1. Developing Real World Tests and Assays Using Gold Particles Arista Biologicals Inc.

2. Developing Real World Tests and Assays 1. Biological Reagent Concerns 2. Assay Characteristics 3. Conjugate Optimization 4. Membrane Optimization 5. Conjugate Pads 6. Sample Pads 7. Problem Solving 8. Clinical and Stability Testing Arista Biologicals Inc.

3. Biological Reagent Concerns Monoclonal antibodies Most reproducible - True since no change in affinity or specificity theoretically Most sensitive and specific - Chosen for best optimal behavior Easiest to conjugate - Since you are dealing with a homogeneous protein Polyclonal antibodies Fastest to produce - Good response in 45-90 days Generally least expensive - Unless affinity purified Least reproducible - Variation from animal to animal and over time in the same animal Most cross-reactivity - Usually not desirable except for looking for variants etc. Arista Biologicals Inc.

4. Biological Reagent Concerns Cont… Antigens Hapten - Carrier conjugates, small molecule assays Recombinant antigens - Very reproducible but may not cover variants and do not always fold as native protein Native antigens - Some variation from lot to lot and usually a mixture of proteins. In many cases less sensitive assay is produced. Many times are denatured Synthetic peptides - Inexpensive but less like native forms. Usually need to be conjugated to a carrier Matching Compatibilities Cross-reactivity - Epitope overlapping, not enough specificity in the reagents, too much specificity in the reagents Interspecies interactions - Usually observed in assay systems containing antibodies from different species Prozoning - Usually observed with non affinity purified antibodies or assays requiring extremely high levels of antigen detection Arista Biologicals Inc.

5. Biological Reagent Concerns Cont… Purity Methods - Protein A and G, ion exchange, affinity purifications, and other chromatographic methods Effects on producing conjugate - Irreproducible sensitivities, aggregation of some conjugates etc. Effects on specificity - Background reactions sometimes immunological in nature and sometimes protein-protein interactions are observed. Effects on sensitivity - Less sensitive due to less binding sites available Testing - Generally electophoresis and chromatographic methods acceptable. HPLC especially useful looking for aggregation of the proteins Arista Biologicals Inc.

6. Biological Reagent Concerns Cont… Buffers Compatibility with conjugation method - Several types of buffers promote aggregation of the conjugate during formation. Many conjugate to the probe directly. Compatibility with membrane coating - Many buffers contain salts or other materials which compete for binding sites with the membrane Arista Biologicals Inc.

7. Biological Reagent Concerns Cont… Concentrations Solubility Issues - Typically problems with recombinant, native, and synthetic peptides because of hydrophobic characteristics Typical Ranges 1-4 mg/ml for antibodies 1 mg/ml for protein antigens 4mg/ml do to ug/ml for Haptens Arista Biologicals Inc.

8. Assay Characteristics Format - Flow through or lateral flow (dipstick, device, or midstream). May require specific components including pads, membranes, pretreatments Serum, Urine, Whole Blood, Feces, Dirt, Air, etc. - Test may be desired to work with several for the same assay. Dilution of sample - Creates another step but allows standardization of the sample Sensitivity - May require large amounts of reagents or slower assay development times Time of assay - If your requirements are for a faster assay, larger amounts of reagents may be required Cost - Limitations on the amounts of reagents, type of components, etc. Arista Biologicals Inc.

9. Conjugate Optimization Type of Conjugate Latex or colloidal gold - Color desired, type of assay, sensitivity, amounts of reagents required, etc. Covalent or passive absorption Size of Particle Type of particle Sensitivity desired - Larger particles generally give stronger signals but more nonspecific interaction problems. Conversely, smaller particles may be used to cure a non-specific binding problem Cost of protein to be conjugated Arista Biologicals Inc.

10. Conjugate Optimization Cont… Sensitivity Desired Coating levels - Passive absorption vs. covalent attachment issues Method to Immobilize Conjugate Dispensing - Can use high concentration with low volumes. Higher conjugation ratios may also be required Soaking - May require vacuum drying or lyophilization depending on how critical conjugate quantity per test is Arista Biologicals Inc

11. Membrane Optimization Quality and Cost Quality and cost vary from manufacturer to manufacturer and from material to material. Concentration Ranges Typically in sandwich assays 1-4 mg/ml, protein antigens usually range from 0.1-1.0 mg/ml, and Hapten assays may require 1 microgram to 4 mg/ml. Sensitivity Desired A higher flow rate membrane will generally develop faster but require more reagents to obtain the same sensitivity. Smaller pore size membranes also bind more capture protein. Arista Biologicals Inc.

12. Membrane Optimization Cont… Flow Rate Generally the higher the flow rate required the larger the pore size needed. Larger pore sizes generally have less chance of aggregation of the reagents causing flow problems Blocking or Not Blocking Blocking takes care of any nonspecific binding and can be used to change flow rates and other characteristics of the native membrane. It is also time consuming in many cases. Several blocking agents may be required to be tested including BSA, PEG, PVA, PVP, etc. Arista Biologicals Inc.

13. Conjugate Pad Optimization Considerations Polyester, rayon, glass fiber materials most widely used Most of these materials are hydrophobic naturally and need to be treated during or prior to conjugate immobilization Flow rates may vary by the material or the treatment Treatment usually includes addition of polymer and surfactant Sugars may or may not be required in the conjugate upon immobilization depending on the method Conjugate can be dispensed up to approximately 3 to 4 microliters per cm. Dispensing is much more accurate than soaking methods Arista Biologicals Inc.

