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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., (Copyright 1998 Academic Press.)

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

The DEAE-cellulose chromatographic procedure which uses glycine-KCN-NaCl solutions as Developers and a NaCl gradient to elute the hemoglobin zones was found to be useful for the quantitation of Hb F in samples containing Hb A, provided the level of Hb F exceeds 2 percent. The method has been evaluted through a study of artificial mixtures and of blood samples from patients with various disorders. The data have been compared with results obtained by four other procedures that are used for the quantitation of Hb F.

The antigenic properties of a number of chromatographically purified primate hemoglobins were compared to those of normal human hemoglobin using a sensitive radioimmunochemical procedure. The degree of inhibition of the antigen-antibody reaction with heterologous hemoglobins appeared to be related to the structural similarity of these proteins to the normal human hemoglobin immunogen. With the exception of the baboon hemoglobin, the antigenicity of the hemoglobins paralleled the phylogeny of the primates. The gorilla and chimpanzee hemoglobins were antigenically identical to normal human hemoglobin, whereas the gibbon and orangutan hemoglobins were substantially more variable. Of the Old World monkey hemoglobins examined, the baboon produced lower inhibition values, suggesting a greater degree of structural dissimilarity than other Cercopithecoidea hemoglobins, which is compatible with a greater rate of evolutionary change occurring in this protein. Using the known amino acid sequences of human and other primate hemoglobins, we have attempted to identify antigenic determinant areas of the proteins.

The carbohydrate structure and complete amino acid sequence of a human lambda-type immunoglobulin light chain, protein Sm lambda has been determined. The protein was isolated from the urine of a patient with a plasma cell dyscrasia resembling gamma-heavy-chain disease. 13 tryptic peptides covering the entire polypeptide chain of 135 residues were isolated from the aminoethylated protein, and 15 chymotryptic peptides, accounting for 131 residues, were recovered from the carboxymethylated protein. The sequence of 18 of these peptides was partially or completely determined by the Edman-dansyl technique or C-terminal analysis, permitting the establishment of the complete primary structure of the polypeptide chain. The sequences established that this light chain possessed an intramolecular deletion of 81 amino acid residues. The N-terminal 30 residues showed considerable homology with other lambda chains of subgroup II. The defect began at position 31, in the first hypervariable region, and encompassed the remainder of the variable region through position 109. The constant region was fully intact and normal synthesis recommenced with a glutaminyl residue at position 110, the first residue of the constant region. This light chain contained carbohydrate in the hypervariable region just preceding the deletion. The precise number and locations of the oligosaccharide chains were established by amino acid sequence analysis of glycopeptides isolated from proteolytic hydrolysates by chromatography on Bio-Gel P-6 columns. These studies showed that protein Sm lambda contains one N-glycosidically-linked chain attached to asparagine-25 and one O-glycosidically-linked oligosaccharide chain attached to serine-21. The structures of the oligosaccharide chains were determined by methylation analysis, gas chromatography and hydrolysis with specific glycosidases. The structure of the N-glycosidically-linked chain was NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 2)Man(alpha 1 leads to 6)[NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 2)Man(alpha 1 leads to 3)]Man(beta 1 leads to 4)GlcNAc(beta 1 leads to 4)[Fuc alpha 1 leads to 6]GlcNAc leads to Asn. The second O-glycosidically-linked chain was a disialylated tetrasaccharide with the structure, Neu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[NeuAc(alpha 2 leads to 6)GalNAc leads to Ser. This mucin-type disialylated tetrasaccharide in close proximity to N-asparagine-linked chains has not been previously observed in the oligosaccharide chains of immunoglobulins.

Hyperimmune antisera to chromatographically purified hemoglobins F and A2 were produced in rabbits and made specific for the immunogen by adsorption with normal human hemoglobin A conjugated to cyanogen bromide-activated agarose. A radioimmunoassay was established that permitted identification and quantitation of each of these two minor hemoglobins in hemolysates containing other hemoglobin components. The quantities of hemoglobins A2 and/or F present in hemolysates of individuals with beta-thalassemia, sickle cell anemia, Hb-C disease, and other hematological disorders were determined immunochemically, and the results were commpared to values obtained by microcolumn chromatography for the measurement of Hb-A2 or with the alkali denaturation technique in quantitating Hb-F. The immunoassay procedure has a greater sensitivity than other commonly employed techniques and can detect as little as 0.05 mug of these hemoglobins.

