A Sensitive and Cost-Effective Chemiluminescence ELISA for Measurement of Amyloid-ß 1-42 Peptide in Human Plasma.

BACKGROUND: Amyloid-ß42 (Aß42) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Studies have suggested the potential utility of plasma Aß42 levels in the diagnosis, and in longitudinal study of AD pathology. Conventional ELISAs are used to measure Aß42 levels in plasma but are not sensitive enough to quantitate low levels. Although ultrasensitive assays like single molecule array or immunoprecipitation-mass spectrometry have been developed to quantitate plasma Aß42 levels, the high cost of instruments and reagents limit their use. OBJECTIVE: We hypothesized that a sensitive and cost-effective chemiluminescence (CL) immunoassay could be developed to detect low Aß42 levels in human plasma. METHODS: We developed a sandwich ELISA using high affinity rabbit monoclonal antibody specific to Aß42. The sensitivity of the assay was increased using CL substrate to quantitate low levels of Aß42 in plasma. We examined the levels in plasma from 13 AD, 25 Down syndrome (DS), and 50 elderly controls. RESULTS: The measurement range of the assay was 0.25 to 500 pg/ml. The limit of detection was 1 pg/ml. All AD, DS, and 45 of 50 control plasma showed measurable Aß42 levels. CONCLUSION: This assay detects low levels of Aß42 in plasma and does not need any expensive equipment or reagents. It offers a preferred alternative to ultrasensitive assays. Since the antibodies, peptide, and substrate are commercially available, the assay is well suited for academic or diagnostic laboratories, and has a potential for the diagnosis of AD or in clinical trials.

Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation.

Convalescent serum with a high abundance of neutralization IgG is a promising therapeutic agent for rescuing COVID-19 patients in the critical stage. Knowing the concentration of SARS-CoV-2 S1-specific IgG is crucial in selecting appropriate convalescent serum donors. Here, we present a portable microfluidic ELISA technology for rapid (15 min), quantitative, and sensitive detection of anti-SARS-CoV-2 S1 IgG in human serum with only 8 µL sample volume. We first identified a humanized monoclonal IgG that has a high binding affinity and a relatively high specificity towards SARS-CoV-2 S1 protein, which can subsequently serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. We then measured the abundance of anti-SARS-CoV-2 S1 IgG in 16 convalescent COVID-19 patients. Due to the availability of the calibration standard and the large dynamic range of our assay, we were able to identify "qualified donors" for convalescent serum therapy with only one fixed dilution factor (200 ×). Finally, we demonstrated that our technology can sensitively detect SARS-CoV-2 antigens (S1 and N proteins) with pg/mL level sensitivities in 40 min. Overall, our technology can greatly facilitate rapid, sensitive, and quantitative analysis of COVID-19 related markers for therapeutic, diagnostic, epidemiologic, and prognostic purposes.

An Intact Cell Bioluminescence-Based Assay for the Simple and Rapid Diagnosis of Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an increase in antibiotic resistance following therapy. To address these shortcomings, rapid bioluminescence assays were developed and evaluated for the detection of UTI using intact, viable cells of Photobacterium mandapamensis USTCMS 1132 or previously lyophilized cells of Photobacterium leiognathi ATCC 33981™. Two platform technologies-tube bioluminescence extinction technology urine (TuBETUr) and cellphone-based UTI bioluminescence extinction technology (CUBET)-were developed and standardized using artificial urine to detect four commonly isolated UTI pathogens-namely, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Candida albicans. Besides detection, these assays could also provide information regarding pathogen concentration/level, helping guide treatment decisions. These technologies were able to detect microbes associated with UTI at less than 105 CFU/mL, which is usually the lower cut-off limit for a positive UTI diagnosis. Among the 29 positive UTI samples yielding 105-106 CFU/mL pathogen concentrations, a total of 29 urine specimens were correctly detected by TuBETUr as UTI-positive based on an 1119 s detection window. Similarly, the rapid CUBET method was able to discriminate UTIs from normal samples with high confidence (p ≤ 0.0001), using single-pot conditions and cell phone-based monitoring. These technologies could potentially address the need for point-of-care UTI detection while reducing the possibility of antibiotic resistance associated with misdiagnosed cases of urinary tract infections, especially in low-resource environments.

