Biomedical subjects

K Pettersson

Publications and source records attributed to K Pettersson.

At least 91 records · Page 5Linked to original sources

PubMed1998

Characterization of serum tartrate-resistant acid phosphatase and development of a direct two-site immunoassay.

Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) to the circulation, where the amount of TRAP is expected to correlate with the bone resorption rate. We have developed two monoclonal antibodies, O1A and J1B, using purified human bone TRAP as antigen. The antibodies recognized different epitopes, allowing us to develop a two-site fluoroimmunoassay. The immunoreactivity in fresh serum specimens was less than 10% of the concentrations measured from the same specimens after 24 h of storage at 4 degrees C, or after addition of 5 mM EDTA or EGTA to them. When fresh serum was gel filtrated using Sephacryl S-200 column, all of the enzyme eluted in the void volume as a complex with a molecular weight of more than 250 kDa. If the serum was treated with EDTA before the gel filtration, the complex was destroyed and the enzyme eluted in fractions corresponding to a molecular weight of 30 kDa, the size of monomeric purified human bone TRAP. The immunoassay was used to measure TRAP concentrations from serum samples that had been stored at 4 degrees C for 24 h. According to the assay, premenopausal women had 13.1 +/- 3.1, postmenopausal women 17.6 +/- 4.2, and children 32.6 +/- 12.2 microg TRAP/l of serum. We conclude that TRAP circulates in the serum as part of a complex, which also contains Ca2+, and that TRAP-immunoassay is a potentially useful method for determining bone resorption rates, as long as the complex is destroyed before the assay.

Acid Phosphatase↗

PubMed1998

A dual-label immunofluorometric assay for human osteocalcin.

Circulating human osteocalcin (hOC) has been shown to be comprised of two main forms: the intact 1-49 form and the proteolytic N-terminal midfragment (N-mid) spanning amino acid residues 1-43 or 1-44. We used three monoclonal antibodies (MAbs) raised against hOC and bovine osteocalcin in developing a dual-label assay for the simultaneous measurement of the proportions of the intact and N-mid forms in serum samples. The assay is based on time-resolved fluorescence utilizing differently labeled trace MAbs. Biotinylated MAb 2H9 is used as a capture antibody for both the intact hOC and the N-mid. Tracer MAb 6F9 labeled with a Europium (III)-chelate binds to the intact the N-mid and the intact hOC, whereas tracer MAb 3G8 labeled with a Terbium (III)-chelate binds to the intact hOC only. The simultaneous binding of the antibodies was tested by comparing full-length hOC purified from human bone and hOC shortened from the C terminus by four amino acid residues with carboxypeptidase Y. Serum hOC measurements with the dual-label assay were in agreement with the corresponding single-label assays (r = 0.96 for intact + N-mid assay and r = 0.81 for intact assays, n = 91). The lower correlation between the intact assays was attributable to proteolytic susceptibility of the intact form due to one additional freezing and thawing cycle in carrying out the dual-label assay. As measured with the dual-label assay, the levels (mean +/- SD) of serum intact + N-mid OC were 6.2 +/- 2.1 ng/ml in the premenopausal group (n = 44), 13.9 +/- 4.9 ng/ml in the postmenopausal group without hormone replacement therapy (HRT; n = 13), and 7.5 +/- 3.4 ng/ml in the postmenopausal group with HRT (n = 13). The levels of intact hOC in the same groups were 4.8 +/- 1.4 ng/ml, 9.8 +/- 2.9 ng/ml, and 5.3 +/- 2.1 ng/ml, respectively. Whether the main forms of OC or their relative proportions in serum can be used for predicting bone diseases or for monitoring the progression and management of diseases awaits further investigations.

Adult↗

PubMed1998

Degradation of cardiac troponin I: implication for reliable immunodetection.

We have analyzed by different immunological methods the proteolytic degradation of cardiac troponin I (cTnI) in human necrotic tissue and in serum. cTnI is susceptible to proteolysis, and its degradation leads to the appearance of a wide diversity of proteolytic peptides with different stabilities. N- and C-terminal regions were rapidly cleaved by proteases, whereas the fragment located between residues 30 and 110 demonstrated substantially higher stability, possibly because of its protection by TnC. We conclude that antibodies selected for cTnI sandwich immunoassays should preferentially recognize epitopes located in the region resistant to proteolysis. Such an approach can be helpful for a much needed standardization of cTnI immunoassays and can improve the sensitivity and reproducibility of cTnI assays.

