Liquid-phase Binding Assay Research Articles

The Use of Serum Survivin and AFP-L3 and their Combination in Improving Hepatocellular Carcinoma Diagnosis Especially in Patients with Low Serum AFP

AbstractBackground: Hepatocellular carcinoma (HCC) is respon-sible for about 4.7% of chronic liver disease in Egypt. Survivin is an essential element in apoptosis inhibition. Survivin is present in fetal and malignant adult tissue and is absent in non malignant tissues. A diagnostic and prognostic HCC marker is the alpha-fetoprotein (AFP) binding to Lens culinaris agglutinin (AFP-L3%) percentage. There is a need for HCC marker that is better than AFP especially in those low AFP.Aim of Study: To detect the role of determination of serum Survivin gene, AFP-L3% and their combination in HCC diagnosis.Subjects and Methods: Fifty HCC patients and 20 healthy control persons were subjected for measurement of serum AFP, survivin gene and AFP-L3%. Reverse transcription-polymerase chain reaction (RT-PCR) was used for detection of serum survivin gene. AFP-L3% was measured by a liquid-phase binding assay on the Wako LiBASys clinical auto analyzer.Results: Serum Survivin gene was positive in 27 (54%) HCC cases and negative in 23 (46%) cases. AFP-L3% was detected in 36 (72%) HCC cases. The combined use of serum servivin gene and AFP-L3% were positive in 43 (86%) of HCC cases. In 22 HCC cases with serum AFP below 200 ng/ml serum servivin gene was detected in 9 (40.9%) cases but AFP-L3% was positive in 13 (59.9%) cases and the combined use of positive serum servivin and AFP-L3 were present in 18 cases (81.18%).Conclusions: The combined use of serum survivin gene and AFP-L3% could have a valuable diagnostic role in HCC especially in those with low AFP.

Clinical Assay for AFP-L3 by Using Multiple Reaction Monitoring-Mass Spectrometry for Diagnosing Hepatocellular Carcinoma.

Lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) is a serum biomarker for hepatocellular carcinoma (HCC). AFP-L3 is typically measured by liquid-phase binding assay (LiBA). However, LiBA does not always reflect AFP-L3 concentrations because of its low analytical sensitivity. Thus, we aimed to develop an analytically sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) assay to quantify AFP-L3 in serum. The assay entailed the addition of a stable isotope-labeled internal standard protein analog, the enrichment of AFP using a monoclonal antibody, the fractionation of AFP-L3 using L. culinaris agglutinin lectin, deglycosylation, trypsin digestion, online desalting, and MRM-MS analysis. The performance of the MRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation. The lower limit of quantification of the MRM-MS assay (0.051 ng/mL) for AFP-L3 was less than that of LiBA (0.300 ng/mL). Thus, AFP-L3, which was not observed by LiBA in HCC samples (n = 39), was detected by the MRM-MS assay, improving the clinical value of AFP-L3 as a biomarker by switching to a more analytical sensitive platform. The method was validated, meeting all the criteria in integrated multinational guidelines. Because of the lower incidence of false-negative findings, the MRM-MS assay is more suitable than LiBA for early detection of HCC.

Development of on-chip fully automated immunoassay system "μTASWako i30" to measure the changes in glycosylation profiles of alpha-fetoprotein in patients with hepatocellular carcinoma.

Glycosylation profiles significantly change during oncogenesis. Aberrant glycosylation can be used as a cancer biomarker in clinical settings. Different glycoforms can be separately detected using lectin affinity electrophoresis and lectin array-based methods. However, most methodologies and procedures need experienced technique to perform the assays and expertise to interpret the results. To apply glycomarkers for clinical practice, a robust assay system with an easy-to-use workflow is required. Wako's μTASWako i30, a fully automated immunoanalyzer, was developed for in vitro diagnostics based on microfluidic technology. It utilizes the principles of liquid-phase binding assay, where immunoreactions are performed in a liquid phase, and electrokinetic analyte transport assay. Capillary electrophoresis on microfluidic chip has enabled the detection of different glycoform types of alpha-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma. AFP with altered glycosylation can be separated based on the reactivity to Lens culinaris agglutinin on electrophoresis. The glycoform AFP-L3 was reportedly more specific in hepatocellular carcinoma. This assay system can provide a high sensitivity and rapid results in 9 min. The test results for ratio of AFP-L3 to total AFP using μTASWako i30 are correlated with those of conventional methodology. The μTASWako assay system and the technology can be utilized for glycosylation analysis in the postgenomic era.

