I did competitive lateral flow immunoassay using gold bridge method.
Antigen-fluorescence bead and immobilized antigen on the PMMA membrane is no problem to measure the optical absorption at 37 degree.
But, intensity of test part was decreased when gold-antibody conjugate was added in sample buffer and incubated at 37 degree.
As I thought the antibody stability is the reason of this problem, the intensity will decreased using incubated gold-antibody conjugate stock solution.
So, I tried to incubate the sample buffer (without gold-antibody conjugate) and gold-antibody conjugate stock solution each other. And then I measured the optical intensity of the lateral flow assay after mixing incubated buffer and gold-antibody conjugate. There is no problem at this time.
I applied two types of antibody-gold conjugation method - passive attraction (optimized pH 6.5) and covalent method (MUA and EDC/NHS).
However, in spite of change of all the experimental conditions, test intensity was always decreased in anytime.
I tried to change the buffer constituent, concentration, and use another antibody, but, any of them was no effect in this problem.
Could you help me, please ?
Hi DKK, couple of questions before I can help you:
How long are you incubating sample buffer and antibody labeled gold nano particles?
What are the components in your sample buffer? Is there some kind of reducing agent?
Are you using a monoclonal or polyclonal, and if monoclonal, what isotope?
What type of tube or well are you using to concubine the sample and beads? Pp or ps?
Dear, Lora Benoit,
1. Sample buffer including antibody-GNP was incubated at 37 deg for 6hours.
2. The components of my sample buffer is;
- 50 mM MES, pH 6.2
- 1 % trisodium citrate
- 0.2 M NaCl
- 2 % sucrose
- 0.01 % Triton X-100
- 10 % antibody stabilizer (stabilguard and Candor Ab stabilizer each)
- 0.1 sodium azide
- some kinds of blocker (0.2 mg/ml Mouse IgG; 0.1mg/ml Heteroblock; 0.6 mg/ml HBR1)
- some kinds of polymer (0.5 % PEG 6K; 0.5 % PVP 360K)
- included Ab-GNP (OD=1)
* I tried to use another buffer pH with tris (pH 7.5) or phosphate (pH 8.0), but the intensity was still decreased.
3. My antibody is IgG1.
4. I use the 1.7 ml tube (PP) for incubating sample buffer with Ab-GNP.
Could you help me, please.
I've been solving this problem for over 3 months...T_T...
Monoclonals are tricky. For starters, the ionic concentration of the sample buffer may be too high for this particular clone, altering the structure so that it does not effectively bind the competitive test line on the lfd as it should. Generally the lfd performance should be assessed in test mode using a matrix of buffer variants to optimize the assay. This is especially true for monoclonals. I would start by reducing the concentration of sucrose and nacl. This will reduce the stringency of the assay immediately and may enhance the conformation of the antibody. The other possibility is the non ionic detergent you are using.
Lastly, I am not sure what your analyte is. But the igg1 may be cross reacting or binding to something in the buffer...I would consider the mouse igg and heteroblock to be a potential risk in this regard.
Dear, Lora Benoit
Thank you for answering my questions!
But, I already tried to change the concentrations of sucrose (with or wothout) and NaCl (0M or 0.1M). Also, i tested this experiment without all kinds of blockers. Those did not affected for my case.
In case of cross-reactivity, my antigen is thyroxine, above all, this trouble is not appeared when using sample buffer included Ab-GNP without incubation. just only when stored at 37deg mixing sample buffer and Au-GNP, this trouble is represented.
The purpose of sample buffer incubation is for my developed proto-type lateral flow assay kit to make more stable when my end-user have this kit.
Thank you so much Lora Benoit
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