Amino Acid Sequence↗

PubMed1997

Dual-label time-resolved immunofluorometric assay for simultaneous determination of pregnancy-associated plasma protein A and free beta-subunit of human chorionic gonadotrophin.

Using time-resolved fluorometry, a simple one-step dual-label immunometric assay has been developed, which allows simultaneous determination of pregnancy-associated plasma protein A (PAPP-A) and free beta-subunit of human chorionic gonadotrophin (beta-hCG) in first-trimester maternal serum samples. Two monoclonal antibodies were biotinylated and immobilized onto the surface of streptavidin-coated microtitration plates, and used to capture PAPP-A and beta-hCG. respectively. Europium (Eu) and Samarium (Sm) chelates were conjugated to two additional monoclonal antibodies acting as detection antibodies for PAPP-A and beta-hCG. The assay was performed using a 4-h one-step format. The within-run precision with buffer-based calibrators was below 8% over the working range of PAPP-A (40-10000 mIU/l) and beta-hCG (7.3-525 micrograms/l) and no hook effect was observed. The intra- and inter-assay coefficients of variation were below 7.1% for serum samples. PAPP-A and beta-hCG concentrations measured by the dual assay in 39 first-trimester serum samples correlated excellently with those obtained by DELFIA single-label PAPP-A (r = 0.997) and the beta-hCG part (r = 0.993) of the DELFIA AFP/beta hCG dual-label assay.

Antibodies, Monoclonal↗

PubMed1997

Immunoreactivity of recombinant human glandular kallikrein using monoclonal antibodies raised against prostate-specific antigen.

The gene encoding human glandular kallikrein (KLK2) was expressed in Escherichia coli, and the corresponding protein (hK2) was produced by fermentation. The hK2 was characterized by Western blotting and epitope map using monoclonal antibodies (MAbs) specific for another protease, prostate-specific antigen (PSA) with high structural identity (80%). MAbs that recognized three different epitopes were bound to hK2, representing 7 out of 23 MAbs tested. One epitope was localized to the sequence region around amino acid position 78, which is believed to be glycosylated in hK2. The affinities of MAbs recognizing hK2 were similar to those for PSA, suggesting that common epitopes seem to contain very conserved structures. The results may help in designing specific diagnostic assays for the assessment of prostate cancer.

Antibodies, Monoclonal↗

PubMed1997

Cloning, sequencing, expression and characterization of three anti-estradiol-17beta Fab fragments.

In order provide data for a basic understanding of the mechanisms of antibody specificity and for the design of antibodies with desired properties, we have sequence-analysed three high affinity anti-estradiol-17beta monoclonal antibodies. All three monoclonal antibodies to estradiol-17beta had been raised by conjugation of the 6-carboxymethyloxime derivative to protein carrier. The genes encoding heavy (Fd) and light (L) chains of these three antibodies were cloned and sequenced. The sequenced antibody chains were found to be from 46.0 to 89.7% sequence identical to a monoclonal antibody (DB3) binding a related steroid, progesterone. The Fd and L chains were paired with all possible Fd-L combinations and the corresponding proteins were expressed in Escherichia coli and characterized for their binding (immunoreactivity) to estradiol-17beta. Under the lac promoter and using the pelB signal sequences the production levels of the soluble (total) heavy and light chain Fab fragment combinations in periplasm and in supernatant varied from 115 to 2207 microg/l, while the immunoreactivity percentages (IR%) varied from < 1 to 45%. The production levels and IR% were dependent on the first constant domain subclasses of the heavy chain as well as the Fd-L chain combination expressed.

Antibodies, Monoclonal↗

PubMed1997

Disc pathology after whiplash injury. A prospective magnetic resonance imaging and clinical investigation.

