Detection of T-2 toxin by an improved radioimmunoassay

T-2 toxin in serum, urine, and saline was analyzed by a modified radioimmunoassay procedure. The specimens were added directly to the assay tubes without extraction steps. The reaction between antibody and ligands was optimal at 1 h. Albumin-coated charcoal was used to separate bound from free radioactivity. Quenching, which occurred with hemolyzed specimens, was corrected by a wet oxidation process with 60% perchloric acid and 30% hydrogen peroxide. The shorter incubation times resulted in an assay that takes less than 6 h to complete. The average affinity constant of the antibody (Km) was 1.75 X 10(10) liters/mol. The sensitivity was 1 ng per assay or 10 ng/ml. Among the other trichothecenes tested, only H-T-2 cross-reacted significantly (10.3%).     

Radioimmunoassay for the detection of hepatitis e antigen (HBeAG) and antibody (anti-HBe).

A solid phase micro-immunoradiometric assay (micro-SPIRA) for the detection of hepatitis e antigen (HBeAg) and antibody has been developed. Chimpanzee anti-HBe/2 was developed by repeated immunizations with purified antigen containing HBeAg/1 and HBeAg/2. An anti-HBe/2 titer of 1:4 was determined by immunodiffusion (ID) analysis. Anti-HBe/1 was not detected. The anti-HBe IgG used in the assay was purified from plasma by a combination of DEAE-cellulose and affinity chromatography. The sensitivity of the micro-SPIRA for antigen and antibody was 193 ng/ml and 65 ng/ml, respectively. By comparing relative endpoint titers obtained by ID to micro-SPIRA, it was determined that micro-SPIRA for antigen and antibody is 320 and greater than 1300 times more sensitive, respectively, than ID. The specificity of the assay was ascertained by the examination of various non-B specimens. The application of the assay to a panel of 50 hepatitis B surface antigen (HBsAg)-positive specimens resulted in an increase in positivity of 18% for antigen and 22% for antibody.     

Growth hormone in urine: development of an ultrasensitive assay applicable to plasma and urine

A radiometric assay for human growth hormone (HGH) was developed based on a polyclonal goat anti-HGH antiserum covalently coupled to nonsedimenting polyacrylamide particles. HGH can be specifically immunoextracted from sample volumes of up to 10 ml. Subsequently, bound HGH is identified and quantitatively measured by a 125I-labelled monoclonal anti-HGH antibody. The assay is insensitive to plasma proteins from 10 to greater than 90%, to changing NaCl and urea molarities and to pH ranges from 6 to 8. The sensitivity in the second incubation is 2 pg/tube, corresponding to a maximum sensitivity of 300 fg/ml of a sample volume of 10 ml (urine) or of 40 pg/ml, if a volume of 50 microliter (plasma) is assayed. In healthy children, a mean HGH excretion of 6.5 ng/24 h was found with a large interindividual range from undetectable to 37.4 ng. An important intraindividual night-to-night variation of HGH excretion was found in several subsequent first morning void samples in healthy children. The mean excretion in 13 HGH-deficient children was 0.9 ng/24 h off therapy and increased to a mean of 6.9 ng/24 h on therapy. In acromegalic patients, the excreted HGH amounted to 73-208 ng/24 h. Preliminary results suggest that the ultrasensitive assay applied to plasma and urine could be a considerable improvement of diagnosis and follow-up of disorders of HGH secretion.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

混合单克隆抗体在固相ELISA双夹心法中的应用研究

在进行固相ELISA双夹心法时,要选择两种配对的单克隆抗体(McAb)殊非易事。本文用不同McAb的混合物与另一种McAb进行配对夹心,获得了较好的效果。实验表明,在心肌肌球蛋白轻链(CM—LC)的固相ELISA双夹心体系中,以抗CM-LCMcAb(1G6)铺底,(2B4及2F6)混合物为后续复盖抗体,最低检出量可低达10ng/mL,其检出率较单独2B4或单独2F6作为后续复盖抗体者高5—10倍。而若反之,以(2B4及2F6)混合物铺底,1G6作为后续复盖抗体,则其最低检出量竟高至200ng/mL,还不如以其中之一铺底为佳。在人绒毛膜促性腺激素(HCG)的检测体系中,用多克隆抗体与单克隆抗体配对的研究中,也获得了类似的实验结果。     