Evaluation of α-fetoprotein-L3 and Golgi protein 73 detection in diagnosis of hepatocellular carcinoma.

Aim of the studyHepatocellular carcinoma (HCC) is common throughout the world. Most HCCs are diagnosed at an advanced stage. There is an urgent need to find new methods for screening and surveillance of individuals at risk for HCC. The aim of this study was to evaluate serum α-fetoprotein (AFP)-L3 and serum Golgi protein 73 (GP73) detection in diagnosis of HCC with different AFP concentration.Material and methodsOne hundred and eighty one patients were involved, including 102 with HCC and 79 with benign liver disease. The serum AFP-L3 and GP73 was measured by a liquid-phase binding assay and quantitative enzyme-linked immunosorbent assay, respectively.ResultsOf the 102 HCC patients, 53 were positive for AFP, 77 were positive for AFP-L3, and 79 were positive for GP73. The maximum area under the curve for AFP-L3% and for GP73 was significantly different from the AUC of 0.5525 for total AFP (p < 0.01). AFP-L3% was not detected for AFP < 20 ng/ml. However, elevated GP73 was detected in 87.50% of the patients. In the HCC patients with total AFP 20–400 ng/ml, elevated AFP-L3 was detected in 26 patients, whereas in 23 patients elevated GP73 could be detected. In the HCC patients with a total AFP > 400 ng/ml, AFP-L3% > 10% was present in 96.23%, and GP73 was detected in 87.50%.ConclusionsThe determination of AFP-L3% and GP73 in combination with AFP can increase the sensitivity and specificity in diagnosis of HCC. α-fetoprotein-L3% and GP73, in combination with AFP, are useful biomarkers to confirm the diagnosis of HCC.

Assessment of prognosis in hepatocellular carcinoma (HCC) using biomarkers and serum measurements.

e15093 Background: Factors that influence prognosis in HCC are well-recognised. They include tumour size and number, the severity of liver dysfunction and the symptomology. Several staging systems combining a number of these factors offer an estimate of prognosis. Some concerned that these staging systems encompass factors that are inherently subjective, e.g. the extent of HCC in CLIP or the use of performance status in BCLC. This have led to the development BALAD score by Toyoda et al, using serum Bilirubin and Albumin and three biomarkers: alpha-fetoprotein-L3 (L3), Alpha-fetoprotein (AFP) and Des-gamma-carboxy prothrombin (DCP). This system was developed in a Japanese population which has mainly HCV-related HCC. Our aim was to develop an objective staging system using the three biomarkers for a western population and compared it to BALAD to assess its performance. Methods: 317 subjects with HCC were recruited from Birmingham, UK between 2007 and 2011. Blood samples were collected at the time of diagnosis. Liver, renal functions plus AFP, L3 and DCP were measured. L3 and DCP were measured by microchip capillary electrophoresis and liquid-phase binding assay on a uTASWako i30 auto analyzer (Wako Pure Chemical Industries Ltd, Japan). Results: Univariable and multivariable regression analyses were performed. Our scoring system (CRCTU score) was developed based on a flexible baseline hazard functions fitted using restricted cubic splines. Suitable fractional polynomials (FP) were used to explain relationship between the outcome and covariates. The level of discrimination achieved using the risk groups was assess by Harrell’s–C statistic. The CRCTU score permitted identification of 4 clearly distinct subgroups as indicated both graphically and by Harrell’s-C values. Both the CRCTU and BALAD scores return Harrell’s-C values of 0.7. Conclusions: We have shown that it is possible to use entirely objective markers to predict prognosis of HCC in a western population. Our model is comparable to the BALAD with the same variables included. However, our treatment of variables with FP is much more robust statistical technique for continuous variable as compared to having artificial categories that were seen in the BALAD system.

