Hugo Moscoso

Generation of a Monoclonal Antibody Specific for Hb G-Philadelphia [α268(E17)Asn→Lysβ2] and Development of an Immunoassay

A murine hybridoma was generated which secreted a monoclonal antibody (Mab) that specifically recognized the α268(E17)Asn→Lysβ2 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 microtitre plate. The ascites fluid containing the Hb G-Philadelphi a Mab was added to the wells followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After the addition of substrate (tetramethylbenzi-dine), a deep blue color developed, signifying a positive reaction. We a...

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.

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

SummaryTo facilitate the screening of blood for the presence of hemoglobins S or C, we devised an enzymelinked immunoassay (ELISA). The ELISA procedure incorporated a murine monoclonal antibody (mAb), βS-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 βS-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 βS-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.

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.

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.

Immunochemical identification of normal and variant hemoglobins after electrophoretic separation and transfer to nitrocellulose membranes

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.