Erythrocyte antigens and antibodies in the Chilean population.

Chile does not have a national registry of immunohematologic test results; there are no data on the prevalence of erythrocyte antigens and the frequency of antibodies in this population. Therefore, foreign references are used for decision-making. In this study, a standard questionnaire was used in 74 laboratories of public and private establishments. The information from tests conducted in 2015 was requested: ABO and D typing, antibody detection, antibody identification, and erythrocyte phenotype. Prevalence for the ABO-D phenotypes were obtained at the country level (D+ [94.4%] and D- [5.5%]) and differ from those recorded in the white population (85% and 15%, respectively). Positive antibody detection results were found in 0.4 and 1.3 percent of blood donors and patients, respectively; the main specificities were anti-Lea, -E, and -D in donors and anti-D, -E, and -K in patients. Inconclusive results were observed in ABO-D typing and antibody identification in donors and patients; these samples were referred to immunohematology reference laboratories for resolution. From this study, it was possible to estimate the prevalence of erythrocyte antigens and the frequency of antibodies at the national level, and this step allows us to characterize Chile's population of blood donors and transfusion recipients and to compare the results and frequencies with other populations or countries.Chile does not have a national registry of immunohematologic test results; there are no data on the prevalence of erythrocyte antigens and the frequency of antibodies in this population. Therefore, foreign references are used for decision-making. In this study, a standard questionnaire was used in 74 laboratories of public and private establishments. The information from tests conducted in 2015 was requested: ABO and D typing, antibody detection, antibody identification, and erythrocyte phenotype. Prevalence for the ABO-D phenotypes were obtained at the country level (D+ [94.4%] and D­ [5.5%]) and differ from those recorded in the white population (85% and 15%, respectively). Positive antibody detection results were found in 0.4 and 1.3 percent of blood donors and patients, respectively; the main specificities were anti-Lea, -E, and -D in donors and anti-D, -E, and -K in patients. Inconclusive results were observed in ABO-D typing and antibody identification in donors and patients; these samples were referred to immunohematology reference laboratories for resolution. From this study, it was possible to estimate the prevalence of erythrocyte antigens and the frequency of antibodies at the national level, and this step allows us to characterize Chile's population of blood donors and transfusion recipients and to compare the results and frequencies with other populations or countries.

Cloning of a tellurite resistance determinant from Bacillus stearothermophilus V in Escherichia coli.

A potassium tellurite-resistance determinant was isolated from Bacillus stearothermophilus V and cloned in Escherichia coli. Transformed cells formed black colonies when grown on solid media containing permissive tellurite concentrations. The resistance determinant was contained in a B. stearothermophilus V chromosomal DNA fragment of 7 kb.

Biochemical characterization of tellurite-reducing activities of Bacillus stearothermophilus V.

Bacillus stearothermophilus V is a naturally occurring Gram-positive rod which exhibits resistance to potassium tellurite. Crude extracts of this bacterium catalyse the NADH-dependent, protease-sensitive reduction of K2TeO3 in vitro. Two fractions which showed the ability to reduce potassium tellurite (H1 and H2) were obtained. Fraction H1 behaved as a macroaggregate exhibiting a very high molecular mass that could not be estimated accurately. Upon electrophoresis in polyacrylamide gels in the presence of SDS, however, it was resolved into three distinct bands of 60, 41 and 37.5 kDa. On the other hand, an M(r) of 121 was determined for fraction H2 by means of gel filtration and high-pressure liquid chromatography. In SDS-PAGE a unique protein band of 60 kDa was observed, suggesting that it is actually a dimer. Both fractions showed pH and temperature optima of 7.5 and 57 degrees C, respectively. Concentrations of 2.5 M NaCl or 0.35 mM SDS inhibited fraction H2 almost completely, while fraction H1 retained 20% of its activity under the same conditions. Concentrations of 5 mM EDTA caused the activity of both fractions to increase 2-fold. In addition to reducing tellurite, they were also able to reduce Na2SeO3 and Na2SO3 in vitro.

Inhibition of HIV-1 replication by an anti-tat hammerhead ribozyme.

