Kind Of Immunoassay Research Articles

Anthracycline-Functionalized Dextran as a New Signal Multiplication Tagging Approach for Immunoassay

The most used kind of immunoassay is enzyme-linked immunosorbent assay (ELISA); however, enzymes suffer from steric effects, low stability, and high cost. Our research group has been developing quinone-linked immunosorbent assay (QuLISA) as a new promising approach for stable and cost-efficient immunoassay. However, the developed QuLISA suffered from low water-solubility of synthesized quinone labels and their moderate sensitivity. Herein, we developed a new approach for signal multiplication of QuLISA utilizing the water-soluble quinone anthracycline, doxorubicin, coupled with dextran for signal multiplication. A new compound, Biotin-DexDox, was prepared in which doxorubicin was assembled on oxidized dextran 40, and then it was biotinylated. The redox-cycle-based chemiluminescence and the colorimetric reaction of Biotin-DexDox were optimized and evaluated, and they showed very good sensitivity down to 0.25 and 0.23 nM, respectively. Then, Biotin-DexDox was employed for the detection of biotinylated antibodies utilizing avidin as a binder and a colorimetric assay of the formed complex through its contained doxorubicin redox reaction with NaBH4 and imidazolium salt yielding strong absorbance at 510 nm. The method could detect the plate-fixed antibody down to 0.55 nM. Hence, the application of Biotin-DexDox in QuLISA was successfully demonstrated and showed a significant improvement in its sensitivity and applicability to aqueous assays.

Microfluidic electrochemical magnetoimmunosensor for ultrasensitive detection of interleukin-6 based on hybrid of AuNPs and graphene.

Cytokines are important factors in the early diagnosis of autoimmune diseases and require high sensitivity, high selectivity and quantitative detection. We proposed a miniaturized electrochemical magneto-immunosensor (EC-MIS) on portable interleukin-6 (IL-6) detection based on this requirement. Firstly, a micro-fabricated working electrode is electrochemically modified with a hybrid of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs). Increased surface area and enhanced charge transfer rate improve the performance of this immunosensor on sensitivity. Secondly, magnetic beads attached with the capture antibody (cAb) are employed in sandwich immunoassay. This kind of immunoassay is immobilized on the working electrode surface by an external magnet to enrich the analyte IL-6. Thirdly, the last two features are combined and integrated on a microfluidic device in order to restrict the sample at certain areas and ease the operation of detection. With our prototypic EC-MIS operated in amperometric mode, we have achieved the detection of IL-6 with a linear range from 0.97 to 250pg/mL and a limit of detection (LOD) of 0.42pg/mL. Real serum samples were demonstrated and compared with benchtop equipment's results.

A Low-Cost Micro-Volume Nephelometric System for Quantitative Immunoagglutination Assays.

Immunoassays have been widely used in scientific research and clinical diagnosis due to their versatile detection capability and high specificity. Immunoagglutination assays are kinds of immunoassay, which can simply and rapidly measure the concentration of analytes. In this work, we developed a low-cost micro-volume nephelometric system for quantitative immunoagglutination assays. We used off-the-shelf components to build the system, and the total cost of key components is only about 20 US dollars. The total detection volume in our system was as low as 3 µL, which could significantly reduce the reagent cost and required sample volume. We further evaluated the system performance via the immunoagglutination assay to measure the concentration of C-reactive protein, a plasma protein with levels rising in response to inflammation. The results demonstrated that our system could measure the concentration of analytes with relatively high sensitivity and precision within four minutes, and has high potential to be applied for clinical diagnostic tests.

Recombinase polymerase amplification combined with a magnetic nanoparticle-based immunoassay for fluorometric determination of troponin T.

The authors describe a method for the improvement of the sensitivity of immunoassays. This was achieved by a combination of immunoassay and an amplification based on the use of a multiplied DNA probe. The immunoassay was enhanced via recombinant polymerase amplification (RPA). It enables fast (35min) isothermal multiplication of DNA at 37°C. This concept was demonstrated for a sandwich immunoassay that is making use of magnetic nanoparticles conjugated to first specific antibodies and then to second specific antibodies linked to reporter DNA via biotin-streptavidin binding. Reporter DNA multiplied by RPA was quantified fluorometrically via the carboxyfluorescein label. Human cardiac troponin T (cTnT), a biomarker for acute myocardial infarction, was used as the target of the immunoRPA (iRPA). The assay can detect cTnT in serum and in plasma within 2h, with detection limits as low as 12.5 ± 1.1pg•mL-1 and 9.4 ± 2.1pg•mL-1, respectively. This represents increased sensitivity and decreased assay time compared to classical ELISAs (3.5 ± 0.3ng•mL-1) or immuno-PCR (4.3 ± 0.8ng•mL-1) that were carried out using the same immunoreagents and reporter DNA. The good performance of this iRPA was underlined by its successful application to the analysis of plasma samples. The iRPA has significant advantages relative to other DNA amplification-based immunoassays due to isothermal conditions and analysis at 37°C. Graphical abstract Schematic representation of a new kind of immunoassay that is enhanced by making use of recombinase polymerase amplification (immunoRPA). Reporter DNA was conjugated with specific antibodies, then amplified and finally quantified fluorometrically. Limit of detection of troponin T in plasma within 2h was 9.4 ± 2.1pg•mL-1.

