Serum Albumin Conjugate Research Articles

Design of iron oxide nanoparticles decorated oleic acid and bovine serum albumin for drug delivery

This study aimed to originally develop a new nanoparticulate drug delivery system of iron oxide nanoparticles (Fe3O4) for biomedical applications. Oleic acid and bovine serum albumin were decorated on the surface of iron oxide nanoparticles in new pattern by conjugation. The decoration was kicked off by the functionalization of arginine on the surface of the iron oxide nanoparticles. It was then followed by the conjugation of oleic acid and bovine serum albumin through the amide bond. Scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction and Fourier transform infrared spectroscopy were used to characterize and determine mechanism of the decorated nanoparticles. Paclitaxel was chosen as the model drug in the study. The nanoparticles demonstrated a potential utility in delivery of anticancer drugs.

Nongenomic effects of estrogen on extracellular signal-regulated kinases through initiating transient calcium flux in endometrial cancer

To study the mechanism on extracellular signal-regulate kinases (ERK) signal transduction by calcium influx initiated by combination of estrogen with calcium channels or estrogen receptor in endometrial cancer cell Ishikawa. Confocal test was used to determine the relative calcium mobilization by stimulation of estrodiol together with and without the inhibition of ICI182780 and nifedipine. Western-blotting was used to detect the protein expression of phosphorylated ERK1/2 (P-ERK1/2) in the same condition. The transient calcium flux initiated by 17β-estrodiol (E2) and a membrane-impermeable conjugate of estrogen and bovine serum albumin (E2-BSA), and the calcium mobilization could be inhibited by ICI182780 and nifedipine in 1 min. In Ishikawa cells, phosphorylation of ERK1/2 was stimulated by E2, and the phosphorylation could not be inhibited by E2 after the combination with ICI182780 in 5 min and in 30 min. The phosphorylation also could not be inhibited by E2-BSA after the combination with nifedipine in 5 min, but in 30 min the phosphorylation was decreased. The phosphorylation of ERK by E2-BSA was decreased by the combination with nifedipine in 30 min. The transient calcium flux initiated by estrogen has an effect on the activation of ERK signal pathway in endometrial carcinoma cells.

Steroid Hormones as Transporters to Carry Exogenous Macromolecules into the Target Cell Nuclei in Vivo

Upon injection into the vascular system of rats, testosterone-bovine serum albumin conjugate (testosterone-BSA) is taken up by cells via the process of endocytosis. When it is taken up by the target cells of testosterone such as spermatogenic cells, it enters the nuclei of the cells. However, testosterone-BSA does not enter the nuclei of the non-target cells such as hepatocytes and thymocytes. Similarly, hydrocortisone-BSA conjugate enters the nuclei of its target cells such as hepatocytes and thymocytes. In the vesicular trafficking of testosterone-BSAs into the nucleoplasm, the vesicle membrane is likely to fuse with a nuclear hemifusion diaphragm. IgG coupled with hydrocortisone also enters the hormone-target cell nuclei, with its antigenicity kept intact. These results suggest that steroid hormones could act as transporters for conveying exogenous macromolecules into the target cell nuclei in vivo. Our studies provide a novel insight to the functions of steroid hormones.

Stress-sensitive organs and blood corticosterone after immobilization of active and passive rats immunized with glutamate-bovine serum albumin conjugate.

We studied stress-induced organ and hormonal responses in behaviorally active and passive rats against the background of immunization with glutamate-BSA conjugate. The relative weight of the adrenal glands after immobilization was lower in rats immunized with the conjugate in comparison with non-immunized animals. The weight of the adrenal glands in behaviorally active rats decreased in parallel with the decrease in blood corticosterone. In behaviorally active and passive rats immunized with the conjugate, ulcer formation in the stomach was slightly intensified after immobilization. It was hypothesized that immunization with glutamate-BSA conjugate suppresses activity of the hypothalamic-pituitary-adrenal feedback mechanism underlying the production of glucocorticoid hormones, which is manifested in slightly increased ulceration due to attenuation of the gastroprotective action of corticosterone under stress.

