IgG is the most popular isotype for conjugation and can be conjugated with a high success rate. The antibody binds to the gold nanoparticles through the Fc region, leaving the Fab region exposed and able to bind the antigen of interest. IgG3 antibodies are more difficult to work with so these should
be avoided if possible.

IgM conjugations have a lower success rate. Cross-linking can occur when they are conjugated, and the Fc regions cannot always be exposed without damaging the integrity of the antibody.

IgE antibodies are raised in an allergic response, these antibodies can be conjugated to gold nanoparticles.

IgY antibodies are functionally similar to IgG antibodies and these can be conjugated to gold nanoparticles with a high success rate. Antigen conjugations are possible, although the success rate is lower. BBI can also conjugate antibody fragments- Fab, Fc, ScFv and limited experience with nanobodies.

The majority of gold conjugates (BA Grade and Custom Conjugates) are presented in a low molarity Borax buffer. EM grade conjugates are presented in a Tris-buffer saline solution containing 30% glycerol.

EM Grade gold conjugates can be frozen for long term storage and thawed for usage as these conjugates contain glycerol in their final buffer.

BA Grade and Custom conjugates cannot be frozen. Freezing of these products will cause the gold nanoparticles to completely and irreversibly aggregate and should be avoided.

It is recommended to store the gold conjugates (BA Grade and Custom Conjugates) at 2-8°C. EM Grade conjugates can be kept frozen at -20°C for long term storage.

BA refers to BioAssay. These conjugates are stock products which are routinely manufactured at BBI Solutions. These are most commonly used in lateral flow diagnostic tests. The particle size is 40nm.

CC refers to custom conjugates. These conjugates are similar to BA Grade conjugates but are usually manufactured on a custom basis by request of a customer. The particle size most
commonly used is 40nm.

EM refers to Electron Microscopy. These conjugates are different to BA and CC conjugates, are usually made with smaller nanoparticles and are most often used in electron microscopy. The particle size most commonly used is 10nm.

In BBIs experience, 40nm is the preferred particle size. 40nm provides good signal visibility whilst encountering relatively little steric hindrance.

Our gold nanoparticles have a high level of single particle dispersion.
Conjugates are tested under the Transmission Electron Microscope (TEM) and have set specifications for % singlets and cluster size.
40nm conjugates are tested using the MonoDispersity Index (MDI). MDI uses UV-Vis readings to assess the ratio achieved between the A520nm (absorbance at which 40nm OD readings are taken) and A600nm (associated with stability) of the conjugate. A pass/ fail criterion is assigned to the ratio which is deemed acceptable to produce a stable product.
Other gold colloid sizes are assessed using TEM and are tested to a set specification.

EM Grade conjugates are available in 0.25ml and 1ml pack sizes.

BA Grade conjugates are available in 5ml, 100ml and 500ml pack sizes. 5ml will be for samples only.

Custom conjugates are available in any pack size. Minimum order volume is usually 5ml. For orders >500ml, individual pack sizes do not exceed 500ml.

The expiry date is stated on the label of each batch. In general BA Grade conjugates have a shelf life of 2 years from the point of manufacture when stored at 2-8°C. BA.GAM40 stability studies are ongoing- this is currently not supplied with an expiry date.

No expiry date is applied to EM Grade conjugates. Anecdotally, these have been used 5+ years after the date of manufacture with no loss of functionality.

No expiry date is applied to Custom Conjugates, unless at request of the customer. A cost would apply for the panel of work required to assign an expiry date.

The gold conjugates are manufactured at our ISO 13485:2015 certified facility in Crumlin, South Wales, UK.

Your inputs, following discussion of your application with our team,

▪ Selected detector protein (antibody/protein). Supplied by customer or BBI can source and recharge the customer.
▪ Identify target sensitivity
▪ Identify particle size
▪ Concentration of final conjugate required
▪ Provide 3-5mg of protein for conjugation and QC materials (capture, target antigen). Antigen no
more than Cat II Biohazard level

BBI outputs

▪ Initial 5-10ml of final conjugate – (enough for 1000 prototype lateral flow strips*)*using a 1:5
dilution of OD10 conjugate per strip
▪ Excess reagents banked for future production
▪ Full laboratory report on performance customised to your requirements
▪ Access to technical team post conjugation

For a custom conjugation to a 40nm particle, BBI request 3mg antibody/protein. Minimum concentration 1.0mg/mL. This allows for a full assessment of conjugation parameters prior to committing material to a batch.

Antibody quantity will be slightly more for smaller particle sizes and for alternative detectors (e.g. antigens).

Initial antibody requirement may be higher than expected but the benefit is gained as conjugate production volume increases. Antibody mg per ml of conjugate is lower with BBI’s manufacture method which is designed with long-term scale-up in mind up to 24L OD10 equivalent.

Antibody pI- this helps with assessment of conjugation parameters

Antibody source- Monoclonal/Polyclonal/Human/Recombinant. This will determine how we assess the antibody pre-conjugation.

Antibody subtype- this helps with assessment of conjugation parameters

Antibody buffer- no thimerosal in antibody buffer- this can affect binding

Antibody Supplier- considerations around robust long-term supply, availability of multiple lots

Assay format- Sandwich/Competitive/Inhibition/Serological/Non Lateral Flow- different assay formats require different considerations around conjugation parameters.

All testing is performed in liquid format.

OD at 520nm- measure of concentration

Dipstick Testing- either anti-species or specific functional system if provided. Determines performance compared to approved past batches.

Free Antibody Check- identify if free antibody is in solution that could interfere with assay.

Mono Dispersity Index (MDI)- for 40nm conjugates. A spectral reading as an indicator of potential conjugate stability.

Cluster Check – for conjugate sizes other than 40nm- indicator of potential conjugate stability. A visual count of particles under TEM to check for clustering of particles that may impact stability, performance, and reproducibility.

It is always best to perform in-process assessment and final QC testing with specific functional materials. This is not always possible if material stocks are low or costs high.

BBI requests 0.5mg capture at minimum concentration of 1.0mg/mL.

BBI request approx. 0.1-0.2mg antigen/analyte at 0.1mg/ml (minimum concentration), along with SDS or details of hazards and disposal.

No materials above Biohazard Level II are permitted.

BBI can perform stability studies at customer request. A charge will apply.

Following assessment of conjugate batches in the intended final format by the customer, further optimisation of parameters and conjugate formulation is often required.

BBI request feedback from conjugation events to determine improvements to conjugate formulation to achieve desired performance. This can take the format of emails, presentations or technical calls.