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Multi-analyte validation in heterogeneous solution by ELISA

Lakshmipriya T, Gopinath SCB, Hashim U, Murugaiyah V

Int J Biol Macromol, 2017 Dec;105(Pt 1):796-800.
PMID: 28732727 DOI: 10.1016/j.ijbiomac.2017.07.115

Enzyme Linked Immunosorbent Assay (ELISA) is a standard assay that has been used widely to validate the presence of analyte in the solution. With the advancement of ELISA, different strategies have shown and became a suitable immunoassay for a wide range of analytes. Herein, we attempted to provide additional evidence with ELISA, to show its suitability for multi-analyte detection. To demonstrate, three clinically relevant targets have been chosen, which include 16kDa protein from Mycobacterium tuberculosis, human blood clotting Factor IXa and a tumour marker Squamous Cell Carcinoma antigen. Indeed, we adapted the routine steps from the conventional ELISA to validate the occurrence of analytes both in homogeneous and heterogeneous solutions. With the homogeneous and heterogeneous solutions, we could attain the sensitivity of 2, 8 and 1nM for the targets 16kDa protein, FIXa and SSC antigen, respectively. Further, the specific multi-analyte validations were evidenced with the similar sensitivities in the presence of human serum. ELISA assay in this study has proven its applicability for the genuine multiple target validation in the heterogeneous solution, can be followed for other target validations.

Matched MeSH terms: Serpins/analysis
Fulltext Dirofilaria immitis possesses molecules with anticoagulant properties in its excretory/secretory antigens

Diosdado A, Simón F, Morchón R, González-Miguel J

Parasitology, 2020 Apr;147(5):559-565.
PMID: 31992384 DOI: 10.1017/S0031182020000104

Dirofilaria immitis is a parasitic nematode that survives in the circulatory system of suitable hosts for many years, causing the most severe thromboembolisms when simultaneous death of adult worms occurs. The two main mechanisms responsible for thrombus formation in mammals are the activation and aggregation of platelets and the generation of fibrin through the coagulation cascade. The aim of this work was to study the anticoagulant potential of excretory/secretory antigens from D. immitis adult worms (DiES) on the coagulation cascade of the host. Anticoagulant and inhibition assays respectively showed that DiES partially alter the coagulation cascade of the host and reduce the activity of the coagulation factor Xa, a key enzyme in the coagulation process. In addition, a D. immitis protein was identified by its similarity to the homologous serpin 6 from Brugia malayi as a possible candidate to form an inhibitory complex with FXa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. These results indicate that D. immitis could use the anticoagulant properties of its excretory/secretory antigens to control the formation of blood clots in its immediate intravascular habitat as a survival mechanism.

Matched MeSH terms: Serpins/metabolism; Serpins/chemistry
Divalent ion-induced aggregation of gold nanoparticles for voltammetry Immunosensing: comparison of transducer signals in an assay for the squamous cell carcinoma antigen

Letchumanan I, Gopinath SCB, Arshad MKM

Mikrochim Acta, 2020 01 14;187(2):128.
PMID: 31938893 DOI: 10.1007/s00604-020-4115-0

A method is described for the electrochemical determination of squamous cell carcinoma (SCC) antigen, and by testing the effect of 30 nm gold nanoparticles (GNPs). Three comparative studies were performed in the presence and absence of GNPs, and with agglomerated GNPs. The divalent ion Ca(II) was used to induce a strong agglomeration of GNPs, as confirmed by colorimetry and voltammetry. Herein, colorimetry was used to test the best amount of salt needed to aggregate the GNPs. Despite, voltammetry was used to determine the status of biomolecules on the sensor. The topography of the surface of ZnO-coated interdigitated electrodes was analyzed by using 3D-nano profilometry, scanning electron microscopy, atomic force microscopy and high-power microscopy. The interaction between SCC antigen and antibody trigger vibrations on the sensor and cause dipole moment, which was measured using a picoammeter with a linear sweep from 0 to 2 V at 0.01 V step voltage. The sensitivity level was 10 fM by 3σ calculation for the dispersed GNP-conjugated antigen. This indicates a 100-fold enhancement compared to the condition without GNP conjugation. However, the sensitivity level for agglomerated GNPs conjugated antibody was not significant with 100 fM sensitivity. Specificity was tested for other proteins in serum, namely blood clotting factor IX, C-reactive protein, and serum albumin. The SCC antigen was quantified in spiked serum and gave recoveries that ranged between 80 and 90%. Graphical abstractSchematic representation of SCC (squamous cell carcinoma) antigen determination using divalent ion induced agglomerated GNPs. Sensitivity increment depends on the occurrence of more SCC antigen and antibody binding event via GNPs integration. Notably, lower detection limit was achieved at femto molar with proper orientation of biological molecules.

