Quantitative determination of erlotinib in human serum using competitive enzyme-linked immunosorbent assay

A selective and sensitive competitive enzyme-linked immunosorbent assay(ELISA) method was developed and validated for the quantification of erlotinib in 50 mL of samples of human serum. Anti-erlotinib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and 3,4-bis(2-methoxyethoxy)benzoic acid using the N-succinimidyl ester method. Enzyme labeling of erlotinib with horseradish peroxidase was similarly performed using 3,4-bis(2-methoxyethoxy)benzoic acid. A simple competitive ELISA for erlotinib was developed using the principle of direct competition between erlotinib and the enzyme marker for anti-erlotinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Serum erlotinib concentrations lower than 40 ng/mL were reproducibly measurable using the ELISA. This ELISA was specific to erlotinib and showed very slight cross-reactivity(6.7%) with a major metabolite, O-desmethyl erlotinib. Using this assay, drug levels were easily measured in the blood of mice after oral administration of erlotinib at a single dose of 30 mg/kg. ELISA should be used as a valuable tool for therapeutic drug monitoring and in pharmacokinetic studies of erlotinib.

Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

Development of a competitive enzyme-linked immunosorbent assay for therapeutic drug monitoring of afatinib

Afatinib is an oral tyrosine kinase inhibitor(TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug monitoring(TDM) in clinical settings. This study sought to develop a simple and sensitive competitive enzyme-linked immunosorbent assay(ELISA) to quantify afatinib in plasma for routine pharmacokinetic applications. An anti-afatinib antibody was obtained using(S)-N-4-(3-chloro-4-fluorophenyl)-7-(tetrahydrofuran-3-yloxy)-quinazoline-4,6-diamine(CTQD), which has the same substructure as afatinib, as a hapten. Enzyme labeling of afatinib with horseradish peroxidase was similarly performed using CTQD. A simple competitive ELISA for afatinib was developed based on the principle of direct competition between afatinib and the enzyme marker for the anti-afatinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Plasma afatinib concentrations below the limit of quantification of 30 pg/mL were reproducibly measurable. Also, the values of plasma afatinib levels measured from 20 patients were comparable with those measured by high-performance liquid chromatography, and there was a strong correlation between the values determined by both methods(Y=0.976 X – 0.207, r=0.975). As indicated by its specificity and sensitivity, this newly developed ELISA for afatinib is an important tool for TDM and studies of the pharmacokinetics of afatinib.

Determnation of ochratoxin A in grain by monoclonal antibody－based enzyme－linked immunosorbent assay

The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.

Enzyme-linked Immunosorbent Assay for Detection of Anti-idiotype Antibodies to Antibodies to Ligand of Nicotinic Acetylcholine Receptor in Sera of Patients with Myasthenia Gravis

Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.

Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

A Comparison of Enzyme-Linked Immunosorbent Assay versus Multiplex Methodology Using an <i>in Vitro</i>Model of Pulmonary Hypertension and Inflammation

Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO2 and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH2O respectively, and 95% FiO2. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.

SERODIAGNOSIS OF CLONORCHIASIS BY ENZYME—LINKED IMMUNOSORBENT ASSAY WITH HRP—SPA

In thes paper the authors used the Horseradish peroxidase labelledstaphylococcal protein A(HRP—SPA)in ELISA,for the detection of Clo-norchis sinensis infection.Serum tests were made on 116 confirmed cases ofclonorchiasis,103(88.8%)of them showed positive,while only 6(4.4%)werepositive among 138 healthy people.Samples were collected on filter paperstrips,111(95.7%)cases were positive among 116 comfirmed cases tested,but only 2(1.5%)were positive out of 138 healthy persons.The resultswere similar to those obtained by sheep antihuman IgG.Animal experimentalso showed that the SPA—ELISA can be used for the diagnosis ofclonorchiasis.In an endemic area,stool egg positive rate was 8.8%(62/703).whenchecked with SPA—ELISA,the rate of conformity in both filter paperstrips and stool examinations was 90.3(56/62).Among 641 serum testsfrom individuals negative in stool examinations,only 35(5.5%)reactedpositively.The authors suggested—that SPA—ELISA with soluble Clo-norchis antigens could be used in a large scale seroepidemiological surveyin endemic areas.

