Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies.

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 microliter of standard serum or serum of patients and 200 microliter of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37 degrees C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantity of myoglobin within the range 2-1000 ng/ml serum. 120 samples can be tested in 3 h.

A novel mutation in the M1S1 gene responsible for gelatinous droplike corneal dystrophy.

PURPOSE: To identify the genetic defect in the M1S1 gene causing gelatinous droplike corneal dystrophy (GDLD) in an Estonian family. METHODS: DNA was extracted from members of a GDLD-affected family and control persons. Polymerase chain reaction followed by direct sequencing was used to detect mutations in the M1S1 gene. Sequencing results were confirmed with restriction analysis. RESULTS: Sequencing of the M1S1 gene revealed a novel mutation and a common polymorphism. All patients with GDLD were found to be homozygous for the insertion of nucleotide C in position 520 in M1S1. The mutation leads to formation of truncated protein. The mutation was excluded in 103 normal, unaffected individuals. Very close to the location where the mutation was identified in the M1S1 gene, a single-nucleotide polymorphism (518A/C) was found, changing aspartic acid to alanine at codon 173. CONCLUSIONS: The data indicate that mutation ins520C in the M1S1 gene is the primary cause of GDLD in the family studied.

Polymorphic glutathione S-transferases as genetic risk factors for senile cortical cataract in Estonians.

PURPOSE: To investigate the possible association between glutathione S-transferase GSTM1, GSTM3, GSTT1, and GSTP1 polymorphism and the occurrence of age-related cataracts in Estonian patients. METHODS: Patients with cortical (155), nuclear (77), posterior subcapsular (120), mixed type (151) of senile cataract and control individuals (202) were phenotyped for GSTM1 and GSTT1 by enzyme-linked immunosorbent assay and genotyped for GSTM3 and GSTP1 by polymerase chain reaction. RESULTS: The frequency of the GSTM1-positive phenotype was significantly higher in the cortical cataract group (60.6%) than in the controls (45.0%) with odds ratio of 1.88 (95% CI, 1.23-2.94; P = 0.004). The cortical cataract risk associated with the GSTM1-positive phenotype was increased in carriers of the combined GSTM1-positive/GSTT1-positive phenotype (OR = 1.99; 95% CI, 1.30-3.11; P = 0.002) and the GSTM1-positive/GSTM3 AA genotype (OR = 2.28; 95% CI, 1.51-3.73; P < 0.001). The highest risk of cortical cataract was observed in patients having all three susceptible genotypes (OR = 2.56; 95% CI, 1.59-4.11; P < 0.001). Also, a significant interaction between the presence of the GSTP1* A allele and cortical cataract was found with prevalence of the GSTP1* A allele among the cortical cataract cases compared with the controls. Ninety-five percent of subjects with cortical cataract had the GSTP1 (AA, AB, or AC) genotype, whereas in controls 87% of persons had a genotype with GSTP1*A allele (OR = 3.1; 95% CI, 1.31-7.35; P = 0.007). In contrast to the GSTP1*A allele, the presence of the GSTP1*B allele in one or two copies leads to decreased cortical cataract risk (OR = 0.09 for GSTP1 BB genotype). CONCLUSIONS. The GSTM1-positive phenotype as well as the presence of the GSTP1*A allele may be a genetic risk factor for development of cortical cataract.

C-band polymorphisms in lymphocytes of patients with ovarian or breast adenocarcinoma.

To establish the significance of the variability of human chromosome constitutive heterochromatin areas (C-band variants) in a risk of malignancy, C-banding pattern study has been performed in 33 female patients with ovarian or breast adenocarcinoma. The control group included 180 healthy women. The following characteristics of C-bands on chromosomes #1, #9, and #16 were studied: (a) size, (b) size heteromorphisms and (c) inversions, using quantitative and semiquantitative methods of analysis. Our data show no significant difference in the presence of C-band size and location variants in chromosomes #1, #9, and #16 between the patients with adenocarcinoma of the ovary or breast and healthy women. From that we conclude that there is no causal association between the presence of C-band variants on chromosomes #1, #9, and #16 and an elevated risk of ovarian and breast adenocarcinoma.

