Antigens In Biological Fluids Research Articles

Development of Quantitative Enzyme Immunoassay of Brain-Derived Neurotrophic Factor on the Basis of Recombinant Antigen

One of the pathophysiological mechanisms of the development of some mental diseases, including depressive disorders, is a decrease in the expression of brain-derived neurotrophic factor (BDNF), mainly in the limbic region and prefrontal cortex of the brain. Immunochemical screening of BDNF in biological fluids showed that the levels of this protein can be considered as a marker of predisposition to depression and a prognostic marker of the effectiveness of therapy. The use of modern technologies for the production of recombinant proteins makes it possible to develop highly standardized ELISA systems for the determination of this antigen in biological fluids. This paper describes a method for creating a test system for quantitative ELISA of brain-derived neurotrophic factor on the basis of recombinant protein BDNF and antibodies obtained as a result of immunization with recombinant BDNF.

Exploiting Photoelectric Activities and Piezoelectric Properties of NaNbO3 Semiconductors for Point-of-Care Immunoassay.

Point-of-care testing (POCT) technology has made major breakthroughs in community medicine and physician office situations, in tandem with the more ubiquitous and intensive usage of highly integrated quick detection equipment for illness diagnosis, personal care, and mobile healthcare. Although the photoelectrochemical (PEC)-based POCT platform offers the benefits of cheap cost and good user engagement, its commercialization is still limited by the photodetection components' downsizing and mobility, among other factors. In this work, a novel highly integrated PEC biosensor aided by piezophototronics to enhance the efficiency of PEC testing was reported for flexible detection of cancer-associated antigens in biological fluids (prostate-specific antigen, PSA, used as an example). Multiple signal enhancement strategies, including a magnetic bead-linked enzyme-linked immune system catalyzing the production of ascorbic acid from the substrate and a piezoelectric-assisted enhancement strategy, were used for sensitive detection of the analyte to be tested in human body fluids. Unlike the electron transfer mechanism in heterojunctions, piezoelectric semiconductors promote the transfer of electrons and holes by generating piezoelectric potentials in the ultrasonic field, thus contributing to the performance of the PEC testbed. Under optimized conditions, the test platform achieves good correspondence for PSA at 0.02-40 ng mL-1. Impressively, the test devices are comparable to or even superior to gold standard ELISA kits in terms of cost approval and batch testing. This research demonstrates the potential of piezoelectric semiconductors for POC applications in revolutionary PECs and offers innovative thoughts for the development of new PEC bioanalytical components.

Antibody mounting capability of 1D/2D carbonaceous nanomaterials toward rapid-specific detection of SARS-CoV-2

Carbonaceous immunosensors are ideal nanoplatforms for developing rapid, precise, and ultra-specific diagnostic kits capable of early detection of viral infectious illnesses such as COVID-19. However, developing a proper carbonic immunosensor requires stepwise protocols to find optimum operating conditions to minimize drawbacks. Herein, for the first time and through a stepwise protocol, activation, and monoclonal IgG antibody mounting capability of multi-walled carbon nanotubes (MWCNTs) at two diverse outer diameters (ODs), viz., 20–30 nm and 50–80 nm, and graphene deriv atives (graphene oxide (GO) and reduced graphene oxide (rGO)) were examined and compared with each other toward finding the prime carbonaceous nanomaterial(s) for maximized antibody loading efficiency along with an ideal detection limit (DL) and sensitivity. Next, the effect of common amplifying agents, i.e., Au nanostars (Au NSs) and Ag nanowires (Ag NWs), on the total performance of the best carbonaceous structure was carefully assessed, and the responsible detection mechanism is investigated in detail. Next, the developed carbonaceous immunosensors were assessed via voltammetric and impedance assays, and their performances toward specific detection of SARS-CoV-2 antigen through immunoreaction were examined in detail. The study's outcome showed the superior performance of conjugated rGO-based immunosensor with Au NSs toward specific and quick (1 min) detection of SARS-CoV-2 antigen in biological fluids compared with other 1D/2D carbonaceous nanomaterials.

A Review on Colorimetric Sensing of Tumor Markers Based on Enzyme-Mimicking Nanomaterials.

