Simple and rapid enzyme‐linked immunosorbent assay for the detection of hemoglobin C[α₂β₂6(A3)Glu → Lys] in cord blood using a monoclonal antibody

Abstract

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.

Peroxidase/monoclonal antibody conjugates for the study of hemoglobinopathies

“…The most likely explanation for these apparent contradictions could reside in the physical properties inherent in each mAb, and/or, as recently shown (18), in the purity of the mAbs. Immunological identification of normal and variant Hbs, although not having received widespread use, has proved to be valuable as a confirmatory test using either polyclonal (6) or monoclonal (7,10,11) antibody-based ELISA. Here we have increased the sensitivity 10-50 fold and decreased the assay time by 1 h as compared with the traditional ELISA (1 0,ll).…”

“…p6-1 recognizes the HbA P-chain position 6 glutamic acid that is the normal counterpart of Hbs S and C (12). p'-1 is a specific for the lysine substitution at position 6 of the HbC P-chain (10). y-1 reacts with the y-chain of human fetal Hb (13).…”

Application of a monoclonal antibody specific for the δ chain of hemoglobin A2 in the diagnosis of β thalassemia

“…As shown in Table 11, certain p thal phenotypes could be further categorized by analyzing the hemolysates with mAbs specific for HbC (PC-I) and HbA (p6-1). The p'-1 mAb recognizes the lysine residue at p6 characteristic of HbC [15], and the p"-1 mAb is specific for the corresponding normal p" glutamic acid residue [18]. The simple p thal heterozygote and the Cp' and Cp" thal double heterozygotes all had elevated levels of Hb A, relative to the normal adult value.…”

Haemoglobin smart plastic antibody material tailored with charged binding sites on silica nanoparticles: its application as an ionophore in potentiometric transduction

“…Some employ chemical reactions and use detectors of optical nature, including uorimetry, 7,8 circular dichroism 9 and UV/visible spectrophometry; 10,11 of separative nature, including electrophoresis 12,13 or high performance liquid chromatography; 14,15 or of electroanalytical nature, including voltammetry 16 or amperometry. 17 An increased selectivity is usually tried out by using biochemical reactions of enzymatic 18 or immunological nature, [19][20][21][22][23][24][25][26][27][28][29][30][31] coupled to different detection modes/strategies, such as polarography, 18 Enzyme-Linked Immunosorbent Assay (ELISA), [19][20][21] radioimmunoassay, [23][24][25][26][27] immunochromatography 28 and surface plasmon resonance. [29][30][31] The antigen/antibody reaction is highly preferred in this context due to the high selectivity of the affinity reaction taking place between these biomolecules.…”