Improved assay for fecal calprotectin.

Fecal calprotectin is a marker of inflammatory and neoplastic disease in the lower gastrointestinal tract. A new fecal sample preparation procedure for the measurement of calprotectin has been developed, with higher calprotectin yield and lower contamination risk. Changes in the new method compared to the original [Roseth AG, Fagerhol MK, Aadland E, Schonsby H. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992;27(9):793-798] are smaller sample size, higher dilution of the sample, presence of dissociating agents in the extraction solution and procedure performed in closed disposable tubes. The extraction yield was 78% (41-100%) of total calprotectin, giving an overall five-fold increase compared to the original method. Samples with high calprotectin values were increased to a slightly higher degree, than low calprotectin samples, thus improving the separation between high and low calprotectin levels. Median calprotectin level in healthy subjects was 26 microg/g. Pathological samples with pancolitis showed levels up to 30000 microg/g. The mean C.V. (coefficient of variation) in blended feces was lower than that of unblended, suggesting uneven distribution of calprotectin. However, no significant difference between spot measurements was found when five samples from each of 47 stools were measured. Thus measurements of calprotectin in fecal samples were accurate and reproducible. No interference with foods or relevant oral pharmaceuticals or nutraceuticals was found.

Colloidal gold conjugated monoclonal antibodies, studied in the BIAcore biosensor and in the Nycocard immunoassay format.

Interactions between immobilized capture monoclonal antibodies (mAbs), analyte molecules and colloidal gold conjugated second monoclonal antibodies have been investigated in the BIAcore biosensor and in the Nycocard immunoassay format. This report focuses on six monoclonal antibodies against human heart myoglobin, although, results with other antigens are also discussed. The BIAcore was used to screen monoclonal antibodies as antigen capture reagents, and for their function as colloidal gold conjugated second antibodies in the Nycocard. Some antibodies with low affinity caused by a rapid antigen dissociation rate, showed high affinity kinetics when used unlabelled or as gold conjugated detector reagents. One gold conjugated mAb with excellent properties in the Nycocard, showed double binding to one epitope, when tested in the BIAcore. The real time visualization of association and dissociation rates was a unique tool in the elucidation of antigen-antibody interactions. Our study confirmed that good antibody candidates selected with the BIAcore must always be tested in their actual conjugation situation before final optimization.

Comparison of three D-dimer assays for the diagnosis of DVT:ELISA, latex and an immunofiltration assay (NycoCard D-Dimer).

Ninety-two consecutive patients referred for suspicion of deep venous thrombosis (DVT) were analyzed for D-dimer using ELISA, latex test, and a new immunofiltration method (NycoCard D-Dimer). Contrast venography verified the diagnosis in 40, and excluded the diagnosis in 52 patients. The sensitivity, negative predictive values, specificity and positive predictive values were, for ELISA 98%, 95%, 38% and 54, for NycoCard D-Dimer 100%, 100%, 42% and 57% and for the latex test 73%, 78%, 75%, and 69%, respectively. Sensitivity and specificity were inversely related with increasing pathological cut-off value. Comparison of test results by concentration category revealed a good agreement between ELISA and NycoCard D-Dimer, but to less extent between latex and the two other tests. It is concluded that NycoCard D-Dimer and D-dimer ELISA are well-suited as exclusion tests for DVT. A plasma sample is tested with NycoCard D-Dimer in less than 2 min. Thus, this test combines advantageous analytical properties comparable to the ELISA-test, with rapidity and simplicity comparable to the latex test.

Assay of D-dimer based on immunofiltration and staining with gold colloids.