Matched MeSH terms: Serpins/analysis*; Serpins/immunology
Fulltext Perceived social support, stress and gender differences among university students: a cross sectional study

Talwar, P., Kumaraswamy, N., Mohd Fadzil, A.R.

Malaysian Journal of Psychiatry, 2013;22(2):42-49.
MyJurnal

Background: The transition from high school to a university is a significant milestone in every student’s life. Stress experienced by students can have a detrimental effect on their academic performance. Social support has generally been found to promote psychological well-being, as well as to buffer the effects of stress. Method: The study was conducted in one of the Universities in Malaysia. The study questionnaire was designed to include questions regarding socio-demographic aspects and stress of the students; in addition, the Multidimensional Scale of Perceived Social Support scale (MSPSS) that was developed by Zimet was administered. The main aim of this study is to investigate the relationship between Perceived Social Support, Stress and Gender differences among University Students. Results: In this study only a small group of the students scored very low on the Perceived social support scale. A low significant inverse correlation was found between MSPSS and stress. Male students’ experienced greater stress than female students’. Female students had a better perception of social support from their families compared to male students. Conclusion: The fact that, as the level of social support increase the level of stress decrease clearly shows that social support acts as a buffer to stress.

Matched MeSH terms: Serpins
Paediatric use of IV magnesium sulphate in severe asthma exacerbation: Report of a case and review of literature

Davendralingam Sinniah

International e-Journal of Science, Medicine & Education, 2014;8(3):45-48.
MyJurnal

Nebulization with B-agonist and administration of systemic corticosteroids are standard treatments for severe asthma exacerbations, but corticosteroids take several hours to become effective. IV magnesium sulphate (MgSO4) acts faster and has both antiinflammatory and bronchodilating properties. It appears to have played a pivotal role in the successful management of a child with severe asthma exacerbation and atelectasis unresponsive to conventional therapy. A literature review reveals that the results of IV MgSO4 are much greater in children than in adults, and can avoid the need to hospitalize 25% of children presenting with severe asthma. Magnesium sulphate appears safe to use.

Matched MeSH terms: Serpins
Fulltext Squamous Cell Carcinoma Biomarker Sensing on a Strontium Oxide-Modified Interdigitated Electrode Surface for the Diagnosis of Cervical Cancer

Wang H, Lakshmipriya T, Chen Y, Gopinath SCB

Biomed Res Int, 2019;2019:2807123.
PMID: 31080815 DOI: 10.1155/2019/2807123

Cervical cancer is a life-threatening complication, appearing as the uncontrolled growth of abnormal cells in the lining of the cervix. Every year, increasing numbers of cervical cancer cases are reported worldwide. Different identification strategies were proposed to detect cervical cancer at the earlier stages using various biomarkers. Squamous cell carcinoma antigen (SCC-Ag) is one of the potential biomarkers for this diagnosis. Nanomaterial-based detection systems were shown to be efficient with different clinical biomarkers. In this study, we have demonstrated strontium oxide-modified interdigitated electrode (IDE) fabrication by the sol-gel method and characterized by scanning electron microscopy and high-power microscopy. Analysis of the bare devices indicated the reproducibility with the fabrication, and further pH scouting on the device revealed that the reliability of the working pH ranges from 3 to 9. The sensing surface was tested to detect SCC-Ag against its specific antibody; the detection limit was found to be 10 pM, and the sensitivity was in the range between 1 and 10 pM as calculated by 3σ. The specificity experiment was carried out using major proteins from human serum, such as albumin and globulin. SCC-Ag was shown to be selectively detected on the strontium oxide-modified IDE surface.

