RESUMEN

A method capable of real-time and label-free monitoring of biomolecular interactions within whole blood, without any sample separation and label process, is described. This was accomplished using silica colloidal crystal (SCC) films, three-dimensionally ordered silica particle arrays whose interference effect is a function of their optical thickness, as interference-sensitive substrates. Interactions between immunoglobulin G (IgG) and protein A from Staphylococcus aureus (SPA) conjugates with changes in the optical thickness of SCC films were monitored spectroscopically. Successful detection of IgG was achieved in the buffer and whole blood. This system constitutes a simple label-free analysis showing great potential in monitoring interactions between biomolecules in complex biological media.

Asunto(s)

RESUMEN

A single-dose study was conducted to characterize the safety, pharmacokinetic, immunogenicity, and pharmacodynamic activity of highly purified Staphylococcal protein A (SPA), a native bacterial protein with immune-modulatory activity. Twenty healthy adults received a single intravenous dose of either 0.3 µg/kg (n = 8) or 0.45 µg/kg (n = 8) of SPA or placebo (n = 4). Changes in C-reactive protein and neopterin were used as markers of immune activation. All treatment-related AEs were of mild severity. Twelve of 16 active-dosed subjects developed detectable anti-protein A antibodies after dosing. These subjects had notably more rapid plasma clearance of SPA even prior to development of detectable titers. A transient post-dose decrease in circulating lymphocytes was observed as a notable pharmacodynamic effect, but was not correlated with plasma clearance or AUC. In peripheral blood mononuclear cells, SPA dosing increased transcription of multiple genes regulated by type-1 interferons, and up-regulation of several of these genes correlated with the degree of lymphopenia seen 24 hours after dosing. This study demonstrates the safety and tolerability of small intravenous doses of SPA and delineates acute and transient pharmacodynamic effects not previously reported.

Asunto(s)

RESUMEN

We evaluated the added benefit of a comprehensive counseling protocol for first-trimester aneuploidy risk assessment. We performed a prospective cohort study surveying patients referred for first-trimester aneuploidy risk assessment. We compared responses between women who underwent serum testing done in advance of their ultrasound such that their final risk assessment was given to them the same day as their ultrasound (comprehensive) versus women who underwent serum testing the same day as their ultrasound and who therefore received their final risk assessment later (standard). Response rate was 94.8%. The comprehensive group was significantly more likely to receive counseling in accordance with recommended American College of Obstetricians and Gynecologists (ACOG) guidelines, had significantly greater reduction in anxiety and increased satisfaction, and was more likely to report an increased understanding of their results. The comprehensive group scored significantly higher on test-style questions about aneuploidy risk assessment. Comprehensive aneuploidy risk assessment counseling including same-day results is associated with increased patient understanding and satisfaction, decreased anxiety, and increased adherence to ACOG guidelines.

Asunto(s)

RESUMEN

OBJECTIVES: PIH and IUGR are serious complications in the third trimester of pregnancy. Many publications claim a connection between false positive prenatal tests and subsequent occurrence of PIH and IUGR. DESIGN: The aim of the study was to estimate the usefulness of the biochemical markers of fetal defects and uterine Doppler examination in predicting PIH and IUGR in the third trimester of pregnancy. METHODS: We examined 156 pregnant patients in The Department of the Fetal Medicine and Gynecology Medical University of Lodz, between 2006-2009. In case of each pregnant woman we estimated biochemical markers in the first (PAPP-A + beta-hCG) and second trimester (AFP, beta-hCG, uE3 - triple test). Each patient underwent three ultrasonographic examinations in the first, second and third trimester (between 11-13, 15-20, and 22-27 weeks gestation, respectively) with uterine artery Doppler examination. We monitored these pregnancies for PIH and IUGR and divided them into three groups: 28 patients with PIH (study group 1), 14 patients with IUGR (study group 2), and 114 patients with uncomplicated pregnancies (controls). RESULTS: In both study groups we observed: higher concentration of beta-hCG, higher percentage of the positive biochemical prenatal tests and abnormal uterine artery Doppler waveform. Positive triple test was the strongest predictor of PIH and IUGR (PPV=60.87% for PIH and PPV = 30.77% for IUGR). CONCLUSIONS: Biochemical markers and abnormal uterine artery Doppler waveform are associated with PIH and IUGR. These parameters can be the base for the test identifying pregnant patients with high risk of PIH and IUGR.