Diagnostic value of AFP-L3 and PIVKA-II in hepatocellular carcinoma according to total-AFP

To evaluate diagnostic value of α-fetoprotein (AFP)-L3 and prothrombin induced by vitamin K absence-II (PIVKA-II) in hepatocellular carcinoma (HCC). One hundred and sixty-eight patients during routine HCC surveillance were included in this study. Of the 168 patients, 90 (53.6%) had HCC including newly developed HCC (n = 82) or recurrent HCC after treatment (n = 8). Sera were obtained during their first evaluation for HCC development and at the time of HCC diagnosis before commencing HCC treatment. HCC was diagnosed by histological examination, appropriate imaging characteristics-computed tomography or magnetic resonance imaging. Control sera were collected from 78 patients with benign liver disease (BLD), which were obtained during routine surveillance with a suspicion of HCC. AFP, AFP-L3 and PIVKA-II were measured in the same serum by microchip capillary electrophoresis and liquid-phase binding assay on a micro-total analysis system Wako i30 auto analyzer. The performance characteristics of three tests and combined tests for the diagnosis of HCC were obtained using receiver operating characteristic curves in all populations and subgroups with AFP < 20 ng/mL. Of 90 HCC patients, 38 (42.2%) patients had AFP < 20 ng/mL, 20 (22.2%) patients had AFP 20-200 ng/mL and 32 (35.6%) patients had AFP > 200 ng/mL. Of the 78 BLD patients, 74 (94.9%) patients had AFP < 20 ng/mL. After adjustment for age and HBV infection status, AFP-L3 levels were higher in HCC than in BLD among patients with low AFP levels (< 20 ng/mL) (P < 0.001). In a total of 168 patients, areas under the curve (AUC) for HCC were 0.879, 0.887, 0.801 and 0.939 for AFP, AFP-L3, PIVKA-II and the combined markers, respectively. The combined AUC for three markers showed higher value than the AUCs of individual marker (P < 0.05). AFP-L3 had higher AUC value than PIVKA-II for HCC detection in entire patients (P = 0.043). With combination of AFP-L3 (cut-off > 5%) and PIVKA-II (cut-off > 40 AU/L), the sensitivity were 94.4% and specificity were 75.6% in all patients. In 112 patients with low AFP levels (< 20 ng/mL), AUCs of AFP-L3, PIVKA-II and combine AFP-L3 and PIVKA-II tests were 0.824, 0.774 and 0.939, respectively. AFP-L3 with a cut-off value of 5% showed sensitivity of 71.1% and specificity of 83.8%, and PIVKA-II with a cut-off value of 40 AU/L had sensitivity of 57.9% and specificity of 95.9% in patients with low AFP levels. The combination of AFP-L3 and PIVKA-II increased the sensitivity and specificity up to 92.1% and 79.7%, respectively, in low AFP group. Combined markers detected 81.8% of early stage HCC (Union for International Cancer Control stage I), 86.7% of small sized tumor (< 2 cm) and 91.7% of single tumor of HCC in the low AFP group. In multivariate analysis, AFP-L3 was correlated with AFP and tumor size, and PIVKA-II was correlated with laboratory tests including serum aspartate aminotransferase, total bilirubin, platelets and albumin levels. PIVKA-II had no correlation with AFP, AFP-L3 or tumor characteristics. Combined determination of AFP-L3 and PIVKA-II could improve the diagnostic value for HCC detection in patients with or without increased AFP levels.

Evaluation of procalcitonin with liquid-phase binding assay in hematological malignancy

BackgroundTo determine procalcitonin (PCT) levels in blood, a newly analyzer based on liquid-phase binding assay (LBA) was developed (micro-total analysis system, μTAS). We evaluated analytical and clinical performance of this system in comparison with autoanalyzer based on chemiluminescence enzyme immunoassay (CLEIA), which is widely-used but takes much longer time than LBA. MethodsA total of 518 serum samples from 28 patients undergoing hematopoietic stem cell transplantation were analyzed. The correlation between the 2 methods and the comparison of the each area under the receiver operating characteristics curve (AUC) for distinguishing bacterial or fungal infections from other events were evaluated. ResultsThe minimum detection limit of serum PCT levels by LBA was 0.02ng/ml. There was an excellent correlation between the two methods in all samples (r2=0.99), the samples obtained on days of fever onset (r2=0.99), and the samples collected in neutropenic state (r2=0.99). The AUC for detection of bacterial or fungal infection on the onset of fever was not significantly different between LBA and CLEIA (0.80 by LBA and 0.82 by CLEIA, P=0.19). ConclusionsThe PCT measurement using LBA was well correlated with conventional method based on CLEIA and had enough clinical performance to detect bacterial or fungal infection in clinical setting.

