The increasing interest in measuring total homocysteine in plasma has led to the development of several different methods (1). Although most publications describe HPLC methods with manual sample preparation, the first fully or partially automated kits were introduced recently: a fluorescence polarization immunoassay on the IMx® analyzer (Abbott Laboratories, Abbott Park, IL), a microtiter plate enzyme immunoassay (Bio-Rad Laboratories), and an HPLC kit with electrochemical detection (BAS). We evaluated the Abbott IMx analyzer automated method for total homocysteine and compared it with our HPLC assay with internal standardization.
The “Abbott Homocysteine (HCY) assay” is a fluorescence polarization immunoassay based on the highly selective enzymatic conversion of homocysteine to S -adenosyl-l-homocysteine, which is then recognized by a monoclonal antibody (2). The assay requires 50 μL of sample, with no sample pretreatment. A batch of 20 samples can be processed within 1 h. We assessed the intra- and interassay variability, the recovery of added homocysteine, the analytical sensitivity, the stability of the calibration curve, the specificity, and the cross-reactivity towards other thiols. EDTA plasma samples were obtained within a maximum of 30 min after blood collection. Serum samples were obtained from whole blood allowed to clot at room temperature for 30–60 min. Blood specimens were collected by the Emory University Hospital Blood Collection Service under an agreement with the Centers for Disease Control and Prevention (including an omnibus informed consent and Human Subjects Review protocol). All serum and plasma samples were stored at −70 °C.
We sequentially analyzed 20 replicates (one carousel) of each of the three serum-based quality-control (QC) pools supplied by Abbott to assess intraassay imprecision …
Conventional methods for conjugation of macromolecules, such as antibodies and reporter groups, typically yield a mixture ranging from unconjugated starting materials to large aggregates. We explored the use of a solid-phase process to allow improved control in conjugation of macromolecules for use in immunodiagnostic reagents.Activated components were sequentially delivered to an immobilized core protein, linking in concentric layers. For immunodiagnostic reagents, proteins with the desired signaling properties were added as interior layers and binding proteins were placed in the final surface layer. After assembly, the conjugates were released into solution by cleaving the linker holding the core protein to the support. Conjugates were prepared with use of three different reporter agents: R-phycoerythrin for microsphere fluorescence flow immunoassay, alkaline phosphatase for enzyme immunoassay, and acridinium for magnetic chemiluminescence immunoassay. For each reporter, six conjugates were prepared with various concentrations of both the reporter and an antibody directed against the alpha-subunit of thyroid-stimulating hormone (TSH), and the complexes were tested in appropriate assay formats for measurement of TSH.Products ranged in mass from approximately 1 to approximately 20 MDa. HPLC analysis of the conjugates on a gel-permeation column showed sizes and chromophore contents highly consistent with the intended structures. In appropriate assay formats, the signal generated by a conjugate increased with incubation time, then plateaued at an intensity approximately proportional to the reporter content but relatively independent of the antibody content of the conjugate. The time required to reach this maximum decreased with increasing antibody content.The high degree of structural control available with solid-phase assembly and the close correlation of structure with desired function of the resulting conjugates make this an attractive method for preparation of an important class of in vitro diagnostic reagents.
We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.
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