Chiral determination of mirtazapine in human blood plasma by high-performance liquid chromatography

A method is described for the determination of the two enantiomers of mirtazapine in human blood plasma by high-performance liquid chromatography. Measurements were performed on drug free plasma spiked with mirtazapine and used to prepare and validate standard curves. Levels of enantiomers of mirtazapine were also measured in patients being treated for depression with racemic mirtazapine. Mirtazapine was separated from plasma by solid-phase extraction using CERTIFY columns. Chromatographic separation was achieved using a Chiralpak AD column and pre-column and compounds were detected by their absorption at 290 nm. Imipramine was used as an internal standard. The assay was validated for each analyte in the concentration range 10–100 ng/ml. The coefficient of variance was 16% and 5.5% for(+)-mirtazapine for 10 and 100 ng/ml control specimens respectively and 15% and 7.3% for mirtazapine for 10 and 100 ng/ml control specimens respectively. This assay is appropriate for use in the clinical range. The range of plasma mirtazapine concentrations from eleven patients taking daily doses of 30–45 mg of racemate was <5 to 69 ng/ml for (+)-mirtazapine and 13–88 ng/ml for (−)-mirtazapine for blood specimens collected 10–17.5 h after taking the dose.     

rmhTNF-α联合抗IL-6抗体对胃癌SGC-7901细胞杀伤作用的研究

目的：研究rmhTNF-α联合抗IL-6抗体对胃癌SGC-7901细胞的杀伤作用。方法：（1）将已培养的SGC-7901细胞株分为a、b、c、d、e组,a、b、c、d组分别加入浓度为10 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml的rmhTNF-α,e组加入等量PBS。ELISA检测不同时间点SGC7901细胞上清液中IL-6的分泌量,MTT法检测细胞培养24 h、32 h、40 h、48 h的凋亡情况。（2）将SGC-7901细胞分为f、g、h、i组,f组加入rmhTNF-α,浓度分别为10 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml;g组加入不同浓度的rmhTNF-α与浓度为100 ng/ml的IL-6,h、i组加入不同浓度的rmhTNF-α与浓度分别为1：62.5和1：125的抗IL-6抗体。MTT法测细胞培养48 h的凋亡情况。荧光显微镜观察细胞凋亡情况。结果：（1）IL-6分泌五组差别具有统计学意义（P〈0.05）,a、b、c、d组IL-6浓度均高于e组（P〈0.05）,a、b、c组相比各时间点IL-6浓度逐渐升高（P〈0.05）,c、d组差别无统计学意义。（2）细胞存活率a、b、c、d组与e组差别具有统计学意义（P〈0.05）,a、b、c、d组内比较差别有统计学意义（P〈0.05）,显示杀伤作用40 h后减弱,c、d组差别无统计学意义。（3）细胞存活率f、g组比较差别具有统计学意义（P〈0.05）,h、i组与f组差别有意义（P〈0.05）,h、i组比较,i组细胞存活率较低。结论：rmhTNF-α可以促进SGC-7901细胞分泌IL-6;抗IL-6抗体可以增强rmhTNF-α对SGC-7901细胞的杀伤作用。     

A monoclonal antibody to T-2 toxin

A specific, high affinity monoclonal antibody to T-2 toxin of Fusarium has been produced. The monoclonal antibody was conjugated to horse-radish peroxidase and employed to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the toxin. The sensitivity of the ELISA was 10 ng/ml with a working range up to 500 ng/ml. The antibody cross-reacted with HT-2 toxin (25%) but did not bind to any other trichothecene tested.     

