Validation of a monoclonal antibody‐based indirect enzyme immunoassay method for picloram detection in soil and plants

Picloram (4‐amino‐3,5,6‐trichloro‐2‐pyridinecarboxylic acid) residues in authentic soil and plant samples were determined by indirect enzyme immunoassay (EIA) and by gas chromatography (GC). Results obtained by EIA methods correlated well with the GC results with coefficients of determination ranging from 0.778 to 0.891. Picloram levels determined by EIA in soil extracts obtained by a potassium hydroxide extraction method (EIA‐1) underestimated picloram levels determined by GC. Conversely, EIA determinations of soil extracts obtained by a more rigorous acetonitrile extraction method (EIA‐2) yielded picloram levels that overestimated levels determined by GC. First order linear regression models using EIA determinations as the independent variable resulted in dependent variable coefficients of 0.325 and 1.42 for EIA‐1 and EIA‐2 respectively. Picloram levels determined in plant extracts were approximately equivalent by EIA and GC methods. Using a point on the standard curve representing 5 ng ml^?1 picloram as a threshold value, the EIA method effectively identified samples as either positive or negative for picloram residues based on the results obtained by GC analysis.     

Are the enzyme immunoassays for antibodies to pneumococcal capsular polysaccharides serotype specific?

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.     

Specificities and Opsonophagocytic Activities of Antibodies to Pneumococcal Capsular Polysaccharides in Sera of Unimmunized Young Children

An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved.     

Specificities and opsonophagocytic activities of antibodies to pneumococcal capsular polysaccharides in sera of unimmunized young children

An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved.     

Are the Enzyme Immunoassays for Antibodies to Pneumococcal Capsular Polysaccharides Serotype Specific?

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.     

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes. Gas chromatographic-mass spectrometric determination of metabolites

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17alpha-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17alpha-methyltestosterone, produced 6beta-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography-mass spectrometry (GC-MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6beta-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6beta-hydroxyl metabolites with testosterone and 17alpha-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6beta-hydroxylation.     

Rapid and sensitive quantification of 8-isoprostaglandin F2alpha in human plasma and urine by liquid chromatography-electrospray ionization mass spectrometry

The isoprostane, 8-isoprostaglandin F2alpha (8-iso-PGF2alpha), is produced non-enzymatically by direct oxidation of arachidonic acid on the cell surface by oxygen radicals. We developed a new assay method for 8-iso-PGF2alpha using 2H4-8-iso-PGF2alpha as the internal standard (I.S.) by high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). For this assay, we established a very simple and rapid pretreatment method using a membrane filter-type solid-phase extraction column (Empore disk cartridge) for human urine extracts or intact plasma. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 353.24 (8-iso-PGF2alpha) and m/z 357.26 (I.S.) with a resolution of 1,500. The imprecision for this method was below 13.7%. Mean inaccuracy was 8.7% for added levels of 8-iso-PGF2alpha up to 5,000 pg/ml of urine and 500 pg/ml of plasma. Determination of plasma and urinary 8-iso-PGF2alpha concentrations in healthy subjects by the present method revealed that its urinary concentration in smokers tends to be higher than that in nonsmokers.     

Detection of lipid peroxidation in light-exposed mouse retina assessed by oxidative stress markers, total hydroxyoctadecadienoic acid and 8-iso-prostaglandin F2alpha

Exposure to excessive light induces retinal photoreceptor cell damage, which may involve lipid peroxidation. Morphological changes and the detection of internucleosomal DNA fragmentation confirmed the retinal damage caused by exposure of the retina of Balb/c mice to white fluorescent light (5000 lux, 2 h). The total amounts of hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) in the retinas obtained from light-exposed mice were assessed after reduction and saponification. In this method, both the free and ester forms of hydroperoxides, hydroxides, and ketones of linoleic acid are measured as tHODE by gas chromatography-mass spectrometry (GC-MS) analysis. When compared with controls, a significant increase in the concentrations of tHODE and 8-iso-PGF2alpha was observed 24 h after light exposure. Furthermore, the stereoisomeric ratio (Z,E)-HODE/(E,E)-HODE decreased after light exposure, suggesting the involvement of free-radical-mediated peroxidation. By the immunohistochemical technique, it was confirmed that 8-iso-PGF2alpha increased in the inner plexiform layer (IPL), outer plexiform layer (OPL), rod outer segment, and choroidal layer, while 13-HODE increased in the OPL and rod inner segment after light exposure. These results demonstrate that tHODE and 8-iso-PGF2alpha assessed by the present method are appropriate biomarkers responding to retinal photooxidative stress in vivo.     

