I need to pipet a low molecular weight antigen (around 20 kDA) on my test line and a secondary antibody of anti-rabbit goat antibody- IgG (around 120-160 kDA) on my control line. The concentrations of each are 1 mg/mL. I will be using a nitrocellulose membrane and this is only my sample test, meaning that I need to prove that this concept would work first before having them produced by a professional company. Because this is a sample test, I would prefer using a limited amount of out the total of 100 microliters of my primary antibody and 1 mg of my secondary antibody. What would be lowest recommended dilution to minimize the amount of antibody I use while also making sure that there is enough to make this reaction successful?
Means you want to design the LF strip on your own? In that case, I'd ask the company who will produce it later for guidance. You still have to titrate, but the first pitfall might be that there are different methods of making the reaction lines: You'll probably pipet a dot and they'll use an inkjet-like or pen-like device to generate lines on the to-be cut membrane, so the composition of the lines will be completely different and development starts again. When your antibody works in ELISA, the test may be transferred to the lateral flow formate, so you don't need another proof of concept. The only problem that may arise is sensitivity and this you can't predict well.
If you should be developing a to-be commercialized test, you might want to contact Karl Schmitt at bioavid.com, they have a lateral flow platform with a unique strip that may be adapted to virtually any analyte, provided the analyte may be detected by an antibody. The only thing that needs to be modified is the specific antibody which later will be added to the reaction vial, not to the strip.
The answer to your question is strictly empirical and can't be predicted well. What I do, when developing a new LF test, is to spot 0.3-0.5 ul of reagent on the membrane and make titrations of it. Make a full-strength spot and see if that gives you signal. If it does, then titrate back . If it does not, then you're in for a lot bigger project. Keep in mind the concentration of the gold conjugate applied to the conjugate pad also plays a role in the overall intensity of your test line and this also must be optimized.
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