One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min

This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml⁻¹ with a limit of detection of 0.4 ng ml⁻¹ and an analytical sensitivity of 0.7 ng ml⁻¹. It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.     

A PMMA microcapillary quantum dot linked immunosorbent assay (QLISA)

The development of a simple and inexpensive quantum dot based immunoassay for detecting myeloperoxidase (MPO) in stool samples is reported (QLISA). The method developed utilizes readily available polymethylmethacrylate (PMMA) microcapillaries as substrates for performing the sandwich assay. High power (80 mW) and low power (10 mW) UV-LEDs were tested for their efficiency in maximizing detection sensitivity in a waveguide illumination or a side illumination mode. The results obtained indicate that both waveguide and side illumination modes can be employed for detecting MPO down to 15 ng/mL, however the high power LED in a side illumination mode improves sensitivity and simplifies the data acquisition process. The protocol and sensor robustness was evaluated with animal stool samples spiked with MPO and the results indicate that the sensitivity of detection is not compromised when used in stool samples. The effect of the ionic strength of the environment on the fluorescence stability of quantum dots was evaluated and found to affect the assay only if long imaging times are employed. Replacing the buffer with glycerol during imaging increased the fluorescence intensity of quantum dots while significantly minimized the loss in intensity even after 2 h.     

Replacement-free mass-amplified sandwich assay with 180-MHz electrodeless quartz-crystal microbalance biosensor

This study describes a sensitivity-amplified detection method in a replacement-free electrodeless quartz-crystal microbalance (QCM) biosensor. A sandwich assay is proposed for detecting C-reactive proteins (CRP), where the biotinated second anti-CRP antibody is weighted by streptavidin for sensitivity amplification. Because the first CRP antibody was immobilized nonspecifically on naked quartz surfaces, the sandwich assay was repeated using the same sensor chip, making possible the replacement-free assay. The mass-amplified sandwich assay detected a CRP solution of 0.1 ng/ml. A methodology for determining the molecular mass of the injected protein is also proposed.     

流式微球一步法快速免疫检测马铃薯A病毒

[目的]建立流式微球一步法快速免疫检测马铃薯A病毒(PVA)的新方法.[方法]以荧光微球为反应载体,通过在微球表面进行双抗夹心免疫反应形成微球-捕获抗体-PVA-标记FITC检测抗体的复合物,利用流式细胞仪荧光检测系统收集荧光信号.[结果]通过实验优化检测条件,最佳捕获抗体工作浓度为4μg/mL、最佳检测抗体工作浓度为1:25倍稀释、最佳反应时间为2h;与马铃薯Y病毒、莴苣花叶病毒、番茄环斑病毒等均未出现交叉反应;阳性样品经64倍稀释后依然可检出,检测灵敏度是传统微孔板ELISA的4倍.[结论]流式微球一步法能灵敏、快速、简便的检测马铃薯A病毒.     

Using an electro-microchip, a nanogold probe, and silver enhancement in an immunoassay

This paper presents a novel immunoassay that uses an electro-microchip to detect the immuno-reaction signal, gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. This study is based on the direct immunoassay (two-layer format) and the sandwich immunoassay (three-layer format). The ANPs were introduced into the electro-microchip by the specific binding of the antibodies-ANPs conjugates and then were coupled with silver enhancement to produce black spots of silver metal. The silver precipitation constructs a "bridge" between two electrodes of the electro-microchip allowing electrons to pass. The variation of impedance can be easily measured with a commercial LCR meter. Various gap sizes (20, 50, 100, and 200 microm) of the electrodes of electro-microchips were designed for the sensitivity study. The experimental data show that a chip with a 20microm gap has the highest sensitivity. There was a significant difference in impedance between the experiment sample and the negative control after 10 min of reaction time. The proposed method requires less time and fewer steps than the conventional enzyme-linked immunosorbent assay (ELISA). In addition, it shows a high detection sensitivity (10 microg/mL of 1st antibody (IgG) immobilized on slides and 1 ng/mL of antigen (protein A)). There is a clear distinction between the signal intensity and the logarithm of the sample concentration. The proposed new immunoassay method has potential applications in proteomics research and clinical diagnosis.     

Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02–200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk

The use of semiconductor quantum dots (QDs) as fluorescent labels to develop a competitive immunoassay for sensitive detection and quantification of progesterone in cow's milk is described. Colloidal water-soluble CdSe/ZnS QDs are conjugated to an antigen derivative (progesterone-BSA conjugate) and a simple methodology is optimised to determine the antigen concentration in the final bioconjugate. The obtained QD-linked antigens were then employed together with unlabelled anti-progesterone monoclonal antibodies, as the biological recognition elements, in the development of the quantitative QDs-based fluorescent immunoassay for progesterone in bovine milk. After optimization, the developed immunoassay proved to cover a progesterone concentration range from 0.3 to 14.5 ng/mL in cow milk. Milk samples were just diluted 10-fold with deionised water and directly analysed with the proposed immunoassay, without additional sample pre-treatment or analyte extraction. The minimum detectable level (IC(10)) of the developed immunoassay turned out to be 0.1 ng/mL of progesterone in bovine milk. The sensitivity (IC(50)) achieved was 2.2 ng/mL with a reproducibility of 3.5% RSD as obtained from the results of the analysis of the triplicate of same samples but in three different days. Applicability of the proposed methodology was evaluated by analyzing cow's milk samples enriched with known concentrations of progesterone and recoveries better than 90% were achieved.     

