Monoclonal Antibody-based Assays Research Articles

Changes in lipoprotein(a), oxidized phospholipids, and LDL subclasses with a low-fat high-carbohydrate diet

Low-fat diets have been shown to increase plasma concentrations of lipoprotein(a) [Lp(a)], a preferential lipoprotein carrier of oxidized phospholipids (OxPLs) in plasma, as well as small dense LDL particles. We sought to determine whether increases in plasma Lp(a) induced by a low-fat high-carbohydrate (LFHC) diet are related to changes in OxPL and LDL subclasses. We studied 63 healthy subjects after 4 weeks of consuming, in random order, a high-fat low-carbohydrate (HFLC) diet and a LFHC diet. Plasma concentrations of Lp(a) (P < 0.01), OxPL/apolipoprotein (apo)B (P < 0.005), and OxPL-apo(a) (P < 0.05) were significantly higher on the LFHC diet compared with the HFLC diet whereas LDL peak particle size was significantly smaller (P < 0.0001). Diet-induced changes in Lp(a) were strongly correlated with changes in OxPL/apoB (P < 0.0001). The increases in plasma Lp(a) levels after the LFHC diet were also correlated with decreases in medium LDL particles (P < 0.01) and increases in very small LDL particles (P < 0.05). These results demonstrate that induction of increased levels of Lp(a) by an LFHC diet is associated with increases in OxPLs and with changes in LDL subclass distribution that may reflect altered metabolism of Lp(a) particles.

Low‐avidity HPA‐1a alloantibodies in severe neonatal alloimmune thrombocytopenia are detectable with surface plasmon resonance technology

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody-based antigen-capture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies. This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time. Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (approximately 490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of low-avidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA. Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies.

The role of domestic allergens.

The documented increase in asthma has been almost entirely in perennial asthma and a large proportion of the cases are allergic to one of the common allergens found all year round in houses, i.e. house dust mites, cats, dogs or cockroaches. In population and case-control studies sensitization to one of these allergens is the strongest risk factor for asthma (adjusted odds ratios > or = 4). Using monoclonal antibody-based assays for the major indoor allergens it has been shown that sensitization to house dust mites is directly related to the concentration of Group 1 mite allergen in dust. This led to the hypothesis that increases in mite allergen secondary to changes in houses were responsible for increases in asthma. However, asthma has also increased in areas of the world where mites do not flourish. In these dry areas sensitization to one of the other indoor allergens is the major risk factor for asthma. Although sensitization of asthmatics reflects the concentration of allergens in their houses, these measurements of exposure do not accurately predict severity of symptoms. Other factors that can contribute to the symptoms of asthma may also have increased. In particular, diesel particulates, ozone, beta 2-agonists, endotoxin and rhinovirus infection have each been shown to enhance the inflammatory response to inhaled allergens. Increases in asthma must relate to some aspect of our predominantly sedentary indoor lifestyle; this could be either increased exposure to allergens or an increase in factors that enhance the response of the lungs to foreign proteins.

Occurrence of Canine Parvovirus Type 2c in the United States

Canine parvovirus (CPV) type 2 (CPV-2) emerged around 1978 as a major pathogen of dogs worldwide. In the mid-1980s, the original CPV-2 had evolved and was completely replaced by 2 variants, CPV-2a and CPV-2b. In 2000, a new variant of CPV (named CPV-2c) was detected in Italy and now cocirculates with types 2a and 2b in that country. The CPV-2c has also been reported from single outbreaks in Vietnam and Spain. This study was conducted to determine if CPV-2c occurs in the United States. Thirty-three fecal samples were collected from dogs in 16 states between April 2006 and April 2007 and were tested for CPV using real-time polymerase chain reaction (PCR). Positive samples were further tested using conventional PCR and minor-groove binding TaqMan PCR assays to determine the viral type and to differentiate vaccine strains from field strains. Twenty-seven samples were positive for CPV, 7 of which were CPV-2c from 5 states: Arizona, California, Georgia, Oklahoma, and Texas. Of the 7 isolates, 4 differed from European CPV-2c isolates by 2 additional single-nucleotide mutations at positions 4076 and 4104, the latter of which produces a ThrAla change at residue 440 located near a major antigenic site. The coast-to-coast geographic distribution of the states in which CPV-2c was detected strongly suggests that this new CPV variant is probably widespread in the United States. The continuous evolution of CPV requires that monoclonal antibody-based and nucleic acid-based diagnostic assays should be periodically checked for sensitivity on prevalent CPV strains.

Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac disease

Background:Coeliac disease is caused by an immune response to gluten. As gluten proteins are proline rich they are resistant to enzymatic digestion in the gastrointestinal tract, a property that probably...

SARS Coronavirus Detection Methods

Using clinical samples from patients with severe acute respiratory syndrome, we showed that the sensitivities of a quantitative reverse transcription–polymerase chain reaction (80% for fecal samples and 25% for urine samples) were higher than those of the polyclonal (50% and 5%) and monoclonal (35% and 8%) antibody-based nucleocapsid antigen capture enzyme-linked immunosorbent assays.

Hev b 5 and Hev b 13 as allergen markers to estimate the allergenic potency of latex gloves

Background Sensitization to natural rubber latex has been linked to proteins from medical latex gloves. Various assays to estimate the amount of residual allergenic proteins extractable from latex gloves to assess their potential exposure hazard have inherent weaknesses. Objective This investigation was aimed at developing 2-site immunoenzymetric assays and identifying appropriate protein markers to assess the allergenic potential of latex gloves. Methods The presence of 6 latex allergens—Hev b 1, 2, 3, 5, 6, and 13—was measured in a cross-section of commercial latex medical gloves by using monoclonal and polyclonal antibody-based 2-site immunoenzymetric assays. The overall allergenic potential of these gloves was assessed by IgE-inhibition assay. Stepwise multiple regression analyses were performed to identify marker allergens that best explained the variation in latex glove allergenicity. Results All 6 latex allergens were detected in at least some of the glove samples. Hev b 5 and Hev b 13 were identified as the marker allergens that combined best to explain the variation in the glove allergenicity. The significant multiple correlation ( R = 0.855) between these 2 markers and glove allergenic potency forms the basis of an assay to gauge latex glove allergenicity. Conclusion The overall allergenic potential of latex gloves can be estimated by using Hev b 5 and Hev b 13 as indicator allergens. The correlation between glove allergenicity and the level of these allergens was maintained for low-protein gloves (<200 μg/g). This estimation of glove allergenicity was superior to that obtained by using total protein readings.

Dust-mite allergen removal from feathers by commercial processing

Individuals with allergies or asthma have had a long-standing bias against the use of chicken, goose, and duck feather pillows, comforters, quilts, and mattresses. We show that raw, unprocessed feathers contain dust-mite allergen that is removed by washing, and that feather pillows, whether covered or not, do not internally accumulate dust-mite allergen when used in mite-infested bedrooms over a 3-month period. Feathers obtained from six companies as unprocessed feathers (n = 8 batches) and processed feathers (n = 16 batches) were analyzed for dust-mite (Der p 1 and Der f 1) using monoclonal antibody-based immunoenzyametric assays with appropriate internal controls. Five pairs of new feather pillows (one covered and one uncovered) were placed in five bedrooms with known dust-mite contamination for 90 days. The pillows were then assayed for mite content. Dust-mite allergen was detected in 7 of 8 unprocessed feather samples; combined Der p 1 and Der f 1 mean = 524 ng/g, range 152 to 1,850 ng/g, whereas all 17 manufacturer-processed feather samples contained no detectable dust-mite. Although 4 of 5 bedrooms contained significant dust-mite allergen (>2,000 ng or Der p 1 and f 1, range 2,500 to 10,300 ng/g), none of the 90-day feather pillows became contaminated by dust-mite allergen even in the absence of a pillow cover. Feathers washed using industrial methods do not contain detectable dust-mite allergen. Pillows manufactured with processed feathers, whether encased in a dust cover or not, do not become contaminated by dust-mite allergen after 90 days of use in environments containing significant dust-mite allergen contamination.

