Biorecognition and detection of antigens from Mycobacterium tuberculosis using a sandwich ELISA associated with magnetic nanoparticles

Highlights

• Recombinant expression of seven MTB proteins (rMTB antigens) in E. coli system.

• Polyclonal antibodies production in rabbits and mice immunized with rMTB antigens.

• Immobilization of antibodies (ab) in functionalized Magnetic nanoparticles (MNP@Si).

• Detection of MTB antigens (patients) using sandwich ELISA associated with MNP@Si@ab.

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the 10 leading causes of death worldwide, especially in low-income areas. A rapid, low-cost diagnostic assay for TB with high sensitivity and specificity is not currently available. Bio-functionalized magnetic nanoparticles (MNPs) which are able to efficiently detect and concentrate biomolecules from complex biological samples, allows improving the diagnostic immunoassays. In this way, a proof-of-concept of MNP-based sandwich immunoassay was developed to detect various MTB protein antigens. The superficial and secretory antigenic proteins considered in this research were: CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B, and MoeX. The proteins were cloned and expressed in an E. coli system. Polyclonal antibodies (ab) against the recombinant antigens were elicited in rabbits and mice. Antibodies were immobilized on the surface of amine-silanized nanoparticles (MNP@Si). The functionalized MNP@Si@ab were tested in a colorimetric sandwich enzyme-linked immunosorbent assay (sELISA-MNP@Si@ab) to recognize the selected antigens in sputum samples. The selected MTB antigens were successfully detected in sputum from TB patients in a shorter time (~ 4 h) using the sELISA-MNP@Si@ab, compared to the conventional sELISA (~15 h) standardized in home. Moreover, the sELISA-MNP@Si@ab showed the higher sensitivity in the real biological samples from infected patients.

Introduction

Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (MTB). According to the last TB report [1], in 2020 around 1.5 million more people died of TB than in 2019, including 214,000 HIV positive patients. Because worldwide covid-19 emergency, it was more difficult to access and provide essential tuberculosis services, while many people were undiagnosed during 2020 [1]. So, a decrease in TB diagnosis was reported from 7.1 million in 2019–5.8 million in 2020. Similarly, treatment of drug-resistant tuberculosis patients decreased by 15%, from 177,000 in 2019–150,000 in 2020 [1].

Smear microscopy, cultures, and molecular methods are the main TB diagnostic tests currently used, although alternative approaches are being developed [2]. Molecular tests based on polymerase chain reaction (PCR) have been preferred as they are relatively fast and have a high sensitivity and specificity [3]. For example, the Cobas Taqman MTB test based on the amplification of the 16 S rRNA gene in real-time PCR, presents 91.5% and 98.7% sensitivity and specificity, respectively [4]. However, it is expensive and not accessible to people with limited economic resources.

In low-income areas, smear microscopy and culture are the main TB diagnostic tests. MTB culture is the conventional ‘gold standard’ for TB diagnosis. Although MTB culture is highly sensitive (limit of detection approx. 10–100 cfu/mL sputum), a long incubation period is required before results are available (4–8 weeks). In addition, the treatment of the sputum is critical, and this process is prone to contamination and requires a biosafety level 3 laboratory with skilled laboratory personnel [5].

On the other hand, smear microscopy is a rapid method but with a low sensitivity [6]. The most used However, the main problem with serodiagnosis for active TB is that the detection of antibodies does not necessarily indicate active infection in the patient [7]. Therefore, it is recommended that efforts should focus on developing methods to directly detect M. tuberculosis or specific superficial or secretory proteins directly from clinical samples [8].

MTB-filtered culture proteins are excellent candidates for biomarkers since they represent the most abundant secretory proteins of MTB. Lee et al. evaluated the production of secretory proteins in MTB culture, finding a production of 10 mg of Antigen 85 (Ag85) complex, 0.56 mg of protein 38 kDa, and 1.81 mg of MTB12, from one liter of the six-week-culture filtrates of M. tuberculosis H37Rv which contained 307.81 mg of culture filtrate protein [9].

