Development of a sandwich ELISA for the 5.9-kDa fibrinogen alpha C chain fragment detected by serum proteome analysis.

PURPOSE: We previously identified novel biomarker candidates in heavy consumers of alcohol using serum proteome analysis. Among several candidates, a 5.9 kDa peptide identified as a fragment of the fibrinogen alpha C chain (FIC5.9) was the most promising. To move FIC5.9 toward potential diagnostic use, we developed an enzyme immunoassay that enables measurement of serum FIC5.9 levels. EXPERIMENTAL DESIGN: Two monoclonal antibodies specific to the N and C-termini of the 5.9-kDa peptide were used to develop a FIC5.9 sandwich ELISA. The assay was evaluated by comparing the results with those obtained by the stable isotope-labeled dilution mass spectrometry (SID-MS) using the ClinProt™ system. RESULTS: The ELISA results correlated with the SID-MS findings (slope=0.795, intercept=-0.011, r(2) =0.908) and the performance of the ELISA was satisfactory in terms of recovery (98.5-103.0%) and within-run (1.4-4.7%) and between-day (2.8-8.4%) reproducibility. The assay was capable of detecting changes in FIC5.9 during abstinence from drinking in patients with alcohol dependency (p<0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum FIC5.9 levels in various pathological conditions, including alcoholism.

Adsorption of urinary proteins on the conventionally used urine collection tubes: possible effects on urinary proteome analysis and prevention of the adsorption by polymer coating.

One possible factor determining recovery of trace amount of protein biomarker candidates during proteome analyses could be adsorption on urine tubes. This issue, however, has not been well addressed so far. Recently, a new technical device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (poly(MPC-co-BMA)) has been developed mainly to prevent the adsorption of plasma proteins. We assessed whether conventionally used urine tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(MPC-co-BMA) can minimize the adsorption. Proteinuric urine samples were kept in poly(MPC-co-BMA)-coated and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or peptides onto urine tubes were analyzed by SDS-PAGE, 2-DE, and the MALDI-TOF MS. It was found that a number of proteins and/or peptides adsorb on the conventionally used urine tubes and that surface coating by poly(MPC-co-BMA) can minimize the adsorption without any significant effects on routine urinalysis test results. Although it remains to be clarified to what extent the protein adsorption can modify the results of urinary proteome analyses, one has to consider this possible adsorption of urinary proteins when searching for trace amounts of protein biomarkers in urine.

Generation of a monoclonal antibody specific for tissue-nonspecific alkaline phosphatase and its use in a clinical diagnostic study.

Tissue-nonspecific alkaline phosphatase (TNSALP) in serum comprises liver alkaline phosphatase (liver-ALP) and bone alkaline phosphatase (bone-ALP). Liver-ALP is a marker of liver disease; thus a specific method for its measurement would be useful. Measurement of ALP by electrophoresis is difficult, although all of the isozymes can be assessed simultaneously. Total ALP can also be measured by automated analyzer, but it is difficult to determine the cause of a high ALP value because bone-, intestine-, placenta-, and tumor-ALP are measured together. Thus, anti-TNSALP monoclonal antibodies that can resolve these problems are needed. Here we have generated an anti-TNSALP monoclonal antibody, 3-29-3R. This clone has specificity to liver-ALP rather than to bone-ALP. In electrophoresis, 3-29-3R reacted with TNSALP and shifted the bands. The use of 3-29-3R enabled easy interpretation of the results. Furthermore, we tested 3-29-3R by developing an immunocapture enzymatic assay (IEA). Preliminary results of the IEA show that this method is effective for measurement of liver-ALP. Thus, the monoclonal antibody that we have established may be a useful tool for clinical diagnosis.

Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulin G.

A convenient method for measuring immune complexes between tissue-nonspecific alkaline phosphatase (TNSALP) and immunoglobulin G (IgG) (i.e., TNSALP-IgG) would be highly useful for routine analyses. Here, we identified a surface-active agent that would dissolve membrane but not dissociate TNSALP-IgG complexes. Next, we developed an enzyme-linked immunosorbent assay (ELISA) method for detecting TNSALP-IgG complexes with two monoclonal antibodies (MoAbs): 3-29-3R was coated on assay plates and captured TNSALP-IgG from a specimen; an horseradish peroxidase (HRP)-conjugated anti-human IgG1 then reacted with captured TNSALP-IgG to form an "immunocomplex sandwich." The immunocomplex was detected via the absorbance of an HRP substrate, resulting in a semiquantitative assay. The mean absorbance of 0.195 (n=5) measured in sera from healthy donors was designated as an arbitrary unit (AU/mL) of TNSALP-IgG concentration. The ELISA values of patient sera known to contain TNSALP-IgG complexes were greater than those of normal sera (normal, 1.86 plusmn; 0.61; patient, 9.30 plusmn; 5.44), and these data were confirmed by electrophoresis. Thus, the ELISA could detect TNSALP-IgG complexes. The intraassay coefficient of variation (CV) was within 7.4% and analytical recovery was excellent. There was no significant interference from hemolytic, lipemic, or icteric serum. In summary, an ELISA using 3-29-3R MoAb and HRP-conjugated anti-human IgG1 constitutes a reliable and convenient method for the semiquantitative detection of TNSALP-IgG complexes in human serum.