Nanomedicine is an arising field that exploits nanotechnology concepts for pioneered therapy and diagnostics. Colorimetric sensors for tumor markers have displayed interesting benefits compared to conventional systems in clinical laboratory diagnosis. Colorimetric immunoassay-based approaches show up-and-coming results since the goal cancer marker is determined with high sensitivity but without the utilization of advanced/- expensive techniques through an effortless optical color change. Also, colorimetric biosensor has the potential to detect proteins in biological fluids swiftly with high sensitivity, and they are anticipated to play a progressively serious role in tumor diagnosis. We reviewed (covering the period 2015-2020) various studies based on colorimetric sensing strategy using nanostructured materials (highly efficient enzyme mimics, artificial enzymes or nanozymes) to detect different tumor antigens in biological fluids. Specifically, we highlighted the recent progress and efforts in the construction of colorimetric immunosensors. Colorimetric immunosensors can be roughly divided into two main categories: transition metal nanozyme-based sensing and noble metal nanozyme-based sensing.

Detection of Mycobacterium tuberculosis serum biomarkers and the relation with previous BCG vaccination

Objectives: Accurate diagnosis of tuberculosis (TB) disease is a cornerstone in the management of disease spread and initiation of proper treatment. Detection of Mycobacterium tuberculosis (MTB) antigens in biological fluids might be a reliable way to directly diagnose TB disease. The aim of the current study is to investigate selected TB antigens as potential diagnostic serum biomarkers. Methods: We have explored the detection of three MTB antigens, namely Rv2324, Rv2435c and Rv1284, in serum specimens from confirmed pulmonary TB patients. In addition, serum specimens from Bacillus Calmette-Guerin (BCG)-vaccinated healthy adults and BCG-unvaccinated infants were also tested as negative controls. The three antigens were recombinantly expressed in Escherichia coli and purified using affinity chromatography. Murine antibodies against the three antigens were raised by mice immunization with corresponding recombinant antigens. TB antigen detection in the serum specimens was carried out by the dot blot test. Results: Serum samples from TB patients showed significant reactivity (94.3-100%) with the antigen-specific antibodies. Surprisingly, the three antigens were also detectable in serum specimens (93.1-100%) from healthy adults. On the contrary, the three antigens were barely detectable in sera from BCG-unvaccinated infants (0-14%). Conclusion: These finding, supported by recent literature, highlight the significant interference of BCG vaccination with the diagnosis of TB disease by direct antigen detection. Nevertheless, the three tested antigens could represent useful TB serum biomarkers in countries where BCG vaccination is not routinely performed.

In vitro Dual Detection of GNPs Conjugated Rabbit IgG Using Anti-IgG Anchored Calcein Green Fluorescent LC Microdroplets

The 4-cyano- $4^{\prime }$ -pentyl biphenyl (5CB) liquid crystals molecules are fascinating materials for the fabrication of sensors as they are able to show a detectable optical signal in presence of small variation in surface forces. The sensitivity of 5CB molecules increased further on using them as microdroplets emulsion as compared to layered structures on plannar solid surfaces. The anchoring of receptor (AIgG) on LC microdroplets makes microdroplets capable to produce species specific optical signal in presence of nonspecific interactions. Gold nanoparticles (GNPs) have increased the interactions of antigen (IgG) with LC microdroplets anchored AIgG. The concomitant emission of calcein green fluorescence on interactions of GNPs anchored esterase with LC microdroplet anchored calcein-AM has provided an evidence to confirm the interactions induced transition from radial to bipolar in LC microdroplets. The AIgG ( $4.2~\mu \text{g}$ , 28 pmol) and calcein-AM ( $4.8~\mu \text{g}$ , 4.55 nmol) anchored LC microdroplets within a size variation of 20- $30~\mu \text{m}$ have shown a lower limit of detection of 0.22 ± 0.001 pmol of IgG in PBS (pH 7.4) within a contact time of 20 minutes. Thus, liquid crystals microdroplets have sufficient scope to develop biosensors for in vitro detection of disease causing antigens in biological fluids.

Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.

Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes.

Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens.

Bioconjugation of peroxidase-like nanostructures with natural enzyme for in-situ amplified conductometric immunoassay of tissue polypeptide antigen in biological fluids

A new conductometric immunoassay based on biofunctionalized prussian blue-gold hybrid nanostructure (PBGN) was designed for sensitive determination of tissue polypeptide antigen (TPA). The immunosensor was prepared by immobilizing anti-TPA capture antibody on the interdigitated transducer, while the PBGN was used for bioconjugation of detection antibody and horseradish peroxidase (HRP). Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the transducer. Accompanying the PBGN, the carried HRP and prussian blue catalyzed iodine ion into iodine relative to H2O2-KI system, thus resulting in the variations of local conductivity. Under optimal conditions, the immunosensor exhibited good conductometric responses toward target TPA, and allowed the detectable concentration as low as 0.28pgmL−1. The precision, specificity and reproducibility were acceptable. Importantly, our strategy was also utilized for analysis of 13 human serum specimens, giving well matched results with those obtained from human TPA ELISA kit.