In this immunofiltration assay of D-dimer in plasma samples, the antigens are captured by a monoclonal antibody on a porous membrane, and labeled with the same antibody conjugated to gold colloids. The assay time is < 2 min, and a color of intensity proportional to the concentration of D-dimer is left on the membrane. The reference range (mean +/- 2 SD) was 0.336 +/- 0.133 mg/L (n = 69). Linearity was found up to 10 mg/L. Comparison with ELISA results (x) for 198 patients' samples demonstrated a linear regression equation of y = 0.99(+/- 0.05)x + 0.68(+/- 0.07) and a mean square error of 0.503. Comparison of visual reading of the color signal (y) vs reflectometric measurements (x) for 220 patients' samples demonstrated a linear regression equation of y = 2.5(+/- 0.06)x -0.22(+/- 0.04) and a mean square error of 0.095. Bilirubin, hemoglobin, fibrinogen, soluble fibrin, and fibrinogen degradation products and freezing/thawing of samples did not interfere. Some interference from rheumatoid factor, heparin, and the presence of cells or large lipid particles was seen. The variance (CV) was 8-12% within run, 10-18% between runs, and 13-20% between persons. The new assay constitutes a rapid and reliable analytical tool combining simplicity equivalent to that of latex tests with analytical information approaching that of ELISA.

On the presence of the chromosomal proteins HMG I and HMG Y in rat organs.

Using antiserum raised against HMG I, we have shown that HMG I and HMG Y are present in perchloric acid extracts of kidney, lung, heart, brain, liver and intestine in the rat, suggesting that the expression of these proteins may not be dependent upon proliferative activity. The results also show that the ratio between HMG I and HMG Y varies between different organs.

The amino acid sequence of the chromosomal protein HMG-Y, its relation to HMG-I and possible domains for the preferential binding of the proteins to stretches of A-T base pairs.

The primary structure of the human high mobility group (HMG) protein HMG-Y has been established except for a few amino acids in the N-terminal and the C-terminal part of the protein. It was found that the sequence was identical to that of HMG-I except for a run of eleven amino acids. Like HMG-I the protein was N-terminally blocked and the palindromic sequence Pro-Arg-Gly-Arg-Pro occurred twice as in HMG-I. The binding of peptides derived from HMG-I (after thermolysin cleavage) to poly (dA-dT).poly(dA-dT) suggested that there are at least two different binding domains in the protein and that binding is not dependent upon an intact protein.

Immunoquantitation and size determination of intrinsic poly(ADP-ribose) polymerase from acid precipitates. An analysis of the in vivo status in mammalian species and in lower eukaryotes.

Antibodies against pig thymus poly(ADP-ribose) polymerase were obtained with enzyme-hemocyanin conjugates and used for immunoquantitation. The quick-blot procedure used allowed the determination of amounts as low as 1 ng of enzyme from whole cell trichloracetic acid precipitates. When applied to analysis of various human, rodent, and bovine cell types, surprisingly similar amounts of polymerase were found (1-5 ng of pig thymus polymerase equivalents/micrograms of DNA, 2 X 10(5) polymerase molecules/HeLa cell). Also, no significant difference was seen between normal and transformed cells. Polymerase tended to decline in several fibroblast cultures upon reaching confluency, which was not reflected by total polymerase activity. Divergence between total activity and immunogenic equivalents was also seen in alkylated cells and in rat liver treated with phenobarbital. Trichloroacetic acid-insoluble fractions dissolved in sodium dodecyl sulfate buffer could also be used to analyze, by Western blotting, the size distribution of poly(ADP-ribose) polymerase in vivo. Application to various cell types revealed that all mouse and rat cells tested had two immunogenic bands (116 and 98 kDa) of similar intensity. A highly conserved structure of poly(ADP-ribose) polymerase may be deduced from the existence of immunogenic and renaturable 116-kDa polypeptide bands even in the low eukaryotes Physarum polycephalum and Dictyostelium discoideum.

The human chromosomal protein HMG I contains two identical palindrome amino acid sequences.

The sequence of 105 amino acids of the human high mobility group chromosomal protein HMG I has been determined. The most striking feature of this sequence is two identical palindrome sequences: pro-arg-gly-arg-pro, which together with a third related sequence: gly-arg-pro-arg, may represent the binding sites of HMG I to clusters of A-T base pairs in DNA.