Reference change values for lens culinaris agglutinin-reactive α-fetoprotein and des-γ-carboxy prothrombin in patients with chronic hepatitis C

Lens culinaris agglutinin-reactive α-fetoprotein (AFP-L3) and des-γ-carboxy prothrombin (DCP) have been routinely used as serological tumor markers of hepatocellular carcinoma (HCC) for surveillance. The aims of this study were: (i) to determine the biological variation of AFP-L3 and DCP in patients with chronic hepatitis C; and (ii) to calculate the reference change values (RCVs) of AFP-L3 and DCP. Ten patients with cirrhosis due to hepatitis C virus (HCV) infection and without HCC were enrolled in the study. Serum samples were collected at 14-day intervals, and 10 samples in total were obtained for each patient. AFP-L3 and DCP levels were measured by microchip capillary electrophoresis and liquid-phase binding assay. Intra-individual (CV(I)) and inter-individual (CV(G)) biological variations and RCVs were estimated from the data generated. The CV(I) was 29.0% for AFP-L3 and 24.6% for DCP, and CV(G) was 63.5% for AFP-L3 and 40.4% for DCP. The RCVs for AFP-L3 and DCP were 68.3% and 58.5%, respectively. Increases in values for AFP-L3 and DCP within 68.3% and 58.5% may be biological variations. Clinician should take these variations into consideration for the management of patients with HCV infection under surveillance of HCC.

Better detection of Hepatocellular Carcinoma by combining Alphafeto-Protein (AFP) and Des-Gamma-Carboxyprothrombin (DCP)

Materials and Methods We performed a single-center, prospective, casecontrol study including 164 HCC patients and 422 controls between 02/2007 and 11/2008. The serum concentration of AFP and DCP, and the percentage of AFP-L3 were determined using the Wako LiBASys clinical auto-analyzer by a liquidphase binding assay. Results AFP, DCP and AFP-L3% were significantly elevated in HCC patients compared to controls (P < 0.0001). AFP and DCP showed equal sensitivities, but in combination the sensitivity was elevated from 28.7% for AFP alone (cutoff level of 400 ng/ml) up to 78.0% for all HCC patients using cutoff levels of 10 ng/ml for AFP and 5 ng/ml for DCP with a specificity of 89.3%. Even within earlystage (BCLC) patients the sensitivity could be elevated to 64.6%. The area under the ROC was 0.88 and 0.87 for AFP and DCP, respectively, and could be elevated to 0.91 for all biomarkers together.

Development of an antibody-lectin enzyme immunoassay for fucosylated α-fetoprotein

BackgroundFucosylation is one of the most important types of glycosylations related to cancer. Our previous studies of the enzymatic basis and structural studies of α-fetoprotein (AFP) samples from liver cancer patients indicated that core-fucosylation by α1,6-fucosyltransferase (FUT8) resulted in the production of fucosylated AFP, and in fact fucosylated AFP allowed differential diagnosis in some types of liver cancer from liver cirrhosis. This served as a predictive biomarker for the development of liver cancer 3 to 18months before it could be detected using imaging techniques. Fucosylated AFP is currently measured by means of a liquid-phase binding assay (LBA) or by an electrokinetic analyte transport assay (EATA). However, these methods require special instrumentation that is currently available only in major medical laboratories. To overcome this problem, we attempted to develop an enzyme immunoassay (EIA) based on the sandwich technique with specific antibody and lectin. ResultsDilute solutions of highly fucosylated AFP in human sera were assayed using a microtiter plate coated with a periodate-oxidized anti-AFP antibody, a fucose-specific biotinylated Aleuria aurantia lectin (AAL), a peroxidase-conjugated streptoavidin, and a chemiluminescent detection system. The technique was able to measure highly fucosylated AFP diluted to 5 to 80ng/ml in human sera using the developed antibody-lectin EIA in combination with the enrichment of AFP. ConclusionA simple method using an antibody-lectin EIA for quantifying fucosylated AFP that does not require special instrumentation was developed. General significanceThe method can be generally applied to the quantitative measurement of various fucosylated glycoproteins using specific antibodies. This article is part of a Special Issue entitled Glycoproteomics.

