One-step Enzyme-linked Immunosorbent Assay Research Articles

Development of alkaline phosphatase-scFv and its use for one-step enzyme-linked immunosorbent assay for His-tagged protein detection.

A histidine (His)-tag is composed of six His residues and typically exerts little influence on the structure and solubility of expressed recombinant fusion proteins. Purification methods for recombinant proteins containing His-tags are relatively well-established, thus His-tags are widely used in protein recombination technology. We established a one-step enzyme-linked immunosorbent assay (ELISA) for His-tagged recombinant proteins. We analyzed variable heavy and light chains of the anti-His-tag monoclonal antibody 4C9 and used BLAST analyses to determine variable zones in light (VL) and heavy chains (VH). VH, VL, and alkaline phosphatase (ALP) regions were connected via a linker sequence and ligated into the pGEX-4T-1 expression vector. Different recombinant proteins with His tags were used to evaluate and detect ALP-scFv activity. Antigen and anti-His-scFv-ALP concentrations for direct ELISA were optimized using the checkerboard method. ZIKV-NS1, CHIKV-E2, SCRV-N, and other His-tag fusion proteins demonstrated specific reactions with anti-His-scFv-ALP, which were accurate and reproducible when the antigen concentration was 50 µg mL-1 and the antibody concentration was 6.25 µg mL-1. For competitive ELISA, we observed a good linear relationship when coating concentrations of recombinant human anti-Müllerian hormone (hAMH) were between 0.78 and 12.5 µg mL-1. Our direct ELISA method is simple, rapid, and accurate. The scFv antibody can be purified using a prokaryotic expression system, which provides uniform product quality and reduces variations between batches.

A handheld testing device for the fast and ultrasensitive recognition of cardiac troponin I via an ion-sensitive field-effect transistor

Cardiac troponin I (cTnI) is an efficient and specific biomarker for the accurate diagnosis of acute myocardial infarction (AMI), one of the diseases with the highest mortality worldwide. Due to the short course and high fatality of this disease, a rapid, accurate and portable device for quantitative detection is urgently needed for early diagnosis and treatment. In this work, we designed a handheld device based on a dual-gate ion-sensitive field-effect transistor (ISFET) for early and accurate warning of AMI through cTnI detection. A one-step enzyme-linked immunosorbent assay strategy was proposed for use in this device to recognize trace cTnI in serum, converting the cTnI concentration to a drain-source current generated by an ultrasensitive ISFET. This portable device exhibited an ultrahigh sensitivity of 132 pA pg−1·mL−1, a wide linear range from 1 to 1000 pg/mL that enabled coverage far exceeding the threshold level (280 pg/mL), and a low detection limit of 0.3 pg/mL for the cTnI assay, which was much lower than the current diagnostic cut-off for a healthy control level for AMI (40 pg/mL). In addition, this handheld device showed satisfactory selectivity and reliable results in the analysis of real serum within 20 min, indicating its potential applications in early screening and diagnosis for the clinical evaluation of AMI.

One-step icELISA developed with novel antibody for rapid and specific detection of diclazuril residue in animal-origin foods

ABSTRACT Diclazuril, a broad-spectrum anticoccidial drug, may be accumulated in edible tissues of animals through illegal use, which poses potential threats to human health through the food chain. In this study, an innovative hapten was designed and an immunogen of diclazuril was successfully synthesised with keyhole limpet haemocyanin as carrier protein; then a monoclonal antibody with high specificity was obtained. Furthermore, based on the novel antibody, a one-step indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for rapid and specific detection of diclazuril residues. Compared with the traditional icELISA method, this method saves at least 0.5 hours and one washing step. Under the optimal conditions, the one-step icELISA for diclazuril exhibited good performance with a 50% inhibition concentration (IC50) value of 0.952 μg/kg. The average recoveries of the icELISA ranged from 73.1% to 115.5% with the coefficient of variation lower than 12.7%, which was evaluated by detecting spiked animal-origin food samples. Finally, the one-step icELISA shows a good correlation with an ultra-high liquid chromatography-tandem mass spectrometry method. Those results demonstrate that the one-step icELISA developed for diclazuril detection is time-saving, low-cost, specific, sensitive, and reliable. It shows good potential for social, environmental, and economic benefits in future use.