The potential role of HCV core antigen testing in diagnosing HCV infection

The potential uses of serological tests that detect HCV core antigens in biological fluids are highlighted. The most common serological tests utilized to detect exposure to HCV rely on the detection of antibodies to HCV. However, these tests cannot distinguish between individuals who have resolved their infection and those who remain actively infected with HCV. By contrast, the HCV core antigen test detects circulating HCV core antigen and identifies individuals who are actively infected with HCV. There is increasing interest in using the HCV core antigen test as a reflex test for seropositive individuals to identify individuals who are actively infected with HCV. In addition, the HCV core antigen test can be utilized to detect the early phase of HCV infection prior to the development of antibodies, both in the blood bank setting and in the diagnostic laboratory. Lastly, quantitative versions of the HCV core antigen test can be used to monitor the effectiveness of antiviral therapy.

Carbon nanotube-based symbiotic coaxial nanocables with nanosilica and nanogold particles as labels for electrochemical immunoassay of carcinoembryonic antigen in biological fluids

A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol–gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO2/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO2/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO2/CNTs as detection antibodies. To embody the advantages of the protocol, the analytical properties of variously modified electrodes were compared in detail on the basis of different nanolabels. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12ngmL−1 in pH 5.5 HAc-NaAc containing 5mM H2O2. At a signal-to-noise ratio of 3, the detection limit (LOD) is 5pgmL−1 CEA. Intra- and inter-assay coefficients of variation were below 9.5%. Meanwhile, the selectivity and stability of the immunosensor were acceptable. In addition, the technique was evaluated by spiking CEA standards in pH 7.4 PBS and with 35 clinical serum specimens, receiving excellent accordance with results from commercially available electrochemiluminescent enzyme-linked immunoassay.

Cancer-associated antigens and antigen arrays in serological diagnostics of malignant tumors

The appearance of antibodies to cancer-associated antigens in biological fluids (particularly, in blood sera) of cancer patients is now a well-established fact, and their detection by immunochemical methods is a promising approach to diagnostics of malignant neoplasms. In this review, we consider some immunobiological aspects of the most extensively studied cancer-associated B-cell antigens, various applications of autoantibodies as cancer biomarkers, and prospects for the use of antigen arrays for improving diagnostic sensitivity.

Monoclonal Antibodies Against LipL32, The Major Outer Membrane Protein of PathogenicLeptospira: Production, Characterization, and Testing in Diagnostic Applications

Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids.

Choices aplenty: antifungal prophylaxis in hematopoietic stem cell transplant recipients.

The incidence of invasive fungal infection (IFIs) in hematopoietic stem cell transplantation (HSCT) recipients ranges from 10 to 25% with an overall case fatality rate of up to 70-90%. Candida and Aspergillus genera remain the two most common pathogens. Although fluconazole prophylaxis in this population has been moderately effective in reducing mortality due to invasive candidiasis, this agent does not have activity against invasive aspergillosis (IA) and other mould. Several new agents such as voriconazole and caspofungin have enhanced potency and broad-spectrum antifungal activity and show promising results against yeasts and filamentous fungi when given as therapy and as chemoprophylaxis. Further, new diagnostic tools to detect circulating fungal antigens in biological fluids and PCR-based methods to detect species or genus-specific DNA or RNA have been developed. Incorporating these techniques along with clinical criteria appear to improve the accuracy of preclinical diagnosis of IFIs. Such approaches may alter the current treatment strategy from prophylaxis to pre-emptive therapy, thereby potentially decreasing cost and toxicity in high-risk patients.

Modelling the kinetics of antigen-antibody reactions at particle enhanced optical immunoassays

Functionalized latexes coated by antibodies are used in diagnostic tests for the detection of antigens in biological fluids. A simple kinetic model is presented which is related to the optical monitoring of the formation of specific complexes between antigen and antibody amplified by latex beads. The antibodies are chemically coupled onto chloromethylstyrene (CMST) particles. The kinetic model is able to describe the immunoprecipitin curves of immunolatex beads. The number of fitting parameters is relatively reduced (only three), and the meaning of these parameters can be interpreted in terms of the chemical equilibrium constant, the percentage of active IgG on the latex beads, and optical response. The model explains very well the optical response of immunolatex prepared by covalent coupling of antibodies on polymer carriers.