Asunto(s)

RESUMEN

OBJECTIVE: The purpose of this study was to investigate the first-trimester placental volume and 3-dimensional (3D) power Doppler vascularization of pregnancies with low serum pregnancy-associated plasma protein A (PAPP-A) levels and to relate these findings to pregnancy outcomes. METHODS: Three-dimensional power Doppler sonography of the placenta was performed at gestational ages of 11 weeks to 13 weeks 6 days in 84 pregnancies with PAPP-A concentrations of less than 0.4 multiple of the median (MoM). With a standardized setting, the placental volume and vascularization index (VI), flow index (FI), and vascularization-flow index (VFI) were calculated and related to pregnancy outcomes. RESULTS: Pregnancy outcomes were as follows: 57 pregnancies with birth weights at or above the 10th percentile (group A), 16 pregnancies with birth weights below the 10th percentile and normal Doppler findings in the umbilical artery throughout gestation (group B), and 11 pregnancies with birth weights below the 10th percentile and abnormal umbilical Doppler findings later in gestation (group C). No differences were found in PAPP-A levels among groups. Placental volume values were significantly lower than reference limits, but no differences were found between groups. In groups A and B, there were no significant differences in 3D Doppler indices. However, these indices were significantly lower in group C (VI mean difference, -1.904; P < .001; FI mean difference, -1.939; P < .001; VFI mean difference, -1.944; P < .001). Placental vascular indices were significantly related to the severity of intrauterine growth restriction (IUGR; VI, r = 0.438; P < .001; FI, r = 0.482; P < .001; VFI, r = 0.497; P < .001) but not to the PAPP-A MoM and placental volume values. CONCLUSIONS: Low serum maternal PAPP-A levels are associated with altered 3D placental Doppler indices, and these changes are related to subsequent development of IUGR and adverse pregnancy outcomes.

Asunto(s)