Characterization of Inhibin Forms and Their Measurement by an Inhibin α-Subunit ELISA in Serum from Postmenopausal Women with Ovarian Cancer

The aim of this study was to characterize the molecular wt forms of inhibins A and B and its free alpha-subunit present in serum from women with ovarian cancer as a basis for developing improved monoclonal antibody-based inhibin assays for monitoring ovarian cancer. Three new inhibin alpha-subunit (alphaC) ELISAs were developed using monoclonal antibodies directed to three nonoverlapping peptide regions of the alphaC region of the inhibin alpha-subunit. To characterize serum inhibin molecular wt forms present in women with ovarian cancer, existing inhibin immunoassays (inhibin A, inhibin B, and pro-alphaC) and the new alphaC ELISAs were applied to sera from women with granulosa cell tumors and mucinous carcinomas previously fractionated using a combined immunoaffinity chromatography, preparative SDS-PAGE, and electroelution procedure. The distribution and molecular size of dimeric inhibins and alpha-subunit detected were consistent with known mol wt forms of inhibins A and B and inhibin alpha-subunit and their precursor forms present in serum and follicular fluid from healthy women. The alphaC ELISAs recognized all known forms of inhibin and the free inhibin alpha-subunit, although differences between alphaC ELISAs were observed in their ability to detect high mol wt forms. To assess which of the alphaC ELISAs was preferred in application to ovarian cancer, the alphaC ELISAs were applied to serum from a range of normal postmenopausal women (n = 61) and postmenopausal women (n = 152) with ovarian (serous, mucinous, endometrioid, clear cell carcinomas, and granulosa cell tumors) and nonovarian (breast and colon) cancers. Despite differences in their ability to detect high mol wt forms of inhibin, the alphaC ELISAs showed similar sensitivity (i.e. proportion of cancer patients correctly detected) and specificity (proportion of controls correctly detected) indexes in the detection of mucinous carcinomas (84% and 95%) and granulosa cell tumors (100% and 95%) compared with earlier inhibin RIA or polyclonal antibody-based immunofluorometric assays. A combination of the alphaC ELISAs with the CA125 assay, an ovarian tumor marker that has a high sensitivity and specificity for other ovarian cancers (serous, clear cell, and endometrioid), resulted in an increase in sensitivity/specificity indexes (95% and 95%) for the all ovarian cancer group. These new monoclonal antibody-based inhibin alphaC ELISAs now provide practical and sensitive assays suitable for evaluation as diagnostic tests for monitoring ovarian cancers.

Lösliche Leukozytenadhäsionsmoleküle bei Vegetariern unterschiedlichen Alters

Upregulation of leukocyte adhesion molecules under atherogenic conditions is accompanied by the release of soluble forms of adhesion molecules into the bloodstream. Vegetarians have a favorable cardiovascular risk profile. The aim of the present study was to assess the levels of circulating E-selectin (cE-selectin), circulating intercellular adhesion molecule-1 (cICAM-1), and circulating vascular adhesion molecule-1 (cVCAM-1) in both vegetarians and subjects of the average population and furthermore to evaluate the age dependence of cell adhesion molecules. 24 male and 59 female vegetarians (mainly members of the German Society of Vegetarians) and 124 male and 179 female control subjects (volunteers, of the ILSE study Leipzig = Interdisciplinary Long Time Study of Health Adult Age and the Lipid-Study Leipzig), 18-89 years old were included in the study. The serum levels of circulating cell adhesion molecules were determined using monoclonal antibody-based ELISA assays (R & D Systems, Abingdon, Europe Ltd.). Vegetarians were characterized by a favorable lipid profile and a low prevalence of common risk factors for atherosclerosis. This group showed a tendency towards lower cICAM-1 levels in comparison with control subjects. Furthermore, significantly lower cE-selectin levels were found in the group of vegetarians. An age-dependent increase of cVCAM-1 and cICAM-1 levels was found both in the group of vegetarians and control subjects. No significant differences were noted regarding the gender of the subjects included in the study. Low cE-selectin levels of vegetarians may reflect the favorable cardiovascular risk profile of this group. Furthermore, the present data indicate that cVCAM-1 and cICAM-1 are age-dependent parameters independent of risk for atherosclerosis.

Experimental infections of pigs with Japanese encephalitis virus and closely related Australian flaviviruses.

