Detection of HbA1c by boronate affinity immunoassay using bacterial magnetic particles

We have developed a boronate affinity immunoassay system using m-aminophenylboronic acid (mAPB) coupling to bacterial magnetic particles (BMPs). Homobifunctional crosslinker, Bis-(succcimidyl)suberate (BS3), was employed for preparation of mAPB-BMPs conjugates (mAPB-BMPs). Quantities of HbA_1c on mAPB-BMPs were evaluated based on luminescence from alkaline phosphatase-conjugated anti-Hb antibody (ALP–antibody) binding to HbA_1c on the BMP surface. The binding of HbA_1c to mAPB-BMPs occurred gradually and was almost completed within 10 mm. The coupling reaction is enhanced due to static electric interaction between the positive charges on HbA_1c and negative charges on BMPs. The amount of HbA_1c binding to mAPB-BMPs increased with increasing sodium chloride concentrations in the range of 0–100 mM. However, the amount of Hb binding to mAPB-BMPs also increased in high concentration of sodium chloride. The Hb binding to mAPB-BMPs was detached from mAPB-BMPs when Hb–mAPB-BMPs were washed with low salt buffer. This indicates that Hb is nonspecifically adsorbed onto the surface of mAPB-BMPs in high concentration of sodium chloride. These results suggest that selective separation of HbA_1c using mAPB-BMPs can be achieved with these conditions. A dose–response curve was obtained between luminescence intensity and HbA_1c concentration using a fully automated boronate affinity immunoassay. A linear relationship between luminescence intensity and HbA_1c concentration was obtained in the range of 10–10⁴ ng/ml.     

Chemiluminescent Enzyme Immunoassay for Measuring Leptin

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1–1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.     

Extended standard curve stability on the CCD magic lite immunoassay system using a two-point adjustment

Ciba Corning Diagnostics has developed a two-point adjustment algorithm for use with the Magic Lite System which allows for extended stability of a full 7–10 point calibration curve over the life of a kit. This adjustment is accomplished by using a master calibration curve established during manufacturing along with two calibrators for each assay. Most conventional non-automated immunoassays contain anywhere from 6 to 10 calibrators which are included with each run of an assay. Eliminating the need to run full standard curves and using the two-point adjustment algorithm results in significant savings in both labour and reagent usage.     

Catch and measure–mass spectrometry‐based immunoassays in biomarker research

Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

A procedure for quantification of cytokinins as free bases involving scintillation proximity immunoassay

Cytokinins occur in a diversity of forms and determination of their individual levels requires extensive purification. However, determination of the total level of each major base in free, riboside and nucleotide forms would often be adequate. Hence, a methanolysis procedure which releases cytokinin bases from 9-ribosyl derivatives was developed and applied to plant extracts. A simple procedure, involving low pressure column chromatography, for purification of the cytokinin bases in treated extracts, and a scintillation proximity immunoassay for their quantification, were developed. The total level of each cytokinin base [N⁶-(2-isopentenyl)adenine, zeatin and dihydrozeatin] in free and ribosylated forms determined by these methods is reported for several plant tissues and the results are compared with those obtained after additional purification by HPLC. Values for zeatin were not changed by HPLC but isopentenyl-adenine and dihydrozeatin levels were usually reduced indicating the presence of unknown compounds which cross-react in the immunoassay. Modifications to the above purification method to quantify O-glucosyl cytokinins are also described.
The methods described facilitate the quantification of the total amount of each cytokinin base in forms closely associated with cytokinin action, and the detection of cytokinin biosynthesis by labelled precursor incorporation.     