Allele-specific monoclonal antibodies against glutathione S-transferase Mu1-1.

IgG1 class mouse monoclonal antibodies (MAbs) were produced against human glutathione S-transferase Mu1-1 (GSTMu1-1). Eight MAbs of 16 are able to recognize only the native form of the enzyme; 4 MAbs bind to native and denaturated enzyme, and the remaining 4 can bind only to partially denatured antigen in direct ELISA or Western blot. The antibodies recognizing the native form of the enzyme bind to six different epitopes. Three overlapping epitopes are responsible for specific binding of MAbs to different allelic variants of GSTMu1-1. Three allele-specific antibodies, 2E1, 11F12, and 7D11, bind to GSTM1a monomer and the other two, 1H8 and 3H10, recognize GSTM1b monomer.

Production and characterization of monoclonal antibodies against class theta glutathione S-transferase T1-1.

The recently discovered human class theta glutathione S-transferase T1-1 (GSTT1-1) is responsible for the GSH-dependent detoxification of naturally occurring monohalomethanes. The detoxifying role of GSTT1-1 has not been investigated in cancer susceptibility and the polymorphism of the protein is unknown in different populations. The purpose of our work was to produce a panel of mouse monoclonal antibodies (MAbs) that could bind to different regions of the GSTT1-1 protein and would help us select suitable MAbs for Western blot analyses and immunohistochemistry, and develop an ELISA assay for detection of GSTT1-1 in whole blood. Six highly specific MAbs were generated against GSTT1-1. Out of six MAbs, one was able to recognize only the native form of the enzyme and possesses two binding sites on the dimeric GSTT1-1 molecule. The other five MAbs bind to both native and denatured GSTT1-1 enzyme in direct and antigen capture ELISA or Western blot. The antibodies recognize at least four different epitopes on the GSTT1-1 molecule. Using MAbs 4G1 and 2D8, a sensitive ELISA assay for determination of GSTT1-1 in whole blood was developed.

Partial short arm deletion of the X chromosome 46,X,del(X)(qter = to p21:).

It is known from the literature that total loss of the short arm causes complete Turner's signs (Hoo, 1975; Therman and Patau, 1974). Partial deletions of the short arm of the X chromosome are in some cases compatible with fertility (Fraccaro et al., 1977; Hoo, 1979), but in other cases they cause a significant ovarial insufficiency with Turner's signs (Giraud et al., 1974) or gonadal dysgenesis (Petrinelli et al, 1978). A common sign for all the patients having the Xp - with the break point in the dark band (p113-p21) seems to be a short stature. The presence of other clinical signs is rather irregular. In this work, a 25-year-old female patient have a Xp deficiency in region p21 (46,X,del(X) (qter = to p21 :)) with short stature, primary amenorrhea, sterility, and clear Turner's is described.

Q- and C-band polymorphisms in patients with ovarian or breast carcinoma.

To establish the significance of the Q- and C-band variants in risk for malignant disease, chromosome analysis was carried out in 37 women with carcinoma of the ovary or breast and in 40 controls. The frequencies of brilliant fluorescence in segments 3p11q11, 4p11q11, 13p11, 13p13, 14p11, 14p13, 15p11, 15p13, 21p11, 21p13, 22p11 and 22p13, the mean numbers of the brilliant fluorescent segments per cell per individual, and the frequency of inversions of the brilliant fluorescent centromeric area of chromosome 3 were established. The absolute and relative lengths of C bands in chromosomes 1,9, and 16 and the frequencies of C-band size heteromorphisms and inversions were determined. The results of the investigation indicate that the presence of Q and C variants is not associated with an elevated risk of ovarian and breast carcinoma.

Polymorphism of Ag-stained nucleolus organizer regions in lymphocytes of patients with ovarian or breast adenocarcinoma.

The Ag-stainability of nucleolus organizer regions (NORs) in the acrocentric chromosomes identified by Q-banding was studied in 45 female patients with adenocarcinoma of the ovary or breast and in 45 healthy females. Significantly higher frequencies of Ag(+)NORs per individual (8.8 and 8.3; P less than 0.05), in the G group chromosomes (3.6 and 3.2; P less than 0.05), and in chromosome 21 (1.9 and 1.7; P less than 0.02) were found in patients, compared with controls. Despite the lack of significant differences in NORs between the groups of patients with ovarian and breast adenocarcinoma, the main difference between the patients and controls was due to the patients with adenocarcinoma of the ovary, where a significantly higher frequency of Ag(+)NORs was found in chromosomes 21 (P less than 0.01) and 13 (P less than 0.05).