Human serum albumin-TRAIL conjugate for the treatment of rheumatoid arthritis.

Albumin conjugation is viewed as an effective means of protracting short in vivo lifespans of proteins and targeting rheumatoid arthritis (RA). In this study, we present a human serum albumin (HSA) conjugate linked with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a bifunctional PEG derivative (HSA-TRAIL). Prepared HSA-TRAIL was found to have a larger molecular size (∼240 kDa, 15.4 nm) than TRAIL (∼66 kDa, 6.2 nm), and its bioactivity (apoptosis, cytotoxicity, and antiproliferation) was well preserved in Mia Paca-2 cells and mouse splenocytes. The enhanced therapeutic efficacy of HSA-TRAIL was demonstrated in collagen-induced arthritis (CIA) mice. The incidence and clinical scores, expressed as degree of erythema and swelling in HSA-TRAIL-treated mice, were remarkably lower than those of TRAIL-treated mice. The serum levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β, and IL-2 in HSA-TRAIL-treated mice were significantly lower than those of TRAIL-treated mice. Furthermore, HSA-TRAIL accumulated in the hind paws of CIA mice, not in naïve TRAIL mice. Pharmacokinetic profiles of HSA-TRAIL were greatly improved in comparison to those of TRAIL (AUCinf: 844.1 ± 130.0 vs 36.0 ± 1.2 ng·h/mL; t1/2: 6.20 ± 0.72 vs 0.23 ± 0.01 h, respectively). The HSA-TRAIL conjugate, which presents clear advantages of targeting RA and long systemic circulation by HSA and unique anti-inflammatory efficacy by TRAIL, has potential as a novel treatment for rheumatoid arthritis.

Peroxidase-mediated conjugation of corn fiber gum and bovine serum albumin to improve emulsifying properties.

The emulsifying properties of corn fiber gum (CFG), a naturally occurring polysaccharide-protein complex, was improved by kinetically controlled formation of hetero-covalent linkages with bovine serum albumin (BSA), using horseradish peroxidase (HRP). The formation of hetero-crosslinked CFG-BSA conjugates was confirmed using ultraviolet-visible and Fourier-transform infrared analyses. The optimum CFG-BSA conjugates were prepared at a CFG:BSA weight ratio of 10:1, and peroxidase:BSA weight ratio of 1:4000. Selected CFG-BSA conjugates were used to prepare oil-in-water emulsions; the emulsifying properties were better than those of emulsions stabilized with only CFG or BSA. Measurements of mean droplet sizes and zeta potentials showed that CFG-BSA-conjugate-stabilized emulsions were less susceptible to environmental stresses, such as pH changes, high K ionic strengths, and freeze-thaw treatments than CFG- or BSA-stabilized emulsions. These conjugates have potential applications as novel emulsifiers in food industry.

Magnetic bead-based colorimetric immunoassay for aflatoxin B1 using gold nanoparticles.

A competitive colorimetric immunoassay for the detection of aflatoxin B1 (AFB) has been established using biofunctionalized magnetic beads (MBs) and gold nanoparticles (GNPs). Aflatoxin B1-bovine serum albumin conjugates (AFB-BSA) modified MBs were employed as capture probe, which could specifically bind with GNP-labeled anti-AFB antibodies through immunoreaction, while such specific binding was competitively inhibited by the addition of AFB. After magnetic separation, the supernatant solution containing unbound GNPs was directly tested by UV-Vis spectroscopy. The absorption intensity was directly proportional to the AFB concentration. The influence of GNP size, incubation time and pH was investigated in detail. After optimization, the developed method could detect AFB in a linear range from 20 to 800 ng/L, with the limit of detection at 12 ng/L. The recoveries for spiked maize samples ranged from 92.8% to 122.0%. The proposed immunoassay provides a promising approach for simple, rapid, specific and cost-effective detection of toxins in the field of food safety.

Immunologic evaluation of patients with cefotetan-induced anaphylaxis.

