anti-AFP Monoclonal Antibodies Research Articles

Multi-marker diagnosis method for early Hepatocellular Carcinoma based on surface plasmon resonance

Early diagnosis of Hepatocellular Carcinoma (HCC) is an important means to raise the survival rate of patients. Multi-marker combined detection is a powerful tool of early HCC diagnosis. Traditional detection methods are not effective and accurate because it is difficult to achieve combined detection of multiple markers. In this paper, we selected Alpha Fetoprotein (AFP) and miRNA-125b as the combined detection markers to improve the simultaneously diagnostic sensitivity and specificity. The anti-AFP monoclonal antibody and the DNA probes paired with the miRNA-125b were modified on the surface of surface plasmon resonance (SPR) sensor respectively to specifically recognize AFP and miRNA-125b in serum. In order to enhance the SPR response signal and detection sensitivity, Double Antibody Sandwich Method (DASM) and S9.6 antibody enhanced method were applied to achieve low detection limit of the two markers. Experimental results showed that AFP (25–400 ng/mL) was accurately detected by DASM and the detection limit of miRNA-125b by S9.6 antibody enhanced method reached 123.044 pM. These results verified the feasibility of the multi-marker detection method in early diagnosis of HCC.

Antitumor effects of brucine immuno-nanoparticles on hepatocellular carcinoma in vivo.

In vitro and in vivo studies have demonstrated that brucine is able to inhibit the proliferation of liver cancer cells and growth of animal tumors, and may be a promising anticancer drug. However, high toxicity, poor water solubility, short half-life, narrow therapeutic window, and similar therapeutic and toxic doses limit its clinical application in the treatment of malignant tumors. In our previous study, brucine immuno-nanoparticles were successfully prepared and added to the culture medium of liver cancer SMMC-7721 cells, and the results indicated that the brucine immuno-nanoparticles were able to target the cell membrane of liver cancer SMMC-7721 cells and significantly inhibit the proliferation, adhesion, invasion and metastasis of SMMC-7721 cells. The aim of the present study was to investigate the antitumor effect of brucine immuno-nanoparticles in vivo by establishing an in situ transplanted liver cancer in nude mice. The results indicated that in vivo application of the brucine immuno-nanoparticles resulted in temporary liver and kidney damage, and significantly reduced the α-fetoprotein (AFP) secretion of tumor cells (Bru-NP-MAb vs. the other groups; P<0.05). The brucine concentration of tumor tissues in the brucine immuno-nanoparticles group was significantly increased compared with that of the brucine nanoparticles group (Bru-NP-MAb vs. Bru-NP group or brucine group; P<0.05). The brucine immuno-nanoparticles were able to inhibit tumor growth and cluster of differentiation 34 expression and angiogenesis of tumor tissues, and induce the apoptosis of tumor cells (Bru-NP-MAb vs. Bru-NP group or brucine group; P<0.05). In conclusion, as a novel type of targeted drug, brucine nanoparticles combined with anti-AFP monoclonal antibodies was more effective compared with brucine nanoparticles or brucine alone in inhibiting tumor growth via the enhancement of apoptosis, and the suppression of proliferation and angiogenesis in vivo. Therefore, the brucine immuno-nanoparticle is a promising targeted drug for the treatment of hepatocellular carcinoma.

Enzyme–antibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein

In this study, we designed a comprehensive strategy for the ultrasensitive detection of alpha fetoprotein (AFP) with high specificity using gold nanoparticle (AuNP)-based enzyme-linked immunosorbent assay (ELISA). A dual-film modified probe was synthesized by coating AuNPs with horseradish peroxidase (HRP) on its surface. Anti-AFP monoclonal antibody (McAb) was immobilized on the surface of the enzyme using glutaraldehyde cross-linking method. AuNPs, employed as support for the immobilization of HRP. HRP was used not only as the enzymatic-amplified tracer but also as a bridge for loading McAb. The limit of detection was 2 ng mL-1. The developed probes can provide an alternative approach with high sensitivity and a simple process similar to that of the traditional HRP-McAb based ELISA for the ultrasensitive detection of AFP in serum.

Fabrication of Carboxylated Silicon Nitride Sensor Chips for Detection of Antigen–Antibody Reaction Using Microfluidic Reflectometric Interference Spectroscopy

In this study, we report label-free detection of alpha-fetoprotein (AFP), which has been used as a biomarker for hepatocellular carcinoma, by a microfluidic reflectometric interference spectroscopy (RIfS) system adopting a simple halogen light source and an inexpensive silicon-based sensor chip. Introduction of carboxy groups on a silicon nitride sensor chip to immobilize anti-AFP monoclonal antibody (anti-AFP) was carried out simply by immersion in aqueous solution containing triethoxysilylpropylmaleamic acid bearing a carboxy group and a silanol group. The RIfS system with the anti-AFP-immobilized sensor chip was found to give a reversible response through 100 on/off cycles using a regeneration buffer with high reproducibility (coefficient of variation (CV) = 5.7%). The limit of detection (LOD) of AFP was 100 ng mL(-1), and the measurement range spanned 3 orders of magnitude. Furthermore, the sensor chip showed no cross-reactivity with human serum albumin, Immunoglobulin G, transferrin, or fibrinogen at 100 μg mL(-1) without the use of blocking reagents such as bovine serum albumin. Consequently, the proposed RIfS system is a potentially effective tool for biomarker detection and in vitro diagnostics.