Determination of sulfamethazine in swine serum with one-step ELISA test kit

Sulfamethazine is a suspected carcinogen. It is widely used as feed additive for swine rations. An effective way of reducing this high incidence of sulfamethazine violations in swine would be to implement a nationwide slaughterhouse surveillance program for the drug. The traditional method for the detection of sulfamethazine spends too much time. A rapid screening test of sulfamethazine residue in swine serum was made by the use of the sulfamethazine one-stop Enzyme-Linked Immunosorbent Assay (ELISA) Kit. The time for sample preparation and reaction in microwells and read in ultrascan meter was about 30 minutes. The detection limit of sulfamethazine was 0.001 ppm. The mean recoveries of added sulfamethazine from serum at levels 0.04, 0.2, and 0.42 μg/ml were 99.8% (coefficient of variation, CV = 2.92%). Among 11 sulfonamide analogus: sulfamethazine, sulfamerazine, sulfadimethoxine, sulfamonomethoxine, sulfathiazole, sulfamethoxypyridazine, sulfaquinoxaline, sulfamethoxazole, sulfapyridine, sulfisoxazole and sulfadiazine with 4 different concentrations: 0.001, 0.01, 0.1 and 1.0 μg/ml had cross reaction to antibodies in the ELISA Kit. The relative reactivity of sulfamethazine, sulfamerazine, sulfisoxazole and sulfapyridine were 100, 10.0, 3.4 and 2.0%, respectively. The concentration of sulfonamide below 0.1 μg/ml, the cross reaction was neglected. This ELISA kit may be used as a rapid screening test for sulfamethazine residue in swine serum.

Clinical, host-derived immune biomarkers and microbiological outcomes with adjunctive photochemotherapy compared with local antimicrobial therapy in the treatment of peri-implantitis in cigarette smokers.

The aim of this study was designed to assess the peri-implant oral hygiene parameters, clinical, radiographic, host-derived immune biomarkers and microbiological levels after photochemotherapy (PCT) and local antibiotic therapy (LAT) in peri-implantitis lesions among cigarette smokers. Fifty current cigarette smokers with peri-implantitis were divided into two groups: PCT and LAT. Test implants received PCT that consisted of toluidine blue photosensitizer and application of 660 nm diode laser with a total of 100 mW power and 124.3 W/cm2 energy using continuous mode of irrigation for 60 s. Control implants received one-unit subgingival application of metronidazole gel in viscous consistency. Clinical measurements included the assessment of plaque scores (PS), bleeding on probing (BOP), probing depth (PD) and clinical attachment level (CAL). Intraoral standardized digital peri-apical radiographs were taken at baseline and at 12 months. Interleukin (IL)-1β and matrix metalloproteinase (MMP)-1 in the PICF were determined using the manufacturers guide from one-step enzyme-linked immunosorbent assay. Real-time polymerase chain reaction (PCR) for Parvimonas micra, Porphyromonas gingivalis and Tannerella forsythia was performed and counts evaluated at baseline, 3, 6 and 12-months. Plaque scores reduced in both groups (p < 0.05). Mean BOP percentage significantly increased in both the groups at 1-month follow-up compared to baseline. Following this period, BOP showed reduction from 1-month to consecutive follow-up periods. The PD significantly reduced in both the groups with no statistically significant difference when compared between PCT and LAT groups at follow-up (p > 0.05). CAL did not change over the period and between both groups (p > 0.05). The differences from baseline to 12 months and between the groups for mesial and distal crestal bone levels did not show any statistical significance (p > 0.05). The levels of IL-1β significantly dropped from baseline to 12 months in the LAT group (p < 0.05). However, for PCT groups, the levels of IL-1β significantly reduced only at 12-month visit follow-up (p < 0.05). MMP-1 showed statistically significant reduction at 9 and 12-months compared with baseline for LAT group. Both P. gingivalis and T. forsythia showed statistically significant reduction in both the groups when values were compared from baseline to 3-, 6- and 12-months follow-up (p < 0.05). However, these differences were not significant when compared between the groups (p > 0.05). Both PCT and LAT showed equal efficacies in improving clinical, host-derived immune biomarkers and microbiological parameters in peri-implant infection in cigarette smokers.