An internal molecular deletion occurring in a human lambda type immunoglobulin light (L)-chain (Sm lambda) has been defined by sequence analysis. The Sm protein was isolated from the urine of a patient with a plasma cell dyscrasia involving the synthesis of an IgG molecule with both deleted gamma and lambda subunits. The Sm lambda polypeptide chain has an approximate molecular weight of 15,000 and contains 135 amino-acid residues. The constant (C) region is fully intact, comprising 105 residues, whereas the variable region (V) has only 30 residues. The V-region segment represents residues 1 through 30 of normal lambda chains and possesses considerable homology (87%) to lambda chains of subgroup II. Since lambdaII proteins normally contain 216 amino-acid residues, the defect represents an intramolecular deletion of 81 residues, which is entirely confined to the carboxyterminal three-quarters segment of the V-region, with a resumption of normal synthesis at a glutaminyl residue at position 110, the initiation point of the C-region. Carbohydrate is attached to an Asx residue at position 25, in the first hypervariable region, associated with the sequence triplet Asx-Ser-Ser, which is postulated to be a common recognition site for glycosylation of immunoglobulins. The carbohydrate moiety is a complex oligosaccharide with a branched chain structure containing sialic acid, fucose, mannose, N-acetylglucosamine, and galactose. These structural studies and other findings suggest that restricted areas in the DNA of immunoglobulin genes, such as the hinge regions of heavy (H) and light (L) chains and the hypervariable regions, are particularly susceptible to breakage and reunion. We postulate that the genetic defect of protein Sm could have originated from a somatic mutational event in the plasmacyte precursor during or after the integration of the V and C genes. These studies provide additional support for the hypothesis and two distinct structural genes encode a single immunoglobulin polypeptide chain.

A rapid and sensitive radioimmunoassay technique for hemoglobin based upon a modification of the Farr test is described. Since hemoglobin is soluble in ammonium sulfate whereas antibody is precipitated, the amount of free and antibody-bound radiolabeled human hemoglobin can be quantitatively measured after ammonium sulfate precipitation. Nanogram quantities of hemoglobin can be detected by the addition of unlabelled human hemoglobin to the antiserum before the labeled hemoglobin, thus establishing a competition reaction between labeled and unlabeled antigen for antibody. If sufficient quantities of unlabeled human hemoglobin are present, all of the antibody combining sites become saturated and the primary interaction between 125-I-labeled hemoglobin and antibody is completely inhibited. The method can be employed to discriminate various normal human hemoglobins, non-human primate hemoglobins as well as sickle-cell hemoglobin (HbS). Other possible applications of the technique are discussed.

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.

Many of the components of amniotic fluid have been found to be valuable in prenatal diagnosis; however, the presence of erythrocytes is usually considered undesirable. The authors have used buoyant density centrifugation on 96 amniotic fluid specimens from 70 subjects to isolate small numbers of erythrocytes from a majority of these specimens. Through immunofluorescence, these specimens were found to have higher levels of fetal hemoglobin-containing cells than the adult, indicating that the erythrocytes were at least in part fetal in origin. Thus, erythrocytes present in amniotic fluid could also be used in prenatal diagnosis.

Antibodies were made to three mutant hemoglobins, each containing different single amino acid substitutions at beta 73:Hb Korle Bu (Asp replaced by Asn), Hb Mobile (Asp leads to Val), Hb Vancouver (Asp replaced by Tyr); and to one mutant hemoglobin, Hb C-Harlem, containing two substitutions in the beta chain (beta 6 Glu replaced by Val, as in Hb S, and beta 73 Asp replaced by Asn, as in Hb Korle Bu). The antiserum to Hb C-Harlem contained two antibody populations, each specific for one mutant amino acid residue. The antiserum to Hb Vancouver was completely specific for this mutant and did not cross-react with Hb Mobile and Hb Korle Bu; however, antiserum to Hb Korle Bu partially cross-reacted with Hb Mobile and to a smaller degree with Hb Vancouver. Antiserum to Hb Mobile exhibited even less cross-reactivity with Hb Korle Bu and C-Harlem and none with Hb Vancouver. These and previous studies indicate the involvement of at least three independent areas in the beta chain as antigenic determinant sites. It appears that the three mutants at beta 73 elicit the formation of antibodies which have a gradation in their specificity due to the nature of the amino acid sidechain.

An erythropoietin-independent virus-induced murine erythroleukemia (FVP) is used to compare the effects of an erythropoiesis inhibitory factor (EIF) isolated from human urine with the effects of prostaglandin F2 alpha. The consequent inhibition of FVP-induced erythropoiesis suggests that EIF and PGF2 alpha have similar effects on the FVP-induced erythropoiesis in mice, and the effect of PGF2 alpha is indirect. The similarity of the actions of EIF and PGF2 alpha may indicate a potential role for prostaglandins in the physiological control of some types of erythrocytosis.