Populational polymorphisms in silver staining of nucleolus organizer regions (NORs) in human acrocentric chromosomes.

The Ag stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q banding of cultured lymphocytes in 41 karyotypically normal persons (33 males and 8 females) originating from southeast Estonia. The data obtained are compared with those established earlier for a combined Vienna-Ulm population of 51 karyotypically normal persons (see Mikelsaar et al., 1977a). Significant differences between the two populations in the frequency and patterns of Ag-positive NORs were found. The following findings were most striking: the frequency of Ag-positive NORs in chromosome 14 and in the totals was significantly lower in the Estonian population than in the Vienna-Ulm population (P less than 0.01). The average modal number of Ag-positive NORs per individual was 7.8 in the Estonian population and 8.7 in the Vienna-Ulm sample (P less than 0.01). If the data of the two populations were combined the frequency of positive NORs was significantly (P less than 0.05) lower in chromosome 22 than in 13, 15, and 21, but not 14.

Comparison of silver staining of nucleolus organizer regions in human lymphocytes and fibroblasts.

Ag-staining of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding in repeated lymphocyte and skin fibroblast cultures from three different individuals. A similar pattern of Ag-stainability of NORs was found in the two tissues in each individual. Small differences concerning, in each case, only one of the acrocentric chromosomes were found between repeated lymphocyte cultures, as well as between lymphocyte and fibroblast cultures of the same individual without indication of any prevalence of one tissue type in a certain direction. The possibility that these differences are caused by different stages of NOR activation is discussed.

Human glutathione S-transferase GSTM1 genetic polymorphism in Estonia.

The distribution of glutathione S-transferase Mu 1 (GSTM1) gene deletion was examined in 151 healthy, unrelated individuals from an Estonian population. The study was carried out using the polymerase chain reaction technique. The frequency of individuals with allele GSTM1*0 in homozygous state in Estonian population was 0.503.

Satellite association frequency and number of nucleoli depend on cell cycle duration and NOR-activity. Studies on first, second, and third mitoses of lymphocyte cultures.

In human lymphocyte cultures the frequencies of satellite associations in first, second, and third mitoses were investigated using the BUDR-method. A marked decrease of the association frequency with increasing numbers of cell cycles was found. The number of nucleoli seen in interphase is correlated with the satellite association frequency in the respective metaphase. Satellite association is positively correlated to Ag-staining intensity of the NORs. Individual differences in satellite association are due to differences in NOR activity and in lymphocyte activation. BUDR diminishes somewhat the Ag-staining intensity of the NORs but has no effect on satellite association frequencies. The main reason for the decrease of satellite association frequency in second and third lymphocyte mitoses is presumably a certain dislocation of the original chromosome position during mitosis and a decreased possibility of association during the short interphases. The high association frequency in first mitosis resembles the chromosome position in the long interphase of G0-lymphocytes.

The frequency of C282Y and H63D mutations in Hemochromatosis gene in native Estonians.

Four hundred and forty two adult individuals of Estonian nationality were examined in different regions of Estonia for the C282Y and H63D HFE mutations to determine the allele and genotype frequencies. The sample consisted only of those people whose at least four grandparents were born in Estonia, and have lived settled in the same region. The study was carried out using the PCR technique and restriction analysis for C282Y and H63D mutations respectively. For the C282Y mutation the frequency of heterozygotes was 6.6% and homozygotes 0.2%, giving allele frequency 0.035. The allele frequency for the H63D mutation was 0.136, and the frequency of homo- and hetero-zygotes 1.6% and 24.0% respectively.

Variant chromosome 3 (inv3) in normal newborns and their parents, and in children with mental retardation.

