Ultimate Guide to Lateral Flow Webinar - Your Questions Answered
Last month we brought to you the first in a series of webinars “The Ultimate Guide to Lateral Flow.”
The event was a great success with attendees from all over the world logging on to hear about lateral flow and considerations when embarking on a development project. Darren Rowles, BBI’s Product Manager for Lateral Flow and Gold Nanoparticles, hosted the session and was joined by Karen Whiting the Global Head of New Product Development whose team were responsible for developing Morffi: Signal Enhancement technology. A live Q&A session unearthed some interesting questions, so we thought we’d summarise them in the article below.
Missed the webinar? Don’t worry you can download the recording here.
1. How small can you make a lateral flow test? (And what are the minimum and greatest dimensions recommended for a blood test).
The dimensions for the majority of LF tests are 4-5mm in width x 60mm in length. It is possible to make the width smaller, as (during manufacture) a cutting machine can consistently cut the strips with very tight tolerances. Any less than 2-3mm would give a line or dot that would be small to interpret accurately, and make cutting more difficult. Most whole blood assays use a whole blood volume of 20-25ul.
2. How do you perform multiplexing? Do you have specialised nitrocellulose membrane with channels, or do you treat them?
BBI introduces multiplexing in LF tests in a number of ways. The simplest way is to have distinct test lines within the assay which are identified by an annotation on the housing cassette. It is then possible to stripe or immobilise different antibodies on the membrane which detect different analytes. It is also possible to detect these analytes with one antibody labelled conjugate or have different antibodies for each of the different analytes in the assay. In some cases, gold nanoparticles or one coloured latex is used as the detector conjugate(s) and so all generated lines are the same colour. In other tests the developer may use different coloured labels to distinguish between the different analytes, such as gold and platinum nanoparticles, providing a red test line and black control line.
3. Do you sell a product to control the conjugation of antibodies on test line in nitrocellulose membrane?
Typically, a capture antibody should be diluted in a phosphate or Tris buffered saline solution of pH 7-8 with/without the addition of blocking agents such as BSA. It is important that each antibody is tested to determine its optimal dilution buffer. This helps preserve antibody activity when immobilised and optimised for its stability when dried on the membrane.
4. Have you faced problems with lateral flow assay development for small molecule detection compared to bigger molecules (e.g. proteins)?
BBI has developed numerous assays where the analyte is small, in the case of drugs of abuse or hormone detection for instance. In this circumstance, developers will typically favour a competitive or inhibition type assay format, as the small molecules will likely have only one distinct epitope for antibody generation.
5. Has BBI used Aptamers?
Yes, BBI has used aptamers in LF test developments. There are some challenges with using aptamers on the detector particle and on the nitrocellulose membrane but BBI have developed conjugation chemistries to allow for conjugation of aptamers to the gold nanoparticle surface that give a stable and sensitive conjugate. Modification of the aptamer also allows it to be successfully immobilised on the nitrocellulose membrane surface.
6. Possible ways to increase assay limit of detection?
There are many ways to do this; through optimisation and titration of the antibody concentration on the detector particle and on the nitrocellulose membrane, increasing the concentration of the detector particle, slowing the flow rate of the nitrocellulose membrane using reagents in the sample pad to speed up release of the dried conjugate from the conjugate pad, and to help with exposure of the analyte to the antibody.
Use of the best particle size and uniformity can also aid in LOD, making the signal more visible. There are also specialised techniques such as BBI’s Morffi signal enhancement technology as well as the use of a streptavidin/biotin conjugate system.
These are only some of the examples that BBI has deployed to improve limit of detection and each assay needs to be carefully optimised to achieve the best performance.
7. Is the background and non-specific binding increasing when Morffi technology is applied?
No, Morffi does not increase the non-specific binding in the assay.
8. Can we find detailed information about Morffi on your website?
Yes, we can also follow up with you separately if you have any further questions after reviewing our website and whitepaper.
9. Where is Morffi added into, conjugate pad, capture line or sample pad?
Morffi is a novel conjugate blocker so is added to the detector label after conjugation to the antibody to block any sites not covered by the antibody. The Morffi blocked conjugate is then added to the conjugate pad, using comparable materials and treatments to BSA blocked conjugates.
10. Is Morffi added before the antibody mixture with the gold particle or after?
The Morffi blocker is added to the particle after conjugation of antibody has occurred. The Morffi blocker will then block any sites on the particle that is not covered by the antibody and so prevents aggregation of the particles and collapse. Using the Morffi blocker in this way BBI has demonstrated up to 10x enhancement in the limit of detection and a stronger signal intensity at all concentrations of the analyte. The rate of appearance of the signal (time to result) is also faster with the Morffi blocked conjugates. Click here to see our newly released Accelerated Results data.
11. Have you seen differences by using aptamers to antibodies in terms of selectivity and specificity? immobilisation on the membrane?
We have not seen data where use of an aptamer is more beneficial than an antibody in direct comparison. Aptamers have been deployed in assay systems where antibodies cannot be developed (e.g. biowarfare type assays). Literature suggests that aptamer assays will be more specific than immunoassays, but BBI do not yet have data to prove this one way or the other. However, we welcome the opportunity to work with customers and partners who wish to develop aptamer based lateral flow assays.
12. Morffi Blocker, is it hydrophilic, and can you adjust hydrophilicity with other additives? in that case, what additives do you recommend?
Morffi is a proprietary blocker to BBI and is a synthetic biological reagent. We are not able to give further details on what the actual Morffi blocker is.