nano flow custom conjugation anti troponin

Abstract

For signal amplification without an additional operation step in a gold nanoparticle (AuNP)-based lateral flow assay (LFA), a new and simple method utilizing two AuNP-antibody conjugates was developed. The 1st conjugate was the AuNP immobilized with an anti-troponin I antibody and blocked with bovine serum albumin (BSA), and the 2nd conjugate was the AuNP immobilized with an anti-BSA antibody and blocked with human serum albumin. The two conjugates were encapsulated in different pads, respectively. A scheme of the LFA system is described in the part A of first figure. The size of the two conjugates was very critical in the detection sensitivity of troponin I. When 10nm for the 1st and 40 nm for the 2nd were used, the detection sensitivity increased about a 100-fold compared to the conventional LFA. We could detect as low as 0.01 ng/mL troponin I in 10 min using the dual AuNP conjugate-based LFA, which was successfully applied in the analysis of serum samples of patients with myocardial infarction.

Introduction

Cardiac troponin I (cTnI) has been used clinically, for years, for diagnosis and risk stratification in patients with suspected acute myocardial infarction (AMI)1. Generally, elevated levels of cTns are conjoined with damage of cardiac muscles and cTnI release to the circulation has specificity for cardiac injury2. Measurement of circulating levels cTnI, particularly observing the rise or fall in cTnI levels, along with the evaluation of patient symptoms, history and electrocardiographic (ECG) abnormalities are current procedures in the triage of suspected AMI patients3,4. The typical challenge is early identification of AMI among the large heterogenous patient population, as only one in three patients, experiencing chest pain, who visit emergency department (ED) receives AMI diagnosis5. Therefore, timely and correct diagnosis to rule-in AMI is important in terms of cost savings and improving the clinical outcomes of AMI patients.

Point-of-care testing (POCT) can facilitate quick turn-around-times (TAT) to promote efficient diagnosis of AMI6. Lateral flow immunoassays (LFIAs) can be used in POCT, particularly in settings where the cost of the high-tech instrumentation may be prohibitive. LFIA in POCT is well-established, mature technology known for its low cost. However, the limitations regarding sensitivity and quantification have hindered its use in many applications7. In our previous work, we have described how the use of upconverting nanoparticle (UCNP) reporters in LFIAs provides quantitative results and improved sensitivity8,9,10.

The use of UCNP measurement technology can enhance the assay sensitivity in comparison to traditional down-converting fluorescent reporters since it enables elimination of the background autofluorescence from the measurement. This is due to the unique photon upconversion luminescence of UCNP reporters converting low-energy excitation (near or at infrared) wavelength into high-energy emission at visible wavelengths11. The UCNP signal can be read with a miniaturized relatively low-cost reader instruments with high performance in POCT settings. Such readers with miniaturized optics for UCNP measurement have previously been described in the literature12,13,14,15.

The performance of LFIAs may suffer from interferences originating from the sample matrix, particularly because of the typical one or two step assay procedure with limited washing. These immunoassay (IA) interferences may be caused by different interfering agents such as circulating heterophilic IgG and IgM antibodies16, autoantibodies targeted against the biomarker of interest17 and human anti-animal antibodies18. In general, IgG antibodies used as binders in IAs are prone to several interfering reactions caused by circulating antibodies.

nano flow custom conjugation anti troponin
nano flow custom conjugation anti troponin

 

If a heterophilic antibody has high affinity for the Fc-region of an IA binder antibodies, an aggregate of binder antibodies with large numbers of Fc-regions close together functions as a binding target for the heterophilic antibody16. Furthermore, it has been shown that complement activation by the classical pathway can occur when several IgG molecules are in close proximity, e.g. on reporter nanoparticle surface19 or on the solid phase20. As a results, complement factors bind to the Fc region of the IA binder antibody20,21 which causes negative interference in the IA. Non-specific binding of autoantibodies such as rheumatoid factor (RF) has been associated with falsely elevated analyte concentrations22. Since IAs utilizing nanoparticle reporters aim at extremely high sensitivities, the effect of matrix interferences should be addressed to fully utilize the potential of nanoparticle IA technologies.

It has been depicted that the effects of interfering agents can be eliminated with sample pre-treatment procedures such as heating or precipitation of interfering antibodies with polyethylene glycol (PEG)20. In addition, pretreatment with Ethylenediamine tetra-acetic acid (EDTA) effectively inhibits the activation of the complement pathway23. However, particularly in the case of POCT, time-consuming sample pre-treatment steps are undesirable. Therefore, in LFIAs the potential solutions should be implemented in the test device itself.

In this study, we report the development of a UCNP-LFIA for cTnI. UCNP reporter technology was utilized to provide the sensitive and quantitative detection of cTnI in LFIA format to overcome the typical limitations of LFIAs. The matrix interference in plasma samples was studied and reduced by incorporating the developed sample pre-treatment steps into the LF strip. The performance of the developed UCNP-LFIA was evaluated with clinical plasma samples (n = 262).

 

 

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