Tat is a virally expressed regulatory protein involved in the replication of HIV-1, the etiological agent of AIDS. To investigate the effect of tat inhibition on HIV replication, we constructed a retroviral vector to express an anti-tat hammerhead ribozyme as part of the 3' untranslated region of beta-galactosidase transcripts. Initial testing of this vector in tat-expressing COS-7 cells reduced tat activity by 85-95% as measured by tat-dependent CAT assays. Amphotropic and HIV-pseudotyped retroviral particles generated with this vector were used in HIV challenge experiments to determine the ability of this reagent to control HIV replication. CD4(+) peripheral blood lymphocytes (PBLs) stably transduced with this vector were subsequently challenged with HIV. These cells were able to resist HIV infection for up to 20 days as measured by cell death and reverse transcriptase activity. These data yield proof of principle that a pseudotyped retroviral vector can target and deliver a protective ribozyme to CD4(+) cells.

The levels of serum myoglobin in cardiac patients with elevated creatine kinase-MB and suspected acute myocardial infarction.

A monoclonal antibody enzyme immunoinhibition assay was used to quantitate serial serum myoglobin (Mb) levels in 121 patients who had > or = 5% creatine kinase-MB (CK-MB) and suspected acute myocardial infarction (AMI). Serum Mb levels higher than 0.16 micrograms/ml were considered abnormal. In 94% of these patients who were finally diagnosed with AMI, Mb levels were higher than 0.16 micrograms/ml, whereas all 30 normal control blood donors had lower Mb levels. Patients with anterior or inferior wall infarcts had higher Mb levels (> or = 0.64 micrograms/ml) than patients with lateral or subendocardial infarction. Only 68% (82/121) of patients evaluated by elevated CK-MB alone had a final diagnosis of AMI. In contrast, 94% (77/83) of patients who in addition showed elevated Mb had AMI. It is suggested that analysis of Mb levels allows a more accurate diagnosis of AMI in patients with elevated CK-MB than does reliance on CK-MB values alone.

Peroxidase/monoclonal antibody conjugates for the study of hemoglobinopathies.

Monoclonal antibodies (mAbs) to normal human hemoglobins (Hbs) A and F and to variant Hbs C and G-Philadelphia were conjugated to horseradish peroxidase (HRP) and used in qualitative or quantitative enzyme-linked immunosorbent assays (ELISAs). Conjugates with output molar HRP/IgG ratios close to 2.0 had higher avidity for the cognate antigens than those with ratios above or below 2.0. The analytical sensitivities of the conjugates ranged from 0.2 to 4 ng of hemolysate containing the target hemoglobin, and it was not related to the input or the output HRP/IgG ratios. The overall imprecision for the qualitative ELISA was below 8%, and the accuracy for the identification of Hbs C and G-Philadelphia was 100% as compared with established methods. Quantitative determinations of HbA based upon direct dose-response curves showed an analytical sensitivity of 1% and an imprecision < or = 11%. The most significant application of the HbA assay was in the differential diagnosis of hemoglobinopathies associated with partial or total suppression of HbA synthesis. Competitive dose-response curves for the HRP/anti-gamma conjugate allowed the quantification of HbF in the clinically significant range of 0.5-10%, with an imprecision < or = 12%. It is concluded that the incorporation of HRP/mAb conjugates into the ELISA technique offers a simpler, more rapid, yet specific alternative for the measurement of hemoglobins.

A monoclonal antibody-linked immunoassay for hemoglobin H disease.

A murine monoclonal antibody (mAb) was generated that recognizes hemoglobin (Hb) H, the tetrameric form (beta 4) of human beta-globin chains. The antibody beta 4-1 (gamma 1, kappa) does not react with Hbs A, F, Bart's, or isolated beta chains, indicating that the antibody recognizes an epitope comprised of multiple beta chains. A simple, rapid, and sensitive enzyme immunoassay was established to detect and quantitate Hb H in hemolysates from subjects with Hb H disease. The delta globin level in these patients was also measured using the monoclonal antibody delta-1, which is specific for delta chains of Hb A2. With these assays, 20 hemolysates from subjects with Hb H disease' ten from normal adults and ten from newborn babies were analyzed. The percent of Hb H ranged from 1.5% to 25% in Hb H patients. There was a significant average reduction (32%) in delta chains in these samples as compared with the normal average adult value. The decreased expression of alpha chains thus results in a reduction of the levels of normal Hbs A and A2 and accumulation of beta 4, causing Hb H disease.