Matched MeSH terms: Serpins/administration & dosage*; Serpins/metabolism
Diagnosing ovarian cancer by identifying SCC-antigen on a multiwalled carbon nanotube-modified dielectrode sensor

Liang T, Qu Q, Chang Y, Gopinath SCB, Liu XT

Biotechnol Appl Biochem, 2019 Nov;66(6):939-944.
PMID: 31468573 DOI: 10.1002/bab.1808

Ovarian cancer starts in the ovaries in its earlier stages and then spreads to the pelvis, uterus, and abdominal region. The success of an ovarian cancer treatment depends on the stage of the cancer and the diagnostic system. Squamous cell carcinoma antigen (SCC-Ag) is one of the most efficient cancer biomarkers, and elevated levels of SCC-Ag in ovarian cancer cells have been used to identify ovarian cancer. Carbon is a potential material for biosensing applications due to its thermal, electrical, and physical properties. Multiwalled carbon nanotubes (MWCNTs) are carbon-based materials that can be used here to detect SCC-Ag. Anti-SCC-Ag antibody was immobilized on the amine-modified MWCNT dielectric sensing surface to detect SCC-Ag. The uniformity of the surface structure was measured with a 3D nanoprofiler, and the results confirmed the detection of SCC-Ag at ∼80 pM. The specific detection of SCC-Ag was confirmed with two control proteins (factor IX and human serum albumin), and the system did not show biofouling. This experimental set-up with MWCNTs a dielectric sensing surface can lead to the detection of ovarian cancer in its initial stages.

Matched MeSH terms: Serpins
Serine protease inhibitor, serpin, is a biomarker following Schistosoma mansoni re-infection in mice

Mohammed ES, El-Dakhly KM

Trop Biomed, 2021 Jun 01;38(2):63-67.
PMID: 33973574 DOI: 10.47665/tb.38.2.039

Schistosomiasis is a chronic parasitic disease affecting mostly low income and resourcelimited countries. Despite the distribution of the curative medicine, praziquantel (PZQ), the frequency of re-infection is commonly reported, thus, making a difficulty to discriminate treatment failure after re-infection. Therefore, assessing Schistosoma mansoni re-infection after praziquantel administration is crucial to prove the treatment efficacy and to break the transmission of infection in endemic areas. The evolution of highly sensitive and specific diagnostic markers, reliable to detect the re-infection and to evaluate the treatment efficacy, is required to control schistosomiasis. In this study, the potential role of serpin recombinant antigen of S. mansoni as a biomarker of re-infection and chemotherapeutic efficacy has been assessed. Therefore, 20 mice were experimentally challenged and re-challenged with 50 S. mansoni cercariae and divided into 4 equal groups; the first included infected mice (control positive), the second group was twice infected with S. mansoni and left untreated, the third included mice twice infected then treated with praziquantel following the last challenge, and the forth one remained uninfected and untreated (control negative). The current findings demonstrated that high levels of IgG and IgG1 bound to serpin were detected following the re-infection and rapidly declined post treatment. In summary, S. mansoni recombinant serpin could be used as a promising marker to discriminate S. mansoni re-infection and evaluated the efficacy of treatment. The translation of such a potential tool in endemic areas will provide a significant support for the elimination and control programs against schistosomiasis.

Matched MeSH terms: Serpins
Fulltext Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

Phang WM, Tan AA, Gopinath SC, Hashim OH, Kiew LV, Chen Y

Int J Med Sci, 2016;13(5):330-9.
PMID: 27226773 DOI: 10.7150/ijms.14341

Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer.

Matched MeSH terms: Serpins/metabolism
Squamous cell carcinoma antigen as an adjunct tumour marker in primary carcinoma of the lung