Novel Lens culinaris agglutinin-reactive fraction of α-fetoprotein: a biomarker of hepatocellular carcinoma recurrence in patients with low α-fetoprotein concentrations

Lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) is a specific marker used to detect hepatocellular carcinoma (HCC). However, its clinical utility is not sufficient in patients with low total AFP concentrations because of limitations in instrument sensitivity. Recent advances have led to the introduction of a highly sensitive AFP-L3% assay (sensitive AFP-L3%), provided by a novel on-chip, liquid-phase binding assay. This cross-sectional study was conducted to evaluate the clinical significance of the sensitive AFP-L3% in determining HCC recurrence in patients with low total AFP concentrations. A total of 370 consecutive patients with HCC were screened within 1-3 months of locoregional treatment, and 215 of the 370 patients showed serum AFP <20 ng/ml. Total AFP, sensitive AFP-L3%, and des-gamma-carboxy prothrombin (DCP) were measured in those 215 patients and HCC recurrence was evaluated by radiological findings. Optimal cutoff values of the markers for detecting HCC recurrence were obtained on the basis of receiver operating characteristic (ROC) curve. The area under the ROC curve of the total AFP, sensitive AFP-L3%, and DCP in HCC patients with serum AFP <20 ng/ml were 0.638, 0.724, and 0.779, respectively. The diagnostic accuracies of the total AFP, sensitive AFP-L3%, and DCP were 60.9% (cutoff value 5 ng/ml), 67.7% (cutoff value 7%), and 64.6% (cutoff value 40 ng/ml), respectively. The new sensitive AFP-L3% assay provides great utility in determining HCC recurrence in patients with low AFP concentrations. Further studies focusing on the combinatorial use of the markers (total AFP, sensitive AFP-L3%, and DCP) are required.

Clinical Advantage of Highly Sensitive On-Chip Immunoassay for Fucosylated Fraction of Alpha-Fetoprotein in Patients with Hepatocellular Carcinoma

Alpha-fetoprotein (AFP) has been widely used as a diagnostic master for hepatocellular carcinoma (HCC), and the fucosylated fraction of AFP (AFP-L3) has been reported to be a specific marker for HCC. However, AFP-L3 has not always been reliable in cases with low serum AFP concentrations. Recently, a novel automated immunoassay for AFP-L3, the micro-total analysis system (μ-TAS), has been developed. The aim of this study is to evaluate the clinical usefulness of μ-TAS AFP-L3. Serum AFP-L3 was measured in 295 patients with HCC and in 350 with benign liver diseases. The diagnostic accuracy of μ-TAS AFP-L3 was compared with that of the conventional assay (liquid-phase binding assay; LiBASys). The relationship between μ-TAS AFP-L3 and clinical features was investigated. When the cutoff value was set at 7%, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of μ-TAS AFP-L3 were 60.0%, 90.3%, 76.4%, 83.9%, and 72.8%, respectively. Its sensitivity was particularly good (41.1%) in HCC subgroups with lower AFP concentrations (<20 ng/ml). The positivity rates for μ-TAS AFP-L3 were higher at each tumor stage than those of LiBASys AFP-L3 (μ-TAS/LiBASys: stage I, 44.2%/16.3%; stage II, 52.9%/37.5%; stage III, 66.4%/44.5%; stage IV, 82.8%/65.5%). μ-TAS AFP-L3 is more sensitive for discriminating HCC than the conventional LiBASys AFP-L3, particularly in subgroups with lower AFP concentrations and early-stage HCC.

Determination of glutamic acid decarboxylase antibodies (GADA) IgG subclasses − comparison of three immunoprecipitation assays (IPAs)

IgG subclasses of glutamic acid decarboxylase (GAD(65)) antibodies (GADA) may reflect the immunological state in the pancreas of GADA-positive patients with autoimmune diabetes. The use of biotin-conjugated antibodies and streptavidin Sepharose are used commonly in immunoprecipitation assays (IPA) based on (125)I- or (35)S-labelled antigens to capture IgG subclasses directed against IA-2 or GAD(65). We have compared three different immunoprecipitation assays for the determination of GADA IgG subclasses. Two of the assays were based on the biotin and streptavidin systems provided in a solid (immobilized) or liquid (mobilized) phase binding environment. The third assay was based on N-hydroxysuccinimide (immobilized) interaction with primary amines (i.e. lysine residues) on the antibody. We found the liquid phase binding assay (LPBA) to be the most stable assay, with a comparatively low coefficient of variation and background.

