[Analytical Method for Melengestrol Acetate in Livestock Products Using LC-MS/MS].

The present study verified that it is possible to analyze melengesterol acetate using the existing multi-residue method. Melengestrol acetate was extracted from livestock products using acidic acetonitrile acidified with acetic acid in the presence of n-hexane and anhydrous sodium sulfate. The crude extracts were cleaned up using an octadecylsilanized silica gel cartridge column. Separation by HPLC was performed using an octadecylsilanized silica gel column with linear gradient elution of 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For the determination of the analyte, tandem mass spectrometry with positive ion electrospray ionization was used. In recovery tests using four livestock products fortified with maximum residue limits levels of melengestrol acetate (0.001-0.02 mg/kg), the truenesses ranged from 82% to 100%, and the repeatabilities for the entire procedure ranged from 0.5 RSD% to 5.6 RSD%. In recovery tests using 11 livestock products fortified with 0.0005 mg/kg of melengestrol acetate, the truenesses ranged from 88% to 99%, and the repeatabilities ranged from 1.3 RSD% to 5.4 RSD%. The limit of quantification for melengestrol acetate in livestock products was 0.0005 mg/kg.

Feasibility and efficacy of newly developed eco-friendly, automatic washer for endoscope using electrolyzed alkaline and acidic water.

INTRODUCTION: As well as preventing nosocomial and healthcare-associated infections, a reliable and eco-friendly washer for medical equipment would also be safe for the global environment. The aim of this study was to evaluate the efficacy of a newly developed automatic washing system (Nano-washer) that uses electrolyzed water and ultrasonication without detergent for washing endoscopes. METHODS: Patients who underwent laparoscopic lobectomy or laparoscopic colectomy at Nagasaki University between 2018 and 2022 were included. A total of 60 cases of endoscope use were collected and classified according to endoscope washing method into the Nano-washer group (using no detergent) (n = 40) and the manual washing group (n = 20). Protein and bacterial residues were measured before and after washing, using absorbance spectrometry and 16S rRNA polymerase chain reaction. The effectiveness of protein and bacterial removal and endoscope surface damage after washing were compared under specular vision between the groups. RESULTS: Nano-washer did not use detergent unlike manual washing. There was no difference in demographic or clinical characteristics between the groups except for the presence of comorbidities in the lobectomy group (Nano-washer, 85%; manual washing, 40%, P = .031). Compared with the manual washing group, residual protein levels in the Nano-washer group were significantly reduced after washing (lobectomy, 0.956 mg/mL vs 0.016 mg/mL, P < .001; colectomy, 0.144 mg/mL vs 0.002 mg/mL, P = .008). Nano-washer group showed a significant reduction in bacteria between before and after lobectomy (9437 copies/cm2 vs 4612 copies/cm2 , P = .024). CONCLUSION: Nano-washer is a promising, effective, and eco-friendly automatic washing device that is safer and more efficient than manual washing.

[Development of Analytical Method and Surveillance ofGibberellic Acid in Banana, Cherry, and Kiwi Fruit].

Gibberellic acid (GA3) is commonly used as a plant growth regulator in many food crops owing to its essential signaling functions during plant growth and development. In Japan, a threshold for administrative action for GA3 content of 0.3 mg/kg applies in produce in which maximum residue limits have not been established. Although the threshold is based on previous studies, the GA3 concentrations in individual foods are still unknown. Thus, we surveyed the concentrations of GA3 in banana, cherry, and kiwi fruit on the Japanese market. We developed and validated a method for the analysis of GA3 using solid-phase extraction and LC-MS/MS in accordance with accepted criteria of trueness, repeatability, and selectivity. The limits of detection and of quantification were determined as 0.005 and 0.05 mg/kg, respectively, in all fruits. Concentrations of GA3 did not exceed 0.3 mg/kg regardless of ripeness, suggesting the reasonability of the current regulation of GA3 in banana, cherry, and kiwi fruit. These findings can support prompt administrative action on these fruits, contributing to the regulation of GA3 in Japan.

[An LC-MS/MS Analytical Method for Moenomycin A in Livestock Products].