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was logY=3.65786+0.43863·logX (R=0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4ngmL−1. By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.

Determination of α-Fetoprotein in Human Serum by a Quartz Crystal Microbalance-based Immunosensor

Increased alpha-fetoprotein (AFP) in adult plasma is considered an early indication of hepatocellular carcinoma and teratoblastoma. The aim of this study was to develop a rapid method to measure AFP in human serum by use of a direct immunosensor based on a quartz crystal microbalance (QCM). A self-assembled monolayer prepared by the cystamine method was applied to immobilize anti-AFP monoclonal antibodies on the gold surface of a quartz crystal. The frequency shifts of the QCM were measured and related to AFP concentrations. The piazoimmunosensor used no labeled reagent and no pretreatment of samples. Ten cycles of measurements could be performed on the gold surface of the same crystal regenerated with a solution of glycine-HCl. A linear relationship existed between the frequency shifts (Hz) and the log values of AFP concentrations from 0.1 to 100 microg/L in buffer and human serum. When used for 15 days, the frequency shifts were all >95% of those on the response at the first day. The regression equation was y = 1.03x - 0.06 (S(y/x) = 3.92; r = 0.9987) for this QCM method and RIA in 29 clinical human serum samples. The QCM sensor can measure AFP in buffer and human serum and offers advantages of high specificity, reusability, low detection limit, no label or sample pretreatment, and low sample requirement. The assay format of the immunosensor was more rapid and simpler than conventional methods.

Alpha-fetoprotein in plasma and serum of healthy adults: preanalytical, analytical and biological sources of variation and construction of age-dependent reference intervals

Alpha-fetoprotein (AFP) is a tumor marker for hepatomas and germ cell tumors, and the serum concentration has prognostic significance in other diseases. We examined the normal serum concentration of AFP in adults and sources of variation in the immunochemical variation of AFP. The serum concentration of the tumor marker alpha-fetoprotein (S-AFP) was log-normally distributed in 284 adult blood donors. S-AFP increased with age (p<10 7), whereas no gender-related difference was found. Reference intervals (95-interpercentile) were constructed for persons 40 years (0.60-9.30kIU/L) and >40 years (1.40-12.60kIU/L). The concentration of AFP was significantly, albeit slightly, higher in serum than in plasma, whereas hemolysis, pretreatment with KCl and food intake did not influence S-AFP. S-AFP only changed 6% when measured twice 2 months apart (p=0.04). Three enzyme immunoassays, using three different anti-AFP monoclonal antibodies for detection, were compared and two assays gave S-AFP values significantly higher, 2.8% (p=0.03) and 19.0% (p<10 4), than the other assay. Thus, the choice of antibody may influence the result of immunochemical concentration determination. This can b eexplained by the existence of conformational variants of AFP with different antibody reactivities, and calls for careful standardization of monoclonal antibodies used in assays for AFP. With broad population reference ranges and slight intra-personal variation, the most effective reference range for S-AFP is previous values obtained in the same person.

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.

Rapid diagnosis of premature rupture of membranes using a new kit employing anti-AFP monoclonal antibody

Rapid diagnosis of premature rupture of membranes using a new kit employing anti-AFP monoclonal antibody

Diagnosis of preterm premature rupture of the membranes using a newly developed AFP monoclonal antibody test kit

Objective: We have developed a new anti-AFP monoclonal antibody kit which is easier to use than other examination methods for detecting AFP in the leaked amniotic fluid. In this study, we investigate the clinical value of this test in the diagnosis of preterm premature rupture of the membranes (PROM). Methods: We employed 103 patients of less than 37 weeks of gestational age for this preliminary study. We compared the clinical usefulness of this new AFP test with that of the nitrazine test and measured the concentration of AFP in vaginal fluid or cervical secretion by EIA. Results: The nitrazine test showed a correct diagnostic rate of 62.1%, in contrast the AFP test kit had a 98.0% rate ( P < 0.001). The reaction time using the kit is 3 min. Conclusion: The AFP test is a simple and non-invasive test which can be easily carried out repeatedly as a bedside examination. This study has confirmed the high clinical efficacy of the newly developed AFP test kit as a method of PROM diagnosis.