Tandem mass spectrometric quantification of 8-iso-prostaglandin F2alpha and its metabolite 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2alpha in human urine

Whole body synthesis of F2-isoprostanes, a family of cyclooxygenase-independent eicosanoids formed by free-radical catalysed peroxidation, should be best assessed by quantifying their urinary metabolites. Two methods for the quantitative determination of F2-isoprostane metabolites in human urine performing either thin-layer chromatography (TLC) (method A) or high-performance liquid chromatography (HPLC) (method B) prior to GC-tandem MS are described. Method A allows for simultaneous quantification of 8-iso-PGF2alpha, one prominent member of the F2-isoprostane family, and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha. Mean excretion was found to be 223 and 506 pg/mg creatinine of 8-iso-PGF2alpha and 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha, respectively (n=14). A tight correlation existed between the urinary excretion of these two isoprostanes (r=0.86). Method B enables quantification of dinor-dihydro metabolites of various F2-isoprostanes including 8-iso-PGF2alpha. 2,3-Dinor-5,6-dihydro-8-iso-PGF2alpha was found to be an abundant dinor-dihydro F2-isoprostane metabolite. Validity of method A was proven by a combination of HPLC with TLC prior to GC-tandem MS analysis. A correlation was observed between the urinary concentrations of 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha measured by GC-MS and GC-tandem MS (r=0.84).     

Relevance of commercial diagnostic tests to detection of enteric adenovirus infections in South Africa

The prevalence of enteric adenoviruses detected by an in-house enzyme-linked immunosorbent assay (the RIVM-ELISA) ranged from 13 to 38%, and subgroup F adenoviruses comprised 86%. All subgroup F adenoviruses reacted with both RIVM anti-adenovirus type 40 (Ad40) and anti-adenovirus type 41 (Ad41) monoclonal antibodies but were not detected by Adenoclone Type 40/41 enzyme immunoassay (EIA). The correlation between the Biotrin EIA and RIVM-ELISA results was low (26%). Immunospecific tests suggest that a significant proportion of enteric adenoviruses, possibly comprising previously unidentified or emerging types, are not detected by commercial diagnostic tests in South Africa.     

Screening of beta-2 agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives

A new method for a comprehensive screening and confirmation of beta-2 agonists in human urine is presented based on gas chromatography-low-resolution mass spectrometry (GC-MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with beta-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other beta-2 agonists remain unchanged. Liquid-liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four beta-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other beta-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.     

Influence of short-chain fatty acids on vascular tone.

Results from research on the influence of short-chain fatty acids (SCFA) (C3-C6) on vascular tone are reported. Isolated vascular smooth muscle strips were studied in vitro and the arterial blood pressure of guinea-pigs injected with SCFA was taken. On the basis of results from these first two methods, the level of PG F2 alpha was determined by radioimmunoassay. The experiments indicate that in vitro the SCFA have spasmogenic effects which are blocked by indomethacin and aspirin and which are reduced by the inhibitor of the activating effect of PG F2 alpha, PG E2, and PG I2-SC19220. The effects of SCFA on smooth muscle strips used as a bioassay are analogous to the effects of PG F2 alpha on the same tissue. The injection of butyric acid into guinea-pigs causes hypertension which is not manifest if indomethacin pretreatment is carried out. Radioimmunoassay results indicate that the level of PG F2 alpha in the blood of animals treated with butyric acid is significantly increased.     

Increased steady-state levels of mRNA coding for extracellular matrix components in kidneys of NZB/W F1 mice.