Cefotetan is a commonly prescribed second-generation cephalosporin that acts against a wide range of bacteria. However, cefotetan-induced hypersensitivity has rarely been reported. We report 2 cases of cefotetan-induced anaphylaxis with immunologic evaluation. The first case was a 70-year-old asthmatic woman who had dyspnea and hypotension during administration of cefotetan, in which high serum-specific IgE to cefotetan-human serum albumin (HSA) conjugate was detected by enzyme-linked immunosorbent assay. The second case was a 63-year-old asthmatic woman who complained of chest tightness and dyspnea during cefotetan infusion, in which high serum-specific IgG1 and IgG4 with no serum specific IgE to cefotetan-HSA conjugate was detected. The basophil activation test using basophils from the patient showed a significant up-regulation of CD63 with the addition of anti-IgG4 antibody compared with that in non-atopic healthy controls. In conclusion, cefotetan can induce anaphylaxis, which may involve both IgE- and IgG4-mediated responses in the pathogenic mechanism.

Establishment of an enzyme-linked immunosorbent assay and application on determination of ginsenoside Re in human saliva.

This work describes an immunochemical approach for the quality control of Panax ginseng and a pharmacological study of ginsenoside Re, a major bioactive constituent in P. ginseng, using an enzyme-linked immunosorbent assay. A hybridoma secreting monoclonal antibody against ginsenoside Re was produced by fusing splenocytes immunized with a ginsenoside Re-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line. The method, at an effective measuring range of 7.8-500 ng · mL(-1) of ginsenoside Re, successfully detected ginsenoside Re in Chinese traditional herb prescriptions. The results demonstrate that we generated a novel and reliable assay system for measuring ginsenoside Re in Chinese medicines more efficiently. Futhermore, we determined the ginsenoside Re concentrations in the saliva of six healthy adults after the oral administration of a ginseng capsule to study the pharmacokinetics of ginsenoside Re in human saliva.

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for sterigmatocystin in cereal and oil products.

Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL−1) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g−1 for wheat, 0.06 ng·g−1 for maize, and 0.1 ng·g−1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products.

Development of antibody-based microarray assay for quantitative detection of aflatoxin B1

BACKGROUND: Aflatoxin B1 (AFB1) is a toxic metaboliteproduced by Aspergillus species that contaminates a wide range ofagricultural products. OBJECTIVES: This study was designed todevelop a rapid and highly sensitive immunoassay method inmicroarray format for quantitative detection of AFB1 to evaluatethe potential of microarray platform for high-throughput screening,which can be beneficial in food and feed industry. METHODS:Following successful optimization, using an indirect competitiveimmunoassay in dot blot format, AFB1-bovine serum albumin(AFB1-BSA) conjugate was contact-printed onto 16 isolated subarrayson multi-pad nitrocellulose coated slides; subsequently usedin competitive binding assays. RESULTS: Using the aforementionedassay, AFB1 was determined from 15 pg/g to 3.04 ng/g workingrange with detection limit (LOD) of 1 pg/g. To evaluate assayperformance in real food matrices, the authors spiked wheatsamples with different concentration of AFB1. After extraction,working ranges of 0.11-4.15 ng/g with detection limit of 30pg/g wasdetermined. Good recoveries (94±9%) were obtained, demonstratingaccurate detection of AFB1 concentrations in wheat samples.Assay procedure completed in 3 hours. CONCLUSIONS: The resultsindicated that the proposed developed assay in microarray formatcould be used for rapid and sensitive detection of AFB1in wheatsamples.

Preparation and sonodynamic activities of water-soluble tetra-α-(3-carboxyphenoxyl) zinc(II) phthalocyanine and its bovine serum albumin conjugate

