Summary
To 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.
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Abbreviations
- ELISA:
-
enzyme linked immunoassay
- mAb:
-
monoclonal antibody
- Hb:
-
hemoglobin(s)
- TBS:
-
tris-buffered saline
- PBS:
-
phosphate-buffered saline
- BSA:
-
bovine serum albumin
References
Abraham EC, Reese A, Stallings M, Huisman THJ (1976) Separation of human hemoglobins by DEAE-cellulose chromatography using glycine-KCN-NaCl developers. Hemoglobin 1: 27–44
Bunn FH, Forget BG (1986) Hemoglobin: molecular, genetic and clinical aspects. Saunders, Philadelphia, pp 502–564
Consensus Conference (1987) Newborn screening for sickle cell disease and other hemoglobinopathies. JAMA 258: 1205–1209
Gardiner MB, Carver J, Abraham BL, Wilson JB, Huisman THJ (1982) Further studies on the quantitation of the hemoglobins A, S, C and F in newborn babies with different hemoglobinopathies using high pressure liquid chromatography. Hemoglobin 6: 1–13
Garver FA, Moscoso H (1985) Identification and quantification of Hb C with an enzyme-linked immunosorbent assay. Hemoglobin 9: 127–136
Garver FA, Moscoso H, Swamy S, Kiefer CR (1988) Generation of a monoclonal antibody specific for Hb G-Philadelphia [α268 (E17)Asn→Lysβ2] and development of an immunoassay. Hemoglobin 12: 125–136
Gaston MH, Verter JI, Woods G, Pegelow C, Kelleher J, Presbury G, Zarkowsky H, Vichinsky E, Iyer R, Lobel JS, Diamond S, Holbrook T, Gill FM, Ritchey K, Falletta JM (1986) Prophylaxis with oral penicillin in children with sickle cell anemia. N Engl J Med 314: 1593–1599
Grenett H, Garver FA (1980) Identification and quantification of sickle cell hemoglobin with an enzyme-linked immunosorbent assay (ELISA). J Lab Clin Med 96: 597–605
International Hemoglobin Information Center Variant List (1989) Hemoglobin 13: 233–297
Jensen RH, Vanderlaan M, Grabske RJ, Branscomb EW, Bigbee WL, Stanker LH (1985) Monoclonal antibodies specific for sickle cell hemoglobin. Hemoglobin 9, 349–362
Kiefer CR, Moscoso H, Shyamala M, Garver FA (1988) Characterization and application of a monoclonal antibody with dual specificity for hemoglobins S and C. J Lab Clin Med 112: 760–764
Milner PF, Gooden HM, General RT (1975) Rapid citrate agar electrophoresis in routine screening for hemoglobinopathies using a simple hemolysate. Am J Clin Pathol 64: 58–64
Moscoso H, Kiefer CR, Kutlar A, Garver FA (1988) Quantification of hemoglobins S, C and F by a magnetic affinity immunoassay. Clin Chem 34: 902–905
Moscoso H, Shyamala M, Kiefer CR, Garver FA (1989) Monoclonal antibody to theγ-chain of human fetal hemoglobin used to develop an enzyme immunoassay. Clin Chem 35: 2066–2069
Pearson HA, O'Brien RT, McIntosh S, Asdnes GT, Yang MM (1974) Routine screening of umbilical cord blood for sickle cell diseases. J Am Med Assoc 227: 420–421
Schneider RG (1978) Methods for detection of hemoglobin variants and hemoglobinopathies in the routine clinical laboratory. CRC Crit Rev Clin Lab Sci 9: 243–271
Serjeant BE, Forbes M, Williams LL, Serjeant GR (1974) Screening cord bloods for detection of sickle cell disease in Jamaica. Clin Chem 20: 666–669
Shyamala M, Kiefer CR, Moscoso H, Garver FA (1990) Simple and rapid enzyme-linked immunosorbent assay for the detection of hemoglobin C [α2β26(A3)GLU→LYS] in cord blood using a monoclonal antibody. Am J Hematol 33: 198–204
Taggart RT, Samloff IM (1983) Stable antibody-producing murine hybridomas. Science 219: 1228–1230
Wilson JB, Headlee ME, Huisman THJ (1983) A new high-performance liquid chromatographic procedure for the separation and quantitation of various hemoglobin variants in adults and newborn babies. J Lab Clin Med 102: 174–186
Wilson JB, Huisman THJ (1986) Detection and quantitation of normal and abnormal hemoglobins in adults and newborn by HPLC. Chap. 2. In: Huisman THJ (ed) The hemoglobinopathies. Churchill Livingstone, Edinburgh, pp 32–46
Wilson JB, Wrightstone RN, Huisman THJ (1986) Rapid cation exchange high-performance liquid chromatographic procedure for the separation and quantitation of hemoglobins S, C, and O-Arab in cord blood samples. J Lab Clin Med 108: 138–141
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Garver, F.A., Kiefer, C.R., Moscoso, H. et al. Screening for hemoglobins S and C in newborn and adult blood with a monoclonal antibody in an ELISA procedure. Blut 60, 334–338 (1990). https://doi.org/10.1007/BF01737848
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DOI: https://doi.org/10.1007/BF01737848