A simple and sensitive method for the determination of moenomycin A residues in livestock products using LC-MS/MS was developed. Moenomycin A, a residual definition of flavophospholipol, was extracted from samples with a mixture of ammonium hydroxide and methanol (1 : 9, v/v) preheated at 50℃. The crude extracted solutions were evaporated and purified by liquid-liquid partitioning between a mixture of ammonium hydroxide, methanol and water (1 : 60 : 40, v/v/v) and ethyl acetate. The alkaline layer was taken, and cleaned up using a strong anion exchange (InertSep SAX) solid phase extraction cartridge. The LC separation was performed on an Inertsil C8 column with liner gradient elution using 0.3 vol% formic acid and acetonitrile containing 0.3 vol% formic acid. Moenomycin A was detected using tandem mass spectrometry with negative ion electrospray ionization. Recovery tests were conducted using three porcine samples (muscle, fat and liver) and chicken eggs. Samples were spiked with moenomycin A at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The trueness ranged from 79 to 93% and precision ranged from 0.5 to 2.8%. The limit of quantification (S/N≥10) of the developed method is 0.01 mg/kg. The developed method would thus be very useful for regulatory monitoring of flavophospholipol in livestock products.

Synthesis of Norgestomet and its 17ß-isomer and evaluation of their agonistic activities against progesterone receptor.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17ß-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

[Analytical Method for Fipronil and Fipronil Sulfone in Livestock Products Using LC-MS/MS].

A rapid and sensitive method for the simultaneous determination of fipronil and fipronil sulfone (metabolite B) in livestock products was developed. The analytes were extracted from samples with acidic acetonitrile. The crude extracts were subjected to clean-up step using neutral alumina cartridge column. The HPLC separation was performed on a C18 column with isocratic elution of acetonitrile and ammonium formate solution. For the determination of the analytes, a tandem mass spectrometry with negative ion electrospray ionization was used. In the recovery tests using 6 livestock products fortified with MRLs levels of analytes, the truenesses for fipronil and fipronil sulfone were 95 to 115 and 94 to 101% with the repeatabilities of 0.8 to 4.1 and 0.9 to 5.1 RSD%, respectively. The limits of quantification for both analytes were estimated to be 0.001 mg/kg. The developed method is considered suitable for regulatory analysis of fipronil and fipronil sulfone.

Total determination of triclabendazole and its metabolites in bovine tissues using liquid chromatography-tandem mass spectrometry.

A reliable LC-MS/MS analytical method for the determination of residual triclabendazole and its principal metabolites (triclabendazole sulfoxide, triclabendazole sulfone and keto-triclabendazole) in bovine tissues was developed, in which triclabendazole and its metabolites are oxidized to keto-triclabendazole as a marker residue. The method involves sample digestion with hot sodium hydroxide, thus releasing the bound residues of various triclabendazole metabolites in bovine tissues. The target compounds are extracted from the digest mixture with ethyl acetate, defatted by liquid-liquid partitioning using n-hexane and acetonitrile, then oxidized with hydrogen peroxide in a mixture of ethanol and acetic acid. The reaction mixture is cleaned up using a strong cation exchange cartridge (Oasis MCX) and the analytes are quantified using LC-MS/MS. The optimal conditions for the complete oxidation of triclabendazole and its metabolites to keto-triclabendazole are an incubation time of 16 h and a temperature of 90 °C. The developed method was evaluated using three bovine samples: muscle, fat, and liver. Samples were spiked with triclabendazole and its principal metabolites at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The validation results show excellent recoveries (81-102%) and precision (<10%) for all target compounds. The limit of quantification (S/N ≥ 10) of the developed method is 0.01 mg/kg. These results suggest the developed method is applicable to quantifying residual triclabendazole in bovine tissues in compliance with the MRLs established by the Codex Alimentarius and EU and Japanese regulations, and thus the proposed method will be a useful tool for the regulatory monitoring of residual triclabendazole and its metabolites.

Total determination of residual flutolanil and its metabolites in livestock products and seafood using liquid chromatography-tandem mass spectrometry.

