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Shukla K.K.,University of Pune | Shukla K.K.,Yashraj Biotechnology Ltd. | Badgujar S.B.,Yashraj Biotechnology Ltd. | Bhanushali P.B.,Yashraj Biotechnology Ltd. | Sabharwal S.G.,University of Pune
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2017

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 23674 Da. 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. © 2017 Elsevier B.V.


PubMed | Tata Memorial Center, Yashraj Biotechnology Ltd and Tata Memorial Hospital
Type: Journal Article | Journal: Clinical biochemistry | Year: 2016

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.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.All the assays were well correlated (r: 0.88-0.98); however there was significant disagreement and bias between the systems, which were more pronounced among the a-PSA assays. The Bland Altman plot showed that the variability was high between the m-PSA assays and the standard Abbott system with mean difference of 3.8-5.8ng/ml. In contrast, the a-PSA had better agreement with mean difference of 0.8-2.3ng/ml. Beckman Coulter showed the best agreement to the institutional reference (slope-1.097; 95% CI: 1.06-1.14; p<0.05, and intercept-0.20; 95% CI-0.38-0.58; p<0.05, Passing Bablok). It led to significant variability in PCa risk stratification and failure to detect biochemical failure in more than 50% cases.The discrepancies between the assays lead to significant clinical misinterpretation with risk group migration and detection of biochemical failure post radiotherapy. There are significant discordances between automated and ELISA based assays.


Bhanushali P.B.,Yashraj Biotechnology Ltd. | Bhanushali P.B.,MGM Institute of Health Sciences | Badgujar S.B.,Yashraj Biotechnology Ltd. | Badgujar S.B.,National Health Research Institute | And 5 more authors.
International Journal of Biological Macromolecules | Year: 2016

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. © 2016 Elsevier B.V.


PubMed | MGM Institute of Health Sciences, National Health Research Institute and Yashraj Biotechnology Ltd.
Type: | Journal: Immunology letters | Year: 2016

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.


PubMed | MGM Institute of Health Sciences, National Health Research Institute, Yashraj Biotechnology Ltd., University of Pune and Tata Memorial Center
Type: | Journal: International journal of biological macromolecules | Year: 2016

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.


Shukla K.,University of Pune | Shukla K.,Yashraj Biotechnology Ltd. | Bhanushali P.,Maharashtra University of Health Sciences | Bhanushali P.,Yashraj Biotechnology Ltd. | And 2 more authors.
International Journal of Pharma and Bio Sciences | Year: 2014

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). Immunological studies in the literature have shown that the use of epitope specific antibodies to measure the level of NGAL from AKI patients has significant effects on the performance of the ELISA, CLIA and other assays, due to the expression of different molecular forms of NGAL. To understand the biochemical similarities and differences between NGALs secreted in different diseased conditions, we have purified and characterized NGAL from the urine of AKI patients (kNGAL) and from neutrophils from healthy humans (nNGAL). In our study 2D electrophoresis followed by quantitative analysis were performed to study the differences between the isoforms of kNGAL and nNGAL. Identification of NGALs of both the sources was confirmed by MALDI-TOF/TOF analysis and further validated using ELISA and western blot. The proteomics analysis in our study reveals that kNGAL is slightly acidic and predominantly monomeric in nature; on the other hand, nNGAL is both monomeric and dimeric and ranges from acidic to far basic forms. Further, we also observed a relatively higher extent of sialylation in kNGAL in comparison to nNGAL suggesting the different roles of kNGAL and nNGAL under different diseased conditions.


Gupta A.K.,Yashraj Biotechnology Ltd | Gupta A.K.,Jagdishprasad Jhabarmal Tibrewala University | Kaur P.,Yashraj Biotechnology Ltd | Sehgal S.,Yashraj Biotechnology Ltd | And 4 more authors.
International Journal of Pharma and Bio Sciences | Year: 2015

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. In this study, we have developed a protein sequence containing 10 Tandem repeats with few approximate repeats out of MUC1 protein expressed in CHO-K1 cells to ensure post translational modifications. We have incorporated 'Gluc' protein (Gaussia luciferase) sequence along with gene of interest for secretion of protein outside the cells. Protein was characterized by western blot using CA15-3 antibody as well as confirmed for the presence of epitopes similar to the ones, as recognized by 115D8 and DF3 on Siemens CLIA based platform. Further validation studies are needed to demonstrate equivalence of the expressed protein with native purified CA15-3 protein to be used to develop diagnostic/prognostic tests.


Gupta A.K.,Yashraj Biotechnology Ltd. | Kaur P.,Yashraj Biotechnology Ltd. | Patil H.,Yashraj Biotechnology Ltd. | Kadam P.,Yashraj Biotechnology Ltd. | And 2 more authors.
Biochemistry Research International | Year: 2015

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. We developed a repertoire of stable hybridoma cell lines producing high-affinity IgG antibodies against recombinant human ST3Gal-I, expressed in E. coli BL21-DE3 strain. In order to demonstrate the diagnostic value of the mAbs, various clones were employed for the immunohistochemistry analysis of ST3Gal-I expression in cancerous tissues. Antibodies generated by 7E51C83A10 clone demonstrated a strong and specific fluorescence staining in breast cancer tissue sections and did not exhibit significant background in fibroadenoma sections. In conclusion, the mAbs raised against recombinant ST3Gal-I recognize cellular ST3Gal-I and represent a promising diagnostic tool for the immunodetection of ST3Gal-I expressing cells. Specific-reactivity of clone 7E51C83A10 mAbs towards ST3Gal-I was also confirmed by immunoblotting. Therefore, our observations warrant evaluation of ST3Gal-I as a potential marker for cancer diagnosis at larger scale. © 2015 Anuj Kumar Gupta et al.


PubMed | Yashraj Biotechnology Ltd.
Type: | Journal: International journal of biological macromolecules | Year: 2016

We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (-FLCs) and kappa free light chains (-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of -FLC and -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 -FLCs and -FLCs. Peptide mass fingerprint analysis of the purified -FLCs and -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.


Gupta A.K.,Yashraj Biotechnology Ltd | Kaur P.,Yashraj Biotechnology Ltd | Patil H.,Yashraj Biotechnology Ltd | Udayakumar K.,Yashraj Biotechnology Ltd | And 2 more authors.
International Journal of Pharma and Bio Sciences | Year: 2015

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. We could develop a repertoire of monoclonal antibodies, which bound to our recombinant protein. However, most of the antibodies exhibited cross reactivity against normal human serum or His Tag. However, only 9A842G6 specifically reacted to recombinant MUC1/Y protein. 9A842G6 monoclonal antibodies exhibited fluorescence specifically with established breast cancer tissue sections and did not bind to fibroid adenoma sections. Specific reactivity of 9A842G6 mAbs towards MUC1/Y was also confirmed by immunoblotting. Therefore, our observations suggest possible use of MUC1/Y as a potential secondary marker for Breast cancer diagnosis along with CA15-3.

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