RESUMEN

OBJECTIVE: To review the obstetrical outcomes associated with abnormally elevated or decreased level of one or more of the most frequently measured maternal serum marker analytes used in screening for aneuploidy. To provide guidance to facilitate the management of pregnancies that have abnormal levels of one of more markers and to assess the usefulness of these markers as a screening test. OPTIONS: Perinatal outcomes associated with abnormal levels of maternal serum markers analytes are compared with the outcomes of pregnancies with normal levels of the same analytes or the general population. EVIDENCE: The Cochrane Library and Medline were searched for English-language articles published from 1966 to February 2007, relating to maternal serum markers and perinatal outcomes. Search terms included PAPP-A (pregnancy associated plasma protein A), AFP (alphafetoprotein), hCG (human chorionic gonadotropin), estriol, unconjugated estriol, inhibin, inhibin-A, maternal serum screen, triple marker screen, quadruple screen, integrated prenatal screen, first trimester screen, and combined prenatal screen. All study types were reviewed. Randomized controlled trials were considered evidence of the highest quality, followed by cohort studies. Key individual studies on which the recommendations are based are referenced. Supporting data for each recommendation are summarized with evaluative comments and references. The evidence was evaluated using the guidelines developed by the Canadian Task Force on Preventive Health Care. VALUES: The evidence collected was reviewed by the Genetics Committee of the Society of Obstetricians and Gynaecologists of Canada. BENEFITS, HARMS, AND COSTS: The benefit expected from this guideline is to facilitate early detection of potential adverse pregnancy outcomes when risks are identified at the time of a maternal serum screen. It will help further stratification of risk and provide options for pregnancy management to minimize the impact of pregnancy complications. The potential harms resulting from such practice are associated with the so called false positive (i.e., uncomplicated pregnancies labelled at increased risk for adverse perinatal outcomes), the potential stress associated with such a label, and the investigations performed for surveillance in this situation. No cost-benefit analysis is available to assess costs and savings associated with this guideline. SUMMARY STATEMENTS: 1. An unexplained level of a maternal serum marker analyte is defined as an abnormal level after confirmation of gestational age by ultrasound and exclusion of maternal, fetal, or placental causes for the abnormal level. (III) 2. Abnormally elevated levels of serum markers are associated with adverse pregnancy outcomes in twin pregnancies, after correction for the number of fetuses. Spontaneous or planned mutifetal reductions may result in abnormal elevations of serum markers. (II-2) RECOMMENDATIONS: 1. In the first trimester, an unexplained low PAPP-A (< 0.4 MoM) and/or a low hCG (< 0.5 MoM) are associated with an increased frequency of adverse obstetrical outcomes, and, at present, no specific protocol for treatment is available. (II-2A) In the second trimester, an unexplained elevation of maternal serum AFP (> 2.5 MoM), hCG (> 3.0 MoM), and/or inhibin-A (> or =2.0 MoM) or a decreased level of maternal serum AFP (< 0.25 MoM) and/or unconjugated estriol (< 0.5 MoM) are associated with an increased frequency of adverse obstetrical outcomes, and, at present, no specific protocol for treatment is available. (II-2A) 2. Pregnant woman with an unexplained elevated PAPP-A or hCG in the first trimester and an unexplained low hCG or inhibin-A and an unexplained elevated unconjugated estriol in the second trimester should receive normal antenatal care, as this pattern of analytes is not associated with adverse perinatal outcomes. (II-2A) 3. The combination of second or third trimester placenta previa and an unexplained elevated maternal serum AFP should increase the index of suspicion for placenta accreta, increta, or percreta. (II-2B) An assessment (ultrasound, MRI) of the placental-uterine interface should be performed. Abnormal invasion should be strongly suspected, and the planning of delivery location and technique should be done accordingly. (III-C) 4. A prenatal consultation with the medical genetics department is recommended for low unconjugated estriol levels (<0.3 MoM), as this analyte pattern can be associated with genetic conditions. (II-2B) 5. The clinical management protocol for identification of potential adverse obstetrical outcomes should be guided by one or more abnormal maternal serum marker analyte value rather than the false positive screening results for the trisomy 21 and/or the trisomy 18 screen. (II-2B) 6. Pregnant woman who are undergoing renal dialysis or who have had a renal transplant should be offered maternal serum screening, but interpretation of the result is difficult as the level of serum hCG is not reliable. (II-2A) 7. Abnormal maternal uterine artery Doppler in association with elevated maternal serum AFP, hCG, or inhibin-A or decreased PAPP-A identifies a group of women at greater risk of IUGR and gestational hypertension with proteinuria. Uterine artery Doppler measurements may be used in the evaluation of an unexplained abnormal level of either of these markers. (II-2B) 8. Further research is recommended to identify the best protocol for pregnancy management and surveillance in women identified at increased risk of adverse pregnancy outcomes based on an abnormality of a maternal serum screening analyte. (III-A) 9. In the absence of evidence supporting any specific surveillance protocol, an obstetrician should be consulted in order to establish a fetal surveillance plan specific to the increased obstetrical risks (maternal and fetal) identified. This plan may include enhanced patient education on signs and symptoms of the most common complications, increased frequency of antenatal visits, increased ultrasound (fetal growth, amniotic fluid levels), and fetal surveillance (biophysical profile, arterial and venous Doppler), and cervical length assessment. (III-A) 10. Limited information suggests that, in women with elevated hCG in the second trimester and/or abnormal uterine artery Doppler (at 22-24 weeks), low-dose aspirin (60-81 mg daily) is associated with higher birthweight and lower incidence of gestational hypertension with proteinuria. This therapy may be used in women who are at risk. (II-2B) 11. Further studies are recommended in order to assess the benefits of low-dose aspirin, low molecular weight heparin, or other therapeutic options in pregnancies determined to be at increased risk on the basis of an abnormal maternal serum screening analyte. (III-A) 12. Multiple maternal serum markers screening should not be used at present as a population-based screening method for adverse pregnancy outcomes (such as preeclampsia, placental abruption, and stillbirth) outside an established research protocol, as sensitivity is low, false positive rates are high, and no management protocol has been shown to clearly improve outcomes. (II-2D) When maternal serum screening is performed for the usual clinical indication (fetal aneuploidy and/or neural tube defect), abnormal analyte results can be utilized for the identification of pregnancies at risk and to direct their clinical management. (II-2B) Further studies are recommended to determine the optimal screening method for poor maternal and/or perinatal outcomes. (III-A).