Latex immunoassays: Comparative studies on covalent and physical immobilization of antibodies. I. F(ab')2 fragments

Colloidal particles coated with antibodies are currently used in diagnostic test systems for the detection of antigens in biological fluids. Immobilization is usually carried out by physical adsorption. Covalent coupling of antibodies to particles, however, offers certain advantages. The present research deals with the study of these possible advantages. A sulphonated polystyrene latex has been used to prepare an immunolatex with physically adsorbed antibodies, while a functionalized latex with chloromethyl groups on the surface has been used for the partly covalent coupling of the antibody (F(ab')2 fragments). The immunoreactivity was studied by measuring the variations in scattered light intensity after mixing a solution of CRP antigen and the sensitized latex. The influence on the immunoresponse of the scattering angle (5, 10, and 20 deg), protein coverage and storage time have been studied for both systems.

A simple kinetic model of antigen-antibody reactions in particle-enhanced light scattering immunoassays

Polymer colloids coated by antibodies are used in diagnostic tests for the detection of antigens in biological fluids. We present in this paper a simple kinetic model for the optical monitoring of the formation of specific complexes between antigen and antibody with amplification by latex beads. The antibodies are physical or chemically adsorbed onto sulphonate polystyrene or chloromethylstyrene particles respectively. This is a very simple model combining the La Mer idea of optimum surface coverage with Langmuir adsorption of antigen molecules. The kinetic model explains reasonably well the optical response of immunolatex prepared by both methods of immobilization of antibodies on polymer carriers. According to the results obtained with this model the percentage of active IgG on latex particles is extremely low, with a maximum of a 5%.

Covalent coupling of antibodies to hydrophilic core-shell particles

Colloidal particles coated with monoclonal antibodies are used in diagnostic test systems for the detection of antigens in biological fluids. Immobilization is usually carried out by physical adsorption. Covalent coupling of antibodies to particles, however, offers, in principle, certain advantages. We present in this paper models for the covalent coupling of antibodies to core-shell particles containing a hydrophilic polymeric shell with functional groups. Clearly two can be distinguished: a repulsion model and an attraction model. Application of core-shell particles coupled with monoclonal antibodies in a latex agglutination immunoassay for the detection of human chorionic gonadotrophin (hCG) appears feasible and opens the opportunity to perform these tests in serum. A detection limit of 200 IU per 1 hCG in urine and in serum is achieved.

Assay of Human Tissue-Type Plasminogen Activator (t-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies to t-PA

An enzyme-linked immunosorbent assay (ELISA) for the measurement of human tissue-type plasminogen activator (t-PA) was developed. Microtiter plates were coated with a mixture of two monoclonal antibodies and bound t-PA was quantitated with a third monoclonal antibody linked to peroxidase. The lower limit of sensitivity of the assay was 0.2 ng of t-PA per ml. The concentration of antigen in citrated plasma of human subjects was found to be 3.4 +/- 0.8 ng/ml. The assay had a good reproducibility with values of 3.8, 6.5 and 4.9 percent respectively for the intra-, inter-assay and inter-dilution variation coefficients. The results of the ELISA assay on plasma samples from patients during thrombolytic therapy with t-PA correlated very well, over a wide concentration range, with those obtained with a previously described two-site immuno-radiometric assay (r = 0.96). This ELISA with monoclonal antibodies constitutes a stable and reproducible set of reagents for the measurement of t-PA antigen in biological fluids, avoiding the disadvantages of the use of radioisotopes and of polyclonal antibodies.

The sensitivities of different immunoassays for detecting leptospiral antigen

The detection of leptospiral antigen in biological fluids is important for the diagnosis of leptospirosis in animals and man. However the sensitivity of dark field microscopy, the usual detection method, is often inadequate. A comparison was made between the sensitivities of several immunological techniques for detecting Leptospira interrogans serovar hardjo. By staphylococcal coagglutination 10(8) leptospires per ml could be detected and by countercurrent immunoelectrophoresis 10(7) per ml. The best sensitivity obtained by enzyme-linked immunosorbent assay was 10(5) leptospires per ml, and by radioimmunoassay 10(4) to 10(5) per ml. Radioimmunoassay offers the prospect of improved diagnosis of leptospirosis through the detection of leptospiral antigen.