Substitution of carbonate buffer by water for IgG immobilization in enzyme linked immunosorbent assay.
The first step of enzyme linked immunosorbent assay (ELISA), namely, adsorption of antigen or antibody to the plastic microtiter well plate, was studied as a function of insolubility of IgG in water. Immobilization efficiency was assessed in terms of number of wells coated per milliliter of primary antiserum. We have compared different coating/immobilization protocols, i.e., direct and indirect immobilization of primary antibody to the plastic microtiter well plate using carbonate buffer and phosphate buffer with glutaraldehyde. We have observed efficient coating when the immobilization of primary antibody through an immunobridge technique was performed, where water was used as a coating medium. It gave a higher number of wells coated per milliliter of anti-serum (primary or secondary) than other compared coating protocols and it allowed the use of serum (non-immune) and anti-serum (primary and secondary antibody) dilutions, avoiding the need for gamma-globulin purification from normal and immunized serum.
Shrivastav
Tulsidas G
TG
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India. nihfw@delnet.ren.nic.in
Basu
Anupam
A
Kariya
Kiran P
KP
D000272:Adipates / Q000737:chemistry; D000327:Adsorption; D000818:Animals; D000918:Antibody Specificity; D002021:Buffers; D002254:Carbonates / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005976:Glutaral / Q000737:chemistry; D006041:Goats; D006735:Horseradish Peroxidase / Q000737:chemistry; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D010710:Phosphates / Q000737:chemistry; D011817:Rabbits; D014867:Water / Q000737:chemistry
D000272:Adipates; D002021:Buffers; D002254:Carbonates; D007074:Immunoglobulin G; D010710:Phosphates; D014867:Water; D006735:Horseradish Peroxidase; C007133:hydrocortisone hemisuccinate; D005976:Glutaral; C010011:adipic dihydrazide; D006854:Hydrocortisone