The present study was carried out to determine how mRNA levels of extracellular matrix (ECM) components including alpha 1(IV) chain, laminin A, B1 and B2 chains, heparan sulfate proteoglycan (HSPG), alpha 1(I) chain, and alpha 1(III) chain are regulated in the kidney of NZB/W F1 mice. Messenger RNA levels for ECM genes except for laminin A chain increased significantly with the progression of nephritis in NZB/W F1 mice. In the NZW kidneys, however, the mRNA levels for alpha 1(IV) chain, laminin B1 and B2 chains, and HSPG declined markedly with age, whereas those for alpha 1(I) and alpha 1(III) chains showed little difference throughout the experimental period. Messenger RNA levels of beta-actin remained constant, and those of laminin A chain could not be detected in either control or diseased kidneys. Immunofluorescent microscopy showed that the intensity and distribution of staining of collagen IV, laminin, and HSPG in the glomeruli of NZB/W F1 mice increased markedly with the progression of disease. Types I and III collagen were not detected in the glomeruli of NZB/W F1 mice by immunofluorescence until 24 weeks of age, after which increased amounts of these collagens were found in the glomeruli and interstitium with progression of disease. These results suggest that increased levels of mRNA coding for ECM components and increased accumulation of these proteins may contribute to a cascade of events, leading to chronic renal injury in lupus nephritis.     

Mouse T-cell associated serine proteinase 1 degrades collagen type IV: a structural basis for the migration of lymphocytes through vascular basement membranes.

We show that CD8+ T-lymphocyte lines perferentially attach to collagen type IV and that mouse T-cell specific serine proteinase 1 (MTSP-1) preferentially degrades native basement membrane collagen type IV. In contrast, the interstitial collagen types I, II, III, V and VI appear not to be affected. The data reveal that MTSP-1 predominantly cleaves the alpha 2(IV) chain, which is found in the native triple helical structure of type IV collagen in a ratio of alpha 1(IV): alpha 2(IV) = 2:1 into small peptides. The cleavage of the alpha 2(IV) chain within the native collagen type IV molecules most likely results not only in a destabilization of single molecules but of the entire collagenous basement membrane scaffold at the site of MTSP-1 secretion.     

Comparison of three different PCR methods for quantifying human papillomavirus type 16 DNA in cervical scrape specimens

We compared real-time LightCycler and TaqMan assays and the GP5+/6+ PCR/enzyme immunoassay (EIA) to assess the human papillomavirus type 16 (HPV16) load in cervical scrape specimens. Both real-time PCR assays determined the HPV16 load in scrape specimens similarly. The level of agreement between these assays and the GP5+/6+ PCR/EIA was low (P = 0.004), suggesting that the latter method is not suited for quantifying HPV16 DNA.     

Conversion of tibolone to 7alpha-methyl-ethinyl estradiol using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry: interpretation and clinical implications

OBJECTIVE: Tibolone, a hormone therapy drug, is used to treat climacteric symptoms. This drug is rapidly metabolized into three major metabolites (3alpha-hydroxytibolone, 3beta-hydroxytibolone, and Delta4-tibolone). One clinical study provided evidence of conversion of tibolone to another estrogenic metabolite, 7alpha-methyl-ethinyl estradiol (MEE). However, no evidence of MEE formation was found in another study using the human aromatase enzyme. Because MEE was analyzed by gas chromatography-mass spectrometry (GC-MS), which requires derivatization, together with the fact that derivatization of some steroids may lead to aromatization, it is feasible that the MEE detected resulted from an artifact generated during the derivatization process. Hence, our objective was to assess whether tibolone is converted to MEE. DESIGN: We assayed MEE formation in a nonbiological system using GC-MS after derivatization and by analyzing MEE formation using liquid chromatography-mass spectrometry (LC-MS) in nonderivatized samples. RESULTS: MEE formation was evident in tibolone samples derivatized with either pentafluoropropionic anhydride or trimethylsilyl and analyzed by GC-MS. The amount of MEE formed increased with increasing amounts of tibolone (0.5, 1, 2.5, and 5 microg) derivatized; however, relative to tibolone, the percentage of MEE formed remained constant and ranged between 0.22% and 0.29% of tibolone. In contrast to GC-MS, no MEE formation was seen when tibolone was analyzed by liquid chromatography-mass spectrometry without derivatization. CONCLUSIONS: Our findings prove that conversion of tibolone to MEE is an artifact that is generated in a GC-MS system and is largely due to the intense heating step involved in GC-MS. Caution should be exercised to extrapolate clinical implications from existing data on MEE formation using a GC-MS system.     