Fractionation and identification of metaphase-specific phosphorylated forms of high-mobility-group proteins.

In the present work chromatography on phosphocellulose and blue Sepharose have been used to fractionate the different phosphorylated forms of the low-molecular-mass high-mobility-group (HMG) proteins from metaphase arrested HeLa cells. The proteins in the different fractions from the blue Sepharose column were analysed by acetic acid/urea gel electrophoresis. Aliquots from the same fractions were also treated with alkaline phosphatase and the dephosphorylated and phosphorylated proteins were then compared by electrophoresis to identify the phosphorylated proteins. It was found that HMG 14 consisted of a mixture of an unphosphorylated and two phosphorylated forms, while HMG Y existed as one homogeneous superphosphorylated form. These findings remove previous uncertainty about phosphorylation of HMG Y and HMG 14. The presence of HMG M and phosphorylated forms of HMG 17 was confirmed. Peptide mapping of HMG I and HMG M gave further evidence that HMG M is a superphosphorylated form of HMG I, and it is suggested that the term HMG Im be used instead of HMG M. The results suggested that HMG I and Y from HeLa cells contained at least three and two metaphase-specific phosphate groups respectively, while HMG 14 and 17 both consisted of an unphosphorylated form and two phosphorylated forms. A protein corresponding to HMG Im from HeLa cells was also found to be present in metaphase-arrested human lymphocytes, while HMG I and from two different rodent species seemed to be less phosphorylated then their counterparts from HeLa metaphase cells.

Method for complete separation of the high mobility group (HMG) proteins HMG I and HMG Y from HMG 14 and HMG 17 and a procedure for purification of HMG I and HMG Y.

A purification procedure which separates the four low-molecular-weight high mobility group (HMG) proteins, HMG 14, 17, I and Y, is described. The procedure includes chromatography on phosphocellulose and Blue Sepharose combined with reversed-phase high-performance liquid chromatography. The blue Sepharose column separates HMG I and Y completely from HMG 14 and 17, and should therefore be an useful tool for the identification of these proteins which in several reports have been confused with HMG 14 and 17. HMG I and Y on the one hand and HMG 14 and 17 on the other exhibited considerable differences in their affinities for Blue Sepharose, probably reflecting fundamental differences in biological function.

A novel, highly phosphorylated protein, of the high-mobility group type, present in a variety of proliferating and non-proliferating mammalian cells.

The present work describes a perchloric-acid-soluble high-mobility-group (HMG)-like protein present in HeLa and Ehrlich ascites cells, rat and calf liver. The protein is designated P1 and has, depending on the source, a molecular mass 48-53 kDa and an amino acid composition which, like the HMG proteins, is characterized by a high content of acidic and basic residues and of proline. The protein contains about 10 mol serine/100 mol amino acid residues, is highly phosphorylated and has, in contrast to the known HMG proteins, an acidic isoelectric point of 5.0. An estimate suggests that protein P1 in HeLa interphase cells contains 25-30 residues of phosphate. Like HMG 1 and 2 it is distributed between the nucleus and the cytoplasm. In HeLa metaphase cells P1 is further modified, resulting in an increase in apparent molecular mass from 53 kDa to 56 kDa.

On the phosphorylation of low molecular mass HMG (high mobility group) proteins in Ehrlich ascites cells.

This paper shows that the low molecular mass HMG proteins 14 and 17 do not seem to be phosphorylated in Ehrlich ascites cells whereas two other small HMG proteins designated HMG I and Y are. Amino acid analysis and peptide mapping of all four proteins demonstrated that HMG I and Y were not phosphorylated modifications of HMG 14 or 17.

Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase.

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them.

On the presence of two new high mobility group-like proteins in HeLa S3 cells.