Application of a monoclonal antibody specific for the delta chain of hemoglobin A2 in the diagnosis of beta thalassemia.

We have developed a murine monoclonal antibody (mAb) specific for the delta chain of hemoglobin (Hb) A2 that does not cross-react with alpha, beta, or gamma chains. The mAb reacted with Hb P-Nilotic (beta delta hybrid), but not with Hb Lepore-Boston (delta beta hybrid), indicating an epitope consisting of positions 116 (Arg) and 117 (Asn) or 125 (Gln) and 126 (Met) of the delta chain. By using this antibody, we have established a simple and rapid enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of Hb A2 in adult, cord, and fetal hemolysates. We analyzed 70 adult, 8 newborn, and 19 fetal hemolysates from normal subjects and those with various hemoglobinopathies. The mean percentage of Hb A2 was 2.5 for normal adults, 5.4 for beta thalassemic (beta thal) heterozygotes, and less than 0.1% in beta thal fetal samples. We were able to distinguish and characterize certain phenotypes of beta thal patients such as beta thal heterozygotes, beta 0 thal homozygotes, and C beta 0 thal, and C beta + thal double heterozygotes with the aid of this and other mAbs we have generated. This technique is a valuable addition to current methods for the diagnosis of beta thal based on quantification of Hb A2.

Quantities of adult, fetal and embryonic globin chains in the blood of eighteen- to twenty-week-old human fetuses.

The prenatal diagnostic program, established at Hacettepe University in Ankara for the purpose of detecting beta-thalassemia (beta-thal), sickle cell anemia (SS), and Hb S-beta-thal, offered the opportunity of evaluating the relative quantities of adult (beta A, beta S), fetal (G gamma, A gamma, A gamma T), and embryonic (epsilon, zeta) chains in 26 fetuses, aged 18-20 weeks. Methodology involved micro high-performance liquid chromatographic (HPLC) procedures and immunology using an mAb, specific for the embryonic epsilon chain. A good correlation was observed between the beta/gamma in vitro chain synthesis ratio and the level of beta A and/or beta S chains determined by reversed-phase HPLC; the combination of these two sets of data strengthens the prenatal diagnostic approach of detecting beta-thal major but not beta-thal trait. The levels of the different gamma chains were about as observed in newborn babies; the frequency of the A gamma T variant in the 26 fetuses was the same as observed for a larger group of Turkish newborn babies. The level of the embryonic zeta chain was higher than seen in full-term babies and varied between 0 and 1.3%; 5 of the 26 fetuses showed the complete absence of zeta. The embryonic epsilon chain was not detectable, not even in babies with beta-thal major. These data indicate that the synthesis of epsilon is completely turned off in fetuses at the age of 18-20 weeks, while that of zeta continues, albeit at a low level.

Negative screening for sickle cell diseases with a monoclonal immunoassay on newborn blood eluted from filter paper.

The most common method of blood sample collection for neonatal screening programs for inherited diseases-blood spots on filter paper--is poorly suited for screening of sickle cell diseases by conventional assays because of the denaturing effects of this medium on hemoglobins that affect their electrophoretic identifications. The monoclonal antibody beta (6)-1 specifically recognizes the hemoglobin A beta-chain residue 6 (glutamic acid), that is, the normal counterpart of hemoglobins S and C, and this recognition is unaffected by changes in hemoglobins induced by filter paper storage. The beta (6)-1 immunoassay analysis of 67 prescreened samples extracted from filter paper permitted unambiguous group identification, by virtue of nonreactivity, of the pathologic sickle cell disease phenotypes SS (sickle cell anemia) and SC (sickle cell-hemoglobin C disease), along with the homozygous hemoglobin C phenotype (hemoglobin CC disease). Other phenotypes identified by beta (6)-1 nonreactivity would include S-beta(0) thalassemia, C-beta (0) thalassemia, and beta(0) thalassemia (Cooley's anemia). As systems for collecting newborn blood specimens on filter paper and their transmittal to centralized laboratories are already established in many states, this assay for sickle cell and hemoglobin C diseases could rapidly be combined with other mass screening programs for inborn errors of metabolism.