Rapid one-step enzyme immunoassay and lateral flow immunochromatographic assay for colistin in animal feed and food

BackgroundColistin (polymyxin E) is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention, treatment, and growth promotion. However, the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings, and its residue in animal-origin food may also pose serious health hazards to humans. Thus, it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food.ResultsA one-step enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic assay (LFIA) for colistin were developed based on a newly developed monoclonal antibody. The ELISA showed a 50% inhibition value (IC50) of 9.7 ng/mL with assay time less than 60 min, while the LFIA had a strip reader-based detection limit of 0.87 ng/mL in phosphate buffer with assay time less than 15 min. For reducing the non-specific adsorption of colistin onto sample vial, the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy. The spiked recovery experiment showed that the recoveries of colistin from feed, milk and meat samples were in the range of 77.83% to 113.38% with coefficient of variations less than 13% by ELISA analysis and less than 18% by LFIA analysis, respectively. Furthermore, actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis.ConclusionsThe developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.

Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin.

A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production of the specific polyclonal antibody, which reacted well with TTX and its analogs, 4-epiTTX, 11-oxoTTX and 5,6,11-trideoxyTTX, except for 4,9-anhydroTTX. The enzyme-linked immunosorbent assay (ELISA) system using this specific antibody was also developed in the present study. This newly developed polyclonal antibody with analytical procedures using direct one-step ELISA is useful to detect TTX and its analogs in toxic organisms and also disclose the mechanisms involved in their metabolic pathways and accumulation of TTX.

Clinical Application of Loop-mediated Isothermal Amplification in Detection of Mycoplasma Pneumoniae

Objective To explore the clinical value of one-step visualization loop-mediated isothermal amplification(LAMP)in the detection of Mycoplasma pneumoniae(Mp). Methods One-step visualized LAMP,polymerase chain reaction(PCR),and enzyme-linked immunosorbent assay(ELISA)were used to simultaneously detect 108 clinical Mp specimens in children,which included 73 cases of Mp infection diagnosed by PCR and 35 cases of other chronic/acute respiratory tract infections.On the first day of admission,one-step visualization LAMP,PCR(fluorimetric method),and ELISA were used to test the throat swab and serum sample obtained from the same patient,and the Kappa value was calculated.The consistence between LAMP and PCR and that between LAMP and ELISA were compared.On the fifth day of admission,40 patients were resampled and the findings of these three tests on the first day and on the fifth day were compared. Results One-step visualization LAMP had a sensitivity of 100% and a specificity of 94.3%,whereas ELISA had a sensitivity of 65.8% and a specificity of 82.9%.The ratio of Kappa camparing one-step visualization LAMP and PCR was 0.956 and the ratio of Kappa camparing one-step visualization LAMP and ELISA was 0.38.The number of positive specimens detected by LAMP was higher than that by ELISA on the first day. Conclusions One-step visualization LAMP has excellent sensitivity and specificity in detecting early acute Mp infection.It has high consistency with PCR and can be applied to detect Mp.

One-step immunoassay for the insecticide carbaryl using a chicken single-chain variable fragment (scFv) fused to alkaline phosphatase

Immunoassays provide a high-throughput method for monitoring pesticides in foods and the environment. Due to easy generation and capable of being manipulated, chicken single-chain variable fragment (scFv) is attractive in the development of immunoassays for pesticides. Two scFvs (X1 and X2) against the insecticide carbaryl were generated from a chicken immunized with hapten C1 conjugated to keyhole limpet hemocyanin and fused with alkaline phosphatase (AP) to develop a rapid one-step enzyme-linked immunosorbent assay for this pesticide. X2-AP showed higher binding affinity to carbaryl than X1-AP. The X2-AP-based ELISA had a half-maximum signal inhibition concentration of 15 ng mL−1 and a limit of detection of 1.6 ng mL−1. This assay showed negligible cross-reactivity with other carbamate pesticides (<0.1%) and low cross-reactivity with 1-naphthol (5%). The average recoveries of carbaryl spiked in soil, apple and pear samples by the one-step assay ranged from 90% to 114% and agreed well with those of high-performance liquid chromatography. The chicken scFv-based assay showed promise as a high-throughput screening tool for carbaryl in environmental and food matrices.

Development of a one-step immunoassay for triazophos using camel single-domain antibody-alkaline phosphatase fusion protein.