Asunto(s)

RESUMEN

2,5-Dihydroxyacetophenone (DHAP) is presented as a matrix which enables highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides, proteins and glycoproteins on AnchorChip targets. Depending on the protein, lower fmol amounts can be detected due to the increased homogeneity and concentration of the crystallization of the analyte/matrix mixture on the anchors. Best results could be generated in the mass range of 8-100 kDa. All sample/matrix preparation steps starting from mixing of DHAP matrix solution with sample solution to the transfer of the mixture to the MALDI-TOF target can be performed manually or automatically allowing low- and high-throughput analyses.

Asunto(s)

Asunto(s)

RESUMEN

OBJECTIVE: To develop an agglutination technique using Staphylococcal protein A (SpA) and Steptococcalprotein G (SpG) to detect human red blood cell antibodies. METHODS: Blood samples were obtained from the National Transfusion Service of Jamaica. SpA, SpG, and anti-IgG, -C3d were commercial preparations. SpA and ApG were incubated with sensitized human red blood cells (RBC) to assess their RBC agglutinating capacity. The Ouchterlony technique was used to determine binding between the bacterial antigens and the IgG in human serum. Polyethyleneglycol (PEG) was used as an agglutination enhancer. Agglutination techniques for a large number of samples were developed, using 96 percent well polystyrene microplates and a microscope for visualizing the agglutination techniques to detect human anti-RBC IgG was compared with the traditional Coombs' test. Sensitivity and specificity were determined. RESULTS: SpA and SpG did not appear to cause agglutination of the red cells sensitized in vivo and in vitro. However, no precipitation bands were formed between human serum and the supernatant obtained after reaction of the sensitized RBC with SpA and SpG (Ouchterlony technique). These results indicated that indeed there was binding of SpA and SpG with the sensitized cells since they were not available for binding with human serum. In additon, SpA and SpG agglutinated the sensitized red blood cells in the presence of the PEG. When compared to the Coombs' test, the following results were obtained with new techniques. For the direct method, sensitivity was 93.8 percent and 95.1 percent for SpA and SpG respectively (n= 81), and specificity was 91.4 percent and 93.5 percent for SpA and SpG, respectivley (n= 93). For the indirect method, sensitivity was 96.3 percent and 97.5 percent for SPA and SpG, respectively (n= 81) and its specificity was 100 percent for both proteins (n= 85). CONCLUSION: Agglutination techniques using SpA and SpG constitute alternative and feasible tests for the detection of human red blood cell antibodies. (AU)

Asunto(s)

RESUMEN

Protein A (SPA), a major cell wall component of Staphylococcus aureus, has occupied numerous investigators from its discovery in the late fifties. Its availability and avid binding to human immunoglobulins have led to extensive usage for diagnostic and research purposes. Today, SPA-based extracorporeal immunoadsorption relies on two rather different systems, namely, SPA-silica (Prosorba), and SPA-Sepharose (Immunosorba). Both systems are approved by the Food and Drug Administration for the core indications of rheumatoid arthritis and idiopathic thrombocytopenic purpura (SPA-silica) or hemophilia with inhibitors (SPA-Sepharose). Off label indications include immune disorders with a conceivable connection between autoantibody titers and disease activity, like forms of glomerulonephritis, systemic lupus erythematodes, myasthenia, and the Guillain-Barré syndrome as well as alloantibody formation in the context of e.g., transplantation. This review summarizes historical developments and important properties of SPA. Indications for extracorporeal therapy are discussed on the basis of available information and personal experience.

