###### Abstract  
The Enzyme Linked Immunosorbent Assay (ELISA) technique has been widely used for the identification and quantification of biochemical markers. The typical ELISA requires a number of washing steps to eliminate the unbound proteins which sometimes cause the desorption of protein due to their weak bonding between protein and well plate. In this study, we have developed a meshed type of plastic membrane in order to increase the reliable binding efficiency between proteins and the membrane surface, and to provide easy steps of washing. The use of our developed solid membrane has significantly increased the binding capacity of the biomolecules because this membrane ELISA (mLLISA) provides 3D binding surfaces which increases the surface area when compared to the conventional 2D surface well plate. The columns were pretreated to form a self-assembled layer (SAM) on the surface for the stable conjugation of a target antibody. The SAM-coated membranes could be stored for one month without any further deterioration of stability. The measured optical density (O.D.) shows a 1.2-fold increase in IgG antigen (25 \(\upmu\)g/mL) from the plastic membrane as compared with the conventional ELISA method. The concentrations of thyroid stimulating hormone were also monitored using the mLLISA method and it shows good linearity against the concentrations. These membranes were immobilized between solid supports and biomolecules by either adsorption or covalent bonding. The typical ELISA requires a number of washing steps to remove the unbound proteins, but these can also cause the desorption of protein due to their weak binding [6]. Because of the weak binding property, immobilization methods have been studied with the aim of improving the bonding of the biomolecule on the solid surface covalently [7]. By contrast, solid surface and biological material can be combined with the highly sophisticated self-assembled layer (SAM). The SAM has been studied by many different and interdependent surface science techniques. Surface treatment has involved treatment with gamma-ray [8] and oxygen plasma for increasing the binding ability between membranes and biomolecules. The ability of ELISA explains the use of an enhanced molecule absorption area for a greater bonding area between the solid support and antibody. The 3D printed fabricated membrane provided enhanced molecule detection sensitivity as compared to that of the conventional ELISA [9]. In addition, gold nanoparticles (AuNPs) were used as probes of the antibody-conjugated with HRP (horseradish peroxidase) in order to enhance the detection of the area of breast cancer biomarker [10]. In this study, we have developed the 3D plastic column type solid phase or membrane structure, on which conjugated antibodies and antigens react. Using this new column type plastic membrane, we performed a typical ELISA by moving and dipping the membrane itself into different wells instead of transferring and washing buffers in the stationary bottom surface, which are typical steps of the ELISA. Interestingly, an improvement in measured signals could be achieved when we used the 3D membrane. In addition, we have applied a covalent bonding method between the antibody and solid surface of the membrane, which accounts for increased optical density (O.D.) results in corresponding conditions. For the immune-reaction test, we performed a characterization test using IgG antigen. For the sensitivity-related test, we have chosen and performed the quantification of thyroid stimulating hormone (TSH). TSH is one of the most significant kinds of hormone and regulates the release of the metabolic hormones such as T3 (triiodothyronine) and T4 (thyroxine). In addition, because of their low concentrations present in serum, it is more difficult to detect without using highly sensitive sensors. In this study, we demonstrated that the developed mELISA method could provide a highly sensitive detection in low levels of TSH (0.01 \(\sim\) 0.1 ng/mL), which is mostly difficult to measure when using the conventional ELISA.