Rapid detection of trace Salmonella in milk and chicken by immunomagnetic separation in combination with a chemiluminescence microparticle immunoassay.

Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 µm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17-1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens. Graphical abstract.

What are the Main Sensor Methods for Quantifying Pesticides in Agricultural Activities? A Review.

In recent years, there has been an increase in pesticide use to improve crop production due to the growth of agricultural activities. Consequently, various pesticides have been present in the environment for an extended period of time. This review presents a general description of recent advances in the development of methods for the quantification of pesticides used in agricultural activities. Current advances focus on improving sensitivity and selectivity through the use of nanomaterials in both sensor assemblies and new biosensors. In this study, we summarize the electrochemical, optical, nano-colorimetric, piezoelectric, chemo-luminescent and fluorescent techniques related to the determination of agricultural pesticides. A brief description of each method and its applications, detection limit, purpose-which is to efficiently determine pesticides-cost and precision are considered. The main crops that are assessed in this study are bananas, although other fruits and vegetables contaminated with pesticides are also mentioned. While many studies have assessed biosensors for the determination of pesticides, the research in this area needs to be expanded to allow for a balance between agricultural activities and environmental protection.

Multiple signal amplification chemiluminescence immunoassay for chloramphenicol using functionalized SiO2 nanoparticles as probes and resin beads as carriers.

A novel, rapid and convenient competitive immunoassay for ultrasensitive detection of chloramphenicol residues in shrimp and honey was established combined with flow injection chemiluminescence. The carboxylic resin beads were used as solid phase carriers to load with more coating antigen due to their larger specific surface area and good biocompatibility. The surface of the silica dioxide nanoparticles was modified with aldehyde group to combine with more horseradish peroxidase and the chloramphenicol antibody. There was a competitive process between the chloramphenicol in solution and the immobilized coating antigen to combine with the limited binding site of antibody to form the immunocomplex. Silica dioxide nanoparticles played an important role in enhancing chemiluminescence signal, because the horseradish peroxidase on SiO2 effectively catalyzed the system of luminol-PIP-H2O2. Under optimal conditions, the chemiluminescence intensity decreased linearly with the logarithm of the chloramphenicol concentration in the range of 0.0001 to 100 ng mL-1 and the detection limit (3σ) was 0.033 pg mL-1. This immunosensor demonstrated acceptable stability, high specificity and reproducibility. The horseradish peroxidase-silica dioxide nanoparticle-chloramphenicol antibody complex successfully prepared in this article was firstly applied to the detection of chloramphenicol, and had extremely important meanings for the application of nanoparticles and enzymatic catalysis in the field of chemiluminescence.

A facile chemiluminescence sensing for ultrasensitive detection of heparin using charge effect of positively-charged AuNPs.

Heparin is a glycosaminoglycan with the highest negative charge density of any known biological molecule. Herein, this highly negative charge structure of heparin and the charge effect from positively-charged AuNPs for luminol chemiluminescence (CL) reaction were combined to build a facile and sensitive CL strategy for detection of heparin. The highly negative charge structure of heparin molecules (four negatively-charged side groups per repeat unit) and the effective signal amplification of charge effect from positively-charged AuNPs make this analysis to display high sensitivity for heparin detection, and the detection limit is as low as 0.06 ng/mL. It is about two orders of magnitude lower than the previously reported colorimetric assay and far lower than the current analysis methods. The established CL strategy is to use the electrostatic interaction between heparin and signal probe (positively-charged AuNPs). Since polyanionic heparin has the highest negative charge in biological system, this CL sensing shows high selectivity for the detection of heparin, and hyaluronic acid (HA), an analogue of heparin, cannot cause interference. This CL sensing succeeded in detecting heparin in human serum samples. Besides, polycationic protamine, heparin antidote, can respond to the system's CL signals through its strong interactions with heparin, thus indirectly detecting protamine. For protamine in serum samples, the detection result was basically consistent with Coomassie brilliant blue assay.