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).

Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay for the Analysis of Jasmonates in Plants

Methyl jasmonate （MeJA） and its free-acid form, jasmonic acid （JA） are naturally occurring plant growth regulators widely distributed in higher plants. In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples, a monoclonal antibody （MAb） （designated as MAb 3E5D7C4B6） against MeJA was derived from a JA- bovine serum albumin （BSA） conjugate as an immunogen. The antibody belongs to the IgG1 subclass with a κ type light chain and has a dissociation constant of approximately 6.07 ×10^-9 M. MAb3E5D7C4B6 is very specific to MeJA. It was used to develop a direct competitive enzyme-linked immunosorbent assay （dcELISA）, conventional and simplified indirect competitive ELISAs （icELISA）. JA was derivatized into MeJA for the ELISA analysis. The IC50 value and detection range for MeJA were, respectively, 34 and 4-257 nglmL by the conventional icELISA, 21 and 3-226 nglmL by the simplified icELISA and 5.0 and 0.7-97.0 nglmL by the dcELISA. The dcELISA was more sensitive than either the conventional or simplified icELISA. The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves. The increased jasmonates content indicated its role in response to the drought stress and pathogens.

Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol

Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.

Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus

Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients’ sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

Tetra-substituted Amino Aluminum Phthalocyanine as a New Red-region Fluorescent Substrate for Horseradish Peroxidase Based Enzyme-linked Immunosorbent Assay

The use of tetra-substituted ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H2O2 could rapidly and significantly react with TAAIPc, thus quenching the fluorescence of TAAIPc. The Michaelis-Menten parameters Km and Vmax were measured to be 2.82 × 10?6 mol/L?1 and 6.0 × 10?9 mol·L?1, respectively. In this paper, TAAIPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5–200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.

Serological diagnosis of nasopharyngeal carcinoma by enzyme linked immunosorbant assay:optimization,standardization and diagnostic criteria

Objectives To produce an enzyme linked immunosorbant assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC).Methods We used a combination of highly purified glutathione transferase fusion proteins of the 40kD carboxy domain of EBNA1 and the 18kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/-20% of this value.Results All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%.Conclusions After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.

含有抗凝剂的羊血液样品对酶联免疫吸附试验结果的影响

为了解肝素钠、柠檬酸钠、乙二胺四乙酸二钾(EDTA-K2)对酶联免疫吸附试验(ELISA)测定羊口蹄疫,小反刍兽疫,布鲁氏菌病病抗体结果的影响,笔者在辖区范围内12个乡(镇)的26个羊场,随机采集480份样本,离心分离得到相关血清、血浆样品,用液相阻断ELISA方法测定FMDO、FMDA抗体,阻断ELISA方法测定小反刍兽疫抗体,竞争ELISA方法测定布鲁氏菌病抗体。结果发现,3种抗凝剂对酶联免疫吸附试验测定羊O型口蹄疫、A型口蹄疫、羊小反刍兽疫、羊布鲁氏菌病均存在一定的影响,不能用含这3种抗凝剂的血液样本来评估以上3种疾病免疫效果。

Enzyme-free photothermally amplified fluorescent immunosorbent assay(PAFISA)for sensitive cytokine quantification