Relationship between gibberellic acid and water amount in the cotton seed

The role of endogenous GA₃ and its application to seed development in two cotton genotypes Hybrid-6 (H-6) (big seeds) and Gujarat cotton 13 (G. Cot) (small seeds) was studied. Kernel and seed coat were subjected to growth analysis in terms of dry weight, water amount, and rates of dry matter accumulation and water uptake. H-6 kernel had manifold higher dry weight and water amount than G. Cot. Seed coat of both genotypes had similar dry weight at maturity, but the maximum rates of dry matter accumulation and water uptake were distinctly higher in H-6. According to growth analysis, development of seed kernel and coat was subdivided into four phases, i.e., cell division, cell elongation, dry matter accumulation and maturation. Endogenous GA₃ level was estimated in kernel and seed coat by indirect ELISA using antibodies raised against GA₃. GA₃ amount per seed components was higher in the seed kernel of H-6 than of G. Cot, except 33 and 36 days after anthesis in kernel. H-6 seed coat had the higher amount of GA₃ during cell division phase than that of G. Cot. Close correlation between in vivo GA₃ level and water amount was recorded in both seed components. With GA₃ or GA₃ + NAA treatments in ovule culture, higher promotion in dry weight, water amount and seed size was noted in G. Cot than in H-6 suggesting that G. Cot is more deficient in endogenous GA₃. The greatest stimulation of parameters studied was obtained in ovule culture with GA₃ + NAA. When GA₃ or GA₃ + NAA was applied, initial significant difference in water amount and seed size was nullified. Data presented in this study indicated that GA₃ regulates cell expansion through the water uptake by cotton seed.     

Future applications of magic lite technology

The features that Magic Lite products offer as an immunoassay delivery system are discussed. The use of paramagnetic particles and acridinium ester labels confers advantages of speed, sensitivity, and stability. The technology has been used to measure analytes of widely varying molecular weights and serum concentrations, indicating its potential to detect the full range of clinically relevant analytes. Initial development efforts have indicated that the advantages of the system can be effectively exploited in an automated instrument.     

The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer

The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0–1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.     

Distribution of parvalbumin isotypes in adult snook and their potential applications as species-specific biomarkers

The highly stable Ca²⁺ binding protein, parvalbumin, is prevalent in fish white muscle tissue. The properties of this protein make it a promising antigen for use as a specific biomarker for fish identification. Parvalbumin was purified from white muscle of an adult common snook Centropomus undecimalis using ammonium sulfate precipitation, size-exclusion chromatography (SEC) and anion-exchange HPLC. Parvalbumins were characterized by the presence of an 11-kDa band following gradient-SDS gel electrophoresis and by their immunoreactivity against mouse anti-parvalbumin antibodies. Anion-exchange chromatography of the parvalbumin fraction separated from the SEC column yielded nine fractions. Subsequent analysis of these fractions by isoelectric focusing gel electrophoresis led to a total of seven parvalbumin isotypes, which may lend themselves as biomarkers in fish identification. The presence of these seven parvalbumin isotypes was confirmed independently by reversed-phase HPLC. A dilution endpoint immunoassay was developed for C. undecimalis parvalbumin using a monoclonal antibody directed against its highly conserved calcium binding site. The utility of parvalbumin isotype distribution and specific monoclonal antibodies against fish parvalbumin in species identification is discussed.     

A SINGLE-CELL IMMUNOASSAY FOR PHOSPHATE STRESS IN THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)

Current techniques for studying phytoplankton physiology in the field, such as measurements of biochemical activities, nutrient addition bioassays, and determination of photosynthetic efficiency, are useful for assessing the physiology of the bulk community but suffer from a lack of specificity. This would be improved by the development of single-cell methods for monitoring in situ physiology. Here we develop and test an antibody-based assay for identifying phosphate stress in the model dinoflagellate Prorocentrum minimum (Pavillard) Schiller. Antiserum was raised against a cell-surface alkaline phosphatase purified from P. minimum. Western screening indicated that the antiserum reacted with phosphate-stressed cells but not nitrate-stressed or phosphate-replete cells in culture. Immunodepletion confirmed the identification of this protein as an alkaline phosphatase. Based on Western blots, the antiserum appeared to be specific for phosphate-regulated proteins in P. minimum because there is no discernible cross-reaction with closely related P. micans. A whole-cell immunofluorescence assay was used to identify phosphate stress in field populations of P. minimum from Narragansett Bay, Rhode Island. The percentage of labeled P. minimum cells in this environment during the summer of 1998 decreased through time as the inorganic phosphate concentration increased. The percentage of antibody-labeled cells significantly correlated with the percentage of ELF-97-labeled cells determined as another single-cell assay of phosphate stress. This is the first antibody-based method developed for monitoring cell-specific physiology in a dinoflagellate, and the method described here may serve as a model for developing similar tools in other species of phytoplankton.