Triazophos is mainly used in Asian and African countries for the control of insects in agricultural production. Camelid variable domains of heavy-chain antibodies (VHHs) show great promise in monitoring environmental chemicals such as pesticides. To improve the rate of success in the generation of VHHs against triazophos, genes specifically encoding VHH fragments from the unique allotype IgG3a of an immunized Camelus bactrianus were amplified by using a pair of novel primers and introduced to construct a diverse VHH library. Five out of seven isolated positive clones, including the VHH T1 with the highest affinity to triazophos, were derived from the allotype IgG3a. A one-step enzyme-linked immunosorbent assay (ELISA) using VHH T1 genetically fused with alkaline phosphatase (AP) had a half-maximum inhibition concentration of 6.6ng/mL for triazophos. This assay showed negligible cross-reactivity with a list of important organophosphate pesticides (< 0.1%). The average recoveries of triazophos from water, soil, and apple samples determined by the one-step ELISA ranged from 83 to 108%, having a good correlation with those by a gas chromatography mass spectrometry (R2 = 0.99). The VHH-AP fusion protein shows potential for the analysis of triazophos in various matrices.

Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice.

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL−1 and a limit of detection of 0.059 ng mL−1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

OR-029 Effects of BCAA Plus Glucose Supplement Timing on Inflammatory Response Indicators after a Resistant Exercise

OR-029 Effects of BCAA Plus Glucose Supplement Timing on Inflammatory Response Indicators after a Resistant Exercise

Production and characterization of a single-chain variable fragment-alkaline phosphatase fusion protein for glycocholic acid detection in a one-step enzyme-linked immunosorbent assay

One-step enzyme-linked immunosorbent assay for glycocholic acid based on single-chain variable fragment-alkaline phosphatase fusion protein.

A Cell Line for Detection of Botulinum Neurotoxin Type B.

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

Development of an automated wax-printed paper-based lateral flow device for alpha-fetoprotein enzyme-linked immunosorbent assay

In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL−1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1–100ngmL−1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA.

Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

ABSTRACTHepatitis B virus (HBV) infection is a worldwide health problem. More than 400 million people are chronic HBV carriers in the world. Infected individuals are at a high risk of developing liver cirrhosis and hepatocellular carcinoma as the main consequences of HBV. The discoveries of fast diagnostic systems and new therapeutic applications are crucial in the fight against viral hepatitis. In this paper we present the generation of a single-chain variable fragment (scFv) from a mouse monoclonal antibody specific to the HBV surface antigen (HBsAg) and demonstrate its expression as a bacterial alkaline phosphatase (AP) fusion protein. In this study, we constructed scFvs from hybridoma cells expressing HBsAg-specific antibody using phage display technology and expressed them in Escherichia coli. The anti-HBsAg scFvs were inserted into pQE-2 vector to produce scFv antibody genetically fused to bacterial AP. Reproducibility of the recombinant HBsAg-scFv fusion protein was tested using Enzyme-linked Immunosorbent Assay (ELISA). Present preliminary findings indicate that the anti-HBsAg-scFv AP conjugate could be further used for the development of one-step ELISA for the detection of HBV.

Sensitivity comparison between rapid one-step test strip and ELISA methods in detection of HBsAg among selected chronic liver disease patients in University of Calabar Teaching hospital, Calabar, Nigeria

Background: Hepatitis B virus (HBV) is the eloquent hepatic infection that leads to hepatocellular carcinoma. This study aims to determine the frequency of HBV by screening selected chronic liver disease (CLD) patients alongside few apparently healthy subjects who might be carriers and pose a high risk in HBV transmission. This study also aims to compare the sensitivity between two diagnostic tests; one step rapid test strip device and Enzyme Linked Immunosorbent Assay (ELISA).Methodology: Serum of 111 subjects comprising of 76 CLD patients and 35 apparently healthy (control) subjects was screened for Hepatitis B surface Antigen (HBsAg).Results: Sero-positivity of the two methods of detecting HBsAg was higher in age group 26-35 years and the male folk was found higher in number than the females. The seropositivity of HBV was 28.8% and 81.1% while seronegativity was 71.2% and 18.9% for rapid test strip test device and ELISA test methods, respectively. All the CLD patients 76(100%) and 14 out of 35 of the apparently healthy subjects was positive for ELISA test method.Conclusion: In conclusion, comparing the two methods, ELISA test is more sensitive than one step rapid test strip device for detecting HBsAg in University of Calabar Teaching Hospital, Nigeria.Therefore, we recommend strongly, thatELISA method be used to confi rm test results obtained from the one step rapid test strip,when screen for CLD.DOI: http://dx.doi.org/10.3126/ajms.v6i5.11851 Asian Journal of Medical Sciences Vol.6(5) 2015 56-60

A simple, rapid one-step ELISA using antibody-antibody complex.

The enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnostic testing and biological research. However, its time-consuming operation is a major drawback. The present work aims to establish a one-step ELISA method through the preparation of a primary antibody (Ab)-secondary Ab complex (Ab-Ab complex) in hopes of realizing more sensitive and faster detection of the trace amount of antigen (Ag). By controlling the mole ratio of the primary Ab to the secondary Ab, one-step ELISA can be successfully achieved. Compared with the traditional ELISA, the one-step ELISA could not only improve the detection sensitivity to 1ngmL(-1) , but also reduce the operating time by 30%. Moreover, the signal intensity can be controlled by adjusting the ratio of the secondary Ab in the complex or by changing the color development time. This technique is further optimized to detect trace amounts of proteins adsorbed onto poly(N-vinylpyrrolidone) (PVP)-modified silicon surfaces (Si-PVP), and the results are close to the radiolabeling method. It is concluded that the simple one-step ELISA can be used for the rapid detection of trace amount of protein. The method holds promise for the clinical detection of trace Ag in solution and on low-adsorption material surfaces.

Negative Interference in Serum HBsAg ELISA from Rheumatoid Factors

BackgroundRF(Rheumatoid factor) is usually thought to cause positive interference in immunoassay. Recently, our study showed that high-concentration RFs caused negative interference as well as positive interference in serum HBsAg(Hepatitis B surface antigen) ELISA(Enzyme-linked immunosorbent assay), but it is unclear that RF causing negative interference is an anomaly produced by a certain ELISA kit or a common property of most of HBsAg ELISA kits.MethodsSerum models were made by blending HBsAg-positive sera and high- or moderate-concentration RFs sera at the ratio of 1: 9, then one-step and two-step ELISA were adopted to determine HBsAg in serum models.ResultsNo matter what kind of kit used, one-step ELISA showed that HBsAg S/CO( sample/cut off) values in serum models were significantly lower than original values. Bivariate correlations tests showed decline rates of HBsAg S/CO Values were not associated to serum RF concentrations ranging from 288 to 3560 IU/mL. HBsAg converted to be negative in 69.80% serum models with original-value ranging from 1.00 to 10.00, and in 2.68% serum models with higher original-value. RF causing decline of HBsAg S/CO value provided by one-step ELISA was more obvious than that provided by two-step ELISA. ConclusionsIt is concluded that susceptibility of all HBsAg ELISA assays to interference from RF, leading to predominantly lower and in some cases "false-negative" results, and moreover, the lower the original HBsAg S/CO Value, the higher the false-negative rate.

Determination of T-2 Toxin and HT-2 Toxin in Milk: A Comparison of Three Formats of Immunoassays

This research paper presents a comparative study of three formats of immunoassays, including conventional enzyme-linked immunosorbent assay (ELISA), one-step ELISA, and reverse enzyme-linked immunosorbent assay (reELISA), for the determination of T-2 toxin (T-2) and HT-2 toxin (HT-2). This comparative study was performed with regard to specificity, sensitivity, matrix effect, accuracy, and precision. Among the three procedures, reELISA exhibited the best sensitivity with limits of detection (LOD) at 3.04 and 2.79 ng mL−1 for T-2 and HT-2. For the other parameters, there were no apparent differences. Good recoveries in negative spiked samples at concentrations of 50, 200, and 500 ng mL−1 were obtained, and the values were 67.9–112.9% for conventional ELISA, 60.8–108.8% for one-step ELISA, and 62.7–88.1% for reELISA. The relative standard deviation (RSD) for each procedure was less than 10.3%, 14.0%, and 11.5%, respectively. Among all three methods, the one-step protocol was the most time efficient, and the reELISA procedure exhibited the best sensitivity.