[The mechanism of TRACP 5b maturation].

Serum tartrate resistant acid phosphatase 5b (TRACP 5b) is an isozyme of osteoclast origin. Indeed, measurement of TRACP 5b activity is used as an index of osteoclast activity. However, the precise mechanism of TRACP 5b maturation is unclear. This study aimed to clarify the mechanism of generation of TRACP 5b. We used a highly sensitive fiber-type DNA chip to investigate the mechanism of generation of TRACP 5b at the genetic level. Genes derived from three related cell types (monocytes, macrophages and osteoclasts) were compared. In addition, at the protein level, posttranscriptional modification was tested by Western blotting using an antiserum specific for the flexible loop region of TRACP 5. Our DNA chip study shows that genes implicated in oligosaccharide construction do not show significant differences in expression levels between the cell types under investigation. Strongly expressed Cathepsin K was observed in osteoclasts. Western blotting demonstrated that TRACP undergoes unique partial degradation during bone resorption, such that serum TRACP 5b lacks the flexible loop found in TRACP 5a. In conclusion, TRACP 5b generated by a specific posttranscriptional modification pathway undergoes partial digestion in resorption lacunae or inside osteoclasts. Serum TRACP 5b lacking the flexible loop differs from TRACP 5a in terms of optimum pH, isoelectric point, sugar chain and antigenicity. The measurement of TRACP 5b could therefore be of great use for monitoring of osteoporosis, rheumatoid arthritis and bone metastasis.

Development of a novel fragments absorbed immunocapture enzyme assay system for tartrate-resistant acid phosphatase 5b.

BACKGROUND: Osteoclastic activity is mainly assessed by measuring urinary markers. To correct for differences in renal clearance, the levels of urinary markers are usually corrected by the urine creatinine concentration. Therefore, alternative serum markers to evaluate osteoclastic activity are required. We developed a novel system for the determination of serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity to evaluate osteoclastic activity. METHODS: Two unique monoclonal antibodies were generated and the specificity was tested using a surface enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI TOF-MS). A novel fragments absorbed immunocapture enzymatic assay (FAICEA) method was developed using 2 monoclonal antibodies. RESULTS: FAICEA gave a sensitivity 0.1 U/l, linearity of 0.1-28 U/l, recovery 92-103%, inter-assay CV 2.95% and intra-assay CV 2.15%. Unlike other TRACP5b assay systems, FAICEA avoided interference from TRACP 5a. CONCLUSIONS: According to the FAICEA, postmenopausal women had higher TRACP5b concentrations than younger women. The results show that TRACP5b is a novel bone resorption marker in serum.

Development and characterization of novel monoclonal antibodies against tartrate-resistant acid phosphatase 5.

Serum band 5 tartrate-resistant acid phosphatase (TRACP 5; EC 3.1.3.2) is a glycoprotein that exists as two very similar isoforms, TRACP 5a and TRACP 5b. The similarity of these two isoforms has made it difficult to establish monoclonal antibodies specific for either isoform. We report here the development of a monoclonal antibody with high specificity for TRACP 5b. We prepared TRACP 5b antigens from four sources: TRACP 5b purified from human bone, recombinant TRACP 5 from Escherichia coli, recombinant TRACP 5 from insect cells, and a synthetic TRACP 5b peptide. Thirty-seven mice were each immunized with 1 of the 4 different TRACP antigens to generate 473 antibody-producing clones. Three of these clones, Trk27, Trk49, and Trk62, reacted with TRACP 5b. These three clones were all established from mice exposed to native bone TRACP 5b antigen. In fact, none of the other antigens were able to generate anti-TRACP 5b monoclonal antibodies in mice. Furthermore, Trk62 interacted more strongly with TRACP 5b than with TRACP 5a. These results suggested that although recombinant proteins can be effective antigens, the native TRACP 5 protein might be more effective at generating monoclonal antibodies of greater specificity due to its more faithful representation of the native three-dimensional structure of the protein.