Sonodynamic therapy (SDT) is a new approach for cancer treatment, involving the synergistic effect of ultrasound and certain chemical compounds termed as sonosensitizers. A water-soluble phthalocyanine, namely tetra-α-(3-carboxyphenoxyl) zinc(II) phthalocyanine (ZnPcC4), has been prepared and characterized. The interactions between ZnPcC4 and bovine serum albumin (BSA) were also investigated by absorption and fluorescence spectroscopy. It was found that there were strong interactions between ZnPcC4 and BSA with a binding constant of 6.83×107M−1. A non-covalent BSA conjugate of ZnPcC4 (ZnPcC4–BSA) was prepared. Both ZnPcC4 and ZnPcC4–BSA exhibited efficient sonodynamic activities against HepG2 human hepatocarcinoma cells. Compared with ZnPcC4, conjugate ZnPcC4–BSA showed a higher sonodynamic activity with an IC50 value of 7.5μM. Upon illumination with ultrasound, ZnPcC4–BSA can induce an increase of intracellular reactive oxygen species (ROS) level, resulting in cellular apoptosis. The results suggest that the albumin conjugates of zinc(II) phthalocyanines functionalized with carboxyls can serve as promising sonosensitizers for sonodynamic therapy.

RAT AIRWAY CHANGES AFTER CHALLENGE WITH TRIMELLITIC ANHYDRIDE: EFFECT OF DIETHYLCARBAMAZINE CITRATE AND OMEGA-3 FATTY ACIDS

Trimellitic anhydride (TMA), an acid anhydride widely used in the plastics industry, can induce occupational asthma in man. This study aims to test the effect of diethylcarbamazine citrate (DEC) and omega-3 fatty acids (ω-3 FAs) as anti-inflammatory agents against pulmonary inflammation induced by TMA challenge. Sensitized albino rats (Rattus albus) were challenged for once with TMA by intra-tracheal instillation of TMA-rat serum albumin (RSA) conjugate. DEC and ω-3 FAs (100 and 600 mg/kg) were given once/day for five days before challenge. After 24 hours of challenge, bronchoalveolar lavage (BAL), lung homogenates and sections for both light and electron microscopy were prepared. Activity of eosinophil peroxidase (EPO) and the quantity of red blood cells (RBCs) in BAL were measured. The EPO activity, nitric oxide (NO) and IL-4 assays in lung tissue homogenates were also measured. The presence of specific IgE was measured by heterologous passive cutaneous anaphylaxis (PCA) assay. Specific stain for collagen fibres was used. EPO in BAL were significantly decreased by both ω-3 FAs and DEC (p<0.01), while RBCs were decreased by ω-3 FAs (p<0.05). In lung homogenates, only DEC decreased (p<0.01) the EPO activity, but both DEC and ω-3 FAs decreased (pp<0.01, respectively) IL-4 level. In contrast, neither DEC nor ω-3 FAs did decrease NO. PCA was high in TMA group, but diminished obviously in ω-3 FAs than DEC groups. Histological and histochemical observations revealed TMA-induced changes, which were ameliorated by both DEC and ω-3 FAs. Ultrastructural observations revealed only changes in pneumocytes type II, which were partially resumed after ω-3 FAs treatment. This study indicated the usefulness of both DEC and ω-3 FAs in treatment of pulmonary inflammation.

Fabrication and sensing property for conducting polymer nanowire-based biosensor for detection of immunoglobulin G

Conducting polymers are excellent sensing materials in the design of bioanalytical sensors because of their electronic conductivity, low energy optical transitions, biocompatibility, and room temperature operation. Among them, Polypyrrole (Ppy) is one of the most extensively used conducting polymers because of a number of properties such as redox activity, rapid electron transfer, and ability to link a variety of biomolecules to pyrrole groups by chemical treatment. In this study, Ppy nanowires were synthesized by an electrospinning method. The nanowires were prepared from a solution mixture of Ppy and poly(ethylene oxide). The method of detection in such a device is based on the selective binding of antigen onto an antibody that is covalently attached to the nanowires. Thus, anti-IgG was immobilized on Ppy nanowires using an EDC {[N-(3-dimethyl aminopropyl)-N2-ethylcarbodiimide hydrochloride]}-NHS(N-hydrosuccinimide) modified technique. Fluorescence images of BSA–FITC (fluorescein isothiocyanate labeling of bovine serum albumin) conjugation demonstrated that antibody was functionalized on the Ppy nanowires without non-specific binding and facilitated selective detection of antigen. Current–voltage (I–V) characterization was used to monitor the change in the conductivity of nanowires while the specific binding interaction occurred. These results of electrical properties enable Ppy nanowire-based biosensors to detect biomolecules in real-time.

