Why test line signal is pretty WEAK in lateral flow immunoassay (LFIA) even high analyte concentration?

Bo-Cheng Huang @Bo_Cheng_Huang

01 January 1970 3 6K Report

Hi everyone!!

I am doing a 1/2 LFIA test strip recently.

However I am suffering the weak test line signal even I apply 40ul 1000ng/ml (in PBS) on my 0.5cm * 6cm test strip. =(

I suffer this problem for 2 month, pretty frustrated.

Detail exp. procedure is listed below.

First conjugate my colloidal gold with antibody

Replace the 40nm colloidal gold solution (OD=1) with 2mM sodium borate ph 9.0 buffer by centrifuge at 12000xg 15mins.

Dilute the antibody by borate buffer above to 0.1mg/ml

Mix the colloidal gold with antibody above at a volume ratio 20:1, and added 10% NaCl to gold-conjugated antibody solution to 2% NaCl concentration for "challenge" the stability.

After I make sure the antibody stability by spectrometer between 520-540nm, I keep this ratio and react 1 hour at RT without salt.

Afterward, BSA was added to 1% for half hour at RT

Centrifuge at 15000xg 30mins to replace the buffer to 20mM pH8.2 tris-HCl in 1%BSA 1%PEG 0.01% sodium azide.

Centrifuge at 15000xg 30 mins again to replace the buffer to 20mM pH8.2 tris-HCL in 1%BSA 1% tween-20 5%sucrose 0.01%sodium azide.

Immobilized antibody on test line

Then I apply my capture antibody and rabbit-specific antibody on my NC membrane by pipette and placed in 37 degree for 1 hour.

Adhere wicking pad at bottom of NC membrane

1/2 test strip

I mixed 40ul 1000ng/ml sample with 10ul gold nanoparticles probe (OD~=4) for 5 mins.

Apply mixure on my NC membrane at the end without wicking pad by pipette.Wait until it totally dry, and control line is visible, but test line is very weak.

I conclude that affinity might be the problem or biodot would obtain more strong signal. But I assume biodot wouldn't affect the signal exceed an order, which means 10-fold or even more.

Any suggestion would be very helpful to me =目

Falk Fish

1. If your antibody is monoclonal and you use the same antibody on both gold particles and on membrane you can expect competition on binding sites.

2. You antigen concentration is too high. Try different dilutions.

3. The capture line is not optimized: try different concentrations of antibody and different buffers. There are no "laws": each antibody is an individual personality. The other issue is stability of the dried capture. You may have to add stabilizers.

4. So goes for the conjugate: different concentrations of the antibody, even if the salt challenge test is OK. Stabilization is also an issue here.

Bo-Cheng Huang

Dear Falk,

Thanks for your reply!!

Correspond to your suggestion, I found I wasn't detailed elaborate my capture/detecion anti. well.

1. Both capture/detection anti. are mono-antibodies. Since test line was formed, so I assume they won't compete the binding site of the antigen. Is it make sense?2. I had tried different concentration from 1 ug/ml and subsequently diluted it to 100ng/ml, 10ng/ml, 1ng/ml. But only 1 ug/ml is visible, so that's why I put this condition on post.

3. I see, different concentration and buffer of capture antibody. The condition of the capture antibody immbolized on the membrane was suspended in ph7.4 PBS (1X), and no stablizer here. If stablizer includes BSA, then 0.1% BSA was included. But the problem is, the antibody couldn't be tuned since we bought it from Abcam in solution, unless we dialysis the antibody.

4.I don't sure how to check the stablization here. Is it means how long I can "stroe" it with same signal performace? If so, all the exp. was done immdiately as I prepared all the membrane and reagents. If I misunderstand it, please advise me =目

Best regard,

B.-C.

Falk Fish

Hi Bo-Cheng,

I find it difficult to understand your response.

Using the same monoclonal for capture and conjugate can work if your antigen features multiple replicates of the antigen moiety. But, concentration of antibodies should be low.

For placing the capture the amount of carrier protein should be carefully limited. Detergent should be avoided.

Try reading this review: http://www.sciencedirect.com/science/article/pii/S131961031400129X

Regds,

Falk

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