Asunto(s)

RESUMEN

In this article, the development of specific adsorbents for extracorporeal blood purification are described. Affinity microparticles were prepared by linking Protein A to crystalline cell surface layers (S-layers) from Thermoanaerobacter thermohydrosulfuricus 1111-69. S-layers were used in the form of cell wall fragments obtained by breaking whole cells by ultrasonification, resulting in cup-shaped structures (average size 0.5 x 1 microm) completely covered with S-layer protein. Protein A was covalently bound to carboxylic acid groups of the S-layer protein after activation with 1-ethyl-3,3'(dimethylamino)propylcarbodiimide. In batch adsorption experiments with fresh frozen human plasma, the resulting S-layer based affinity microparticles showed a high adsorption capacity for IgG (40 mg IgG were bound per g wet pellet of S-layer based affinity microparticles). Fractions eluted from the microparticles were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contained only IgG demonstrating that adsorption was specific. In biocompatibility tests, preparations of the S-layer microparticles showed no low-density lipoprotein-reactivity, no cytotoxicity, and no cytokine inducing activity.

Asunto(s)

RESUMEN

Encapsulation of therapeutic molecules in a new noncationic multilamellar vector (Spherulites), composed of phosphatidylcholine, cholesterol, and polyoxyethylene alcohol, is described here. Spherulites with entrapped drugs were prepared by shearing a phospholipidic lyotropic lamellar phase using a recently discovered method. The average size of these vesicles is approximately 300 nm. Our formulation did not show cytotoxicity to human cells and could be used as a drug delivery system. Our previous experiments showed that this new multilamellar vector is stable in many different buffers such as serum, acidic or basic buffers, and enzymatic buffers and may deliver drugs in vivo. We describe two ways of administration for drug delivery. The tissue biodistribution of radiolabeled Spherulites entrapping 125I protein A was studied after intravenous injection in Wistar rats using the major organs of the body. Approximately 70% of the radioactivity was found in the spleen 60 min after injection and about half this percentage was found in the liver. By 6 hr, only 52% remained in the spleen. The other tissues accumulated <30% of the dose throughout the duration of the study. On the other hand, oral administration of Spherulites, entrapping111 In-NTA, in fasting rats showed a significant increase of radioactivity in blood.

Asunto(s)

RESUMEN

OBJECTIVE: To examine the value of first trimester maternal serum free beta human chorionic gonadotrophin (beta hCG) and pregnancy associated plasma protein A (PAPP-A) as predictors of pregnancy complications. DESIGN: Screening study. SETTING: Antenatal clinics. POPULATION: Singleton pregnancies at 10-14 weeks of gestation. METHODS: Maternal serum free beta hCG and PAPP-A were measured at 10-14 weeks of gestation in 5,584 singleton pregnancies. In the 5,297 (94.9%) pregnancies with complete follow up free beta hCG and PAPP-A were compared between those with normal outcome and those resulting in miscarriage, spontaneous preterm delivery, pregnancy induced hypertension or fetal growth restriction and in those with pre-existing or gestational diabetes. RESULTS: Maternal serum PAPP-A increased and beta hCG decreased with gestation. The multiple of median maternal serum PAPP-A was significantly lower in those pregnancies resulting in miscarriage, pregnancy induced hypertension, growth restriction and in those with pre-existing or gestational diabetes mellitus, but not in those complicated by spontaneous preterm delivery. The level was < 10th centile of the reference range in about 20% of the pregnancies that subsequently resulted in miscarriage or developed pregnancy induced hypertension or growth restriction, and in 27% of those that developed gestational diabetes. Maternal serum free beta hCG was < 10th centile of the reference range in about 15% of the pregnancies that subsequently resulted in miscarriage or developed pregnancy induced hypertension or growth restriction, and in 20% of those that developed gestational diabetes. CONCLUSION: Low maternal serum PAPP-A or beta hCG at 10-14 weeks of gestation are associated with subsequent development of pregnancy complications.