Succinylated Jeffamine ED-2003 coated polycarbonate chips for low-cost analytical microarrays.

Analytical microarrays feature great capabilities for simultaneous detection and quantification of multiple analytes in a single measurement. In this work, we present a rapid and simple method for bulk preparation of microarrays on polycarbonate sheets. Succinylated Jeffamine® ED-2003 was screen printed on polycarbonate sheets to create a polyfunctional shielding layer by baking at 100 °C. After microdispension of capture probes (antibodies, oligonucleotides, or small molecules) in a microarray format, chips were assembled with a flow cell from double-sided tape. It was shown that the shielding layer was firmly coated and suppressed unspecific binding of proteins. Universal applicability was demonstrated by transferring established flow-based chemiluminescence microarray measurement principles from glass slides to polycarbonate chips without loss of analytical performance. Higher chemiluminescence signals could be generated by performing heterogeneous asymmetric recombinase polymerase amplification on polycarbonate chips. Similar results could be shown for sandwich microarray immunoassays. Beyond that, lower inter- and intra-assay variances could be measured for the analysis of Legionella pneumophila Serogroup 1, strain Bellingham-1. Even surface regeneration of indirect competitive immunoassays was possible, achieving a limit of detection of 0.35 ng L-1 for enrofloxacin with polycarbonate microarray chips. Succinylated Jeffamine ED-2003 coated polycarbonate chips have great potential to replace microtiter plates by flow-based chemiluminescence microarrays for rapid analysis. Therefore, it helps analytical microarrays to advance into routine analysis and diagnostics. Graphical abstract ᅟ.

Development of a cost effective and robust AlphaScreen® platform for application.

The use of AlphaScreen® detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many labs have focused on miniaturization, while there have been limited efforts to scale down Alpha reagents. Thus, we describe the optimization of an AlphaScreen detection platform by systematically reducing the Alpha reagents down to 2.5 µg/ml beads, without compromising assay integrity. We demonstrate that reducing bead concentration reduces detection costs substantially while yielding robust statistics. We believe this simple new protocol will enhance the future utilization of AlphaScreen technology in high-throughput screening.

Efficient label-free chemiluminescent immunosensor based on dual functional cupric oxide nanorods as peroxidase mimics.

Dual-functional cupric oxide nanorods (CuONRs) as peroxidase mimics are proposed for the development of a flow-through, label-free chemiluminescent (CL) immunosensor. Forming the basis of this cost-efficient, label-free immunoassay, CuONRs, synthesized using a simple hydrothermal method, were deposited onto epoxy-activated standard glass slides, followed by immobilization of biotinylated capture antibodies through a streptavidin bridge. The CuONRs possess excellent catalytic activity, along with high stability as a solid support. Antigens could then be introduced to the sensing system, forming large immunocomplexes that prevent CL substrate access to the surface, thereby reducing the CL signal in a concentration dependent fashion. Using carcinoembryonic antigen (CEA) as a model analyte, the proposed label-free immunosensor was able to rapidly determine CEA with a wide linear range of 0.1-60ngmL-1 and a low detection limit of 0.05ngmL-1. This nanozyme-based immunosensor is simple, sensitive, cost-efficient, and has the potential to be a very promising platform for fast and efficient biosensing applications.

Usefulness of a single-assay chemiluminescence test (Tularaemia VIRCLIA IgG + IgM monotest) for the diagnosis of human tularemia. Comparison of five serological tests.