14. Conjugate Pad Optimization Cont… Considerations Cont… Treatments can be varied to make the product more or less sensitive to humidity and temperature conditions Conjugate can be formulated to vary the rate of release of the conjugate during actual assay. This is sometimes key in determining sensitivity. It also can save on reagent Blocking reagents may be included in this pad if membrane is not blocked Assay buffer may be included in the pad if compatible with conjugate stability and resolubilization. Arista Biologicals Inc.

15. Sample Pad Optimization Is a Sample Pad Required? If the conjugate is stable and resoluble in the assay buffer a one bottom pad system can be utilized. The material must also have acceptable flow and liquid capacity characteristics Arista Biologicals Inc.

16. Sample Pad Optimization Cont… Material Requirements Flow characteristics - Does it flow at the proper rate in order not to inhibit the rate of assay development Volume capacity - Does it hold enough buffer for the components to be in high enough amounts to have the proper effect for the entire range of clinical samples Nonspecific binding of antigen - Does the analyte interact with the material nonspecifically Ease of handling - Can the material be processed with out damaging it during the normal manufacturing conditions Arista Biologicals Inc.

17. Sample Pad Optimization Cont… Buffer Components pH buffering capacity - the pad must control the pH of the assay during use. Ionic strength control - the pad must contain proper amounts of salts to control the ionic strength of the assay The pad material - must be blocked of any nonspecific binding of the analyte if the material does not already have that characteristic The pad material- must contain any surfactants required for specificity requirements of the assay. It must reduce these type of nonspecific interactions The pad material - must contain any polymers or protein needed to block either immunological reactions from occurring nonspecifically or protein-protein interactions not desired Note: If membrane blocking reagent is required the sample pad normally will contain this reagent and/or the conjugate pad Arista Biologicals Inc.

18. Typical Problem Observations and Solving Nonspecific Binding Heterophile Reactions absorption addition of IgG addition of surfactants antibody fragmentation pH changes addition of “hard” buffers Arista Biologicals Inc.

19. Typical Problem Observations and Solving Cont… Nonspecific Binding Cont … General Nonspecific Reactions blocking changing blocking agent on membrane change blocking agent on conjugate surfactant addition smaller particle production pH changes buffer changes decreasing reagents or strength change in conjugation procedure (method) Arista Biologicals Inc.

20. Typical Problem Observations and Solving Cont… Lack of Sensitivity Increase capture material Increase conjugate Increase strength of conjugate Increase particle size Decrease or change blocking agent Decrease flow rate decrease pore size increase blocking agent Decrease rate of conjugate release Use higher affinity antibodies or stronger antigens Increase activity of present reagents through repurification Add activity enhancer - such as PEG Decrease or change surfactants Check for analyte absorption onto assay component Arista Biologicals Inc.

21. Typical Problem Observations and Solving Cont… Large Volumes of Reagent Required Increase particle size Decrease flow rate Increase activity or purity of reagent Decrease rate of conjugate release Dispense conjugate Lack of Specificity (Immunological) Repurify (affinity chromatography, absorption) Scavenger antibodies and antigens Find new reagents Arista Biologicals Inc.

22. Typical Problem Observations and Solving Cont… Migration Problems Remake conjugate smaller particle different particle change protocol for production (chemistry changes) change or increase blocking agent add surfactants block membrane directly look for leaching of reagents from one material to another Arista Biologicals Inc.

23. Typical Problem Observations and Solving Cont… Stability Problems Change conjugate pad material Change preparation of conjugate pad increase or add surfactants increase or add polymers change pH or buffer add or increase sugars to conjugate change sugars in conjugate Look for moisture problems Change membrane material Add preservatives to membrane block Add preservatives to capture protein prior to dispensing Look for leaching of reagents from one material to another Look for degradation of materials on sample pad Arista Biologicals Inc.

24. Clinical and Stability Testing Clinical Testing Highly probable problem samples should be tested first. Suspected crossreactant containing samples, heterophile type samples, low sensitivity samples, variants, lipemic, hemolyzed, etc. samples should be tested early before large complete final clinical tests are performed. Reformulation should be performed as required If new formulations or materials are used than before especially new blocking formulations, conjugate pad formulations, membranes, etc. then some accelerated stability studies should be performed. Both biological stability and changes in physical characteristics such as flow should be observed closely During clinical testing several technicians should be used especially when performing in house testing looking for slight variations in users techniques Enough clinical testing should be performed using challenging samples which would be expected to be encountered in the field Arista Biologicals Inc.

25. Clinical Testing and Stability Cont… Stability Proper stability testing should be carried out. If real time stability cannot be used the product should be tested at several different temperatures. At least three different temperatures is recommended. These assays are multicomponent biological systems and cannot be treated a simple chemical reactions. However, we have observed using identical formulations and procedures for key components as a rule do follow a pattern We have also found that fresh product sometimes does not perform the same as slightly aged product. Several drying or curing steps seem to take a few days to complete. In some cases performance increases. This is especially important when producing assays which require high precision optimization for every batch such as DOA tests Arista Biologicals Inc.