Research Articles

Purification of Recombinant Human Thyroid Peroxidase (hTPO) from AD293 Mammalian Cells

Parvinder Kaur, Harshada Patil, Paresh Bhanushali, Shamkant Badgujar and Anuj Gupta

Human thyroid peroxidase (hTPO) has been secretory expressed in AD293 mammalian cells. cDNA sequence of ‘Gluc’ (Gaussia luciferase) protein from Gaussia princeps was incorporated at the amino terminal of hTPO gene for secretion of targeted protein outside the mammalian cells. Augmentation of TPO clone in serum free mediums was investigated and a simplified purification procedure of hTPO has been reported here. Purified hTPO was further analyzed by SDS-PAGE and immunoblotting (western blotting). The relative molecular mass of hTPO was found to be 105kDa. This is the first report with respect to cost effective and simplified purification approach to get highest yield and purity of recombinant hTPO.

Simplified Approach for In Vitro Production and Purification of Cell Derived Cancer Antigen 15-3

Shoaib Haidar, Paresh Bhanushali, Kunal Shukla, Deepak Modi, Chander Puri, Shamkant Badgujar and Manoj Chugh

Cancer antigen 15-3 (CA15-3) is a key biomarker, currently used for understanding the onset and prognosis of breast cancer. In present investigation, CA15-3 has been purified from the culture supernatant of breast cancer T47-D cell line with 76% yield and 3350 fold purification. Isolated CA15-3 was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting (western blotting), chemiluminescence immunoassay (CLIA) and Fourier-transform infrared spectroscopy (FTIR). CA15-3 is a monomeric protein with an apparent molecular mass in between ∼250-350kDa. The FTIR spectroscopy revealed similar profiles of T47-D derived CA15-3 and commercially available CA15-3 protein. With the easy availability of T47-D cell line and a simple purification approach described here will support for the large scale production of CA15-3 to be used for various clinical and diagnostic applications.

Simplified Purification Approach of Urinary Neutrophil Gelatinase-associated Lipocalin by Tangential Flow Filtration and Ion Exchange Chromatography

Kunal Shukla, Shamkant Badgujar, Paresh Bhanushali, Sushma Sabharwal

This investigation reports a simplified approach for the purification of urinary siderocalin known as neutrophil gelatinase-associated lipocalin (NGAL). Urinary NGAL was purified by tangential flow filtration and ion exchange chromatography. Isolated NGAL was analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular mass of NGAL is 23674Da. Peptide mass fingerprinting of the purified NGAL yielded peptides that partially matched with known sequence of P80188 (NGAL_HUMAN). The tryptic digestion profile of isolated NGAL infers that it may be unique and additive molecule in the dictionary of urinary proteins. This is the first report of purification and validation of urinary NGAL from large volume sample by using tangential flow filtration and peptide sequencing respectively. This cost-effective and simplified approach to purification of NGAL, together with the easy availability of urine sample makes the large-scale production of NGAL possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

A Novel Approach for the Chromatographic Purification and Peptide Mass Fingerprinting of Urinary Free Light Chains

Bhupesh Mali, Shamkant Badgujar, Kunal Shukla and Paresh Bhanushali

We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

Development of an Indirect Immunofluorescence Based Assay for Diagnosis of Ulcerative Colitis in Indian Population

Allan Rodrigues, Chander Puri, Paresh Bhanushali, Kunal Shukla, Sampurna Roychoudhury and Shamkant Badgujar

The prevalence of Ulcerative Colitis (UC), once thought to be negligible, has increases exponentially in the Indian population. The development of novel, cost effective and time efficient Indirect Immunofluorescence (IIF) based assay for detection of anti-neutrophil cytoplasmic antibodies (ANCA) and diagnosis of UC in the Indian population is discussed. A novel IIF based assay was developed using intact nuclei from human neutrophils to detect atypical p-ANCA in patients suffering from UC. Sera from 45 patients diagnosed with UC, 45 healthy controls and one related disease control were tested using a novel UC-ANCA assay and validated by commercially available ANCA IIF assay. Prevalence of ANCA amongst UC patients in the Indian population was determined. Atypical p-ANCA was detected in 86.6% of the patients using the UC-ANCA assay as compared to 71.1% using the commercial ANCA assay. The validation of UC-ANCA assay with a commercially available ANCA IIF assay resulted in higher sensitivity. The UC-ANCA assay proved to be not only enhanced in terms of performance but also comparatively economical and rapid. The novel UC-ANCA assay may prove to be very useful in identification and differentiation of UC patients from typical ANCA positive subjects suffering from other autoimmune diseases at one tenth the cost of clinically available ANCA IIF tests which will immensely benefit the cost constrained diagnostic field of developing countries.

Development of Glycan Specific Lectin Based Immunoassay for Detection of Prostate Specific Antigen

Paresh Bhanushali, Shamkant Badgujar, Mukesh Tripathi, Sanjeev Gupta, Vedang Murthy, Musti Krishnasastry and Chander Puri

We describe an analytical approach for the detection and verification of glycosylation patterns of prostate specific antigen (PSA), a key biomarker currently used for understanding the onset and prognosis of prostate cancer. PSA has been purified from the human seminal plasma and total PSA from prostate cancer sera. PSA is a monomeric glycoprotein with an apparent molecular mass 28040.467 Da, which exhibits a characteristic protease activity against casein and gelatin. Its optimal protease activity is centered on neutral pH. Peptide mass fingerprint analysis of the purified PSA has yielded peptides that partially match with known database sequences (Uniprot ID P07288). Tryptic digestion profile of isolated PSA, infer the exclusive nature of PSA and may be additive molecule in the dictionary of seminal proteins. Surface plasmon resonance and lectin immunoassay revealed direct interaction between a newly developed anti-PSA monoclonal antibody (C4E6) and PSA. A lectin based immunoassay is reported here which was achieved with the C4E6 anti-PSA antibody and biotinylated plant lectins. This investigation provides an alternative method to isolate and quantify PSA with altered glycosylation which might be seen in the prostate cancer and developing a lectin based immunoassay to detect PSA in serum of prostate cancer patients.