The aim of this work was to ascertain the usefulness of a new commercially-available single-assay chemiluminescence test (CHT) for the diagnosis of human tularemia (Tularaemia VIRCLIA IgG + IgM monotest, Vircell, Santa Fe, Granada, Spain). A total of 773 sera from 773 patients including 364 initial sera from patients with diagnosed tularemia, patients with suspected tularemia not confirmed (100), healthy people (152), patients with serology positive to Brucella (97), patients diagnosed with other infectious diseases (30), and patients diagnosed with autoimmune diseases (30) were included. All sera were tested by CHT, "in-house" microagglutination test (MAT), immunochromatographic test (ICT) (Virapid Tularaemia, Vircell, Santa Fe Granada, Spain), and "in-house" ELISA IgG, and ELISA IgM. Of the total initial sera, 334 (sensitivity 91.8%) were positive in the CHT, 332 (sensitivity 91.2%) in the MAT, 330 (sensitivity 90.7%) in the ICT, and 328 (sensitivity 90.1%) in the ELISA IgG and ELISA IgM tests. The specificity of the CHT was 96.7%; of the MAT, 100%; of the ICT, 98.7%; and of the ELISA IgG and ELISA IgM, 97.4%. In the group of patients with serology positive to Brucella, at least 12.4% of sera were positive in tularemia tests (12.4% in ELISA IgM, 13.4% in MAT, 14.4% in ICT, and 15.5% in CHT and ELISA IgG). In conclusion, CHT presents a sensitivity and specificity in early diagnosis of human tularemia, similar to MAT, ICT, and ELISA IgG and ELISA IgM. Its single assay design allows lower costs, especially in areas of low endemicity or inter-epidemic periods.

GloSensor assay for discovery of GPCR-selective ligands.

G protein-coupled receptors (GPCRs) are modulators of almost every physiological process, and therefore, are most favorite therapeutic target for wide spectrum of diseases. Ideally, high-throughput functional assays should be implemented that allow the screening of large compound libraries in cost-effective manner to identify agonist, antagonist, and allosteric modulators in the same assay. Taking advantage of the increased understanding of the GPCR structure and signaling, several commercially available functional assays based on fluorescence or chemiluminescence detection are being used in both academia and industry. In this chapter, we provide step-by-step method and guidelines to perform cAMP measurement using GloSensor assay. Finally, we have also discussed the analysis and interpretation of results obtained using this assay by providing several examples of Gs- and Gi-coupled GPCRs.

Strategies to improve the efficiency of celiac disease diagnosis in the laboratory.

The demand for testing to detect celiac disease (CD) autoantibodies has increased, together with the cost per case diagnosed, resulting in the adoption of measures to restrict laboratory testing. We designed this study to determine whether opportunistic screening to detect CD-associated autoantibodies had advantages compared to efforts to restrict testing, and to identify the most cost-effective diagnostic strategy. We compared a group of 1678 patients in which autoantibody testing was restricted to cases in which the test referral was considered appropriate (G1) to a group of 2140 patients in which test referrals were not reviewed or restricted (G2). Two algorithms A (quantifying IgA and Tissue transglutaminase IgA [TG-IgA] in all patients), and B (quantifying only TG-IgA in all patients) were used in each group, and the cost-effectiveness of each strategy was calculated. TG-IgA autoantibodies were positive in 62 G1 patients and 69 G2 patients. Among those positive for tissue transglutaminase IgA and endomysial IgA autoantibodies, the proportion of patients with de novo autoantibodies was lower (p=0.028) in G1 (11/62) than in G2 (24/69). Algorithm B required fewer determinations than algorithm A in both G1 (2310 vs 3493; p<0.001) and G2 (2196 vs 4435; p<0.001). With algorithm B the proportion of patients in whom IgA was tested was lower (p<0.001) in G2 (29/2140) than in G1 (617/1678). The lowest cost per case diagnosed (4.63 euros/patient) was found with algorithm B in G2. We conclude that opportunistic screening has advantages compared to efforts in the laboratory to restrict CD diagnostic testing. The most cost-effective strategy was based on the use of an appropriate algorithm.