Screening for hemoglobins S and C in newborn and adult blood with a monoclonal antibody in an ELISA procedure.

To facilitate the screening of blood for the presence of hemoglobins S or C, we devised an enzyme-linked immunoassay (ELISA). The ELISA procedure incorporated a murine monoclonal antibody (mAb), beta s-1, which recognized both Hb variants but did not react with Hb A, Hb A2 or Hb F. Hemoglobins in cord or adult hemolysates were coated on the surface of wells of polystyrene microtiter plates and treated with beta s-1 mAb, followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After addition of tetramethylbenzidine substrate solution, a deep blue color developed, signifying the presence of Hb S or Hb C. The beta s-1 mAb ascites fluid could detect purified Hb S and Hb C when diluted to over 1/512,000 and cord blood hemolysates containing Hb/S or Hb C when diluted to 1/128,000. Although maximal reactivity was achieved using undiluted hemolysates, the ELISA system could easily detect Hb S and Hb C in cord blood hemolysates when diluted 10(-4). The sensitivity of the ELISA was 1%, which exceeds the lowest quantities of these variants normally found in cord blood. In addition, we found that the ELISA procedure was suitable for detecting Hb S/Hb C in whole blood as well. The entire assay could be conducted on multiple samples in less than 1 h, thus providing a specific, sensitive, rapid and simple screening technique for Hb S and Hb C in cord or adult blood.

Simple and rapid enzyme-linked immunosorbent assay for the detection of hemoglobin C[alpha 2 beta 2 6(A3)Glu----Lys] in cord blood using a monoclonal antibody.

We have generated a murine hybridoma that secretes a monoclonal antibody (mAb) that is highly specific for hemoglobin C (HbC) [alpha 2 beta 2 6(A3)Glu----Lys] and shows no cross reactivity with HbA, HbA2, HbF, HbS, HbE, or Hb O-Arab. Using this antibody, we developed a simple and rapid enzyme linked immunosorbent assay (ELISA) technique for the detection of HbC in both adult and cord blood. The assay can be carried out using either whole blood samples or hemolysates. With as little as 10 microliters/well of whole blood or 5 micrograms Hb/well of hemolysates, and, with dilutions of the antibody up to 10(-5), we were able to detect HbC unequivocally in cord blood samples. The ELISA procedure could detect HbC in proportions as low as 0.01%. This simple diagnostic test represents a technological advance in Hb identification and can easily be used for mass screening (96 samples in less than 45 min) to detect HbC. Furthermore, this assay, when employed in conjunction with an mAb specific for beta 6GLU of HbA, allows the discrimination between HbC homozygotes, heterozygotes, and normals.

Enzyme immunoassay for the identification of hemoglobin variants.

We have prepared monospecific antibodies to Hbs D-Los Angeles, J-Baltimore, O-Arab and J-Paris-I and developed an enzyme immunoassay (ELISA) for their identification in hemolysates. Hbs in adult or cord blood hemolysates were coated to the wells of microtiter plates and reacted with the appropriate antisera followed by the detection system which contains anti-rabbit IgG/peroxidase conjugate and the substrate tetramethylbenzidine. Sixty-nine samples were tentatively considered to contain the above hemoglobin variants by isoelectrofocusing and the identity of 83% of them was confirmed by ELISA. Some of the non-reacting hemolysates were shown by amino acid sequence analysis to contain Hbs Korle-Bu, D-Ibadan, G-Copenhagen and the new variant Chandigarh. This ELISA offers specificity and simplicity for the confirmatory identification of hemoglobin variants.

Monoclonal antibody-based immunoassays for serum myoglobin quantification in acute myocardial infarction.