The flavivirus Japanese encephalitis (JE) virus has recently emerged in the Australasian region. To investigate the involvement of infections with related enzootic flaviviruses, namely Murray Valley encephalitis (MVE) virus and Kunjin (KUN) virus, on immunity of pigs to JE virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. Antibody responses to primary and secondary infections were evaluated using panels of monoclonal antibody-based blocking enzyme-linked immunosorbent assays and microtiter serum neutralization tests (mSNTs). Identification of the primary infecting virus was possible only using the mSNTs. Following challenge, unequivocal diagnosis was impossible due to variation in immune responses between animals and broadened and/or anamnestic responses. Viremia for JE virus was readily detected in pigs following primary infection, but was not detected following prior exposure to MVE or KUN viruses. Boosted levels of existing cross-neutralizing antibodies to JE virus suggested a role for this response in suppressing JE viremia.

Asthma severity, atopic status, allergen exposure and quality of life in elderly persons.

Although asthma can be associated with significant airflow obstruction in those over the age of 65, it is often underdiagnosed and undertreated. To describe severity of asthma, allergy skin test sensitivities, indoor allergen exposures, and the impact on quality of life (QOL) and health status in elderly persons with asthma. A cross-sectional data analysis with 80 elderly persons with asthma recruited from medical, geriatric, and allergy/immunology tertiary care centers. Asthma severity was determined by symptoms and measurements of lung function. House dust specimens were collected from mattresses and bedroom carpets and analyzed separately for the major allergens of house dust, using monoclonal antibody-based immunoenzymetric assays. QOL was measured using Juniper's Asthma Quality of Life Questionnaire. Health status was measured using the Short Form Health Survey Medical Outcome Questionnaire which included Ferrans and Powers' Quality of Life Index subscales. Two-thirds of participants had either moderate or severe persistent asthma. Skin tests to a battery of common airborne allergens were positive to at least one allergen in 56 of the 75 participants tested (74.7%). Reservoir dust allergen levels were often high enough to place participants at risk of symptoms or at risk of developing sensitization. Increased asthma severity was associated with significantly lower QOL and a trend toward decreased health status. Asthma is a significant chronic problem in the elderly. Atopy was common. Asthma severity impacts on these participants' QOL and health status. Results support interventions aimed at identifying allergens precipitating attacks and reducing them in the home.

Falsely Increased Values for Rabbit Immunoglobulin-based Nephelometric Immunoassays Attributable to Human Anti-Rabbit Antibodies

A large number of serum proteins are routinely measured by automated immunonephelometric assays utilizing antisera derived from rabbits, goats, or sheep. Because this type of immunoassay uses polyclonal reagents derived from animals other than mice, it is free from interference by the human anti-mouse antibodies that commonly plague monoclonal antibody-based sandwich assays and competitive immunoassays (1). Nonspecific antibodies against murine immunoglobulins can be detected in the serum of a significant proportion of patients, with a prevalence estimate as high as 80% (2). The incidence of anti-rabbit antibodies in serum samples is considerably lower, with estimates between 0.1% and 5% (1)(3)(4)(5)(6). Interference from human anti-rabbit antibodies has been documented for two-site immunoassays and radioimmunoassays for several hormone assays as well as for creatine kinase MB (3)(4)(5)(6)(7)(8). It recently was reported that anti-rabbit antibodies produced falsely increased C-reactive protein values when measured by nephelometric methods utilizing rabbit reagents (9). In this …

DNA repair: counteragent in mutagenesis and carcinogenesis-- accomplice in cancer therapy resistance.