A diagnosis of chronic lymphatic leukemia (CLL) was made in an 83-year-old man on the basis of marked lymphocytosis (131.1 X 10(9)/L) and infiltration of the marrow (77%) by small lymphoid cells. The hemoglobin was 11.8 g/dL and the platelet count was 427.5 X 10(9)/L. With minimal and unsustained treatment, abrupt resolution of the lymphocytosis occurred with reciprocal emergence of a classic picture of chronic myelogenous leukemia (CML). During the hematologic evolution, blood lymphoid cells were isolated for surface marker, cytogenetic, and functional studies. E-rosette receptors, Fc-receptors, and membrane immunoglobulins were found on only 7.5%, 7.4%, and 10.0% of these cells. These cells also failed to express a CLL-associated antigen we have detected on approximately equal to 95% of the cells of all CLL patients studied, regardless of cell phenotype. In addition, the patient's lymphoid cells responded poorly to leucoagglutinin (LPHA) stimulation, showed increased spontaneous metabolic activity and exhibited the Philadelphia chromosome. These observations suggest that the patient's transient initial lymphocytosis was not due to CLL, but perhaps represented myeloid precursors in circulation prior to terminal differentiation.

The presence and quantity of hemoglobins Barts and A2 in hemolysates from normal donors and individuals with alpha- and beta-thalassemia trait, respectively, were determined with an enzyme-labeled immunosorbent assay (ELISA). This technique requires the incorporation of monospecific antisera capable of specifically reacting only with these hemoglobins, e.g., with the delta chain of Hb A2 and gamma 4 chains of Hb Barts. By the ELISA, the mean percentage of Hb Barts in hemolysates from normal persons and persons with alpha-thalassemia was 0.25 (SD 0.07) and 6.1 (SD 0.40), respectively. Corresponding values for Hb A2 in hemolysates from normals and persons with beta-thalassemia were 3.1 (SD 0.22) and 5.9 (SD 0.21), respectively. The results obtained by the ELISA procedure were in good agreement with those determined by radioimmunoassay or microcolumn chromatography. The ELISA technique is more sensitive and specific than biochemical assays currently used to measure these hemoglobins and can detect 250 ng of Hb Barts in 100 micrograms of hemoglobin or 50 ng of Hb A2 in 5 micrograms of hemoglobin.

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.

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%.

The experimental details of ELISA for the identification and quantitation of Hb S are presented; the assay is based upon the passive adsorption of Hb S top a solid phase (polystyrene tubes) and the addition of monospecific rabbit antibodies capable of recognizing the (beta 6 Glu leads to Val) substitution in Hb S. After the addition of alkaline phosphatase-conjugated goat antibody to rabbit IgG and substrate, the yellow color produced by hydrolysis of substrate is measured spectrophotometrically. For the identification and quantitation of Hb S in unknown samples, the hemolysate is added to the Hb S-coated tubes before the addition of antibody to Hb S, thus causing an inhibition of the antigen-antibody reaction as evidenced by an absence or reduction of color formation. With this procedure, there is no cross-reactivity with normal hemoglobins, and the immunoassay has a sensitivity in detecting 50 ng quantities of the abnormal hemoglobin in a 5 microgram hemolysate. The assay can be performed on multiple samples in 1 day and offers many advantages over other techniques currently used for the identification and quantitation of Hb S and other abnormal hemoglobins in the clinical laboratory.

Antisera were produced in rabbits to the three known types of Lepore hemoglobins, which contain hybrid delta-beta non-alpha-chains, and to hemoglobin Kenya, which has a hybrid gamma-beta non-alpha-chain. By using a sensitive radioimmunoassay technique, the absorbed antisera were shown to contain an antibody population that was specific for the hybrid hemoglobin and did not cross-react with normal hemoglobins. However, with the absorbed Lepore-specific antisera, the three known types of Lepore hemoglobins were antigenically indistinguishable from each other, suggesting that antibodies are not produced to the primary structural differences which define the three non-alpha-chains of the Lepore hemoglobins. These studies demonstrate that the non-alpha-subunits of hemoglobins Lepore and Kenya possess unique antigenic determinant sites, evidently resulting from an altered polypeptide conformation.

The variable domain (V) sequence of the lambda-light chain of the Gar IgG2 human myeloma protein was determined from enzymatically generated peptides that were isolated, characterized, and ordered by overlap and/or by subgroup homology. The sequence consisted of the amino terminal 107 residues of the light chain, and shared subgroup V lambda III-specific residues as well as a pattern of CDR deletions unique to most V lambda III sequences. The structural role of the light V region in the high affinity for flavins and the low affinity for 2,4-dinitrophenyl haptens characteristic of the intact IgG molecule was coarsely assessed by way of model building and by comparison with the refined combining site model of MOPC 315, an Ig with relative affinity values for DNP and riboflavin the converse of those observed in Gar.

