Determination of Human IgG by Enzyme-linked Fluorescence Immunoassay with pH-Sensitive Phase Separation Polymer as Carrier

Lin Peng 12, Zheng Hong, Yang Huanghao, Yang Wei, Zhang Changgong 3, Xu Jinguo 1 (1. Key Laboratory of Analytical Sciences, Ministry of Education, Xiamen University, Xiamen 361005, China; 2. College of Fisheries, Jimei University, Xiamen 361021, China; 3. Research Center of Cancer Research, Xiamen University, Xiamen 361005) Due to its unique pH-sensitive properties, pH-sensitive polymers have been used in the controlled release of drugs, molecular separation, and biosensors. When applied to immunoassay, pH-sensitive polymers are required to respond when the solution is fluctuating around pH 7.4. Excessive deviation from physiological pH can cause varying degrees of damage to the antigen-antibody complexes produced by the immune response. At present, pH-sensitive polymers are not widely used in immunoassays, which is mainly due to the fact that the phase transition pH of polymers is mostly around 4 or 10. We have synthesized a 37*C pH-sensitive polymer with a phase transition pH of about 5.6 and used it as an immunoreaction carrier to establish an analysis system for hepatitis B surface antigen, although its phase-change pH is higher than that of conventional macromolecules. Close to neutral, but did not reach the physiological pH of 7.4 range, the separation may still cause some damage to immune complexes.

pH-sensitive polymers contain either acidic groups (such as carboxylic acids, sulfonic acids) or basic groups (such as ammonium salts) that can change their solubility by receiving or releasing protons in response to changes in the external environment. It has been reported that the monomer N-isopropylacrylamide (NIP), which is a temperature-sensitive polymer, can be polymerized with a monomer having an acidic group or a basic group, resulting in the formation of an ionizable or protonatable group. On the basis of temperature sensitivity, the copolymer is added with pH-sensitive characteristics. The phase transition pH value is related to the monomer ratio and the ionization or protonation constant of the introduced group. It was found that by copolymerizing NIP with amino-containing N,N*-dimethylaminopropyl methacrylamide (DMAPM), a sensitive polymer with a phase transition pH of about 7.4 at 37*C can be synthesized. For the first time, we used this base-containing pH-sensitive polymer as a carrier for immunoassay. When the solution was at pH 7.4, the polymer quickly precipitated out of solution. This can be complemented with sensitive polymers containing acidic groups and is suitable for the determination of labile substances in acidic conditions. When P(NIP-DMAPM) is used for enzyme immunofluorescence analysis of human IgG content, the phase change pH is neutral, so that the damage to the antigen-antibody complexes during separation is minimized, and the reaction temperature is balanced by the increase of the reaction temperature. Only 4 min. 1 Experimental section 1.1 Reagents and instruments NIP, N, N, N*, tetramethylethylenediamine (TEMED) and p-Hydroxyphenylacetic acid (p-HPA) (Analytical, Kasei); DMAPM (Aldch Company), before use by vacuum distillation; N hydroxysuccinimide acrylate (NAS) according to the PH value of the change, the light transmittance mutation PH value is the phase change pH. From the visible, the phase change pH with The increase of the DMAPM mole fraction in the copolymer increases, when the molar fraction of DMAPM reaches 5*, the phase transition pH just reaches the physiological pH of 7.4, and the precipitation and dissolution changes appear near this pH value to the antigen-antibody complexes. Minimal damage, suitable for immunoassays. It is reported that the phase-to-phase pH of the copolymer is closely related to the balance of the hydrophilicity and hydrophobicity of the polymer chain, and a precipitation-dissolving mutation will occur only when the hydrophilic and hydrophobic groups reach a fixed ratio. The polyNIP showed complete hydrophobicity above the critical solution temperature (about 31C), with precipitation of 141, and the protonation of DMAPM undoubtedly increased its hydrophilicity. The higher the DMAPM content, the more protonated the degree of protonation must be reduced in order to produce a fixed ratio of the hydrophilic and hydrophobic groups required to change the light transmittance of the copolymer solution at 37C. The pH (ie phase change pH) increases accordingly.

22 Immunoassay principle The “sandwich type” immunoassay is used for the determination of human IgG. The macromolecule is coupled with Ab under the action of active ester NAS. After adding human %G, an excess of HRP-Ab is added to form a sandwich compound Ab. Human IgG/HRP-Ab, after isolation, fluoresce by adding HRP fluorescence substrate to generate fluorescence signal for human IgG antigen quantification The use of pH-sensitive macromolecule as an immunoreaction carrier instead of traditional carrier can be performed at physiological temperature of 37C. The reaction is performed to further increase the speed and efficiency of the immune response.

23 optimization of immune conditions for 1:500 dilution of the labeled complex, the appropriate amount of HRP-Ab for 40 ~ 300ML, selected in this article for the 80MLP (NIP-DMAPM)-Ab the optimal amount of 30ML. The immune response can be in the organism The physiological temperature of 37C, the increase in temperature 4min to reach equilibrium, the experiment was selected for 5min. 24 working curve and sample determination in the optimal immune response conditions, human IgG mass concentration in the range of 0 ~ / mL, and the system The relative fluorescence intensity is in a good linear relationship, and the linear equation is F = - the standard deviation of the white chemistry determination in chemical school of the college. 9) The detection limit of the method divided by the slope of the working curve is 2.2 ng/mL. The above method is used for 3 persons. Serum human IgG concentrations were determined and human serum samples were diluted 1×1000 times with PBS. Each time a sample of 50 ML was obtained, the results are shown in Table 1. The measured values ​​were in the normal human serum IgG content (10–14 mg/kg). In the range of mL), simultaneous verification with the standard recovery experiment was carried out with satisfactory results.