wnt signaling and gene expression related to cancer development. Therefore, it is critical to develop an LGR5 antibody with great sensitivity and specificity to detect endogenous LGR5 expression in cancer stem cells.In this research, several LGR5 mouse monoclonal antibodies were generated for multiple applications. These LGR5 antibodies are also highly sensitive for flow application on LGR5 stable expression cells and proved to be highly specific with our proprietary high density protein microarray chip assay. Furthermore, we also performed immunohistochemistry assays on colon cancer tissue blocks for diagnostic application. These newly generated LGR5 antibodies could provide new tools for earlier diagnosis and therapeutic guidance on colon cancers.

Evaluating anti‐β‐Catenin monoclonal antibodies for cancer diagnosis and prognosis

Colorectal cancer is one of the most common cancers and ranks second in mortality in the US. Wnt/β‐Catenin signaling pathway plays an important role in colorectal cancer, and the mutation events have been studied the most for colorectal carcinogenesis. However, other studies have shown the nuclear translocation of β‐Catenin could be an additional factor for clinical diagnosis of colorectal cancer. Therefore, it is essential to develop the monoclonal antibody with the highest sensitivity and specificity. Even though there are many monoclonal antibodies being used for β‐Catenin studies, they have not been carefully examined for potential cross‐reactivity which could cause false diagnosis.To develop the most suitable monoclonal antibody for β‐Catenin studies, we used different immunoassays to validate the specificity and sensitivity of the commercially available antibodies. By the high density protein microarray chip technology and other assays, we identified that β‐Catenin UltraMAB antibody can be used for different applications in basic research. Furthermore, we used immunohistochemistry with tissues from colorectal cancer patients and showed that β‐CateninUltraMAB antibody is a great tool for potential cancer diagnosis and prognosis.

Development of a Highly Specific Met Antibody for Screening Met Overexpression in NSCLC

Approximately 5%‐22% of non‐small cell lung cancer (NSCLC) patients with secondary resistance to EGFR TKIs had evidence of amplification of the Met oncogene. This dysregulation of Met signaling was implicated in the proliferation and eventual metastasis of NSCLC, and therefore represents an attractive target for anticancer drug development.Currently, there are a number of clinical trials for Met targeted small molecule and monoclonal antibody therapies. Immunohistochemistry (IHC) analysis of the Met protein will be very important for these Met targeted therapies and requires a highly specific antibody to prevent false positives. To develop a highly specific monoclonal antibody against Met for IHC analysis in NSCLC, we generated 97 monoclonal antibodies. IHC evaluation showed that 8 of the antibodies had suitable performance in Met positive NSCLC patient tissues. The 8 Met monoclonal antibodies were further tested on a high density protein microarray chip containing 17,000 human proteins. The microarray analysis showed that clone 3G5 was highly specific as evidenced with it only reacting with Met on the microarray. This new 3G5 Met monoclonal antibody will be a promising candidate as a diagnostic reagent for detecting Met protein in NSCLC with minimal false positives.

Using Protein Microarray Technology to Screen Anti‐E‐Cadherin (ECAD) Monoclonal Antibodies for Specificity and Applications in Pathology

E‐cadherin (ECAD) is a calcium‐dependent cell‐cell adhesion molecule playing a crucial role in establishing epithelial architecture and maintaining cell polarity and differentiation. Reports showed that decreased ECAD expression has been associated with more advanced tumor stage and grade for lung tumors, gastric tumors, breast tumors and etc. Other research discovered that staining for ECAD may be useful for the differentiation between ductal and lobular breast carcinomas. Thus, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is required to evaluate ECAD protein levels in formalin‐fixed paraffin‐embedded tissue samples.To develop the a suitable monoclonal antibody for ECAD IHC analysis, 49 monoclonal antibodies were generated, of which 10 were selected for screening in IHC on breast and prostate cancer tissues. Further evaluation with a high density protein microarray chip containing 17,000 human proteins indicated that clone 3C11 is highly specific and only detected ECAD. Comparison to the monoclonal 4A2C7, showed that 3C11 is more sensitive on breast, prostate, and prostate cancer tissues. The new monoclonal antibody 3C11 will be a promising candidate to detect the expression and aberrant alterations of ECAD for the diagnosis, prognosis, and therapy of cancer.

Abstract 869: Evaluate breast cancer diagnostic antibody specificity by using high density protein microarray technology

Abstract Detection sensitivity and target specificity are the two key features for a good diagnostic monoclonal antibody. There are a number of well-established technology platforms to quantitatively measure antibody affinity. However, there is no complete technology solution to evaluate antibody target specificity. Antibody cross-reactivity could lead to false diagnostic reports. Currently, high density protein microarray technology is the best technology platform available for antibody specificity evaluation. Here we will showcase how we utilized protein microarray technology to evaluate target specificity for a couple of commonly used breast cancer IHC diagnostic antibodies. Our data suggested that cross-reactivity is one of the reasons to create off-target staining for breast cancer diagnostics. We will also show how we can utilize OriGene protein microarray chip technology to develop UltraMABs, the Ultra-specific monoclonal antibodies for anatomic pathology application. Citation Format: Donghui Ma, Jian Chen, Wei Fu, Julie McDowell, Kehu Yuan. Evaluate breast cancer diagnostic antibody specificity by using high density protein microarray technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 869. doi:10.1158/1538-7445.AM2014-869

Influence of material properties upon immobilization of histidine-tagged protein on Ni–Co coated chip

In protein research, protein microarray facilitates high-throughput study of protein abundance and function. An appropriate microarray surface that can be used to immobilize protein samples is a prerequisite for the investigation of molecular interactions. Ni–Co alloy coated protein microarray chip has been found to adsorb histidine-tagged proteins effectively based on the method of immobilized metal affinity chromatography. Due to the ingredient of bi-metallic elements, different electroplating conditions resulted in distinct binding affinities. Therefore, the influence of Ni–Co material properties on the immobilization of histidine-tagged protein was systematically investigated in this study. In the experiments, the contact angle measurement suggested that no strong relationship can be established between the wettability of chip surface and its corresponding protein immobilization. ESCA test demonstrated that the major ingredients of the Ni–Co alloy coated protein microarray chip were Ni and Co. In addition, the XRD test concluded that a Ni–Co protein chip that consists mostly of hcp lattice has better binding capability. SEM micrographs provide direct image evidence. These material tests summarize that the Ni–Co alloy coated protein microarray chip adsorbs His-tagged proteins through its surface morphology. Therefore, it can provide specific binding due to the affinity adsorption between the intermediate metals and the protein.

Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach

BackgroundLeptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease.MethodologyIn order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results.Principal findingsWe found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients.ConclusionsOur study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the utility of these antigens in more deployable diagnostic platforms.