We have developed a simple and sensitive LC-MS/MS analytical method for the determination of residual flutolanil and its principal metabolites, including α,α,α-trifluoro-3'-hydroxy-o-toluanilide (M-4) and its conjugates, in livestock and seafood products. Both flutolanil and its metabolites contain the 2-(trifluoromethyl)benzoic acid (2-TFMBA) moiety. In this method, flutolanil and its metabolites are converted to 2-TFMBA by hydrolysis. The method involves direct hydrolysis with sodium hydroxide at 200°C, acidification, partitioning into a mixture of ethyl acetate-n-hexane (1:9, v/v), clean-up using a strong anion exchange cartridge (InertSep SAX), and then quantification using LC-MS/MS. The optimal conditions for the complete hydrolysis of flutolanil to 2-TFMBA are an incubation time of 6 h and a temperature of 200°C. The developed method was evaluated using seven types of food: bovine samples of muscle, fat, liver and milk, as well as egg, eel, and freshwater clam. Samples were spiked both at 0.01 mg/kg and at the Japanese maximum residue limit (MRL) established for each food type. The validation results show excellent recoveries (88-107%) and precision (< 10%) for flutolanil and M-4. The limit of quantification (S/N ≥ 10) of the developed method is 0.01 mg/kg. The developed method is applicable to the definition of residual flutolanil for animal-based food commodities and MRLs established by the Codex Alimentarius, and will be useful for the regulatory monitoring of residual flutolanil and its metabolites in food products.

Analytical Method for Nitroimidazoles and Their Major Metabolites in Livestock and Fishery Products Using LC-MS/MS.

A sensitive and reliable method for the simultaneous determination of four nitroimidazoles (ipronidazole (IPZ), dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ)) and three metabolites (IPZ-OH, MNZ-OH and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI)) in livestock and fishery products was developed. The analytes were extracted from samples with acetone containing acetic acid. The crude extracts were defatted by liquid-liquid partition using acetonitrile and n-hexane followed by solid-phase extraction using a cartridge column packed with divinylbenzene-N-vinylpyrolidone copolymer bearing sulfo groups. The analytes in the eluate from the cartridge column were extracted with ethyl acetate after addition of ammonium sulfate. The solvent was removed from the extract, and the residue was dissolved in 0.1 vol% formic acid. The HPLC separation was performed on a C18 column with a gradient formed from water containing 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For detection of the analytes, tandem mass spectrometry with positive ion electrospray ionization was used. The recovery tests were performed on 10 livestock and fishery products. The truenesse ranged from 74.6 to 111.1%, with repeatability of 0.5-8.3 RSD% for the entire procedure. The limit of quantification was 0.0001 mg/kg for IPZ, IPZ-OH, MNZ and MNZ-OH, and 0.0002 mg/mg for DMZ, RNZ and HMMNI.

Quantitative analysis of veterinary drugs in bovine muscle and milk by liquid chromatography quadrupole time-of-flight mass spectrometry.

A simple and reliable multiresidue method for quantitative determination of veterinary drugs in bovine muscle and milk using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was developed. Critical MS parameters such as capillary voltage, cone voltage, collision energy, desolvation gas temperature and extraction mass window were carefully optimised to obtain the best possible sensitivity. Analytical samples were prepared using extraction with acetonitrile and hexane in the presence of anhydrous sodium sulphate and acetic acid, followed by ODS cartridge clean-up. The developed method was validated for 82 veterinary drugs in bovine muscle and milk at spike levels of 0.01 and 0.1 mg kg-1. With the exception of cefoperazone and phenoxymethylpenicillin, all these compounds exhibited sufficient signal intensity at 0.01 µg ml-1 (equivalent to 0.01 mg kg-1), indicating the high sensitivity of the developed method. For most targets, the determined accuracies were within 70-120%, with repeatability and reproducibility being below 20% at both levels. Except for sulfathiazole in bovine muscle, no interfering peaks at target compound retention times were detected in the blank extract, indicating that the developed method is highly selective. The absence of sulfathiazole in bovine muscle was confirmed by simultaneous acquisition at low and high collision energies to afford exact masses of molecular adduct and fragment ions. Satisfactory linearity was observed for all compounds, with matrix effects being negligible for most targets in bovine muscle and milk at both spike levels. Overall, the results suggest that the developed LC-QTOF-MS method is suitable for routine regulatory-purpose analysis of veterinary drugs in bovine muscle and milk.

Total determination of chloramphenicol residues in foods by liquid chromatography-tandem mass spectrometry.