Cytokine monitoring has attracted great attention due to its significance in the diagnosis and treatment of many diseases,such as tumors,microbial infections,and immunological diseases.Enzyme-linked immunosorbent assay(ELISA)is one of the most popular methods in cytokine detection,ascribing to the lavish signal amplification methods in the ELISA platform.In addition to classical enzymes,other signal amplifiers such as fluorescent probes,artificial nano-enzymes,and photothermal reagents have been applied to reduce the detection limit and produce more sensitive ELISA kits.Due to the accumulative effect of heat,photothermal reagents are promising materials in the signal amplification of ELISA.However,the lack of efficient photothermal generation material at an aggregate scale may delay the further development of this area.In this contribution,based on an efficient organic photothermal aggregate material,an enzyme-free photothermally amplified fluorescent immunosorbent assay system consisting of an assay microfluidic chip and detecting platform was developed.The photothermal nanoparticles with highly efficient photothermal conversion by harvesting energy via excited-state intramolecular motions and enlarging molar absorptivity were successfully prepared.The detection concentration at 50 pg/mL of interleukin-2 was achieved,realizing a signal improvement of detection limits by 20-fold compared to that of previously reported photothermal ELISA.The microscopic imaging integrated with plane sweeping technology provided high spatial resolution and precision,indicating the potential of achieving high throughput profiling at the microscale.Moreover,as an alternative excitation source,light-emitting diode not only provided a more affordable and miniaturized detection system but also revealed the great feasibility of intramolecular motion-induced photothermy nanoparticles for biological analyses.

细菌培养液中卡那霉素的酶联免疫试剂盒检测法

采用自制酶联免疫试剂盒检测细菌培养液中的卡那霉素含量。方法学验证结果表明,酶联免疫试剂盒能够准确检测细菌培养液中的卡那霉素含量,标准品浓度在0.0~16.2μg·L^(-1)范围内与吸光度线性关系良好,线性系数为0.9959,加标回收率为97.0%~102.8%,批内变异系数为3.66%,批间变异系数为5.05%,满足酶联免疫试剂盒要求。

Development and evaluation of immunoassay for zeranol in bovine urine

A high affinity polyclonal antibody-based enzyme linked immunosorbent assay （ELISA） was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1：5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml, and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation （CVs） were in the range of 82%-127% and 3.5%-8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol （ranging from 0.2 to 10 μg/ml） were detected by the ELISA and liquid chromatography （LC） method, and good correlations were obtained between the two methods （R^2=0.9643）. We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an altemative for the conventional LC method for zeranol in bovine urine.

Von Willebrand Factor Antigen and ADAMTS13 Activity Assay in Pregnant Women and Severe Preeclamptic Patients

The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia.Thirty healthy women of childbearing age,22 second trimester pregnant women,30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study.ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay.The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women,between third and second trimester pregnant women (P【0.05).The third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women (P【0.05).Nevertheless,no significant differences in plasma ADAMTS13 activity were found between severe preeclamptic patients and the third trimester pregnant women (P】0.05).In conclusion,plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels.Prothrombotic state is involved in the pathogenesis of severe preeclampsia,as a result of endothelial injury.

Simple and sensitive determination of sparfloxacin in pharmaceuticals and biological samples by immunoassay

Plasma quinolone concentrations are not routinely measured in clinical practice.However,in order to optimize quinolone treatment,monitoring of plasma concentrations could sometimes be useful particularly in critically ill patients.In this study,anti-sparfloxacin antibody was obtained by immunizing rabbits with sparfloxacin conjugated with bovine serum albumin using isobutyl chloroformate method.After the assay procedure was optimized,the standard curve of sparfloxacin was established.The practical measuring range of the competitive ELISA extended from 5 ng/mL to 2 mg/mL.The recovery rates and coefficients of variation for rat plasma,urine and tissues were 87.7-106.2% and 4.8-15.3%,respectively.To demonstrate the potential of the ELISA,a preliminary pharmacokinetics and tissue distribution study of sparfloxacin in rats and quantitative analysis of sparfloxacin in several pharmaceuticals were performed and compared with high-performance liquid chromatography(HPLC).The experimental data indicated that the proposed method would be a valuable tool in therapeutic drug monitoring(TDM) for sparfloxacin.