Among other important MTB markers are: MTC28, a 28 kDa proline-rich secretary and conserved antigen of MTB which is a potential candidate for vaccine design and for the immunodiagnosis of TB [10]; MoeX, Molybdopterin biosynthesis protein, which has been clinically validated as a diagnostic biomarker for active pulmonary TB [11]; Ag85B which is essential for the intracellular survival of MTB within the macrophage [12]; and MPT64, a 24 kDa protein of MTB which is a highly specific to the MTB complex and represents 8% of the total proteins found in a filtered MTB culture. Furthermore, MPT64 is one of the predominant proteins actively secreted during MTB growth and is expressed early when MTB is in active division [13]. ESAT6/CFP10, the 6 kDa early secretory antigenic target and 10 kDa culture filtrate antigen, are the most abundant secretory proteins found in filtered MTB culture, and both are considered important virulence factors. They are not present in M. bovis bacillus Calmette–Guerin (BCG) strain or in most non-tuberculous mycobacteria [14].

Magnetic nanoparticles (MNPs) are widely used in several applications such as cell separation, gene targeting, drug delivery, magnetic resonance imaging, and hyperthermia [15]. This is due to the optical, magnetic, and electric properties of the nanoparticles. Otherwise, stabilization of nanoparticles is an important parameter to consider their use in biomedical applications [16]. In addition, the functionalization of the MNP surface using biomolecules had been widely studied. For example, antibody-immobilized nanoparticles are used to improve specific bio-separation of antigens, detection of biological markers or cells from corporal or environmental samples [17]. Practical applications include the detection of pathogens associated with infectious diseases in food [18]. This method is based on the capture of antigens using magnetic nanoparticles coated with antibodies. Due to the size and shape of the MNPs these can be dispersed uniformly in the sample, increasing the capture of antigens and, consequently, the sensitivity of the test [19].

Likewise, the use of MNP has been used in the development of methods to improve the diagnosis of tuberculosis. For example, Gupta et al., 2021, proposed a giant magnetoresistance (GMR) to detect the early stage of TB, where functionalized MNPs bind the GMR sensor proportionally to ESAT6 protein concentration (range of pg/mL), leading to the change in overall resistance of the sensor [20]. Similarly, Mohd Azmi et al., 2021, reported a portable electrochemical immunosensor for the detection of MTB secreted protein CFP10-ESAT6 in clinical sputum samples. They used iron/gold magnetic nanoparticles conjugated with specific antibodies to detect CFP10-ESAT6 proteins. The detection of MTB antigens was achieved using a differential pulse voltammetry technique in a linear range from 10 to 500 ng/mL. The method was validated to detect MTB from sputum samples, using culture and smear microscopy as gold standard, obtaining 100% sensitivity and 91.7% specificity [21]. In the same way, Cheon et al., 2019, developed a colorimetric biosensing system to detect anti-MPT64, using DNA aptamer adsorbed magnetic nanoparticles [22]. In addition, a magnetophoretic immunoassay (MPI) was designed to detect CFP10 antigens effectively from early growth of MTB in liquid culture. Two specific monoclonal antibodies against CFP10, including gold nanoparticles for signaling and magnetic particles for separation were used. The detection limit was 0.3 pM. Using clinical samples MPI shows robust and reliable sensing while monitoring MTB growth (3–10 days), it was comparable to that with the commercial mycobacteria growth indicator tube (MGIT) test [23].

Previously, our group also reported the synthesis and characterization of amino-silanized magnetic nanoparticles to improve the diagnosis of tuberculosis [24]. However, the assay was performed only to detect the recombinant MTB Hsp16.3 antigen, and biological samples from TB patients were not used. So, this work is the continuation of our previous research. Here we present a sandwich ELISA assay which incorporates MNPs functionalized with anti-MTB specific polyclonal antibodies (sELISA-MNP@Si@ab) to detect MTB in sputum from TB patients. For this purpose, superficial and secretory MTB antigenic proteins (CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B and MoeX) were expressed in an E. coli system and used for polyclonal antibody production in rabbits and mice. Iron MNPs were synthesized using a co-precipitation method, previously described. The surface of the iron magnetic nanoparticles, coated with amine silane groups, was functionalized with polyclonal antibodies, previously produced in mouse. These sELISA-MNP@Si@ab complexes were successfully used in a sandwich ELISA assay to detect native antigens from positive sputum samples and allow to increase the sensitivity of the diagnosis of active tuberculosis.

The novelty of this research is the comparative detection of different MTB antigens, some of them evaluated for the first time in this diagnostic device. Furthermore, as the introduction of bio-functionalized MNPs in a colorimetric sELISA increases the amount of captured antigens from biological samples, we demonstrated that sELISA-MNP@Si@ab performed better than classical sELISA.