Monoclonal antibody immunoassay for the identification of hemoglobin variants in neonatal screening

M. A. Rosenthal, C. N. Ou, W. Hoffman, S. Fucharoen, C. Little, K. Pass, B. L. Therrell, N. Epstein

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Background: Four monoclonal antibody assay kits (HemoCard) for the identification of hemoglobins A, S, C and E were evaluated. Current methods used to screen newborns for hemoglobin variants are based on charge separation techniques. Unlike the charge separation methods, these kits require no instrumentation. The suitability of HemoCard for the confirmation of results from charge separation screening methods was examined. Methods: Neonatal filter paper blood samples, previously analyzed in several newborn screening labs, were reanalyzed using the various HemoCard kits. These samples included homozygotes and heterozygotes for hemoglobins A, S, C and E. Also, over 20 rare variants were used to test the specificity of the HemoCard assays. Other evaluations included tests for detection limit, sample concentration, reproducibility and temperature. Sample stability was also examined. Results: All results (n = 2305) from the filter paper blood samples were correctly confirmed by HemoCard. Differentiation of heterozygotes and homozygotes for hemoglobin A was possible by use of HemoCard A, except in samples containing hemoglobin E. None of the rare hemoglobin variants tested interfered with the HemoCard S, C and E assays. These kits gave a positive result when the variant concentration was at least 1.5% of the total hemoglobin present. The HemoCard assays detected various hemoglobins over a wide range of concentrations. All of the assay kits gave positive results on the 15 replicates of all samples tested for reproducibility. There was no effect of temperature in the range of 4-37°C. All samples tested for room temperature stability continued to exhibit the same results for at least 6 months. Conclusions: The monoclonal antibody conjugates used in the HemoCard assays were specific for the targeted variants. Since the HemoCard A monoclonal antibody is directed to the β6 epitope, it also binds to hemoglobin variants that have intact β6 epitopes which include Hb E. The HemoCard assays were found to be extremely beneficial in confirming the absence and presence of Hb S, C and E in neonatal blood spot samples.

Original languageEnglish (US)
Pages (from-to)67-76
Number of pages10
Issue number2
StatePublished - Aug 1994
Externally publishedYes


  • Confirmation assay
  • Hemoglobinopathy
  • Immunoassay
  • Neonatal screening

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health


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