Clinical Impact of Prostate Specific Antigen (PSA) Inter-assay Variability on Management of Prostate Cancer

Vedang Murthy, Anupam Rishi, Sanjeev Gupta, Sadhana Kannan, Umesh Mahantshetty, Hemant Tongaonkar, Ganesh Bakshi, Kumar Prabhash, Paresh Bhanushali, Bhoopal Shinde, Nitin Inamdar, Shyamkishore Shrivastava

Purpose: To evaluate the inter-assay variability of six commercially available prostate specific antigen (PSA) assays, its clinical impact in prostate cancer (PCa) and comparison of automated versus manual assays. Patients and methods: Sera from 495 patients (425 with PCa and 70 men with Benign Prostatic Hyperplasia (BPH), were measured with six different assays [three automated assays (a-PSA) and three manual ELISA based assay (m-PSA)]. Variability, agreement and bias were measured and compared among assays using Bland Altman plots and Passing and Bablok regression analysis. The possible impact of inter-assay variability on important clinical scenarios was also studied.

Development of Monoclonal Antibodies Against MUC1/Y Recombinant Protein Expressed in E. Coli

Anuj Gupta, Parvinder Kaur, Harshada Patil and Udayakumar K.

Cancer antigen 15-3 (CA 15-3) is a MUC1 peptide fragment widely used for the diagnosis of breast cancer. However, because of a lack of sensitivity and specificity, especially during early stages, CA 15-3 alone has not proved to be a reliable marker for early diagnosis of breast cancer. High preoperative concentrations of CA 15-3 are, however, associated with adverse patient outcome. Therefore, an emergent need still exists to find out secondary markers to enhance specificity of serum based detection of breast cancer. MUC1/Y a trans-membrane protein (Non-polymorphic), and a part of MUC1 gene devoid of tandem repeats array and its immediate flanking sequences has also been reported to be expressed in vivo by tumor cells and suggested to be a potential target both for epithelial tumor diagnosis and immunotherapy. In this study, we have expressed and purified recombinant human MUC1/Y from Escherichia coli BL21 (DE3) strain.

Multi Functional Diagnostic Exploitation of Human Galectin-3

Anuj Gupta, P. Kaur, Paresh Bhanushali and Prashant Khadke

Galectin-3 is a member of the family of animal lectins, which selectively binds β-galactoside residues. Galactin-3 regulates a number of biological processes, including cell differentiation, growth, angiogenesis, embryogenesis, inflammatory responses, cell progression and metastasis. Several studies have shown a correlation between levels of Galectin-3 and tumor progression in liver, thyroid, colon, gastric and breast carcinomas, making Galectin-3 an emerging cancer marker.

Secretory Expression of CA15-3 like Recombinant Antigen in Mammalian Cells

Anuj Gupta, Parvinder Kaur, S. Sehgal and Pranali Patil

Breast cancer is most common life-threatening malignant lesion in women all over the world. Cancer antigen 15-3 (CA 15-3) is a widely used prognostic marker for breast cancer. CA 15-3 is a glycoprotein that is recognized with a match pair of DF3 and 115D8 monoclonal antibodies. DF3 antibody detects 8 amino acids sequence (DTRPAPGS) of tandem repeats in MUC1 protein, serves as a detection antibody in a sandwich assay while 115D8 monoclonal antibody binds to peptide- carbohydrates epitope on same repeat acts as the capture antibody in sandwich assay.

Development of Monoclonal Antibodies Against CMP-N-Acetylneuraminate-Beta-Galactosamide-Alpha-2,3-sialyltransferase 1 (ST3Gal-I) Recombinant Protein Expressed in E. Coli

Anuj Gupta, Parvinder Kaur, Harshada Patil, Pallavi Kadam, Paresh Bhanushali and Manoj Chugh

Aberrant glycosylation is one of the major hallmarks of cancer with altered gene expression signatures of sialyltransferases. ST3Gal-I, a sialyltransferase, is known to play a crucial role in sialylation of T antigen in bladder cancer and it has reported elevated expression in breast carcinogenesis with increased tumor progression stages. The aim of the current study is to develop new monoclonal antibodies (mAbs) against human ST3Gal-I and evaluate their diagnostic potential.

Purification and Comparative Study of NGAL Isoforms from Neutrophil and Kidney Origin Suggests Its Different Roles Under Different Stress Conditions

Kunal Shukla, Paresh Bhanushali, Anuj Gupta and S. Sabharwal

Neutrophil gelatinase associated lipocalin (NGAL) is a member of the lipocalin family and binds with iron in the presence of enterochelin. The role of NGAL is not clear but earlier experiments suggest that it acts as a bacteriostatic agent and is dramatically up regulated during acute kidney injury (AKI).

A Novel Chromatographic Purification Method for High Pure CA 15-3

Paresh Bhanushali, Kunal Shukla, Prerana Rathod and M. M. Tripathi

Carcinoma antigen 15-3 (CA 15-3) is the most widely used tumor marker for breast Cancer (Schmidt-Rhode et al. 1987). It is derived from Mucin1 (MUC1) protein. CA 15-3 is a glycosylated protein of molecular weight around 400 KDa (Steger et al. 1989). It is secreted by the surface epithelia of cancer tissue and shed in to the blood stream. Free floating CA 15-3 at high level is detected in the blood of breast cancer patient (Ogawa et al. 1986; Gang et al. 1985).