Determination of phosphinothricin acetyltransferase in genetically transformed canola seed by a two-antibody sandwich enzyme immunoassay

We report a quantitative, two-antibody sandwich immunoassay for phosphinothricin acetyltransferase (PAT), the selectable marker protein. The method yielded a standard curve with a working range of 0 to 5 ng PAT per mL extract. Replicate absorbance values for standards within a single assay showed a coefficient of variation typically less than 5 percent. Over three separate assays, the coefficient of variation for the slope and y-intercept of the standard curve was 3.8 and 3.7 percent, respectively. TransgenicBrassica napus L. seed was used to demonstrate the utility of the assay. Non-transgenic seed extracts did not show a positive immunoassay signal. Determinations of the PAT enzyme conducted on spiked non-transgenic seed extracts repeated in three separate assays fell in the acceptable range of 80 to 120 percent recovery. Transgenic canola seed, analyzed in three separate assays, showed a mean PAT enzyme content of 403 ng/g with a standard error of 19 ng/g and a coefficient of variation of 8.1 percent. The method has also been applied to several other tissues and processed products of canola, maize, and tobacco.     

Sensitive detection of cardiac biomarker using ZnS nanoparticles as novel signal transducers

C-reactive protein (CRP), a 115 kDa pentameric protein, is one of the important cardiac biomarkers that are indicative of coronary heart events. Sensitive detection of CRP in human serum is critical for the diagnosis of coronary heart disease. This work presents a sensitive sandwich immunoassay for the detection of CRP in human serum using zinc sulfide (ZnS) nanoparticles as novel fluorescence signal transducers. In this assay, monoclonal anti-CRP antibodies are used to capture CRP in human serum, and then the captured CRPs are incubated with biotinylated monoclonal anti-CRP and Neutravidin coated ZnS nanoparticle to form sandwich immunocomplexes. Quantification of CRP occurs when zinc ions released from ZnS nanoparticle labels are mixed with zinc-ion sensitive fluorescence indicator Fluozin-3 for fluorescence generation. The developed assay presents a detection limit around 10 pM and a detection range with more than two orders of magnitude.     

Quantitative HPLC-UV method for the determination of firocoxib from horse and dog plasma

A sensitive reversed-phase HPLC-UV method was developed for the determination of firocoxib, a novel and highly selective COX-2 inhibitor, in plasma. A 1.0 mL dog or horse plasma sample is mixed with water and passed through a hydrophobic-lipophilic copolymer solid-phase extraction column to isolate firocoxib. Quantitation is based on an external standard curve. The method has a validated limit of quantitation of 25 ng/mL and a limit of detection of 10 ng/mL. The validated upper limit of quantitation was 2500 ng/mL for horses and 10,000 ng/mL for dogs. The average recoveries ranged from 88-93% for horse plasma and 96-103% for dog plasma. The coefficient of variation in all cases was less than 10%. This method is suitable for the analysis of clinical samples from pharmacokinetic and bioequivalence studies and drug monitoring.     

Development of a bead-based aptamer/antibody detection system for C-reactive protein

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10 mg/L in diluted serum with acceptable recoveries (extrapolated values of 70–130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer–target systems could increase the number of analytes measurable using xMAP-type assays.     

Sandwich electrochemical immunoassay for the detection of Staphylococcal enterotoxin B based on immobilized thiolated antibodies

A new approach for the sensitive detection of Staphylococcal enterotoxin B (SEB) is presented based upon an electrochemical enzymatic immunoassay that utilizes thiolated antibodies immobilized on a gold surface. This method relies on the use of amine- or sulfhydryl-reactive heterobifunctional cross-linkers for the introduction of 2-pyridyl-disulfide groups to the antibody. The disulfide-containing linkages are subsequently cleaved with a suitable reducing agent, such as dithiothreitol (DTT), and the thiolated antibody–gold bond is covalently formed on a gold working electrode. Various cross-linking agents for immobilization of the capture antibody onto the gold electrode were investigated and compared. Factors influencing the thiolation and immobilization were investigated and optimized. The feasibility of such antibody immobilization and the subsequent sandwich enzyme immunoassay is demonstrated for the sensitive detection of SEB. The detection limit estimated from a representative dose–response curve is 1 ng/mL, corresponding to 5 pg in a 5-μL sample. Coupling the specificity of immunoassays with the sensitivity and low detection limits of electrochemical detection shows real promise for future sensing technology in enabling the development of single-use disposable devices.     