A simple and sensitive analytical method for the determination of chloramphenicol (CAP) and chloramphenicol glucuronide (CAPG) residues in foods such as livestock products, seafood, honey and royal jelly was developed. The method comprises solvent extraction with methanol, enzymatic hydrolysis with ß-glucuronidase and clean-up using a hydrophilic lipophilic balanced copolymer solid phase extraction column. To determine the optimal conditions for the complete enzymatic hydrolysis of CAPG, we examined the effect of enzyme concentration and incubation time on the hydrolysis. The detection of CAP using LC-MS/MS was optimized and determined by SRM. The developed method was validated using ten food products at a spiked level of 0.5µg/kg. The validation results show excellent recoveries (79-109%) and precision (<15%) for CAP and CAPG. The limit of quantification (S/N≥10) of the developed method was 0.5µg/kg. The proposed method would be useful for the regulatory monitoring of CAP and CAPG residues in foods.

[Determination of pindone in animal products, fishery products, and honey by LC-MS/MS].

A sensitive and selective analytical method for the determination of the rodenticide pindone in animal products, fishery products, and honey by LC-MS/MS was developed. Pindone was extracted with acidified acetone, and the crude extract was purified by liquid-liquid partitioning, followed by silica gel and ODS column chromatography. LC separation was performed on an ODS column with methanol/water containing ammonium acetate as the mobile phase, and detection was carried out using tandem mass spectrometry (MS/MS) with electrospray ionization (ESI) in the negative mode. The average recoveries from fortified bovine muscle, bovine liver, bovine fat, chicken muscle, salmon, eel, freshwater clam, egg, milk, and honey spiked at 0.001 mg/kg were in the range of 76-92%, and the relative standard deviations were 4-8%. The limit of quantitation (S/N≥10) of the developed method was 0.001 mg/kg for all the tested foods.

[Determination of vedaprofen in livestock products and seafoods by liquid chromatography-tandem mass spectrometry].

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the trace residue determination of vedaprofen (VPF) in livestock products and seafoods. VPF was extracted from each sample with acidified acetone, and the crude extract was re-extracted with ethyl acetate and NaCl solution. Clean-up was performed using a weak anion exchange cartridge (Bond Elut DEA). The LC separation was performed on a C18 column using acetonitrile-0.0025 mol/L formic acid (3 : 2) as the mobile phase and MS was run in the negative ion electrospray ionization mode. The calibration curve was linear in the range of 0.001-0.1 µg/mL VPF. The mean recoveries from equine muscle, cattle muscle, cattle liver, cattle fat, salmon, eel, corbicula, milk, egg and buckwheat honey were 72-94%, and the relative standard deviations (RSDs) were 1.1-2.0%. Limits of quantitation (LOQs) ranged from 0.001 to 0.007 µg/g.

[Determination of 4-hydroxycoumarin rodenticides in animal products, fishery products, and honey by liquid chromatography-tandem mass spectrometry].

A sensitive and selective method for the determination of 4-hydroxycoumarin-type rodenticides (warfarin, coumatetralyl, bromadiolone, and brodifacoum) in animal products, fishery products, and honey was developed. 4-Hydroxycoumarin rodenticides were extracted with acidified acetone, and the crude extract was purified by liquid-liquid partitioning followed by PSA column cleanup. Gradient liquid chromatographic separation was performed by using an Inertsil ODS-4 column, with methanol and water containing ammonium acetate as the mobile phase. Detection was carried out on a tandem mass spectrometer with electrospray ionization in the negative mode. Average recoveries from bovine muscle, bovine liver, bovine fat, swine muscle, salmon, eel, freshwater clam, egg, milk, and honey spiked at 0.0005-0.001 mg/kg were in the range of 79-108%, and the relative standard deviations were 2-8%. The limits of quantitations of the developed method were 0.0005 mg/kg for brodifacoum, 0.001 mg/kg for warfarin, coumatetralyl, and bromadiolone.

[Surveillance of perchlorate level in leafy vegetables and bottled water].

Perchlorate (ClO(4)(-)) is both a naturally occurring and artificial compound, and it inhibits iodide uptake into the thyroid gland and disturbs thyroid function. It has been detected in many foods in the United States. In order to investigate perchlorate contamination in foods in Japan, perchlorate level in 82 leafy vegetable samples and 20 bottled mineral water samples was measured using a procedure based on the FDA's procedure, employing IC-MS/MS with (18)O(4)-labeled perchlorate as an internal standard. Among 82 leafy vegetable samples tested, perchlorate levels were under the LOQ (0.3 ng/g) in 3 samples and ranged from 0.3 ng/g to 29.7 ng/g in 79 samples. In 20 bottled water samples, perchlorate was under the LOQ (0.1 ng/mL) in 14 samples and ranged from 0.14 ng/mL to 0.35 ng/mL in 6 samples.

