Preparation And Preliminary Assessment Of Monoclonal Antibody Against Human Procalcitonin

At present,the complications of sepsis,hospital infection and multiple organ failure syndrome(MODS)in clinical are the most common cause of death in critically ill patients.As a result of the growth of the elderly population,the number of immunocompromised patients and the quantity of resistant microorganisms,the mortality rate of severe sepsis is still in the range of 28%to 50%or even higher.Procalcitonin(PCT)has been approved as a detection index of sepsis by U.S.food and drug administration(FDA).Sepsis caused by surgical infection brings great risk to patients after surgery in ICU and newborn babies.Therefore,early diagnosis and treatment for these infection complications are particularly important.In recent years,it was found that PCT has certain superiority compared with the commonly used indexes of infection,such as blood culture,CRP and IL-6 etc.Continuing detection of PCT will help to guide the use of broad-spectrum antibiotics to avoid the abuse of antibiotics and shorten the course of treatment of anti-infection.In this study,the recombinant human PCT protein was expressed in E.coli and purified by subsequential fractional precipitation of proteins by ammonium sulfate,his tag column purification and molecular sieve chromatography assay.The purity of the recombinant protein reached 99%.By mass spectrometry,Western blot and indirect ELISA analyses confirmed that the purified protein was PCT.The recombinant PCT had excellent immunoreactivity with anti-native PCT polyclonal antiboby from Proteintech,suggesting that it could be used as antigen for the preparation of monoclonal antibody.We used the purified recombinant human PCT protein as immunogen to immunize the BALB/c mice.The antibody titers in the serum were investigated by indirect ELISA.After 4 rounds of immunization,the million times diluted serum was ELISA positive,suggesting that the spleen cells could be fused with myeloma cells to produce the hybridoma.At the fourth day before fusion,the spleen immunization and intravenous injection were performed to strengthen the immune effect.The spleen cells were fused with the SP2/0 myeloma cells using the standard protocol and the myeloma cells were selected by indirect ELISA.The positive myeloma cells which secreted the anti-PCT antibody were cloned by 4 rounds of limiting dilution assay.We had acquired seven strains of the cell line which stably secrete the anti-PCT monoclonal antibody.The subtype,relative affinity and epitope were identified for each antibody.The results showed that the antibodies have high affinity and recognize three different epitopes.The seven antibodies were used to establish the HRP labeled and FITC labeled sandwich ELISA methods to detect the PCT concentration for rapid diagnosis of sepsis in clinical.We identified that 8E6-HRP-7F9,8E6-HRP-2D3 and 2D3-HRP-4G9 antibody pairs are suitable to be used in the HRP labeled sandwich ELISA,and 7F9-FITC-2D3 is the optimal antibody pair in the FITC labeled sandwich ELISA.All the above antibody pairs have high sensitivity and specificity with PCT.The HRP matched antibody pairs were subjected to test the PCT concentration in the serum of the patients with severe bacterial infection.Three independent experiments showed that the correlation coefficient between the 8E6-HRP-7F9 based ELISA and imported commercial kits used in hospital were 0.998,0.974 and 0.976 respectively,illustrating that the HRP labeled sandwich ELISA we had established using the monoclonal antibodies produced by ourselves had potential clinical application value.Overall,our work laid the foundation for the future development of PCT diagnostic kit with independent intellectual property rights.