DNA-reactive carcinogens and anticancer drugs induce many structurally distinct mutagenic and cytotoxic DNA lesions. The varying capability of normal and malignant cells to recognize and repair specific DNA lesions influences both cancer risk and the relative sensitivity or resistance of cancer cells towards cytotoxic agents. Using monoclonal antibody-based immunoanalytical assays, very low amounts of defined carcinogen-DNA adducts can be quantified in bulk genomic DNA, in individual genes, and in the nuclear DNA of single cells. DNA repair kinetics can, thus, be measured in a lesion-, gene-, and cell type-specific manner, and the DNA repair profiles of malignant cells can be monitored in individual patients. Even structurally very similar DNA lesions may be repaired with strongly differing efficiency. The miscoding DNA alkylation products O(6)-methylguanine and O(6)-ethylguanine, for example, differ only by one CH(2) group. These lesions are formed in DNA upon exposure to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea, both of which induce mammary adenocarcinomas in female rats at high yield. Unrepaired O(6)-alkylguanines in DNA cause G:C-->A:T transition mutations via mispairing during DNA replication. O(6)-methylguanines are repaired at a similar slow rate in both transcriptionally active (H-ras, beta-actin) and inactive genes (IgE heavy chain; bulk DNA) of the target mammary epithelia (which express the repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) at a very low level). In contrast, O(6)-ethylguanines are repaired approximately 20 times faster than O(6)-methylguanines in both DNA strands of the transcribed genes selectively via an AGT-independent, as yet unclarified excision mechanism. Accordingly, G:C-->A:T transitions resulting from the misreplication of an O(6)-methylated guanine at the second position of codon 12 (GGA) of H-ras represent a frequent "signature" mutation in rat mammary adenocarcinomas that develop after exposure to N-methyl-N-nitrosourea. However, this mutation is not observed when these tumors are induced by N-ethyl-N-nitrosourea, due to the fast repair of O(6)-ethylguanines in the H-ras gene. The key importance of "conventional" and "conditional" gene knockout technology for resolving the intricacies of the complex network of DNA repair pathways is briefly discussed.

Human Anti-Animal Antibody Interferences in Immunological Assays

The scope and significance of human anti-animal antibody interference in immunological assays is reviewed with an emphasis on human anti-animal immunoglobulins, particularly human anti-mouse antibodies (HAMAs). Anti-animal antibodies (IgG, IgA, IgM, IgE class, anti-isotype, and anti-idiotype specificity) arise as a result of iatrogenic and noniatrogenic causes and include human anti-mouse, -rabbit, -goat, -sheep, -cow, -pig, -rat, and -horse antibodies and antibodies with mixed specificity. Circulating antibodies can reach gram per liter concentrations and may persist for years. Prevalence estimates for anti-animal antibodies in the general population vary widely and range from <1% to 80%. Human anti-animal antibodies cause interferences in immunological assays. The most common human anti-animal antibody interferent is HAMA, which causes both positive and negative interferences in two-site mouse monoclonal antibody-based assays. Strategies to prevent the development of human anti-animal antibody responses include immunosuppressant therapy and the use of humanized, polyethylene glycolylated, or Fab fragments of antibody agents. Sample pretreatment or assay redesign can eliminate immunoassay interferences caused by anti-animal antibodies. Enzyme immunoassays, immunoradiometric assays, immunofluorescence, and HPLC assays have been designed to detect HAMA and other anti-animal antibodies, but intermethod comparability is complicated by differences in assay specificity and lack of standardization. Human anti-animal antibodies often go unnoticed, to the detriment of patient care. A heightened awareness on the part of laboratory staff and clinicians of the problems caused by this type of interference in routine immunoassay tests is desirable. Efforts should be directed at improving methods for identifying and eliminating this type of analytical interference.

ENVIRONMENTAL CONTROL AND THE MANAGEMENT OF ALLERGIC DISEASES

ENVIRONMENTAL CONTROL AND THE MANAGEMENT OF ALLERGIC DISEASES

Risk levels for mite allergens. Are they meaningful?

Allergens found in house dust are among the most common environmental antigens to which man is naturally exposed. Standardized methods for measuring allergen exposure are essential for assessing the relationship between exposure, sensitization, and the severity of asthma. Monoclonal antibody-based assays are the most widely used method for assessing allergen exposure. In the effort to define the best "index of exposure" to mite allergens, several factors need to be investigated, including: 1) whether allergen should be measured in reservoir dust or airborne 2) whether the results of reservoir measurement should be expressed as recovered allergen per unit weight or per unit area. As yet, airborne sampling is insufficiently sensitive to produce reliable and repeatable results. Therefore, measurement of house-dust-mite allergen concentration in reservoir dust should be regarded as the best-validated index of exposure. The results should be expressed and reported both per unit weight (concentration) and per unit area. The strongest predictor of chronic symptoms and acute exacerbation of asthma is sensitization to indoor allergens. A simple dose-response relationship between IgE-mediated hypersensitivity and allergen exposure has been established. For example, exposure to more than 2 microg Group 1 mite allergen/g dust should be regarded as a risk factor for the development of IgE antibody and asthma in susceptible children. The quantitative relationship between exposure and symptoms in patients already sensitized is complex due to a number of possible confounding factors (e.g., other allergens, viruses, asthma medication). A simple threshold level for provocation of asthmatic symptoms has not been clearly defined.