Immunoassay for serum amyloid A using a glassy carbon electrode modified with carboxy-polypyrrole, multiwalled carbon nanotubes, ionic liquid and chitosan

We report on a highly sensitive electrochemical immunoassay for the serum inflammation marker amyloid A (SAA). It is making use of a glassy carbon electrode that was modified with carboxy-endcapped polypyrrole (PPy-α-COOH), multiwalled carbon nanotubes (MWCNTs), ionic liquid and chitosan acting as the support platform. The nanocomposite increases the sensitivity and stability of the assay. Antibody against SAA was immobilized on a monolayer surface consisting of PPy-α-COOH. The electrode material was characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectra, cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. The calibration plot for this assay, when operated at 0.16 V (vs. SCE) and applied to spiked serum samples, is linear in the 0.001 to 900 ng mL−1 SAA concentration range, and the detection limit is as low as 0.3 pg mL−1 (at an S/N ratio of 3). The electrode is stable and highly sensitive. The detection scheme is likely to be applicable to numerous other kinds of immunoassays.

Computational study on the interactions and orientation of monoclonal human immunoglobulin G on a polystyrene surface

Having a theoretical understanding of the orientation of immunoglobulin on an immobilized solid surface is important in biomedical pathogen-detecting systems and cellular analysis. Despite the stable adsorption of immunoglobulin on a polystyrene (PS) surface that has been applied in many kinds of immunoassays, there are many uncertainties in antibody-based clinical and biological experimental methods. To understand the binding mechanism and physicochemical interactions between immunoglobulin and the PS surface at the atomic level, we investigated the binding behavior and interactions of the monoclonal immunoglobulin G (IgG) on the PS surface using the computational method. In our docking simulation with the different arrangement of translational and rotational orientation of IgG onto the PS surface, three typical orientation patterns of the immunoglobulin G on the PS surface were found. We precisely analyzed these orientation patterns and clarified how the immunoglobulin G interacts with the PS surface at atomic scale in the beginning of the adsorption process. Major driving forces for the adsorption of IgG onto the PS surface come from serine (Ser), aspartic acid (Asp), and glutamic acid (Glu) residues.

Comparison of thyroid function tests results determined by two kinds of immunoassay in uncomplicated pregnant women

Objective To compare thyroid function test (TFT) results and reference range of normal pregnant women in different gestational stages between two kinds of automation chemiluminescence immunoassay kit commonly used.Methods Serum of 693 normal pregnant women attended antenatal clinics in the International Peace Maternity & Child Health Hospital Affiliated to the Medical School of Shanghai Jiaotong University from Feb.17th to June 9th,2011 was collected,and were divided into five groups according to their gestational age,9-13,16-20,24-28,32-34 and 37-40 weeks.Thyroid stimulating hormone (TSH),free thyroxine (FT4) and free triiodothyronine (FT3) levels were determined by 2 kinds of detection reagents:Abbott Architect I2000 and Roche Cobas Eleesys 600.According to National Academy of Clinical Biochemistry (NACB),the reference range of the TFT indexes was calculated.The results were analyzed by one-way ANOVA,t-test and Spearman relation analysis.Results During pregnancy,TSH level at different stage of pregnancy detected by the two reagents showed linear correlation and the changing pattern was also consistent (r =0.833-0.973,P =0.000).In 9-13 gestational weeks group,TSH level were significantly lower than that of other groups; and in 37-40 gestational week group,it was higher than that of other groups (FAbbott =18.830,FRoche =21.012,all P=0.000).And TSH levels and its reference range determined by Roche detection reagent in each group were higher than those by Abbott detection reagent (|t| =3.002,4.948,6.353,4.636 and 4.391,P＜0.01,respectively).In 9-13 gestational weekgroup,FT4 level was higher than that of other groups (F A b bott =17.230,FRoche =14.439,P=0.000).In 9-13 week group,FT4 level determined by Roche reagent was higher than that detected by Abbott reagent (|t| =4.964,P=0.000).While in 24-28 and 37-40 weeks group,they were lower (|t| =4.183,P=0.000; |t| =2.417,P=0.016).In 9-13 and 16-20 gestational weeks group,FT:3 levels,determined by both Abbott and Roche reagents,were higher than those of other groups (FAbbott =46.142,FRoche=68.149,P=0.000).The FT3 level in each group detected by Roche reagent were higher than that detected by Abbott reagent (|t| =31.762,26.411,28.877,22.710 and 22.736,P=0.000).Only in 9-13 weeks group,the TSH level was related to FT4 and FT3 level determined byboth reagents (Abbott:r=-0.319 and -0.361,P=0.000; Roche:r=-0.352 and -0.329,P=0.000).Conclusions TSH serum level is the most accurate indicator of thyroid function.The upper limit of the empirical TSH reference range during pregnancy recommended by America Thyroid Association might not be appropriate for Chinese.Different reference range for pregnant women should be established according to different reagents applied. Key words: Pregnancy; Thyrotropin; Thyronines; Reagent kits, diagnostic; Reference values