Efficacy of benzalkonium chloride against bioluminescent P. aeruginosa ATCC9027 constructs.

Whole cell biosensors have been seldom used in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET). According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to assess the applicability of Pseudomonas aeruginosa ATCC9027 and its validated bioluminescent strains for preservative efficacy tests using benzalkonium chloride (BKC). Applicability of five constitutively-expressed bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication, bioluminescence and fluorescence in a three-way study. PET using BKC showed no significant difference between bioluminescence and enumeration. Good correlations between bioluminescence, colony-forming units (CFU) count and fluorescence were obtained for BKC concentrations (R>0.9) between 0.0003-0.0025% against strains containing the constructs lys-pMElux, lpp-pMElux and tat-pMElux. Furthermore, two-way ANOVA analysis showed that the bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003-0.01%) during preservative efficacy tests. PET testing with BKC showed that tat-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence; P. aeruginosa ATCC9027 tatH5-pMElux is the best construct for testing various antimicrobial agents.

Determination of veterinary penicillin antibiotics by fast high-resolution liquid chromatography and luminescence detection.

A chromatographic method based on the use of a fused-core column and luminescence detection is described for the determination of six penicillin antibiotics used in veterinary practice, namely amoxicillin, ampicillin, penicillin G, oxacillin, cloxacillin and nafcillin. The use of this column provides the separation of these antibiotics with retention times lower than 4.5min. The tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)32+] - Ce(IV) system has been used as post-column derivatization reagent, obtaining a luminescence signal (λem 610nm) proportional to the analyte concentration when the system is excited at 450nm. The dynamic ranges of the calibration graphs are 100-10,000ngmL-1 for all the antibiotics assayed and the limits of detection are in the range of 44-51ngmL-1. The precision, established at two concentration levels of each analyte and expressed as the percentage of the relative standard deviation is in the range of 6.9-9.8%. The method has been satisfactorily applied to the analysis of water and pharmaceutical samples, with recoveries ranging from 88.6% to 108.5%.

Simultaneous electrochemiluminescence determination of galanthamine, homolycorine, lycorenine, and tazettine in Lycoris radiata by capillary electrophoresis with ultrasonic-assisted extraction.

After ultrasonic-assisted extraction, four lycoris radiata alkaloids: galanthamine, homolycorine, lycorenine, and tazettine were determined by capillary electrophoresis electrochemiluminescence. Polyvinylpyrrolidone was added to the running buffer (RB) to obtain better resolution. Experimental conditions influencing the determination were examined, including the additives, detection potential, separation voltage, injection voltage and time, and RB pH and concentration. Under optimal experimental conditions, the baseline separation of the four alkaloids occurred within 16min. The proposed method displayed the following linear ranges (in ng/mL): galanthamine [60-5000], homolycorine [40-5000], lycorenine [5.0-1500], and tazettine [8.0-2500]. The detection limits in ng/mL, (S/N=3), were galanthamine [14], homolycorine [11], lycorenine [1.8], and tazettine [3.1]. Intra-day and inter-day RSDs for the four alkaloids of the six replicates were less than 2.7% and 3.1%, respectively. The recoveries in% were: tazettine [102.5], lycorenine [98.20], galanthamine [97.30], and homolycorine [98.33].

Video approach to chemiluminescence detection using a low-cost complementary metal oxide semiconductor (CMOS)-based camera: determination of paracetamol in pharmaceutical formulations.