Transdermal delivery of proteins using a combination of iontophoresis and microporation.

This study aimed to investigate transdermal delivery of proteins using combination of microporation and iontophoresis (ITP). Materials & methods & results: Delivery of model protein, Alexa Fluor 555 bovine serum albumin conjugate (AF-BSA) using ITP alone, microneedle (MN) alone, and ITP plus MN combination was assessed using confocal microscopy. Compared to MN alone, combination of MN plus ITP significantly increased skin's penetration depth of AF-BSA (300 vs 110 μm) and achieved lateral distribution of the model protein. Average fluorescence intensity quantified around each microchannel was 23.7-fold (8.2-fold, in vivo) higher for combination treatment compared with MN alone, in vitro. After 1 h in vitro permeation study, the unlabeled BSA amount delivered across skin was found to be 0, 1.4, 0.63 and 14 μg by passive, MN alone, ITP alone and ITP plus MN combination delivery, respectively.

Gold nanocatalyst-based immunosensing strategy accompanying catalytic reduction of 4-nitrophenol for sensitive monitoring of chloramphenicol residue

A new competitive-type immunosensing system based on gold nanoparticles toward catalytic reduction of 4-nitrophenol (4-NP) was developed for sensitive monitoring of antibiotic residue (chloramphenicol, CAP, used in this case) by using ultraviolet–visible (UV–vis) spectrometry. Gold nanoparticle (AuNP) with 16nm in diameter was initially synthesized and functionalized with CAP–bovine serum albumin (CAP–BSA) conjugate, which were used as the competitor on monoclonal anti-CAP antibody-coated polystyrene microtiter plate (MTP). In the presence of target CAP, the labeled CAP–BSA on the AuNP competed with target CAP for the immobilized antibody on the MTP. The conjugated amount of CAP–BSA–AuNP on the MTP decreased with the increase of target CAP in the sample. Upon addition of 4-NP and NaBH4 into the MTP, the carried AuNP could catalytically reduce 4-NP to 4-aminophenol (4-AP), and the as-produced 4-AP could be monitored by using UV–vis absorption spectroscopy. Experimental results indicated that the absorbance at 403nm increased with the increment of target CAP concentration in the sample, and exhibited a dynamic range from 0.1 to 100ngmL−1 with a detection limit (LOD) of 0.03ngmL−1 at the 3sblank level. Intra- and inter-assay coefficients of variation were lower than 5.5% and 8.0%, respectively. In addition, the methodology was evaluated for CAP spiked honey and milk samples, respectively. The recovery was 92–112%.

A Magnetic Nanoparticle Stabilized Gas Containing Emulsion for Multimodal Imaging and Triggered Drug Release

To develop a multimodal imaging guided and triggered drug delivery system based on a novel emulsion formulation composed of iron oxide nanoparticles, nanoscopic bubbles, and oil containing drugs. Iron oxide paramagnetic nanoparticles were synthesized and modified with surface conjugation of polyethylenimide (PEI) or Bovine Serum Albumin (BSA). Both particles were used to disperse and stabilize oil in water emulsions containing coumarin-6 as the model drug. Sulfur hexafluoride was introduced into the oil phase to form nanoscopic bubbles inside the emulsions. The resulted gas containing emulsions were evaluated for their magnetic resonance (MR) and ultrasound (US) imaging properties. The drug release profile triggered by ultrasound was also examined. We have successfully prepared the highly integrated multi-component emulsion system using the surface modified iron oxide nanoparticles to stabilize the interfaces. The resulted structure had distinctive MR and US imaging properties. Upon application of ultrasound waves, the gas containing emulsion would burst and encapsulated drug could be released. The integrated emulsion formulation was multifunctional with paramagnetic, sono-responsive and drug-carrying characteristics, which may have potential applications for disease diagnosis and imaging guided drug release.