STUDY DESIGN: This study was used to evaluate the relationship between magnetic resonance imaging findings and clinical findings after whiplash injury. OBJECTIVES: To identify initial soft-tissue damage after whiplash injury, the development of disc pathology, and the relationship of disc pathology to clinical findings. SUMMARY OF BACKGROUND DATA: Although a few studies have reported pathological magnetic resonance imaging findings after whiplash injuries, there is no prospective study published to our knowledge. METHODS: Thirty-nine patients, 20 women and 19 men with a mean age of 32 years, were treated for whiplash injury. Magnetic resonance imaging and clinical examination were performed in a blinded manner at a mean of 11 days after trauma. The procedure was repeated at a 2-year follow-up visit. Two patients could not be examined with the second magnetic resonance imaging because of claustrophobia and pregnancy, respectively. RESULTS: The authors found 13 patients (33%) with disc herniations with medullary (six cases) or dura (seven cases) impingement over the 2-year follow-up period. At the follow-up examination all patients with medullary impingement had persistent or increased symptoms, and three of 27 patients (11%) with no or slight changes on magnetic resonance imaging had persistent symptoms. No ligament injuries were diagnosed. CONCLUSION: Although disc pathology seems to be one contributing factor in the development of chronic symptoms after whiplash injury, it may be unnecessary to examine these patients in the acute phase with magnetic resonance imaging; correlating initial symptoms and signs to magnetic resonance imaging findings is difficult because of the relatively high proportion of false-positive results. Magnetic resonance imaging is indicated later in the course of treatment in patients with persistent arm pain, neurologic deficits or clinical signs of nerve root compression to diagnose disc herniations requiring surgery.

Adolescent↗

PubMed1997

Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research. International Collaborative Research Group.

OBJECTIVE: To determine the worldwide frequency of a common immunological LH variant because of two point mutations in the LH beta-subunit gene (Trp8Arg and Ile15Thr). DESIGN: Cross-sectional study on LH status (variant and wild-type) in serum (or DNA) samples from Finland (Finns and Lapps), Estonia, Poland, Sweden, The Netherlands, United Kingdom, Italy, South Africa (blacks), Thailand, China, Japan, and the United States (Hispanics and blacks). SETTING: Academic research environment. PATIENT(S): Ambulatory adult men and women (n = 2,936) with minor illnesses and no known endocrinological disorders. INTERVENTION: A single blood sample was collected from each subject. MAIN OUTCOME MEASURE(S): The LH status was determined by two immunofluorometric assays using monoclonal antibodies. One (assay 1) only recognizes the wild-type LH, the other (assay 2) recognizes equally variant and wild-type LH. The ratio of assay 1 to assay 2 indicates the LH status: wild-type, > 0.9; heterozygote, 0.2 to 0.9; and homozygote, < 0.15. One population (Lapps) was studied by DNA analysis using polymerase chain reaction and allele-specific oligonucleotide hybridization. RESULT(S): The carrier frequency of the variant LH beta allele varied from 7.1% in U.S. Hispanics to 41.9% in Lapps of northern Finland. The variant LH beta allele tended to be more common in populations from Northern Europe as compared with those from Asia. CONCLUSION(S): The high frequency of the LH beta variant worldwide makes it an important confounding factor when obtaining disproportionately low LH levels with some immunometric assays. The LH variant may contribute to some pathologies of the pituitary-gonadal function.

Adolescent↗

PubMed1997

Epitope mapping of human prostate specific antigen and glandular kallikrein expressed in insect cells.

Two human prostate gland proteases were expressed in insect cells using recombinant baculovirus expression system. Prostate specific antigen (PSA) is an established serum marker of prostate cancer whereas the clinical utility of its close homologue, human glandular kallikrein (hK2) is presently unknown. The production levels using Trichoplusia ni cells were roughly 300 &mgr;g/l and 6 mg/l for hK2 and PSA, respectively. On western-blot we estimated the size for both proteins to be approximately 33 kDa which was consistent with PSA purified from seminal plasma. Nine anti-PSA monoclonal antibodies (Mabs) out of 26 tested, representing five independent epitopes, also reacted with hK2. The results obtained in this study may help in designing more accurate diagnostic assays for detection and monitoring of prostate cancer.

Journal Article↗

PubMed1997

Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vitro, radioreceptor activity and immunoactivity.