Foxp3-expressing regulatory T cells expanded with CD28 superagonist antibody can prevent rat cardiac allograft rejection.

BACKGROUND: It is well known that CD4(+)CD25(+) regulatory T (Treg) cells play a central role in the suppression of autoimmunity, inflammation and allograft rejection. Therefore, therapeutic agents that capable of enhancing the number and activity of this T-cell subset are highly desirable. METHODS: The present study was designed to investigate the effects of superagonistic CD28-specific monoclonal antibody (supCD28 MAb) on preferentially expanded rat naturally occurring CD4(+)CD25(+) Treg (nTreg) cells and its applicability in cardiac transplantation. RESULTS: A single administration of supCD28 MAb preferentially proliferated nTreg cells. The increase of Foxp3 expression and polarization toward a Th2 cytokine profile correlated with decreased production of interferon-gamma and increased production of interleukin-4 and -10 in the expanded CD4(+)CD25(+) Treg subset, which was capable of suppressing CD4(+)CD25(-) T-cell proliferation after purification. Furthermore, supCD28 MAb administration revealed that nTreg cells were preferentially proliferating in vivo and recruited into the grafts, resulting in significant prolongation of full MHC-mismatch cardiac graft survival. CONCLUSIONS: Our data demonstrate that supCD28 MAb targets expansion of nTreg cells in vivo and maintains and enhances their regulatory functions, which represents a major advance toward the therapeutic use of polyclonally activated Treg cells as cellular therapy for treatment of allograft rejection.

Determination method for ractopamine in swine and cattle tissues using LC/MS.

Simple and reliable methods using LC/MS have been developed for the determination of the beta-agonist ractopamine in swine and cattle tissues. Ractopamine was extracted with ethyl acetate from muscle and liver, and the ethyl acetate layer was evaporated to dryness. The residue was purified by partition with acetonitrile/n-hexane. In the case of fat, ractopamine was extracted and purified by partition with acetonitrile/n-hexane. The resulting acetonitrile solutions were evaporated to dryness. The residue was dissolved in methanol, and subjected to LC/MS. The LC separation was performed on a Wakosil-II 3C18HG column (150 x 3 mm i.d.) in isocratic mode with 0.05% trifluoroacetic acid-acetonitrile (80:20) as a mobile phase at a flow rate of 0.4 mL/min. The MS detection was performed in the selected ion recording (SIR) mode, with detection of the M + H+ ion of ractopamine (m/z 302) produced by electrospray ionization (ESI). The mean recoveries of the drug from swine muscle (0.01 microg fortified), fat (0.01 microg fortified) and liver (0.04 microg/g fortified) were 99.7%, 99.5% and 100.8%, and those from cattle samples were 108.3%, 97.0% and 109.4%, respectively. The relative standard deviations (RSDs) ranged from 0.1% to 9.5%. The limit of quantification (LOQ) of the drug was 1 ng/g.

Surface plasmon resonance immunosensor for highly sensitive detection of 2,4,6-trinitrotoluene.

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (< or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor.

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-beta-alanine-ovalbumin (TNP-beta-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-beta-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3x10(-11) to 3x10(-7)g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1x10(-11)g/ml.

Sensitive detection of glycyrrhizin and evaluation of the affinity constants by a surface plasmon resonance-based immunosensor.

A sensitive and selective determination of glycyrrhizin (GC) based on surface plasmon resonance (SPR) was performed by using an anti-GC monoclonal antibody (GC-MAb) and GC-bovine serum albumin (GC-BSA) conjugate (antigen). GC-BSA was immobilized on an Au thin film of the SPR sensor chip by physical adsorption, and GC determinations were performed by an indirect competitive method. The addition of GC into the GC-MAb solution (5 microg/ml) was found to decrease the incident-angle shift sharply because of an inhibition effect of GC. The RSDs (n = 3) of each point were less than 4%. The lowest detection limit for GC by SPR was almost the same as that by ELISA, 60-75 ng/ml. An evaluation of the affinity constant between GC-MAb and GC using the data from ELISA and those from SPR measurements was performed. The values of the association constant (KA) from three different analyses of ELISA data and from SPR measurements are discussed in detail. As a whole, the affinity constant (KA) between GC-MAb and GC was on the order of 10(7) M(-1).