Fucosylated fraction of alpha‐fetoprotein, L3, as a useful prognostic factor in patients with hepatocellular carcinoma with special reference to low concentrations of serum alpha‐fetoprotein

The aim of the present study was to establish L3 fraction before initial treatment as a useful prognostic factor in a prospective fashion in hepatocellular carcinoma (HCC) where the alpha-fetoprotein (AFP) was very low. From 1990 to 2004, 298 HCC patients in whom L3 could be measured were examined in the present study. Enrolled patients with HCC underwent operation, transcatheter arterial chemoembolization and percutaneous ablation therapy. The current patient status was confirmed as of the end of March 2005. L3 was determined by crossed immuno-affinoelectrophoresis when AFP was >/=30 ng/mL. It was carried out by liquid-phase binding assay system on cases where AFP < 30 ng/mL. The tentative discriminating line of L3 was set at 15%. The HCC group included four subgroups: 110 patients with AFP concentrations </=100 ng/mL, 70 with AFP</= 50 ng/mL, 38 with AFP </= 30 ng/mL and 29 with AFP </= 25 ng/mL. The mean survival rate in the HCC group, whose L3 was >15% (high L3), was significantly lower than that in the HCC group whose L3 was </=15% (low L3). There were also statistically significant differences in survival rates between high and low L3 in the four HCC subgroups. The statistically significant differences were more distinct in the subgroups with low AFP concentrations. The present study indicates that the L3 fraction before treatment serves as a useful prognostic indicator when the serum concentrations of AFP were very low.

Liquid-Phase Binding Assay of α-Fetoprotein Using DNA-Coupled Antibody and Capillary Chip Electrophoresis

An immunoassay using DNA-coupled antibody for bound/free separation in a liquid-phase binding assay format is described. Anti-alpha-fetoprotein monoclonal antibody was conjugated with DNA, mixed with alpha-fetoprotein (AFP), and incubated, and then 1 muL of the mixture was applied to capillary electrophoresis on a microchip. The DNA molecule of the antibody-DNA conjugate and the DNA-conjugated immune complex peak were detectable fluorophotometrically using intercalator dye within 90 s, whereas the Alexa-labeled antibody was detected as a broad and slower migrating peak. The electrophoretic mobility of the immune complex could be optimized for resolution and sharpness by changing the length of the DNA coupled to the antibody. The detection limit of AFP was approximately 300 pM in a sample. This immunoassay method utilizing a liquid-phase binding assay format is simple and convenient for antigen measurements on microchips.

Prenatal trisomy 21 screening using the Lens culinaris agglutinin-reactive alpha-fetoprotein ratio.

For the purpose of improving the clinical efficacy of alpha-fetoprotein (AFP)-L3% in prenatal screening for trisomy 21, we calculated the multiple of the median (MoM) of AFP-L3% (L3 MoM) and the ratio of L3 MoM to AFP MoM (L3 MoM/AFP MoM) in maternal serum. Maternal serum samples from 1822 women (maternal age 37.3 +/- 3.8 years, and weeks of gestation 16.0 +/- 1.0; mean +/- SD) with unaffected pregnancies and 28 women (37.6 +/- 4.6 years, 16.6 +/- 3.1) pregnant with of trisomy 21 fetuses were obtained. The AFP concentration and AFP-L3% in maternal serum were measured using a liquid-phase binding assay. The areas under the receiver operating characteristic curves (AUCs) of AFP MoM, AFP-L3%, L3 MoM, and L3 MoM/AFP MoM were 0.750, 0.868, 0.949 and 0.946, respectively. The AUCs of L3 MoM and L3 MoM/AFP MoM were significantly higher than AFP-L3% (P < 0.05) and AFP MoM (P < 0.0005). However, no statistical difference was observed between the AUCs of L3 MoM and L3 MoM/AFP MoM. In conclusion, the L3 MoM should be an effective replacement for AFP-L3% in prenatal trisomy 21 screening.

Interaction of human salivary mucin MG2, its recombinant N-terminal region and a synthetic peptide with Actinobacillus actinomycetemcomitans.