To establish whether sialic acid content is responsible for an observed 7-8-fold variability in bioactivity in vitro of highly purified human pituitary luteinizing hormone (hLH) isoforms, the bioactivity in vitro, radioreceptor activity and immunoactivity of hLH isoforms were determined before and after enzymatic desialylation. Three immunofluorometric assays with different hLH specificities allowed characterization of 13-24 pituitary hLH isoform preparations of pI 7.03-8.98 in terms of sialic acid content (1-5 sialic acid residues per LH molecule), bioactivity in vitro (4030-30,000 I.U. mg[-1]), radioreceptor activity (6420-25,400 I.U. mg[-1]) and hLH immunoactivity (2900-4400 to 18,300-27,300 I.U. mg[-1]). Significant positive correlations between sialic acid content and either immunoactivity or in vitro bioactivity were observed, whereas radioreceptor activity showed a curvilinear response. Following more than 90% removal of sialic acid, both in vitro bioactivity and radioreceptor activity were increased, although specific activity still differed between isoforms; immunoactivities were unaffected. It is concluded that the presence of the sialic acid residue(s) on hLH isoforms does partially contribute to the in vitro bioactivity and radioreceptor activity of the isoforms, but that hLH immunoactivity is independent of sialic acid content.

Animals↗

PubMed1997

Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha.

The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-beta (ER beta) and the functional characterization of mouse ER beta protein. ER beta is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-alpha (ER alpha) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ER beta and ER alpha subtypes was demonstrated. Furthermore, ER beta and ER alpha form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ER beta/ER alpha heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ER beta in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.

Amino Acid Sequence↗

PubMed1997

A two-site delfia immunoassay for measurements of the N-terminal peptide of pro-atrial natriuretic peptide (nANP).

A rapid, sensitive and reliable two-site immunoassay for measurements of the N-terminal peptide of pro-atrial natriuretic peptide (nANP) is presented. The method uses one monoclonal antibody, directed against the N-terminal part of nANP, as catcher antibody and another monoclonal antibody, directed against the C-terminal part of nANP, as detector antibody. The catcher antibody is biotinylated and is bound to streptavidin pre-coated microtiter strips. The detector antibody is labelled with Europium, which is measured in a Wallac DELFIA time-resolved fluorometer. Blood collected in plain Vacutainer tubes gave same measured amounts of nANP as blood collected in heparinised tubes. Blood collected in tubes containing EDTA gave same measured amounts of nANP as the plain tubes, provided that a 2-step assay protocol was used. Based upon 100 healthy blood donors, a reference interval was calculated to < 450 pmol/L. Within the reference group there was a significant increase of serum nANP with age. Based on 42 patients with different degree of impaired renal function, a significant correlation of nANP and serum creatinine was found. Assay performance, given as total assay variation was 12%, 10% and 9% respectively at serum levels of 140, 970 and 3500 pmol/L. It is concluded that this method is fast, sensitive and reliable for clinical measurements of nANP.

Adult↗

PubMed1997

Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex.

Fourteen monoclonal antibodies (mAbs) against human cardiac troponin I (cTnI) were generated by commonly used experimental techniques. All these antibodies, as well as antibody 414 (HyTest), were specific for human cTnI. Fifteen antibodies thus obtained were tested in a sandwich cTnI immunofluorescence assay (altogether 196 combinations). Ten pairs giving the highest sensitivity were selected for further investigation. The effect of TnI-TnC complex formation on antibody interaction with antigen was analyzed. The formation of TnI-TnC complex results in a significant decrease of the interaction of mAbs with TnI for seven of 10 analyzed pairs of antibodies. Using two pairs of cTnI-specific mAbs, one that recognized only free cTnI but not cTnI complexed with cTnC, and another that could be used for measurement of total cTnI (free cTnI and cTnI in complex with cTnC), we demonstrated that the main part of cTnI in serum collected from acute myocardial infarction patients is presented in the complex from. We concluded that effective and reliable immunological detection of TnI is possible only when antibodies used for assay development recognize both free TnI and TnI complexed with other troponin components.

Antibodies, Monoclonal↗

PubMed1997

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Chelating Agents↗

PubMed1997

Double-monoclonal immunofluorometric assays for pregnancy-associated plasma protein A/proeosinophil major basic protein (PAPP-A/proMBP) complex in first-trimester maternal serum screening for Down syndrome.