We have developed a monoclonal antibody-based enzyme immunoassay and a solid-phase radioimmunoassay for human myoglobin. Both assays are based on competition for the monoclonal antibody between the free myoglobin present in the standards or serum samples and the myoglobin coated to the wells of microtiter plates. Consequently, the absorbance at 630 nm and the radioactivity are inversely related to the concentrations of free myoglobin. The sensitivity of both assays was 10 micrograms/L with linearity up to 1,000 micrograms/L. There was no interference with other serum proteins, as judged from analysis of specimens with high concentrations of lactate dehydrogenase, creatine kinase, or hemoglobin. The average serum myoglobin concentration in 30 normal individuals was 67 micrograms/L. Five patients with cardiac arrhythmias had normal values (average, 63 micrograms/L) while four patients with myocardial infarction had abnormally high concentrations of myoglobin (300-1,000 + micrograms/L). In a typical case of myocardial infarction, serum myoglobin rose 21 hr earlier and peaked 12 hr earlier than creatine kinase and its cardiac isoenzyme. These rapid immunoassays appear to be useful for the early detection of increased serum myoglobin indicative of myocardial infarction.

Monoclonal antibody to the gamma chain of human fetal hemoglobin used to develop an enzyme immunoassay.

A monoclonal antibody (mAb) that recognizes the gamma chain of human fetal hemoglobin (Hb F) has been produced by cell hybridization techniques. The mAb reacts with Hb F (alpha 2 gamma 2), Hb Bart's (gamma 4), and Hb Kenya (gamma-beta hybrid), but does not cross-react with Hb A (alpha 2 beta 2) or Hb A2 (alpha 2 delta 2). We describe a direct enzyme-linked immunoassay (ELISA) for measurement of Hb F, in which hemoglobins from standards or from unknown hemolysates are covalently bound to the wells of microtiter plates. The antigen is quantified by addition of the gamma-specific mAb, followed by anti-mouse IgG conjugated with horseradish peroxidase, and incubation with the substrate, tetramethylbenzidine. Absorbances at 630 nm are directly proportional to the amount of Hb F present in the standards or samples. Results for Hb F in 53 hemolysates agreed well with values obtained by "high-performance" liquid chromatography, RIA, alkali denaturation, and magnetic affinity immunoassay. This ELISA can detect a 0.5% proportion of Hb F in 1 h and offers distinct advantages over other techniques currently in use.

Characterization and application of a monoclonal antibody with dual specificity for hemoglobins S and C.

In an effort to develop a rapid screening immunoassay for the presence of hemoglobin S (Hb S) in cord blood, we have produced a hybridoma (beta s-1) secreting a monoclonal antibody (MAb) with strict specificity for Hb S over hemoglobin (Hb A). A reactivity was observed for hemoglobin C (Hb C) that was weaker than that for Hb S but still greater than 10(3) times greater than that seen for Hb A. Application of this antibody in a dot blot assay provided for a rapid (50-minute) single-step confirmatory test for Hb S, Hb C, or both in cord blood hemolysates. The sensitivity of the test would allow for mass screening of cord blood hemolysates (40 mg total hemoglobin per milliliter) and detection of Hb S, Hb C, or both at concentrations greater than or equal to 1%.

Quantification of hemoglobins S, C, and F by a magnetic affinity immunoassay.

This magnetic affinity immunoassay (MAIA) quantifies hemoglobins (Hb) S, C, and F in hemolysates from adults or newborns. Monospecific antisera to the hemoglobins are covalently conjugated to magnetic beads and reacted with the corresponding 125I-labeled hemoglobin. After centrifugation to separate the free and antibody-bound 125I-labeled hemoglobin, the amount of radioactive hemoglobin in the pellet is measured. To determine the concentration of the Hb under study, the percent inhibition of the reaction is quantified. The standard curve is established by adding known quantities of unlabeled hemoglobin before adding 125I-labeled hemoglobin. The amount of Hb S, Hb C, or Hb F present in hemolysates is determined by measuring the percentage of inhibition and extrapolating the concentration from the standard curve. Results agree well with values for Hb S and Hb C obtained by "high-performance" liquid chromatography and RIA and for Hb F as measured by alkali denaturation and RIA. This assay can be completed in 1 h and is more sensitive than enzyme immunoassay or RIA: we can detect proportions of Hb S and Hb C as low as 1% of total Hb, and Hb F as low as 0.05%.

Identification of abnormal hemoglobins in cord blood by an immunoblot technique.