Rapid induction of the Growth Hormone Gene Transcription by Glucocorticoids In Vitro: Possible Involvement of Membrane Glucocorticoid Receptors and Phosphatidylinositol 3‐Kinase Activation

The regulation of transcription of the growth hormone (GH) gene by glucocorticoids was studied in MtT/S cells, a cell line derived from an oestrogen-induced mammotrophic tumour in the rat, and in the primary culture of the anterior pituitary gland of adult mice. The levels of the GH heteronuclear RNA (GH hnRNA), which are mainly determined by the transcription rate, increased by 25-fold during 24 h in response to dexamethasone (DEX; 1 μM) in MtT/S cells that were cultured in the medium containing charcoal-stripped serum for 7 days. The stimulatory effect of DEX on the GH hnRNA levels was detectable as early as 30 min. This rapid effect of DEX did not require on-going protein synthesis, whereas it was considered that DEX requires the presence of unknown cellular proteins produced independently of DEX stimulation. By contrast, on-going protein synthesis was required for DEX action when incubated for 6 h, as has been observed in the previous studies. The specific inhibitor of glucocorticoid receptor, RU486, inhibited both rapid (30 min) and delayed (6 h) the effects of glucocorticoids on GH hnRNA levels. Membrane impermeable glucocorticoid, corticosterone-bovine serum albumin conjugate (CSBSA), was found to have effects similar to those of DEX and free corticosterone (CS), suggesting that glucocorticoids regulate GH gene transcription at least in part through the membrane bound receptors. From pharmacological studies, it was suggested that phosphatidylinositol 3-kinase (PI3K) activation is involved in the rapid effects but not in the delayed effects of glucocorticoids. This also suggests that the delayed effects of glucocorticoids depend on mechanisms other than the activation of PI3-kinase. Finally, both rapid and delayed effects of CS and CSBSA were observed not only in MtT/S cells, but also in the mouse pituitary cells in primary culture. Therefore, it is possible that the membrane initiated action of glucocorticoids is involved in the regulation of GH transcription in normal pituitary cells, as well as in pituitary tumour cells.

A Direct Enzyme Immunoassay to Detect Erythrosine in Foods

As a synthetic food dye, erythrosine is associated with serious toxicity in inducing thyroid tumors, and the use of erythrosine is strictly regulated in most counties including China. In this study, a direct enzyme-linked immunosorbent assay (ELISA) has been developed for analysis of erythrosine in food products. A highly specific monoclonal antibody (MAb) for erythrosine was produced using erythrosine–bovine serum albumin (BSA) conjugate as an artificial antigen, and horseradish peroxidase (HRP)-labeled MAb for erythrosine was utilized as the detection antibody. Under the optimized condition, the UV absorbance in microplate related well with erythrosine concentration in the range of 0.1–10.0 μg/g. The proposed method could be applied to determine erythrosine in beverage and cookie, with good recoveries (80 –103 %) for the three spiked levels (30, 50, and 100 μg/g), and the relative standard deviations of detected amount were <12.3 %.

Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications

An enzyme-linked immunosorbent assay (ELISA) was developed, and its application in immunoaffinity column chromatography was studied using a monoclonal antibody (MAb) against puerarin. Splenocytes isolated from a female BALB/c mouse immunised with a puerarin-bovine serum albumin (BSA) conjugate were fused with SP2/0 myeloma cells. The hybridoma cell line secreting MAb against puerarin (AA9) was acquired by screening and limiting dilution. The antibody generated was highly specific for puerarin with <0.01% cross-reactivity with over 50 structurally related chemicals, except for baicalein (51.8%). Using AA9, we developed an immunoassay for puerarin with a linear detection range of 10ng/ml to 1μg/ml. This assay system was further validated using intra- and inter-assays and recovery experiments. In addition, puerarin levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. Finally, we developed and validated protocols for knocking puerarin out of its parent medicine completely. In conclusion, we successfully developed a reliable ELISA and an immunoaffinity column for puerarin detection and knockout, which are useful tools for exploring the role of puerarin in formulated Chinese medicines.