A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi

A rapid and sensitive gold-nanobioprobe based immunoassay format has been presented for the detection of capsular Vi polysaccharide of Salmonella enterica serovar Typhi (surface antigen) using anti-Vi antibodies. The Vi antigen was extracted from serovar Typhi cells, under the optimised growth conditions for its over-expression. Anti-Vi antibodies were produced and conjugated with gold nanoparticles (GNPs) of definite size (~30 nm), which served as the nano-bioprobe in the detection system. A sandwich immunoassay was developed using nitrocellulose dot blot comb (8/12 wells) membranes immobilized with anti-Salmonella antibodies at the optimal concentration (43 ng spot(-1)). The Vi antigen in the clinical isolates, spiked samples and also in the standard strain (serovar Typhi Ty2) was detected by measuring the colour intensity of GNPs and correlating it with the concentration of serovar Typhi in samples. Using this developed immunoassay technique Vi positive serovar Typhi strains could be detected with a sensitivity of up to 10(2) cells mL(-1) in the clinical isolates as well as in the spiked samples. The developed immunoassay technique could be useful for the detection of typhoid fever and may be important from an epidemiological point of view.     

A colloidal gold probe-based silver enhancement immunochromatographic assay for the rapid detection of abrin-a

High-affinity anti-abrin-a monoclonal and polyclonal antibodies were used to develop a sandwich immunochromatographic assay and silver enhancement technology was used to further increase the sensitivity. Using a matrix of double distilled water or soybean milk with added abrin-a, the visual detection limit was found to be 10 ng mL(-1). The detection limit was 0.1 ng mL(-1) for abrin-a, an increase in sensitivity of 100-fold when the silver enhancement technology was employed. The assay was portable and very simple to perform and the detection was completed within 20 min without complicated handling procedures. There was no significant cross-reactivity with several homologous toxins and associated agglutinin. The assay reagents could be stored for 12 weeks at 4°C without significant loss of activity. These characteristics make the strip assay to be an ideal candidate for the development of a rapid toxin detection kit.     

Chemiluminescence enzyme immunoassay (CLEIA) for the determination of chloramphenicol residues in aquatic tissues.

A competitive indirect chemiluminescence enzyme immunoassay (ic-CLEIA) for chloramphenicol (CAP) residues in shrimp has been developed. After optimization (incubation time, concentration of Tween-20, concentration of PBS and pH), the method gave a limit of detection of 0.01 ng/mL and a detection range of 0.03-23.7 ng/mL, with an ED(50) of 0.47 ng/mL. The method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively) and accuracy (mean recovery 95-123%). The assay performance is better than the ELISA method which is widely used to detect chloramphenicol and indicates that the CLEIA method can be used to test aquatic samples instead of ELISA.     

Cysteine-free mutant of aequorin as a photolabel in immunoassay development

The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 x 10(-12) M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay.     

Rapid method for detection of Salmonella in milk by surface plasmon resonance (SPR)

The Plasmonic surface plasmon resonance (SPR) device was used to develop a rapid, simple and specific immunoassay for detection of Salmonella in milk. Rapid detection of Salmonella contamination is a major challenge for the food industry. Salmonella contamination is well known in all foods including pasteurised milk. The SPR assay was developed as a sandwich model using a polyclonal antibody against Salmonella as capture and detection antibody. Milk spiked with Salmonella typhimurium cells, killed by thimerosal (1%, w/w) treatment was used. Using the Plasmonic SPR assay it was possible to detect S. typhimurium down to a concentration of 1.25 x 10(5) cells ml(-1) in both milk and buffer system. The results obtained are comparable with existing, approved rapid Salmonella detection techniques. No negative effects on the sensitivity of the assay are encountered due to the milk matrix. Hence, no sample preparation or clean-up steps are required. The sample volume requirement for the assay is only 10 microl. Using the assay S. typhimurium was detected in milk within 1h, whereas the cultural techniques require 3-4 days for presumptive positive isolates and further time for confirmation. The rapid tests require at least 24h for the results. The Plasmonic SPR device operates on the Kretschmann configuration and is a cuvette-based system with the advantage of having eight channels on one single SPR chip.     

Protein chip for the parallel quantification of high and low abundant biomarkers for sepsis

We present herein a protein chip for diagnosis of sepsis that combines both a sandwich and a binding inhibition format in order to quantify high (CRP) and low abundant proteins (cytokines, PCT, neopterin) in parallel. Using the combined assay format the lowest detectable concentrations for CRP, IL-6, IL-8, IL-10, TNFα, PCT, and neopterin are 3 mg/L, 15 ng/L, 26 ng/L, 65 ng/L, 40 ng/L, 78 ng/L, and 0.46 μg/L. Four different combined assay formats are tested, using separate or joint incubation steps of analytes and detection antibodies. Yet, low limit of detection (LOD) and short processing time are contradictory: while the combined assay performed in a multistep protocol is extremely sensitive (e.g., the LOD for IL-6 is 15 ng/L), but more time-consuming (4 h), the all-in-one protocol takes only 2.5 h, but suffers from lower sensitivity compared with the multistep protocol (e.g., the LOD for IL-6 is up to 40 times enhanced). Reproducibility is good in both cases (CV 5–20%).     

A novel sensitive immunoassay method based on the Invader technique

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-α (hTNF-α). Detection limits obtained were 0.1 pg/ml hTNF-α when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-α enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.