Asunto(s)

RESUMEN

OBJECTIVE: To evaluate the introduction of a one stop multidisciplinary clinic for screening for fetal chromosomal abnormalities in the first trimester by a combination of maternal serum biochemistry and ultrasonography providing a risk of chromosomal abnormalities within a one hour clinic visit. DESIGN: One year retrospective review of screening performance. POPULATION: All women attending for routine antenatal care. The population included 4,190 singleton pregnancies in women of all ages screened between 10 weeks and 3 days and 13 weeks and 6 days of gestation between the periods 1 June 1998 and 31 May 1999 in a district general hospital antenatal clinic. METHODS: All women booked into the clinic were offered screening by a combination of maternal serum free beta human chorionic gonadotrophin (hCG) and pregnancy associated plasma protein A (PAPP-A) and fetal nuchal translucency thickness. Women at increased risk of carrying a fetus with trisomy 21 or trisomy 18/13 (> or =1 in 300 at sampling) were offered counselling and an invasive diagnostic procedure. Follow up of the outcome of all pregnancies was carried out. MAIN OUTCOME MEASURES: The detection rate for trisomy 21, trisomy 18/13 and all aneuploides, false positive rate, uptake of screening, uptake of chorionic villus sampling in women identified at increased risk and fetal loss after chorionic villus sampling. RESULTS: Overall 97.6% of the women (4,088/4,190) accepted first trimester screening. The rate of detection of trisomy 21 was 86% (6/7), for trisomy 18/13 100% (9/9) and for all aneuploides 95% (18/19). Fetal death at presentation was found in 1.6% of pregnancies (69/4,088). Of women who accepted screening, 6.1% (257/4,088) presented too late for fetal nuchal translucency measurement and 6.5% of the women (271/4,088) presented too early. The false positive rate was 6.7% (253/3,762). Uptake of invasive testing was 83% (207/253). CONCLUSION: First trimester prenatal screening for chromosomal abnormalities using a combination of maternal serum biochemistry and fetal nuchal translucency thickness can achieve detection rates in excess of 90%. These services can be provided in a one stop multidisciplinary clinic.

Asunto(s)

RESUMEN

There are several unsolved clinical findings in patients with idiopathic pulmonary fibrosis (IPF); (i) predominance of fibrosis in the lower lung fields, (ii) digital clubbing, and (iii) patchy distribution of pulmonary fibrosis. To explain these unsolved problems, we hypothesized that regenerated or premature bronchoepithelial cells may circulate in the blood in patients with IPF. To prove this, we performed the reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK19) and pulmonary surfactant protein A (SPA) in peripheral blood in patients with IPF and pulmonary fibrosis associated with collagen vascular disorders. In addition, 20 patients with chronic pulmonary emphysema as a disease control and 19 normal volunteers were also evaluated for the existence of circulating bronchoepithelial cells. RT-PCR analysis showed that CK19 was expressed in 12 of 38 blood samples (31.6%) of IPF and pulmonary fibrosis associated with collagen vascular disorders, seven of 20 (35.0%) blood samples of chronic pulmonary emphysema, and four of 19 (21.1%) blood samples of normal volunteers. mRNA for SPA was positive in eight of 38 (21.1%) blood samples of IPF. In contrast, SPA expressing cells were not detected in any blood samples obtained from patients with chronic pulmonary emphysema or normal volunteers. This evidence suggests that there were some circulating bronchoepithelial cells expressing mRNA for SPA in peripheral blood of patients with IPF and pulmonary fibrosis associated with collagen vascular disorders.