The antimicrobial properties of human salivary mucin MG2 against the periodontal pathogen, Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), were investigated using purified MG2, rNMUC7 (a recombinant polypeptide containing residue 1-144 of MG2) and synthetic peptides PEP1 (residue 1-17) and PEP2 (residue 47-63). MG2 and rNMUC7 bound to A. actinomycetemcomitans strains SUNY75, SUNY465, SUNY523, 652 and JP2 in a liquid phase binding assay. The bactericidal activities of rNMUC7, PEP1 and PEP2 against A. actinomycetemcomitans SUNY523 were examined in a colony forming unit killing assay. The LD50 for rNMUC7 was 9 microM, for PEP2 was 20 microM and PEP1 did not exhibit bactericidal activity. The primary structure of these polypeptides was analyzed and a direct relationship between net positive charge and bactericidal activity was found. Screening of saliva samples from 60 individuals on Western blots probed with an anti-MG2 antibody against PEP2 revealed that a 20 kDa MG2 fragment was present in 66% of subjects and that this fragment was not present in glandular secretions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides derived from the 20 kDa fragment confirmed that this fragment contained a portion of the amino terminal region of MG2. The present study showed that the N-terminal region of MG2 and a subdomain within this region are microbicidal against A. actinomycetemcomitans and that a 20 kDa fragment of MG2 occurs in whole saliva. This suggests that cleavage of MG2 in vivo may produce fragments with microbicidal properties and that this may represent a novel mechanism of host defense.

Automatic and simultaneous analysis of Lens culinaris agglutinin-reactive alpha-fetoprotein ratio and total alpha-fetoprotein concentration.

An automated analytical method for analyzing alpha-fetoprotein (AFP) carbohydrate chain microheterogeneity based on competitive reaction between lectin and anti-AFP monoclonal antibody in liquid phase is described. The antibody used binds to all species of AFP molecule without Lens culinaris agglutinin (LCA); however, its binding reaction to LCA-reactive AFP was inhibited by LCA. Sulfated tyrosine octamer was conjugated to the antibody, and sulfated tyrosine pentamer and peroxidase were conjugated to other monoclonal antibodies, respectively. Serum reacted with three anti-AFP monoclonal antibodies and LCA in liquid phases, and two types of immune complex were observed. The two types were separated directly by the liquid-phase binding assay system equipped with an anion-exchange column. Peroxidase activity of immune complex was determined fluorophotometrically. Total AFP concentration and the ratio of LCA-reactive AFP in samples were calculated simultaneously, using the sum of the two peaks and the ratio of peaks obtained by LCA inhibition to sum of two peaks. The results correlated well with conventional methods. The method is simple and convenient for routine clinical assays.

Determination of α-fetoprotein concentration based on liquid-phase binding assay using anion exchange chromatography and sulfated peptide introduced antibody

A new immunoassay for α-fetoprotein based upon liquid-phase binding reactions is described. In this procedure, a sample that contains α-fetoprotein is mixed with a solution of two anti- α-fetoprotein monoclonal antibodies complexed with peroxidase and sulfated peptide, respectively. After incubation, immune complex is separated from other components by anion exchange column chromatography. Immune complex is quantified using fluorometric detection by peroxidase enzymatic activity. Peroxidase activity correlated with a α-fetoprotein with a 1:1 relationship. The stoichiometric immunoreaction allowed a large analytical range (0.4–7500 ng/ml) with a linear dose–response relationship, high sensitivity and good precision. Endogenous substances did not interfere with assay performance. Assay results showed good correlation with other established methods. These results indicate that the method is useful for clinical α-fetoprotein determinations.

Liquid-phase binding assay of alpha-fetoprotein using a sulfated antibody for bound/free separation.

A rapid immunoassay using a sulfated antibody for bound/free separation in a liquid-phase binding assay is described. A first anti-alpha-fetoprotein monoclonal antibody was labeled with peroxidase (Fab'-POD) and a second monoclonal antibody was conjugated with polysulfated tyrosine peptide (Fab'-YS). The monoclonal antibodies and alpha-fetoprotein (AFP) were mixed, incubated, and analyzed directly by anion-exchange column chromatography. The amount of POD activity in the column effluent was determined fluorophotometrically. The bound (Fab'-POD + AFP + Fab'-YS) and free (Fab'-POD) forms of the conjugate were clearly and easily separated by ionic charge, and the free sulfated antibody (Fab'-YS) was not detectable fluorophotometrically. The elution position of the bound conjugate was adjusted by varying the length of the polysulfated tyrosine peptide. This method is convenient for antigen measurement because (1) only two modified antibodies are used in a buffer solution, (2) the concentration of antibodies and other assay conditions are easily set, (3) no solid phase is required, and (4) no washing is necessary.