Four double-monoclonal time-resolved immunofluorometric assays (TrIFMAs) have been developed for the specific determination of pregnancy-associated plasma protein A/proeosinophil major basic protein (PAPP-A/ proMBP) complex in first-trimester maternal serum samples. The assays have a functional sensitivity of < 4 mIU/L and a working range from 4 to 1000 mIU/L. These 4 assays, together with a polyclonal sandwich TrIFMA, were compared for their ability to discriminate between normal pregnancies (n = 149) and pregnancies carrying a Down syndrome fetus (n = 36) in maternal serum screening samples from gestational weeks 4-13. In 26 Down syndrome pregnancies from gestational weeks 7-12, the median PAPP-A multiples of the median concentration in controls (MoMs) determined by monoclonal antibody combinations 234-3/234-2*, 234-4/234-2*, 234-4/234-5*, and 234-5/234-6* were 0.35, 0.37, 0.42, and 0.44, respectively, whereas the median MoM determined by the polyclonal assay was 0.56. ROC curve analysis also showed that better overall diagnostic accuracy and detection rates were achieved by the monoclonal TrIFMAs than by the polyclonal TrIFMA. This report is the first to describe assays that specifically measure PAPP-A/proMBP complex without possible interference from other proMBP-containing complexes.

Antibodies, Monoclonal↗

PubMed1996

Epitope mapping of nine monoclonal antibodies against osteocalcin: combinations into two-site assays affect both assay specificity and sample stability.

Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length+large NH2-terminal fragment as stimulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks gamma-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to gamma-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of gamma-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at -20 degrees C in an undercooled state was detectable as loss of immunoreactivity (ranging from -42 +/- 17 to -50 +/- 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length+large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.

Animals↗

PubMed1996

Structural investigation of the alpha-1-antichymotrypsin: prostate-specific antigen complex by comparative model building.

Prostate-specific antigen (PSA), produced by prostate cells, provides an excellent serum marker for prostate cancer. It belongs to the human kallikrein family of enzymes, a second prostate-derived member of which is human glandular kallikrein-1 (hK2). Active PSA and hK2 are both 237-residue kallikrein-like proteases, based on sequence homology. An hK2 model structure based on the serine protease fold is presented and compared to PSA and six other serine proteases in order to analyze in depth the role of the surface-accessible loops surrounding the active site. The results show that PSA and hK2 share extensive structural similarity and that most amino acid replacements are centered on the loops surrounding the active site. Furthermore, the electrostatic potential surfaces are very similar for PSA and hK2. PSA interacts with at least two serine protease inhibitors (serpins): alpha-1-antichymotrypsin (ACT) and protein C inhibitor (PCI). Three-dimensional model structures of the uncleaved ACT molecule were developed based upon the recent X-ray structure of uncleaved antithrombin. The serpin was docked both to PSA and hK2. Amino acid replacements and electrostatic complementarities indicate that the overall orientation of the proteins in these complexes is reasonable. In order to investigate PSA's heparin interaction sites, electrostatic computations were carried out on PSA, hK2, protein C, ACT, and PCI. Two heparin binding sites are suggested on the PSA surface and could explain the enhanced complex formation between PSA and PCI, while inhibiting the formation of the ACT-PSA complex, PSA, hK2, and their preliminary complexes with ACT should facilitate the understanding and prediction of structural and functional properties for these important proteins also with respect to prostate diseases.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli.

Human osteocalcin (hOC) is a 49-amino-acid peptide produced mainly by bone osteoblasts. The amount of hOC in the circulation reflects the status of bone metabolism and it is used to monitor various bone-related diseases. The aim of this study was to produce recombinant human osteocalcin (rhOC) in Escherichia coli and use it for designing new osteocalcin fluorescence immunoassays. Recombinant DNA technology was used to fuse synthetic hOC coding sequences to an affinity handle system based on glutathione S-transferase (GST) gene. GST-rhOC fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The affinity-purified fusion protein was cleaved with activated protease factor X releasing the rhOC portion. The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing. The fusion protein and its proteolytic cleavage product proved to be immunoreactive as shown by Western blotting analysis and by a new osteocalcin immunoassay based on time-resolved fluorescence. When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no differences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be important only in oriented high-affinity binding.

Amino Acid Sequence↗