Asunto(s)

RESUMEN

First-trimester screening for Down syndrome has been proposed as a significant improvement with respect to second-trimester serum screening programs, the current standard of care, because of apparently higher detection rates and an earlier gestational age at diagnosis. First-trimester nuchal translucency on ultrasonography forms the basis of this new form of screening, although studies of its efficacy have yielded widely conflicting results, with detection rates ranging from 29% to 91%. Studies of first-trimester serum screening with measurements of pregnancy-associated plasma protein A and free beta-human chorionic gonadotropin serum concentrations have been much more consistent, with Down syndrome detection rates of 55% to 63% at a 5% false-positive rate. The combination of first-trimester ultrasonographic and serum screening has the potential to yield a Down syndrome detection rate of 80% at a 5% false-positive rate, although this approach has not been adequately studied. There have been no studies performed to date to directly compare the performance of first-trimester and second-trimester methods of screening. Two major trials are underway that will address this issue, one in the United Kingdom and one in the United States. Until the results of these trials are available, the current standard of care with respect to Down syndrome screening should not be changed, and first-trimester screening should remain investigational.

Asunto(s)

RESUMEN

Immunoassays designed to measure low concentrations of staphylococcal protein A (SPA) that have been leached into antibody preparations intended for therapeutic use are subject to differing degrees of interference. Methods established to quantify SPA in murine antibody preparations are not accurate in the presence of human or humanized IgG. We report the development of an enzyme-linked immunosorbent assay (ELISA) for SPA with a detection limit of 7 pg/ml and the optimization of a method that permits complete dissociation of SPA-immunoglobulin-complexes. This assay is a modification of our heat-mediated dissociation (HD-SD) treatment with sodium dodecyl sulfate (SDS) and diethylenetriaminepentacetic acid (DTPA) for total immune-complex dissociation, in which the heat treatment has been prolonged and the diluent is characterized by increased protein content and buffering capacity. The diluent developed contains SDS, DTPA and bovine serum albumin dissolved in a 0.1 M phosphate buffer (pH 7.2). To validate the efficiency of this novel method, a series of samples have been assayed, including samples reconstituted in vitro, samples of purified antibodies, and plasma from patients. The described method has been shown to be generally efficient in quantitating all native and recombinant SPA in samples containing up to 50 mg/ml of human IgG. These data demonstrate the utility of this technique in determining SPA contamination of recombinant immunoglobulin therapeutic products.

Asunto(s)

Asunto(s)

RESUMEN

The reaction of the bacteriosorption of immune complexes (RBIC), based of on the avidity of horse IgG (T) to protein A in interaction with complement antigen, is proposed. The possibility of using RBIC for the evaluation of the titer of native antitoxic sera and the degree of the enzymolysis of specific antitoxins with pepsin has been shown.

Asunto(s)

RESUMEN

Infectious diseases of wild animals are of increasing importance, both from an economic viewpoint and because several of these diseases are pathogenic to man. However, serosurveys to determine the circulation of infectious organisms in wildlife are complicated by the fact that antibodies to species-specific immunoglobulins are not available for use in serological assays such as enzyme-linked immunosorbent assays (ELISAs) or immunofluorescence assays. To determine the binding potential of four commercially available antibody conjugates with the sera of wild animals, sera from 27 species of small terrestrial mammals were allowed to react with alkaline phosphatase-labelled protein A, anti-rabbit IgG, anti-mouse IgG and anti-human IgG by by the use of an ELISA. It was found that sera from some species of the order Lagomorpha bound optimally to anti-rabbit IgG, while anti-mouse IgG could be used for most species of Rodentia. For all Carnivora, Insectivora, Macroscelidea, Hyracoidea and other Rodentia, staphylococcal protein A demonstrated optimal binding. None of the sera that was tested bound to anti-human IgG. These results demonstrate that commercial conjugates can be used in serological assays in which wild animal sera are used, and should be useful for future serosurveys to determine the circulation of infectious agents in small terrestrial mammals.

Asunto(s)