Search

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.

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.

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.

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.

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 variant of fetal hemoglobin (Hb F-Malta-I) has been detected and quantitated in adult blood with a sensitive radioimmunoassay employing monospecific antisera. The concentration of Hb-F-Malta-I was 0.002-0.05%, with an average value of 0.011%. The ratio of Hb F-Malta-I/Hb F in adults was about 4.8%, compared to a ratio of about 27% in the newborn. Since the F-Malta-I variant is a product of a mutated Ggamma locus, which is one of the nonallelic structural genes directing the gamma chain synthesis, its presence in blood of adults shows that the synthesis of this gene is not completely suppressed after birth, as was previously suggested.

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.

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.