Cheah PL, Liam CK, Yap SF, Looi LM

J Clin Pathol, 1994 Jun;47(6):535-7.
PMID: 8063936

AIMS: To determine (1) the detection rate of primary carcinoma of the lung by serological assay of CEA (carcinoembryonic antigen); and (2) whether addition of seroassay of squamous cell carcinoma related antigen before treatment improves detection sensitivity.
METHODS: A prospective study spanning 27 months was conducted at the University Hospital, Kuala Lumpur. Serum CEA (Abbott IMx) and serum squamous cell carcinoma antigen (Abbott IMx) from patients clinically suspected of having primary carcinoma of the lung, were assayed using the microparticle enzyme immunoassay method.
RESULTS: Thirty seven cases of histologically confirmed primary lung carcinoma were studied. Of these, 17 were squamous cell carcinomas, 10 adenocarcinomas, nine small cell carcinomas, and one large cell carcinoma. The patients' ages ranged from 34-82 years. The male:female ratio was 3.6:1. Squamous cell carcinoma antigen was raised above the cutoff value of 1.5 ng/ml in 94.1% of squamous cell carcinomas, 20.0% of adenocarcinomas, and 11.1% of small cell carcinomas. By comparison, CEA was raised above the cutoff value of 3.0 ng/ml in 70.6% of squamous cell carcinomas, 77.8% of small cell carcinomas, and 100% of adenocarcinomas. CEA and squamous cell carcinoma antigen were not raised in the patient with large cell carcinoma and in 14 healthy volunteers. None of 15 patients with a variety of benign lung diseases showed a rise of CEA, while two patients--a 25 year old Indian woman with pneumonia and a 64 year old Malay man with bronchial asthma--had raised squamous cell carcinoma antigen values above the cutoff. Serum CEA and squamous cell carcinoma antigen values did not seem to correlate with stage or degree of differentiation of the tumours.
CONCLUSIONS: The findings suggest that CEA is a good general marker for carcinoma, particularly adenocarcinoma. In contrast, squamous cell carcinoma antigen is more specific for squamous carcinoma.

Matched MeSH terms: Serpins*
Fulltext Inherited Anti-Thrombin Deficiency in A Malay-Malaysian Family: A Missense Mutation at Nucleotide g.13267C>A aka anti-thrombin Budapest 5 (p.Pro439Thr) of the SERPINC 1 gene

Norlelawati AT, Rusmawati I, Naznin M, Nur Nadia O, Rizqan Aizzani R, Noraziana AW

Med J Malaysia, 2014 Feb;69(1):27-30.
PMID: 24814625 MyJurnal

OBJECTIVE: Inherited anti-thrombin deficiency is an autosomal dominant disorder which is associated with increased risk for venous thromboembolism (VTE). This condition is very rare in Malaysia and there has been no documented report. Thus, the aim of the present study is to investigate the type of an inherited anti-thrombin deficiency mutation in a 25-year-old Malay woman who presented with deep vein thrombosis in her first pregnancy.

METHODS: DNA was extracted from the patient's blood sample and buccal mucosal swabs from family members. Polymerase chain reaction(PCR) assays were designed to cover all seven exons of the serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) gene; and the products were subjected to DNA sequencing. Sequences were referred to NCBI Reference Sequence: NG_012462.1.

RESULTS: A heterozygous substitution mutation at nucleotide position 13267 (CCT->ACT) was identified in the patient and two other family members, giving a possible change of codon 439 (Pro→Thr) also known as anti-thrombin Budapest 5. The genotype was absent in 90 healthy controls.

CONCLUSION: The study revealed a heterozygous antithrombin Budapest 5 mutation in SERPINC 1 giving rise to a possible anti-thrombin deficiency in a Malay-Malaysian family.

Matched MeSH terms: Serpins
Fulltext Key drug-targeting genes in pancreatic ductal adenocarcinoma

Sakharkar MK, Dhillon SK, Mazumder M, Yang J

Genes Cancer, 2021;12:12-24.
PMID: 33884102 DOI: 10.18632/genesandcancer.210

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal type of cancer. In this study, we undertook a pairwise comparison of gene expression pattern between tumor tissue and its matching adjacent normal tissue for 45 PDAC patients and identified 22 upregulated and 32 downregulated genes. PPI network revealed that fibronectin 1 and serpin peptidase inhibitor B5 were the most interconnected upregulated-nodes. Virtual screening identified bleomycin exhibited reasonably strong binding to both proteins. Effect of bleomycin on cell viability was examined against two PDAC cell lines, AsPC-1 and MIA PaCa-2. AsPC-1 did not respond to bleomycin, however, MIA PaCa-2 responded to bleomycin with an IC50 of 2.6 μM. This implicates that bleomycin could be repurposed for the treatment of PDAC, especially in combination with other chemotherapy agents. In vivo mouse xenograft studies and patient clinical trials are warranted to understand the functional mechanism of bleomycin towards PDAC and optimize its therapeutic efficacy. Furthermore, we will evaluate the antitumor activity of the other identified drugs in our future studies.

Matched MeSH terms: Serpins

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