Methods of making active antibodies from biological fluids

A method of making an antibody by identifying a circulating antibody with activity from a subject comprising:

• i) subjecting biological fluid selected from the group consisting of blood, plasma and serum and combinations thereof from the subject to one or more rounds of affinity chromatography to purify the circulating antibody;
• ii) optionally further subjecting the circulating antibody to isoelectric focusing to purify the circulating antibody based on charge;
• iii) testing the purified circulating antibody for activity;
• iv) digesting the purified circulating antibody from parts i) or ii) to create an antibody fragment;
• v) subjecting the antibody fragment to mass spectrometry to generate a mass assignment and a deduced amino acid sequence of the antibody fragment;
• vi) comparing the deduced amino acid sequence with an amino acid sequence of an antibody generated from the subject's B-cells to identify an antibody sequence that matches the deduced amino acid sequence;
• vii) generating an antibody comprising light chain and heavy chain CDR sequences of the B-cell antibody that matches the deduced amino acid sequence of part vi);
• and
• viii) testing the antibody of part vii) for activity;
• wherein the circulating antibody with activity binds an HIV antigen selected from the group consisting of gp120, p24, gp41, p31, p17, p55, RT, rev, nef, vpu, and tat;
• and
• wherein the B-cells comprise bone marrow cells exhibiting a long or short-lived phenotype, wherein the B-cells have a phenotype that is CD38hi, CD19+ or CD19−, and CD138+ or CD138−.