Colonies derived from erythroid burst-forming units (BFU-E) synthesize fetal hemoglobin (HbF) in amounts that far exceed in vivo levels. There is some evidence that HbF synthesis is controlled at the level of a primitive erythroid precursor cell. Dexamethasone may potentiate the development of BFU-E. Since a means of augmenting HbF production in sickle cell anemia or severe beta-thalassemia would be of great therapeutic value, we studied the effects of dexamethasone on HbF and gamma-globin chain synthesis in BFU-E from patients with sickle cell anemia and controls. HbF was measured by radioimmunoassay of BFU-E lysate and gamma-chain synthesis by the incorporation of 3H-leucine into globin, which was then purified by gel filtration and column chromatography. Dexamethasone (10(-9) M) produced an increase in the number of BFU-E in 16 of 19 subjects when compared with numbers of BFU-E cultured with only erythropoietin. The individual BFU-E were larger and contained more subcolonies. Dexamethasone did not increase HbF or gamma-chain synthesis, and there was no relationship between proliferation of BFU-E and augmented HbF production. Thus, although dexamethasone augmented the development of erythroid bursts, there was no increment in HbF.

A sensitive and specific radioimmunoassay has been developed for the identification or quantification of the human hemoglobin variants S, C, D-Los Angeles, E, G Philadelphia, Russ, O Arab, Beograd, J Paris I, G San Jose, Q Iran, Korle Bu, and F Malta I. In the immunoassay, monospecific antibody preparations are used which recognize the single amino acid substitution in the variant polypeptide chail and do not cross-react with normal hemoglobins or hemoglobin variants containing a different amino acid exchange at the same position.

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.

A highly specific method for the conclusive identification of normal and variant human hemoglobins is described in this communication. The method employs the standard electrophoretic technique in combination with the immunoblot technique using monospecific antisera raised in rabbits. Various hemoglobins such as Hb, S, C, A2, and F were separated on an alkaline polyacrylamide gel or a cellulose-acetate membrane, and transferred to a nitrocellulose membrane and the individual hemoglobins were identified by the immunoblot procedure. With this method hemoglobins with similar or identical electrophoretic mobilities can be definitively identified with the use of monospecific antisera.

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.

An inhibitory factor of erythropoiesis, obtained from normal human urine, is indicated to be a complex of a fragment of alpha 1-acid glycoprotein and prostaglandin F2 alpha. Immunoelectrophoresis reveals two protein components in the EIF complex which separate during acrylamide gel electrophoresis. A gamma-globulin (MW 185,000) is a carrier of the complex. A fragment of alpha 1-acid glycoprotein (MW 9300) retains the inhibitory factor, PGF2 alpha. Noncovalent forces bind the PGF2 alpha to the protein, and PGF2 alpha can be extracted with benzene.

The complete variable region sequence of the glycosylated Bence-Jones protein Wh has revealed that the most hypervariable segments of the first and third complementarity-determing regions (CDR) are identical with those of another glycosylated Bence-Jones protein (Nei) of the same lambda-subgroup. The carbohydrate is attached in both proteins at the identical sequences in the third CDR, and the sequences of the first CDR differ at only one position in fourteen. Because CDR sequence comparisons more sensitively reflect overall germ-line V gene similarities, proteins Wh and Nei could possibly reflect a "set" relationship analogous to the mouse V kappa isotype. Cluster analyses of currently available V lambda sequences supported this suggested relationship.

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%.

Escherichia coli strain chi 1776 carrying recombinant DNA plasmids containing cDNA copies of human beta or gamma globin mRNAs has been shown by radioimmunoassay to synthesize polypeptides antigenically related to the beta and gamma chains of human hemoglobin. The gamma and beta polypeptides have been enriched from lysates on immunoabsorbent columns containing hemoglobin antibodies and shown to specifically inhibit the antigen-antibody binding between 125I-labeled hemoglobin and the homologous antibody but not other hemoglobin-antihemoglobin reactions. Clone JW151, which is known to contain a complete copy of the coding portion of the gamma globin mRNA, has been shown to produce a protein that reacts specifically with antibody to the chain of fetal hemoglobin, hemoglobin Kenya, and hemoglobin Bart's.

Hemopexin is a normal serum glycoprotein that functions as a carrier for intravascularly liberated free heme. Although its role is well established in the reutilization of hemoglobin-derived heme, there have been no previous clinical data to support its suspected interaction with heme released in the degradation of myoglobin. In a patient with fulminant rhabdomyolysis, we found depletion of serum hemopexin in the presence of high serum levels of myoglobin with normal levels of haptoglobin and hemoglobin. This combination of laboratory findings is evidence for an interaction between myoglobin-derived heme and hemopexin and implies a role for hemopexin in the catabolism of myoglobin. These findings support the proposed induction mechanism for observed increases of serum hemopexin in Duchenne muscular dystrophy and in polymyositis.

Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500-3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform-methanol-insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNP1 protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform-methanol-insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.