小麦中脱氧雪腐镰刀菌烯醇酶联免疫吸附测定方法的研究

用B淋巴细胞杂交瘤技术制备了能稳定分泌抗脱氧雪腐镰刀菌烯醇(DON)单克隆抗体的杂交瘤细胞株,命名为3D7.3D7的亚类为lgG_1.运用该抗体,建立了检测小麦中DON的间接竞争性酶联免疫吸附测定法.该法最低检出量为5ng/ml,敏感范围为5—1000ng/ml;平均回收率为96.6—107.3%;精密度为6.2—13.3%.运用该方法,检测了10份小麦样品.均为阳性,其含量为56.4—1002.0ppb.     

Daily changes in blood serum levels of 17 beta-estradiol and 11-ketotestosterone in the mature carp Cyprinus carpio L

Daily changes in blood serum levels of 17 beta-estradiol and 11-ketotestosterone in the mature carp Cyprinus carpio were investigated. Fish were kept under natural light conditions (LD = 16:8) water temperature about 19 degrees C. Females and males were divided into 2 groups respectively. Each group was sampled every 4 h during 24 h beginning at 08:00 (group 1) and 10:00 (group 2). The 17 beta-estradiol assay included a chromatographic step which increased the specificity of the measurement while in 11-ketotestosterone assay a specific antibody was used, having a low cross reactivity with testosterone (1.1%). Data obtained in this work were statistically analyzed using cosinor circle with error ellipses. The highest levels of estradiol (0.46-0.58 ng/ml) were observed between 09:00 and 11:00 and of 11-ketotestosterone (2.09-3.00 ng/ml) between 10:00 and 12:00. It was proved statistically that changes in the estradiol and 11-ketotestosterone levels during 24 h are of circadian rhythm type. The problem of hormonal changes during 24 h, connected with sexual maturation of fish, is discussed.     

Evaluation of the Bio-Rad Geenius HIV 1/2 Confirmation Assay as an Alternative to Western Blot in the Korean Population: A Multi-Center Study

Recently updated recommendations for diagnosis of HIV infection suggest a new diagnostic algorithm including HIV-1/HIV-2 antibody differentiation immunoassay instead of western blot (WB) as a confirmatory testing. We evaluated Bio-Rad Geenius HIV1/2 confirmation assay as a simple and reliable alternative to WB in the Korean population with low HIV prevalence. The Geenius HIV1/2 was performed in a total of 192 serum specimens (140 reactive and 52 nonreactive specimens by ARCHITECT HIV Ag/Ab Combo assay) that were prospectively collected from five institutions. HIV-1 nucleic acid amplification test (NAT) was performed in negative or indeterminate specimens by Geenius HIV1/2 or WB. Among 140 reactive specimens by HIV Ag/Ab assay, 82 (58.6%) were positive for HIV-1 Ab by Geenius HIV1/2. Among 58 negative or indeterminate specimens by Geenius HIV1/2, four specimens (6.9%) were positive by HIV-1 NAT. The sensitivity and specificity of Geenius HIV1/2 were 95.3% and 100.0%, respectively. When we considered only WB, the sensitivity and specificity of Geenius HIV1/2 were 100.0% and 99.1%, respectively. Agreement between Geenius HIV1/2 and WB was excellent (weighted Kappa = 0.89). The Geenius HIV1/2 is simple and time-saving compared with WB. It has an excellent performance and can be a reliable alternative to WB. HIV-1 NAT should be performed in negative or indeterminate specimens by Geenius HIV1/2 to detect acute HIV infection as recommended in new HIV testing algorithms.     

