I am currently developing a Lateral Flow Assay, and I have an antigen with a concentration of 1 mg/mL and a molecular weight of 20.7 kDA. I also have an antibody with a concentration of 2 mg/mL with a molecular weight of around 140 kDA. I would like to use as little of the antibody and antigen as possible, so I am wondering if I could dilute it to be a lower concentration.
Lola, you'll need to titrate. From the protein concentration you cannot deduct anything. Dilute the analyte to a little more than the concentration you need to be able to detect and test how much antibody you need to detect it.
I don't have a fixed protocol, sorry. What is the chemistry / principle of the LF strip that you are using?
ELISA is generally more sensitive than LF, and as such, the test line concentration on a LF test might be 50-100x greater than what would be required to coat an ELISA well. I've worked on tests that required mg quantities on the test line (maximum binding capacity of the nitrocellulose is around 2-3 mg/ml, depending on the nitrocellulose characteristics), whereas others required just microgram quantities of protein. The answer to your question is strictly empirical and difficult to predict. A good starting place in your case would be to try spotting 0.1, 0.3, and 0.5 mg/ml on different strips and see if a spot develops when you run the test. You can also try a full-strength strength (undiluted) spot. Spot about 0.5 ul on the membrane and let it dry at 37C for 30 minutes. This will be the best way for you to empirically determine this without sacrificing large quantities of reagent since you have very little. I do not think you'll have good success with hand striping. I've never been able to do it. You'll get better consistency with a spot.
It depends on the nc membrane you're using. Many of them today are already pretreated and no treatment is necessary. You'll also want to test different nc membranes to see which works best for your particular assay. Different nc membranes have different porosities which affect the flow rate, which in turn affects the sensitivity and specificity based on how fast or slow the transit time of the sample is over the deposited analyte.
Pl. let me first know on which format or principle you are developing LFA. It is competitive format or non-competitive format? What label you are going to use (colloidal gold or any thing else). Which immunoreagent will be used as label (either antibody or antigen). Generally immobilization of antigen or antibody on nitrocellulose membrane depend on the titre of antibody. If the antibody generated has high titre then less amount of antigen or antibody are required to be immobilized. Generally what I experienced is that the concentrations of immunoreactant required for immobilization is more than 2 mg per millilitre. But the safe way is to titrate on nitrocellulose membrane. Generally LFA are completed with in 10 minutes therefore there is no need to block the membrane. If you block the membrane than the flow rate of membrane will change (decrease).
For any more clarification kindly get back to me.
With best wishes
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