Two phosphorylated HMG-like proteins with Mr approximately 10 000 have been isolated from HeLa S3 cells, one being present in metaphase and one in interphase cells. The amino acid compositions of these proteins are very similar but differ from the known HMG proteins. However, they exhibit similarities being rich in proline, basic and acidic amino acids. A possible role in chromatin condensation of the HMG-like protein characteristic for metaphase cells is suggested.

ADP-ribosylation in permeable HeLa S3 cells.

ADP-ribosylation in permeabilized metaphase and interphase cells using [32P]NAD at pH 8.0 have been compared. Incorporation into trichloroacetic acid insoluble material was 4-5-times greater in metaphase cells. 17-22% was in the soluble fraction which contained material released from the cells, 16-22% in the 0.2 M HCl extract (histones) of the cell ghosts and the remaining activity in the residual fraction. Fractions were analyzed using dodecylsulphate/polyacrylamide gel electrophoresis at pH 6.0. The soluble fractions from metaphase and interphase cells exhibited three common unidentified ADP-ribosylated proteins corresponding to 78 000, 54 000 and 36 000 Da. In addition metaphase cells contained several other ADP-ribosylated proteins not present in interphase cells. The 0.2 M HCl extracts gave from metaphase cells radioactivity in the 32 000-39 000-Da region suggesting ADP-ribosylation of histone H1 with up to 10 residues of ADP-ribose and in the 17 000-20 000-Da region indicating ADP-ribosylation of core histones. The pattern of ADP-ribosylation of core histone in metaphase and interphase cells was qualitatively similar whereas the number of ADP-ribose residues per H1 molecule was higher in metaphase cells. The residual fraction contained free poly(ADP-ribose) and oligo(ADP-ribose). The results do not lend support to a special function of ADP-ribosylated histones in the mitotic event while certain ADP-ribosylated non-histone proteins may be specific for metaphase cells.

The inhibitory effect of Zn2+ on poly(ADP-ribose) polymerase activity and its reversal.

Zn2+ inhibits purified poly(ADP-ribose) polymerase (50% inhibition at 10 microM). Furthermore poly (ADP-ribose) polymerase present in nuclei and metaphase chromosome clusters is also inhibited by Zn2+. The inactivated enzyme could be re-activated by dithiothreitol. The concentration of Zn2+ needed to affect the enzyme activity in the organelles is sufficiently low for it to have a possible role in controlling the activity of this chromatin-bound enzyme.

A comparison of purified poly(ADP-ribose) polymerases from Ehrlich ascites tumor cells, pig thymus, and HeLa S3 cells.

Poly(ADP-ribose) polymerases from Ehrlich ascites tumor cells, pig thymus, and HeLa S3 cells were purified by chromatography on DNA-agarose and Blue Sepharose. A molecular mass of 112000 Da was found for all three polymerases. Fragmentation of polyacrylamide-gel-embedded polymerases with cyanogen bromide, and subsequent analysis of the fragments by polyacrylamide gradient gel electrophoresis, showed great similarities with regard to fragment sizes. The amino acid composition of the pig thymus enzyme was very similar to that of the polymerase from Ehrlich ascites tumor cells, and the terminal amino group appeared to be blocked. The HeLa polymerase electrofocused in two peaks at pH 8.8 and 5.5, while the Ehrlich ascites tumor cells and the pig thymus enzyme focused in single peaks at pH 9.4 and 9.6 respectively. Removal of residual DNA by treatment with hydroxyapatite abolished these differences in apparent isoelectric points; all three polymerases focused at pH 9.8. No important differences were found with regard to the effect of a number of substances on the synthesis of poly(ADP-ribose). Apparent Michaelis constants for NAD of 41 microM, 48 microM, and 34 microM were found for polymerase from Ehrlich ascites tumor cells, pig thymus, and HeLa S3 cells, respectively. All these results indicate that the three polymerases, which represented the major poly(ADP-ribose) polymerase activity in the organisms investigated, are closely related proteins.