Polydispersity of liposome preparations as a likely source of peak width in capillary zone electrophoresis

The tetrapeptide AcSDKP, a natural and specific substrate of angiotensin I-converting enzyme (ACE), is a negative regulator of hematopoiesis. AcSDKP has been measured in various biological media using an enzyme immunoassay (EIA), but its presence in human plasma and urine has not been formally established. By using immunoaffinity extraction and liquid chromatography-electrospray mass spectrometry, we demonstrate that AcSDKP-like immunoreactivity measured with EIA in plasma and urine samples from untreated, captopril- (an ACE inhibitor) and AcSDKP-treated subjects corresponds to AcSDKP. The present study confirms that AcSDKP is naturally present in human plasma and urine and that EIA is reliable for its measurement in such media.     

Differential determination of recombinant human interleukin-1 alpha and its deamidated derivative by two sandwich enzyme immunoassays using monoclonal antibodies. Comparison with a polyclonal antibody-based competitive enzyme immunoassay

Using three different monoclonal antibodies (McAb no. 374, no. 964 and no. 1190) to human interleukin-1 alpha (rHu-IL-1 alpha), we have established two sandwich enzyme immunoassays (EIA) to differentiate rHu-IL-1 alpha and its deamidated derivative (rHu-Asp36-IL-1 alpha) where the asparagine at position 36 (counting from the N-terminus) of rHu-IL-1 alpha is converted to Asp. The McAb no. 1190 reacts specifically with rHu-IL-alpha and not with the rHu-Asp36-IL-1 alpha whereas both no. 374 and no. 964 can react with the two different forms of rHu-IL-1 alpha. The first EIA (S-EIA I) which uses the McAb no. 964 labelled with horse-radish peroxidase and the McAb no. 1190 fixed to the microtiter plate, only measure rHu-IL-1 alpha. The second EIA (S-EIA II) which uses enzyme labelled no. 964 and no. 374 fixed to the plate, can detect both rHu-IL-1 alpha and rHu-Asp36-IL-1 alpha and this assay of total rHu-IL-1 alpha is comparable to a competitive EIA using an enzyme-labelled rHu-IL-1 alpha and an anti-rHu-IL-1 alpha polyclonal antibody. Thus, the level of rHu-Asp36-IL-1 alpha in the samples containing the two IL-1 alpha s can be calculated by subtracting the level measured by S-EIA I from that measured by S-EIA II. The two EIA systems with an assay range of 1.5-100 ng/ml do not recognize IL-1 beta, IL-2, rHu-TNF alpha, IFN-alpha and IFN-gamma of human origin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Typing of herpes simplex virus isolates with monoclonal antibodies and by nucleic acid spot hybridization

Fifty-one clinical isolates of herpes simplex virus (HSV) were typed by an enzyme immunoassay (EIA) using mouse monoclonal antibodies, by DNA spot hybridization, and by restriction enzyme analysis using restriction endonuclease Eco RI. Extracts of VERO cells infected with the isolates were used for coating microtitre plates or denatured and spotted onto nitrocellulose filters. Viral antigens passively adsorbed to microtitre plates were detected by an indirect EIA using mouse monoclonal antibodies specific for HSV type 1 (HSV-1) or HSV type 2 (HSV-2). Spotted DNA was hybridized with 32P-labeled probes containing Hind III/Sal I-fragments of either HSV-1 or HSV-2 DNA and bound radioactivity was detected by autoradiography and counted in a liquid scintillation counter. All the three methods gave identical results for the 51 isolates studied. Twenty-six isolates were identified as HSV-1 and 25 as HSV-2. An additional 30 specimens were tested only by EIA and hybridization. Results by both techniques were in complete agreement.