Rapid diagnosis of premature rupture of membranes using a new kit employing anti-AFP monoclonal antibody

A kit using mouse monoclonal antibody against human alpha-fetoprotein (anti-AFP kit) has been developed. Cervical secretion (n = 34) and vaginal fluid (n = 15) was obtained from 30 pregnant volunteers. All 31 specimens obtained from women with intact membranes tested negative while all 18 specimens from women with premature rupture of membranes (PROM) tested positive. The newly developed kit allowed rapid and accurate diagnosis. We compared the kit results with those obtained by pH determination (nitrazine, bromthymol blue test) and measurement of AFP concentrations by enzyme-linked immunoassay (EIA).

Immunotargeting chemotherapy for AFP-producing pediatric liver cancer using the conjugates of anti-AFP antibody and anti-tumor agents

The effect of immunotargeting chemotherapy for hepatoblastoma (HB) and hepatocellular carcinoma (HCC) following the application of adriamycin (ADM) or cis-platinum conjugated with anti-alpha-fetoprotein (AFP) antibody was evaluated experimentally and clinically. The conjugate was made from mouse monoclonal antihuman AFP antibody linked to ADM or CDDP, with a weight ratio of 2.5:1 via a dextran bridge. Experimentally, AFP-producing human HCC transplanted subsequently on nude mice was used. A mixture of the antibody and ADM or CDDP was prepared with the same ratio. Each drug was injected intraperitoneally, three times at the total dose of 14.4 mg/kg as ADM and one time at the dose of 8 mg/kg as CDDP. Tumor growth was inhibited significantly in the conjugate group compared with the other mixture group, the ADM or CDDP group, and the control group. Clinically, the conjugates were administered intraarterially in 4 cases (2 HBs and 2 HCCs) and intravenously in one case (1 HB). ADM and CDDP conjugated with anti-AFP antibody were used in 2 cases and 3 cases, respectively. Antitumor effects from the viewpoint of volume suppression rate showed partial response in 2 cases and no change in 3 cases. The immunotargeting chemotherapy using anti-AFP monoclonal antibodies may be a promising method for treatment of malignant epithelial liver cancer in children.

FIRST TRIMESTER MATERNAL SERUM ALPHA-FETOPROTEIN (MSAFP) SCREENING FOR CHROMOSOME DEFECTS

Second trimester MSAFP screening for low values is proving valuable in the detection of fetal chromosome defects in women < 35 yrs. of age. Detection prior to 16 wks. would be desirable. To determine whether low MSAFP values also occur in the first trimester in association with a fetal chromosomal abnormality, we used a “simultaneous-sandwich” radioimmunoassay with polystyrene beads coated with anti-AFP monoclonal antibodies, using solid phase support. The coefficient of variation was<5% for this assay which is about 10 x more sensitive than conventional RIAs. Blood was sampled in 359 cases just before CVS (chorionic villus sampling) in Milan. Fetal age was usually assessed by 2 ultrasound studies± 1 wk. apart, and chromosomes of the villi analysed. Sera were sent blind & only after AFP assay results were sent to Milan were karyotypes released. MSAFP≤0.6 MOM occurred in 5/17 (45.5%) significant fetal chromosome defects (trisomy 21, 18 [2], 13 & unbal. 13/14 translocation) while 2 (18.2%) were<0.5 MOM. 11/17 (64.7%) affected fetuses had MSAFP<median. 1/5 trisomy 21 fetuses had MSAFP< 0.5 MOM. About 75% of trisomy 21 offspring are delivered by women< 35 yrs. of age. These results suggest that first trimester MSAFP screening could identify a significant number of these pregnancies early enough for CVS. Further study is necessary to determine the optimum week for efficient screening.

1305 FIRST TRIMESTER MATERNAL SERUM ALPHA-FETOPROTEIN (MSAFp) SCREENING

Second trimester MSAFP screening is valuable in the prenatal detection of neural tube defects and in the recognition of other problems. Our screening leads to amniocentesis in 1:400, but not infrequently leads to a later conclusion of prenatal studies with options to abort often between 20-24 wks. DB has established an extremely sensitive “simultaneous-sandwich” radioimmunoassay (M-RIA) with polystyrene beads coated with anti-AFP monoclonal antibodies and using solid phase support. Comparative assays to available polyvalent commercial kits showed M-RIA to be 4-10 times more sensitive. In addition, inter- and intra-assay variation and reproducibility were assessed over a wide range of values. Results were noted to be accurate with a coefficient of variation < 5%. MRIA studies of 400 non-pregnant females showed serum AFP undetectable in 99.6%, others having MSAFP values < 3 ng/ml. We have analysed 449 first trimester MSAFP samples, from apparently normal pregnancies. We report that AFP was detectable between 8-9 wks, significant (p <0.001)AFP “elevations” were noted at 10 wks, AFP rose linearly 10-24 wks, SEM's were small, higher AFP levels were found & M-RIA was accurate, precise, & reproducible. 3/4 patients have also had a 2nd trimester MSAFP screen. Correlations between ultrasound studied pregnancies, 1st and 2nd trimester AFPs, and outcome will be presented. These preliminary data suggest that MSAFP screening may be possible earlier than 16 wks and even in the 1st trimester.