Effect of anti-immunoglobulin antibodies produced in cattle infected with Trypanosoma evansi on antigen detection ELISA

The possibility of interference with the antigen-detection ELISA for trypanosomosis by anti-rodent IgG antibodies produced in cattle infected with Trypanosoma evansi was investigated. Two different ELISA for detection of trypanosome antigen and three different systems for anti-rodent IgG antibody detection were established. The former two were respectively polyclonal antibody-based and a combination of monoclonal and polyclonal antibody-based assays. The latter three were also adapted for detection of anti-mouse IgG, anti-rabbit IgG and anti-IgG antibodies cross-reactive with both rabbit and mouse IgGs. A total of 170 samples were collected from a dairy cattle farm where an outbreak of T. evansi infection was reported. One hundred and two cattle (59%) were found to be positive for trypanosome antigens by the polyclonal antibody-based assay and 86 (51%) were positive by the combination-based system. On the other hand, 51 (30%) and 10 (6%) of cattle had anti-rabbit and anti-mouse IgG antibodies respectively but none had antibodies cross-reactive with both IgGs. Of the 102 cattle positive for trypanosome antigens in the polyclonal antibody-based ELISA, 48 (47%) were also anti-rabbit IgG antibody positive. It is concluded that antigen detection ELISA based on a single-species immunoglobulin for capture and detection might misdiagnose T. evansi infection. Results indicate that this bias will be avoided if reagents for capture and detection are derived from different species.

Relevance of DNA repair to carcinogenesis and cancer therapy.

DNA-reactive carcinogens and anticancer drugs induce many structurally distinct cytotoxic and potentially mutagenic DNA lesions. The capability of normal and malignant cells to recognize and repair different DNA lesions is an important variable influencing the risk of mutation and cancer as well as therapy resistance. Using monoclonal antibody-based immunoanalytical assays, very low amounts of defined carcinogen-DNA adducts can be quantified in bulk genomic DNA, individual genes, and in the nuclear DNA of single cells. The kinetics of DNA repair can thus be measured in a lesion-, gene-, and cell type-specific manner, and the DNA repair profiles of malignant cells can be monitored in individual patients. Even structurally very similar DNa lesions may be repaired with extremely different efficiency. The miscoding DNA alkylation products O6-methylguanine (O6-MeGua) and O6-ethylguanine (O6-EtGua), for example, differ only by one CH2 group. These lesions are formed in DNA upon exposure to N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), both of which induce mammary adenocarcinomas in female rats at high yield. Unrepaired O6-alkylguanines cause transition mutations via mispairing during DNA replication. O6-MeGua is repaired at a similar slow rate in transcribed (H-ras, beta-actin) and inactive genes (IgE heavy chain; bulk DNA) of the target mammary epithelia (which express the repair protein O6-alkylguanine-DNA alkyltransferase at a very low level). O6-EtGua, however, via an alkyltransferase-independent mechanism, is excised approximately 20 times faster than O6-MeGua from the transcribed genes selectively. Correspondingly, G:C-->A:T transitions arising from unrepaired O6-MeGua at the second nucleotide of codon 12 (GGA) of the H-ras gene are frequently found in MeNU-induced mammary tumors, but are absent in their EtNU-induced counterparts.

Monoclonal antibody-based dual-label time-resolved fluorometric assays in a simplified one-step format

We have performed a one-step all-in-one dual-label dry reagent time-resolved fluorometric assay measuring free and total prostate-specific antigen (PSA). All assay-specific components were predried in an ordinary microtitration well, including the Eu and Tb chelate-labeled monoclonal antibodies for the recognition of free and total PSA, respectively. Only the sample and assay buffer needed to be added. Optimally, equilibrium was reached in <15 min. New intrinsically fluorescent Eu and Tb chelates were used, which made it possible to read the fluorescence directly from the solid phase. The developed dual-label assay with the simple protocol is ideal for easy automation, as the reaction wells can be of alternative design. The performance characteristics are equivalent to those of the present state-of-the-art single-label assays. The detection limits for free and total PSA were 0.03 and 0.02 microg/L, respectively, and the assays were linear over the range tested (up to 300 microg/L).