Variant hemoglobins S, C, and FHull were identified in newborn cord blood hemolysates with an immunoblot procedure. The abnormal hemoglobins were originally detected in cord blood hemolysates by cellulose acetate electrophoresis and presumptively identified according to their mobilities. Hemolysates that contained a hemoglobin variant again underwent electrophoresis and were transferred to nitrocellulose membranes. For identification, the membrane was treated with the appropriate rabbit monospecific antiserum and developed with peroxidase-conjugated anti-rabbit IgG or peroxidase-conjugated protein A. After the addition of substrate, a violet reaction developed at the site of the hemoglobin band, signifying an antigen-antibody reaction or a positive reactivity. The use of monospecific antisera in an immunoblot technique allows for confirmed identification of variant hemoglobins in cord blood samples in as little as 5 hours. In some cases in which the variant is near the limits of detectability by protein staining, the immunoblot allows an unequivocal identification.

Generation of a monoclonal antibody specific for Hb G-Philadelphia [alpha 2(68)(E17)Asn----Lys beta 2] and development of an immunoassay.

A murine hybridoma was generated which secreted a monoclonal antibody (Mab) that specifically recognized the alpha 2(68)(E17)Asn----Lys beta 2 substitution of Hb G-Philadelphia. Hybridomas were produced by fusion of RBF/DnJ immune splenic lymphocytes with FOX-NY murine myeloma cells and selected in adenine-aminopterin-thymidine (AAT) medium. Culture fluids were screened by ELISA for antibody reacting with Hb G-Philadelphia but not Hb A. One such culture was cloned by limiting dilution, expanded and injected into pristane-primed, cyclophosphamide-suppressed BALB/c mice for ascites production. An enzyme-linked immunoassay was developed by conjugating hemoglobin in hemolysates or purified hemoglobins to the plastic surface of wells of a microtiter plate. The ascites fluid containing the Hb G-Philadelphia Mab was added to the wells followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After the addition of substrate (tetramethylbenzidine), a deep blue color developed, signifying a positive reaction. We analyzed 58 hemolysates (17 adult, 41 cord) containing a G-variant along with 28 control hemolysates (12 cords comprising FA, FAC, FAS, FSS, FCC phenotypes; 16 adults consisting of AA, AS, SS, SC, S-beta thal, AD-Los Angeles phenotypes). Of the 58 hemolysates containing a G-variant, 53 were positive by ELISA and confirmed by radioimmunoassay (RIA). Four of the five hemolysates negative for Hb G-Philadelphia were shown to be Hb G-Montgomery by RIA. None of the control hemolysates were positive. The assay could be completed in 1 hr and represents a technological advance in hemoglobin identification.

Identification and quantification of Hb C with an enzyme-linked immunosorbent assay.

A highly specific enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification and quantification of hemoglobin C in hemolysates. The procedure involves coating the surface of microtiter wells with Hb C and then addition of monospecific rabbit antibodies that recognize the unique beta 6 GLU----LYS substitution in Hb C. Next, an antibody to rabbit gamma-globulin conjugated with alkaline phosphatase is added, followed by substrate; a yellow color is formed due to the enzymatic hydrolysis of the substrate, which can be measured spectrophotometrically. For quantification purposes, a hemolysate containing Hb C is introduced just prior to the addition of the Hb C antibody. This results in blocking the attachment of the anti-Hb C to the Hb C coated to the plastic surface. Upon addition of anti-rabbit gamma-globulin conjugate and substrate, there is a consequent reduction or elimination of color formation. Since the degree of diminution of color formation is dose-dependent, standard curves can be developed for quantification of Hb C in unknowns. Of the total hemoglobin, the amounts of Hb C in heterozygotes averaged 27.3 +/- 5.7% by ELISA and 25.1 +/- 3.9% by radioimmunoassay (RIA). In SC individuals the corresponding values were 30.2 +/- 10.1% by ELISA and 24.7 +/- 10.9% by RIA. In homozygotes, Hb C values averaged 83.2 +/- 4.2% by ELISA and 85.0 +/- 6.6% by RIA. Subjects with Hb C beta(+)-thalassemia had 66.5 +/- 3.7% Hb C as measured by ELISA and 63.5 +/- 9.1% as determined by RIA. The ELISA procedure offers distinct advantages for Hb C identification and quantification over other techniques in parameters such as specificity, sensitivity, and rapidity.