This is essential to achieve the required sensitivities along with dynamic ranges appropriate for biological relevance for each of the analytes

This is essential to achieve the required sensitivities along with dynamic ranges appropriate for biological relevance for each of the analytes. 76C111.2?%. MI compared well with the established immunoradiometric assay (IRMA) with r?=?0.98, em p /em ? ?0.01 (n?=?41). No cross-reactivity was seen between the immobilized antibodies. Although two hormones are addressed in this report, MI using PC TEM and isotopic/non-isotopic tracers has the potential for highly automated multiplexed analysis. strong class=”kwd-title” Keywords: Microarray immunoassay, Track-etched membranes, Antibody chip, Thyroid stimulating hormone, Thyroglobulin Introduction The profiling of analytes in serum for a given disease is often required for both clinical diagnosis and management of patients. The quantitation of analytes, present in serum in minute concentrations, e.g., hormones and tumour markers, is mostly done using immunoassays, which currently, are developed to assay one analyte at a time. Since the initial description by Ekins, [1, 2] and early advances by Huang [3], Schweitzer et al. [4], Tam et al. [5] and Knight et al. [6], MI (also referred to as multi-analyte immunoassay) has been demonstrated to be promising in area of clinical diagnosis [7C10] and several other areas such as environmental monitoring and food safety [11, 12]. Also, by creating disease-associated analyte panels, sensitivity and specificity of clinical diagnosis and/or prognosis is usually increased by the weight of evidence derived from comparing associated analytes. Although several MI have been developed, but most of them are for targeting immunoglobulin or cytokines and MI for routine use in endocrinology is limited to that from Randox for thyroid hormones [13C15]. Technical and operational considerations have hindered implementation Niraparib tosylate of MI in clinical settings [14]. One of the important considerations while developing MI, is usually a process for the immobilization of OCP2 the antibodies at high density while maintaining its functionality. This is essential to achieve the required sensitivities along with dynamic ranges appropriate for biological relevance for each of the analytes. Moreover, immobilization procedure should provide low variability to achieve good reproducibility of the assay. Other considerations in developing MI include the elimination of assay cross-reactivity under highly multiplexed condition to achieve high sensitivity and specificity. This report describes the Niraparib tosylate development of MI for simultaneous estimation of TSH and Tg useful in monitoring patients with differentiated thyroid cancer. PC-TEM, which is usually highly microporous (108 pores per cm2) and very thin (10 micron), was used as an immobilization support. TEMs were earlier described by us, for the first time, as an optimal support providing efficient immobilization of antibodies with low background [16C18]. Capture antibodies, at high density, for both the analytes were immobilized on PC-TEM as small spots (~1?mm diameter). Detection, which is the key step in development of MI, was accomplished using 125I because of its easy availability at our Centre and detection. Using a mixture of 125I labeled detection antibodies against both TSH and Tg, a non-competitive immunoassay approach was used for development of MI. MI for TSH and Tg,?as an analytical technique, was validated with respect to sensitivity, accuracy, precision and reproducibility, which are important parameters for any bioanalysis [19]. The performance of MI was compared with individual IRMA using linear regression analysis. The designed MI has scope for multiplexing several assays. Materials and Methods Materials Reagents Bovine serum albumin (BSA) was purchased from Sigma, USA. Human recombinant TSH (thyrogen) was purchased from Genzyme Corporation, USA. Tg standards were obtained along with human Tg IRMA kit from M/s Izotop, Budapest, Hungary. All other chemicals and reagents required for this study were purchased locally and were of analytical or comparative grade. Low conductivity deionised water was used wherever required. TEM PC TEMs having 25?mm Niraparib tosylate Niraparib tosylate diameter with pore size of 0.4 micron and pore density of 108/cm2 were procured from Millipore, USA. Antibodies and Tracers Monoclonal antibodies to TSH were purchased from Biodesign International, USA. Polyclonal antibodies against Tg were produced.