A new system for continuous flow chemiluminescence detection, based on the use of a simple and low-priced lens-free digital camera (with complementary metal oxide semiconductor technology) as a detector, is proposed for the quantitative determination of paracetamol in commercial pharmaceutical formulations. Through the camera software, AVI video files of the chemiluminescence emission are captured and then, using friendly ImageJ public domain software (from National Institutes for Health), properly processed in order to extract the analytical information. The calibration graph was found to be linear over the range 0.01-0.10 mg L-1 and over the range 1.0-100.0 mg L-1 of paracetamol, the limit of detection being 10 µg L-1. No significative interferences were found. Paracetamol was determined in three different pharmaceutical formulations: Termalgin®, Efferalgan® and Gelocatil®. The obtained results compared well with those declared on the formulation label and with those obtained through the official analytical method of British Pharmacopoeia. Graphical abstract Abbreviated scheme of the new chemiluminescence detection system proposed in this paper.

A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3 O4 @SiO2 magnetic nanoparticles.

A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe3 O4 @SiO2 magnetic nanoparticles (Fe-MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe-MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA-CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe-MNPs (amine-functionalized Fe3 O4 @SiO2 and carboxylic acid-functionalized Fe3 O4 @SiO2 ). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC50 ) values of the two methods were about 0.024 ng ml-1 and 0.046 ng ml-1 respectively and LODs were approximately 0.0002 ng ml-1 and 0.001 ng ml-1 respectively. These methods were much more sensitive than that of any traditional enzyme-linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe-MNPs.

A low-cost smartphone-based platform for highly sensitive point-of-care testing with persistent luminescent phosphors.

Through their computational power and connectivity, smartphones are poised to rapidly expand telemedicine and transform healthcare by enabling better personal health monitoring and rapid diagnostics. Recently, a variety of platforms have been developed to enable smartphone-based point-of-care testing using imaging-based readout with the smartphone camera as the detector. Fluorescent reporters have been shown to improve the sensitivity of assays over colorimetric labels, but fluorescence readout necessitates incorporating optical hardware into the detection system, adding to the cost and complexity of the device. Here we present a simple, low-cost smartphone-based detection platform for highly sensitive luminescence imaging readout of point-of-care tests run with persistent luminescent phosphors as reporters. The extremely bright and long-lived emission of persistent phosphors allows sensitive analyte detection with a smartphone by a facile time-gated imaging strategy. Phosphors are first briefly excited with the phone's camera flash, followed by switching off the flash, and subsequent imaging of phosphor luminescence with the camera. Using this approach, we demonstrate detection of human chorionic gonadotropin using a lateral flow assay and the smartphone platform with strontium aluminate nanoparticles as reporters, giving a detection limit of ≈45 pg mL-1 (1.2 pM) in buffer. Time-gated imaging on a smartphone can be readily adapted for sensitive and potentially quantitative testing using other point-of-care formats, and is workable with a variety of persistent luminescent materials.

Nitrogen-doped graphene-chitosan matrix based efficient chemiluminescent immunosensor for detection of chicken interleukin-4.

Chicken interleukin-4 (ChIL-4), which is released by activated type 2 helper (Th2) cells following their stimulation in vitro, is an important indicator for the study of cell-mediated immunity in chickens after infection or vaccination. In this work, the first ChIL-4 chemiluminescent (CL) immunosensor was developed via the immobilization of monoclonal ChIL-4 antibodies on a nitrogen-doped graphene (NG)-chitosan nanocomposite matrix. NG nanosheets were used for the first time in the CL immunoassay to provide a biocompatible microenvironment for the immobilized capture antibody. The ChIL-4 immunosensor was characterized systematically. The proposed immunosensor displayed a wide linear range from 0.05 to 70ngmL-1 and a low detection limit of 0.02ngmL-1 at a signal-to-noise ratio of 3. Compared to traditional assay methods, this system was more flexible, simple, rapid, and sensitive. Moreover, this CL immunoassay system had an excellent detection and fabrication reproducibility, a high specificity, an acceptable accuracy, and a high stability. This work enables the specific detection of ChIL-4 and the further study of its role in the immune responses of poultry.