The chromosomes of 102 normal newborn babies (51 boys and 51 girls) born at term, their parents, and 45 nonrelated children with mental retardation at the level of imbecility were investigated by fluorescence microscopy using propyl quinacrine mustard. In each of the 11 families, one of the parents had a variant chromosome 3 that was interpreted as resulting from a pericentric inversion of the brilliant band (or C band) only (inv3). In four cases inv3 was transmitted to the child. The frequency of inv3 in newborn boys and girls was 2 and 6% respectively, and in adult men and women 5 and 6% respectively. In children with mental retardation of unknown etiology the inv3 was detected in five cases (11.1%). This difference from normal persons was not significant.

The nature of the Ag-staining of nucleolus organizer regions. Electron- and light-microscopic studies on human cells in interphase, mitosis, and meiosis.

Electron micrographs reveal that the Ag-stainable substance is located on the outside of NOR's or around them but not in the chromosomes themselves. In association figures, the Ag-positive material lies between the acrocentric chromosomes. Light-microscopic studies show that the Ag stainability of the nucleolus in interphase is correlated with the function of the NOR, as seen from inactive and activated lymphocytes. Much more Ag-positive material is seen in prophase than in meta- and anaphase. It starts to increase again in late telophase. In male meiosis the NOR's remain Ag-positive until pachytene. First and second metaphase figures are negative. Experiments using RNase, TCA, and trypsin indicate that the Ag-stainable substance is an acidic protein. The precipitation of Ag granules in interphase nuclei seen in the electron microscope is greatest over the fibrillar component of the nucleolus. The most likely interpretation is that the Ag-stainable material is a component of ribonucleic protein accumulating around active NOR's. In mitosis some of this material remains at the NOR's. In first meiosis it is completely removed before diakinesis.

DIPI and DAPI: fluorescence banding with only negliglible fading.

DIPI and DAPI produce distinct fluorescent bands in human chromosomes similar to quinacrine banding patterns. Additionally, the AT rich secondary constrictions in the chromosomes Nos. 1, 9 and 16 are brightly fluorescent. On the other hand the brilliantly fluorescent regions after staining with quinacrine mustard in the chromosomes Nos. 3 and 4, satellites and some other regions in the acrocentric chromosomes are less striking. The distal part of the Y, however, is clearly discernible. Thus DIPI and DAPI seem to be strictly AT specific fluorochromes like Hoechst 33258. In interphase nuclei the Y chromosome can be identified. However, quinacrines are superior for Y-body analysis in buccal, hair cell and sperm smears. BrdU labeled chromatids show reduced fluorescence intensity. The difference, however, is less apparent than after staining with Hoechst 33 258. DAPI and especially DIPI are highly resistant to UV-irradiation; there is almost no fading within 30 min when using DIPI. Moreover, fluorescence intensity is stronger than in quinicrines. When photographing, exposure times may be reduced to about one quarter compared to quinacrine mustard.

Inheritance of Ag-stainability of nucleolus organizer regions. Investigations in 7 families with trisomy 21.

The Ag-stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding in cultured lymphocytes from seven children with trisomy 21 and their parents. The observed Ag-NOR patterns were in accordance with chromosomal inheritance except for a slight intraindividual variation which might be explained mainly by technical causes. In two cases the meiotic nondisjunction could be attributed to one of the parents, once to the father, and once to the mother. It is concluded that the Ag-stainability of the NORs is in general a heritable characteristic of the acrocentric chromosomes in maximally activated cells as, e.g., cultured lymphocytes. It may reflect individual differences in the amount of rDNA as well as differences in the capacity for NOR activation.

Frequency of Ag-stained nucleolus organizer regions in the acrocentric chromosomes of man.

The Ag-stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding of cultured lymphocytes in 51 karyotypically normal persons (31 males and 20 females). A consistent pattern of Ag-positive NORs was found in each individual. Ninety percent of individuals have a model number of 8--10 Ag-positive NORs per cell. The frequency of Ag-positive NORs is similar in all five acrocentrics. A statistically nonsignificant lower frequency is found in chromosome 22. Ag-negative NORs on both homologues were found in four cases. The observed frequency distribution of individuals with homozygous NOR-positive, heterozygous, and homozygous negative acrocentric chromosomes was in accordance with the Hardy-Weinberg law in all five pairs of the acrocentric chromosomes as well as in total. No sex difference was observed on our material.