Microplate and In Vitro Diagnostic Kit for HPV 16/E7 Oncoprotein Detection and Preparation Method Thereof

A preparation method of an in vitro diagnostic kit for an HPV type 16 E7 oncoprotein detection, comprising:

• 1) preparing a microplate pre-coated with an HPV type 16 E7 antibody which comprises
• taking the HPV type 16 E7 antibody, adding a coating buffer to microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h);
• washing the microwells with a washing solution five times at an amount of 300 μl/microwell;
• adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2 h for blocking;
• suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing,
• wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na2CO3 and 2.93 g/L of NaHCO3;
• the washing solution is comprised of 1.44 g/L of Na2HPO4, 0.24 g/L of KH2PO4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300;
• the blocking solution is comprised of 1.44 g/L of Na2HPO4, 0.24 g/L of KH2PO4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300;
• 2) preparing calibrators and quality control samples which comprises
• preparing HPV type 16 E7 oncoprotein detection calibrators 0, 1, 2, 3, 4 and 5:
• taking lyophilized powder as the calibrators, and respectively adding 1 ml ddH2O for dissolving, wherein linear concentrations of calibrator solutions are 0, 10, 20, 40, 80, and 160 ng/ml, respectively;
• preparing HPV type 16 E7 oncoprotein detection quality control samples 1 and 2:
• taking lyophilized powder as the quality control samples, and respectively adding 1 ml ddH2O for dissolving, wherein concentrations are 10 and 80 ng/ml, respectively;
• 3) preparing an HRP-labeled HPV type 16 E7 antibody which comprises
• labeling with a sodium periodate method, adding an HRP stabilizer, mixing thoroughly and storing at 2-8° C., wherein a concentration of the HRP-labeled HPV type 16 E7 antibody is 0.05-0.4 μg/ml;
• 4) preparing a human cervical exfoliated lysis buffer which comprises
• preparing 0.05%-1% (vol/vol) of surfactant with 10 mM-1 M of buffer and adding a protease inhibitor with final concentration of 1 mM-10 mM before use,
• wherein the surfactant is one or more of Tween 20, Triton-100 or sodium dodecylsulfonate;
• the buffer is phosphate-buffered saline, Tris-HCl or a carbonate solution, and the protease inhibitor is one or more of cystatin, PMSF or Antipain;
• 5) preparing a concentrated washing solution (20×) which comprises adding 28.8 g of Na2HPO4.12H2O, 4.8 g of KH2PO4, 160 g of NaCl, 4 g of KCl, 10 ml of Tween-20, and 6 ml of Proclin 300 to ddH2O, diluting to 1000 ml, mixing thoroughly and storing at room temperature;
• and
• 6) directly obtaining a substrate solution and a sealing film by purchasing.