Hi as per me, i would like to suggest that you first calculate the biomass(liquid or solid) that you used for extraction of antibody, then check the activity. same method you just follow for dialysed sample that you purified through affinity chromatography. then check the activity. finally you just calculate from biomass used and antibody recovered from chromatography this will give you the yield. impurities can be eliminated by affinity chromatography. i think ELISA can give you the qualitative and quantitative result.
Since your starting material is a mixture of proteins and antibodies including the specificity you are purifying I would use activity as my first measurement for fold purification, % recovery and yield. I conduct such evaluations with ELISA as follows. My immunogen is used to coat all plates employing a standardised method. My crude antibody containing 100% activity is titrated against target immunogen and results are expressed as the reciprocal of the dilution which produces an absorbance of (for example) 0.5 under assay conditions. So, for example, a 1: 25,000 dilution of your starting antibody (100%) produces an absorbency of 0.5 under assay conditions. Assign the starting antibody a unit activity as measured i.e. 100 % = 25,000 units/ml. Use your starting antibody to produce a standard curve for all future assays as the absorbency may change slightly between assays. For example, make dilutions of 1:10,000, 1:25,000, 1:50,000, 1:75,000 & 1:100,000. You can now read your unknown directly from the standard curve to obtain dilution which = units. Make sure you check your starting antibody pre and post affinity column. In my experience there will be a lot of specific antibody left in the starting material. It may take 3 or more passes to remove 95% of starting antibody activity. I pool the eluates from each specific antibody elution, measure activity calculate recovery, concentrate to desired concentration and retest activity. Recovery = activity of recovered fraction x volume / activity of starting material x volume. Remember that elution buffers are harmful to antibody activity so if you are using low pH to elute you must neutralise the antibody fraction as it comes off the column. If you are using chaotropic agents you must dialize your eluate against a buffer compatible with antibody activity. I use PBS 0.15 M NaCl 0.02 M phosphate buffer pH 7.3. Fold purification = total protein of affinity purified antibody x volumn / protein or total IgG of starting material (depending on what you are working with) x volumn.
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