Production and characterization of a generic antibody against group A trichothecenes

An antibody against group A trichothecenes was produced after immunization of rabbits with an immunogen prepared by conjugation of T-2 toxin to bovine albumin at the C-8 position. T-2 toxin was first converted to 3-acetylneosolaniol (3-Ac-NEOS) and then to its hemisuccinate (HS) before conjugation to the protein. The rabbits showed a quick immune response after immunization of the new conjugate. The antibody produced bound with tritiated T-2 toxin, T-2 tetraol tetraacetate, and diacetoxyscirpenol (DAS) and showed good cross-reactivities with most of the group A trichothecenes. The concentrations causing 50% inhibition of binding of 3H-T-2 toxin to the new antibody by unlabeled T-2, acetyl-T-2, 3'-OH-T-2, DAS, 3-Ac-NEOS-HS, 3'-OH-Ac-T-2, T-2 tetraol tetraacetate, iso-T-2, 3-Ac-NEOS, Ac-DAS, and 3,4,15-triacetyl-7-deoxynivalenol were found to be 0.34, 0.34, 0.6, 2.5, 4, 10, 18, 24, 100, 200, and 300 ng/assay, respectively; for HT-2, T-2 triol, and T-2 tetraol, the concentration was greater than 1000 ng/assay. Nivalenol, deoxynivalenol (DON), 15-acetyl-DON, and triacetyl-DON, did not inhibit the binding at 1000 ng/assay. The practical application of using this new antibody for radioimmunoassay (RIA) of trichothecene was tested by spiking T-2 toxin to corn. T-2 toxin was then extracted with acetone, subjected to a simple Sep-Pak C-18 reversed-phase treatment, and analyzed by RIA. The overall recovery for 18 samples spiked with 10 to 50 ppb of T-2 toxin was 94.22%.     

A chimera antibody erythroimmunoassay for detecting HBsAg in human sera

A highly efficient chimera antibody, a monoclonal anti-hepatitis-B-surface (anti-HBs) antibody coupled with polyclonal anti-sheep-red-blood-cell (anti-SRBC) antibody was prepared using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithiopropionate) (SPDP). Using SRBC as a marker, we established a sensitive solid-phase chimera antibody erythroimmunoassay (CAEIA) according to Guesdon's method. The sensitivity of this assay was 2–20 times higher than the reverse passive haemagglutination assay (RPHA) for detecting HBsAg in serially diluted sera from 10 hepatitis B patients. The weakest quantity of HBsAg detected by this assay was 4.5 ng/ml, while RPHA was unable to detect less than 75 ng/ml of HBsAg. The assay was as sensitive as the enzyme-linked immunosorbent assay (ELISA) and gave more accurate, reproducible and stable results than ELISA; specificity was also satisfactory.     

A specific and potent immunotoxin composed of antibody ZME-018 and the plant toxin gelonin

Murine monoclonal antibody ZME-018 recognizes a 240 Kda glycoprotein present on the surface of most human melanoma cells and on over 80% of human biopsy specimens tested. Gelonin is a ribosome-inactivating plant toxin similar in nature and rivaling the activity of ricin A chain. ZME-018 was coupled to purified gelonin using the reagents SPDP and 2-iminothiolane. The ZME-gelonin conjugate was purified by S-300 Sephacryl and Blue Sepharose chromatography, removing unreacted gelonin and antibody, respectively. PAGE analysis showed that ZME was coupled to 1, 2, or 3 gelonin molecules. The ZME-gelonin conjugate was 10(6)-fold more active than gelonin itself in inhibiting the growth of log-phase human melanoma cells in culture. The immunoconjugate was not cytotoxic to antigen negative T-24 (human bladder carcinoma) cells. Treatment of melanoma cells with recombinant IFN-alpha or TNF substantially augmented the cytotoxicity of the immunoconjugate while treatment with IFN-gamma had a minor effect. Using the human tumor colony assay of melanoma cells obtained from fresh biopsy specimens, greater than 90% growth suppression was observed in 2 of 4 samples tested at a concentration of 250 ng/ml. In addition, 25% growth suppression was observed with a third sample tested, and no growth suppression was observed in 1 sample. Thus, clonogenic melanoma cells are sensitive in vitro to the cytotoxic activity of this immunotoxin at concentrations which we presume are pharmacologically relevant.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed,Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5⁺/6⁺ (BSGP)5⁺/6⁺. To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.     

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.