Solid supports for microarray immunoassays.

Stimulated by the achievements of the first phase in genomics and the resulting need of assigning functions to the acquired sequence information, novel formats of immunoassays are being developed for high-throughput multi-analyte studies. In principle, they are similar in nature to the microarray assays already established at the level of nucleic acids. However, the biochemical diversity and the sheer number of proteins are such that an equivalent analysis is much more complex and thus difficult to accomplish. The wide range of protein concentration complicates matters further. Performing microarray immunoassays already represents a challenge at the level of preparing a working chip surface. Arrays have been produced on filter supports, in microtiter plate wells and on glass slides, the last two usually coated with one-, two- or three-dimensionally structured surface modifications. The usefulness and suitability of all these support media for the construction and application of antibody microarrays are reviewed in this manuscript in terms of the different kinds of immunoassay and the various detection procedures. Additionally, the employment of microarrays containing alternative sensor molecules is discussed in this context. The sensitivity of microspot immunoassays predicted by the current analyte theory is not yet a reality, indicating the extent of both the technology's potential and the size of the task still ahead.

Optimal Design for ELISA and Other Forms of Immunoassay

In enzyme-linked immunosorbent assay (ELISA), as well as in many other kinds of immunoassay, a log-logistic or similar-shaped calibration curve is fit using standards at a series of known levels and then used to transform the measured values for the unknowns into estimated concentrations. The choice of the number of standards, the concentration of the standards, and the number of replicates of the standards and of the unknowns all affect the precision of the measurement. This article develops an optima1 design paradigm for this type of problem and shows how optimal choices can be calculated so that the system achieves the maximum precision of which it is capable. Although exact calculation of optimal designs requires use of a computer program, close approximations to the optimum can be derived from simple rules for hand calculation.

Optimal Design for ELBA and Other Forms of Immunoassay

In enzyme-linked immunosorbent assay (ELISA), as well as in many other kinds of immunoassay, a log-logistic or similar-shaped calibration curve is fit using standards at a series of known levels and then used to transform the measured values for the unknowns into estimated concentrations. The choice of the number of standards, the concentration of the standards, and the number of replicates of the standards and of the unknowns all affect the precision of the measurement. This article develops an optima1 design paradigm for this type of problem and shows how optimal choices can be calculated so that the system achieves the maximum precision of which it is capable. Although exact calculation of optimal designs requires use of a computer program, close approximations to the optimum can be derived from simple rules for hand calculation.

Quantification of platelet-bound alloantibodies by radioimmunoassay: a study on some variables.

Several variables that may affect accurate measurement of platelet-associated IgG (PA-IgG) were studied using a radioimmunoassay of the consumption type. The amount of PA-IgG of washed, unfixed normal donor platelets was 1.0 +/- 0.9 fg IgG/platelet (mean +/- 2 SD). Upon storage of washed platelets in a buffer containing EDTA, the amount decreased significantly to 0.2 +/- 0.2 fg IgG/platelet. Simultaneously, an increase in modal platelet volume was observed. Similar results were obtained when platelets were fixed with paraformaldehyde (PFA). We postulate that this decrease in PA-IgG is caused by the release of plasma IgG entrapped by the surface-connected canicular system of the platelet, when the platelets swell during storage in EDTA or fixation with PFA. This presence of varying amounts of entrapped plasma IgG may cause the wide discrepancies in PA-IgG found in normal donor platelets as well as platelets from ITP patients by other investigators. A good quantification of platelet-bound alloantibodies was possible with our assay when platelets were routinely fixed to diminish the amount of nonspecific PA-IgG. This was demonstrated with different anti-Zwa (= anti-PlA1), anti-Baka and anti-HLA sera. We also observed that fragments of platelets as well as fragments of cells of other types can cause aspecifically increased Pa-IgG values and can thus interfere with the proper measurement of platelet-bound antibodies in all kinds of immunoassays in general.