The Establishment And Validation Of ELISA For HbA2and HbF

Human hemoglobins are divided into two groups of globin chains, one of which is a chain(a, Q, other is non-a chains ((3, γ,8or ε). The main hemoglobin ingredient in normal adult is hemoglobin A(HbA, which is constituted of2a-chains and2β-chains i,e α2β2and accounts for96.5-97.5%of the whole hemoglobins, another ingredient is hemoglobin A2(HbA2), constituted by the α2δ2, accounting for2.5-3.5%of the whole hemoglobins, and the last one is fetal globin (HbF, α2γ2), accounting for less than1%in the whole hemoglobins. The abnormal ratio of the globin chains can happen in some hemoglobin disorders,such as aandp.thalassemia and their gene carriers.β thalassemia as a serious monogenic inheired disorder with β globin gene defects can manifest low pigment anaemia [1-3], which is widely distributed in many parts of the world incluing Southeast Asia, especially in southern China, such as Guangxi, Hainan and Guangdong Province. The very high incidence of carriers with thalassemia in southern China leads to serious birth defiecency, and serious anemia eveneasily die before adult [1-5].The conventional way to prevent the birth of βthalassemia infant is to perform prenatal diagnosis, in which both pregnant woman and her husband should be detected. However, the more effective prevention or urgently strategy would be screening of β thalassemia carriers from people in reproductive age, since the patients with β thalassemia is homozygous from two heterozygous gene carriers.Therefore, it is very important to develop a simple, rapid, independence of expensive equipments or special technique facilities, which should be applicated in high throughput screening.In general, there are two kinds of techniques for diagnosis of thalassemia, which are the gene detection and phenotype screening. The gene diagnosis is a golden standard for inherited diseases and the gene carriers [6-9], in which the specialized skill, equipment and qualition should required. The phenotype screening for βthalassemia includes determine of HbA2and HbF, especially dependence of HbA concentration or percentage. At present, both of HbA2and HbF are measured by high-performance liquid chromatography (HPLC), which is difficult for extensively used in common hospital in small town or countryside, and is not suitable for high throughput screening. Therefore, it is rather urgent to develop a screening system that should be simple, fast and suitable for high throuput screening. As determination of protein, ELISA is considered as very specific, sensitive and convenient method compared with HPLC and phenotype screening. Therefore, we have tried to establish sandwich ELISA to determine HbA2and HbF level in hemolysates from red blood cells, which based on the generation of mAbs specific to HbA2and HbF, and polyclonal antibody against HbF. In addition, we also observed the expression of HbF in13tumor cell lines. Based on the generation of monoclonal antibodies (mAbs) against HbA2in our previously work, a sandwich ELISA for detection of HbA2were established. By cross-pairs of4strains mAbs for HbA2, mAbs2H4and1H11specific to Hbδ were used as capture antibody, and mAb2C9with reaction to Hbδ and Hbβ was used as detector antibody conjugated with HRP. The optimalization for sandwich ELISA system were performed by selection of the concentration of capture and detection antibodies, microplates, coating and blocking condition, reaction temperature and time etc. Finaly, specificicity and sensitivity of the sandwich ELISA for detection of HbA2were identified and proved. This system can recognize HbA2speicifically, and without any cross reaction to HbA, HbF and Hb zeta. Moreover, this system is very sensitive to detect about20ng/ml HbA2, much more than the requirement of HbA2in blood. Therefore, we have to decrease the sensitivity to avoid the inconvenient due to high dilution of blood samples. Now, this ELISA system still require to improve and optimalize some conditions, such as the selection of hemolysis buffer, hemolysis time and temperature as well as storage condition of blood sample, which would influence stability and fidelity of measurement.Based on combination test of3strains mAbs with rabbit anti-HbF polyclonal antibody purified by affinity chromatography, the mAb2C8was proved as sensitive and specific capture antibody. We also have optimalized the conditions for this sandwich ELISA system, such as the concentration of capture and detector antibody, selection of coating buffer, blocking buffer and temperature etc, especially, Tris-HCI buffer instead of PBS was used. Moreover, the specificity, sensitivity, measurement range and stability of the sandwich ELISA for HbF were well characterized. The results show that this ELISA system can detect HbF specifically, and can not react with HbA and HbA2, the sensitivity is about0.039-24.66ug/ml. Finally,100blood samples were measured and showed a good correspondence with HPLC.According to literature reported, the expression of embryo antigen in cells and serum of patiens with some cancers can be defined as tumor associate antigen and used as tumor biomarker[10-14]. We prepared FITC labeled anti-HbF mAb and tested the expression of13tumor cell lines (such as K562, HL-60and some solid tumor cell lines) by flow cytometry and q-PCR..The results show that HbF only expressed on K562cell line. In the results of immunohistochemistry of the primary tumor tissues, we can find that there are HbF expression-positive cells in microvessels of hepatocellular carcinoma, cervical cancer and peritoneal B-cell lymphoma tumor, but failed to check in the tumor cytoplasm and the interstitial spacesee.