First check the conjugation has occurred or not by:

1. UV spectroscopy of conjugated and non-conjugated gold. Conjugated will show shift in spectra.

2. DLS spectrum or XRD of conjugated and non-conjugated gold. Conjugated will show increase in size as compared to non-conjugated one.

3. Zeta potential of conjugated and non-conjugated gold. Conjugated will show Zeta potential to approx zero (neutralisation of charges).

Titrate the amount of antigen coating along with conjugated gold in dot blot before developing LFIA.

If more clarification is needed please get back.

With best wishes.is

Dear Shyam Kokkattunivarthil Uthaman,

There can be a lot of issues with the assay. How did you conjugate the mAb with the AuNPs? Did you do a covalent immobilization? This is more preferable compared to passive adsorption. Also: You said the mAb was successfully tested in ELISA: Did you do apply the sandwich format there as well? So that the mAb is used as capture ab and as prim ab? if so, it should be transferable to LFIA, but for sandwich-assays, it is usually better to use two different antibodies, because two different epitopes of the antigen need to be recognized at the test line. I think the latter is your problem: All of the antigen epitopes are recognized by the mAb, that are lableled with the AuNPs, but then these epitopes cannot be retained at the test line anymore.

In general, the NCM and buffer compositions can highly affect your assay as well. Make sure, that your antibodies on the test line are properly immobilized. You can use for example a AuNP labelled secondary ( =anti-IgG-AuNP) Antibody to make sure, they are properly immobilized. If you apply them to a membrane where your mAb is immobilized, simple use it as a test, without any antibody. The test line should appear red.

I hope that helps you for a first try!

best regards

Peter

Hi Peter Carl

Thank you very much for your comments..!!

1. We conjugated the mAb with AuNP using Turkevich-Frens protocol. We haven't done the covalent immobilization instead utilized the passive physical adsorption. But the problem was found only with one LFIA diagnostic test, means other such LFIA detecting another molecules produced via the same method and technique are working pretty well.

2. We have used simple indirect ELISA but not the sandwich one. We had tried with two mAbs in LFIA. One in AuNP conjugation and another one targeting different epitope of the same antigen on test line. There was also faced the same problem. So, I think if that was the real problem, test should get positive when we used two mAb, right?

3. Antibody immobilization on test line is to be checked. But during the buffer screening phase, some of the buffers showed test line coloured (false positive). This is indicating the test line antibodies are well immobilized to bind their target?

Anyway, I will check again the immobilization problem. Thanks for your informative comments Dr. Carl.

Merry Christmas !!

Regards

Shyam

Hello, I agree with the previous recommendation, but are you sure that you dint have a technical problem, fist it is did you block your membrane with Casein/ BSA. Are sure that the concentration of the antigin it is really high, sometimes you have to concentrate it 10-20 times for LFIA. More over, if you do the dot analysis the spot maybe not similar like with C line of your secondary Ab. Because of viscosity. And about buffer, I prefer Borat Buffer.

First check the conjugation has occurred or not by:

1. UV spectroscopy of conjugated and non-conjugated gold. Conjugated will show shift in spectra.

2. DLS spectrum or XRD of conjugated and non-conjugated gold. Conjugated will show increase in size as compared to non-conjugated one.

3. Zeta potential of conjugated and non-conjugated gold. Conjugated will show Zeta potential to approx zero (neutralisation of charges).

Titrate the amount of antigen coating along with conjugated gold in dot blot before developing LFIA.

If more clarification is needed please get back.

With best wishes.is

Antibodies which work in Elisa not always working in LF. The LF is faster & differen temp. Try add blockers to the SAP, slow down the reaction by using different pore size membrane, adding PEG.

If you use the same MAB or capture and conjugate and your antigen has only a single copy of the related epitope on each molecule, a sandwich cannot be generated. Try a different antibody for either capture and conjugate.

Have you checked that the antibody is attached to the gold? Absorb antibody at different pHs and concentrations: centrifuge and then challenge with 0.1 M NaCl: if the gold aggregates then it is not "protected" by the antibody ,i.e. not enoug his attached. Check the activity of those that survive the salt challenge at pHs with the minimal concentration of antibody.

As above, passivate the nitrocellulose, NC with e.g. BSA, casein +/- Tween 20, in case your antigen is sticking to the NC, which is hydrophobic and slightly negatively charged.

Perhaps this paper can also help....:: Article Chemical Modification of Antibody Enables the Formation of S...

As Falk Fish stated, typically you cannot use the same Mab as the detector and capture for the reason stated. You need to use a test line Mab that identifies a separate antigen epitope to the conjugate antibody. Alternatively, you can use a polyclonal antibody on the test line which will provide a more robust signal.

Hi

Peter Carl Anna Raysyan Tulsidas G Shrivastav Oded Babai Falk Fish John Clarkson Angel R Vargas

Thank you all for your valuable and informative comments and suggestions. I will incorporate all these points and will do my experiments accordingly.

Once again, Thanks for your active discussion

Happy New Year to All !!!

Regards

Shyam

No problem Shyam. For general LFIA troubleshooting and protocols, you can vist our NCX-U knowledge-base: https://nanocomposix.com/pages/introduction-to-lateral-flow-rapid-test-diagnostics

Hi. If the problem yet not resolved, You can contact IRIS NANOTECH and we can develop a custom technology for you. Find out more about us at www.irisnanotech.com or contact us at [email protected].

Thanks.

Hitesh Chauhan.