Major Aspects of Detector Reagents Arista Biologicals Inc.

Types of Conjugates Enzymes - Alkaline phosphatase and horse radish peroxidase most commonly used Latex Particles - Dyed, magnetic, and fluorescent types most commonly used Colloidal Metals - Gold, silver, selenium, iron, copper, platinum used. Colloidal gold most popular Disperse Dyes - Organic dyes with hydrophobic and hydrophilic characteristics Arista Biologicals Inc.

Criteria For Selecting A Conjugate Sensitivity - High or low level sensitivity required or desired to be detected Color Desired - Specific color or multiple color requirements Amount of Antibody or Protein Required for Conjugation – Cost and availability Format - Flow thru or lateral flow type tests desired Size - Difficulty in processing and pore size restrictions Membrane Desired to Use in Device - Pore size, material, etc. Ease of Preparation and Reproducibility - Volumes required and scaleup issues Multiple Analyte Testing - Mixing conjugates or common conjugate Stability - Ability to dry and rehydrate over time. Sensitivity to moisture Arista Biologicals Inc

Criteria For Selecting A Conjugate Cont… Dry or Wet conjugate - Will material remain active and stable over time under conditions stored and utilized Scale Issues - What level of success can be obtained during process scaleup Arista Biologicals Inc

Enzyme Conjugates Positive Aspects Highly Sensitive - picogram level and lower detection possible Relatively low antibody or protein requirements - low cost per test for antibody and antigen and lack of need to overload system. Easy to Rehydrate - No complicated formulations required to maintain stability and ability to rehydrate in order to achieve sensitivity. Relatively inexpensive - Enzymes are inexpensively purchased or purified. Conjugation procedures relatively well documented and easy to scale-up - These reagents have been produced for the microtiter assays for several decades. Arista Biologicals Inc

Enzyme Conjugates Negative Aspects Require multiple steps - Normally wash steps and substrate steps are required. Higher level of technical ability required from end user. Require multicomponent substrates - They can be difficult to stabilize Generally require longer incubation times - In order for both immunological and enzyme reactions to take place. Arista Biologicals Inc

Latex Particles Positive Aspects Choice of Colors - Variety of colors and intensities available General conjugation procedures well documented - Latex for agglutination assays and other formats have been produced for over a generation Arista Biologicals Inc

Latex Particles Negative Aspects Generally require high amounts of antibody - Intensity of color less than other conjugates Materials and larger particles generally used for purposes of ease in handling More difficult to reproduce lot to lot - Raw material needs to be qualified Large particle size can cause aggregation problems on membrane - Generally use 0.3micron and larger particles for membranes used Relatively low dye levels require more particles for visual detection, can make sensitivity requirements difficult to obtain Arista Biologicals Inc

Colloidal Metal Dyes Positive Aspects Very high dye content - 100% dye essentially. Scatters light which increases signal intensity. Visual signal increases with increasing particle size. Small particles easy to work with - Particle sizes typically used range from 10 to 60nm Require relatively lower protein amounts for conjugation and assay - Typically use less than 1 microgram of antibody per test Conjugation protocols somewhat documented - Immunohistochemical reagents have been produced for several decades and procedures documented Arista Biologicals Inc

Colloidal Metal Dyes Negative Aspects Lack of color choices - Red only Relatively unstable - Exists as a colloidal mixture. Not a true solution. Requires maintaining colloidal stability. Difficult to resuspend once dried - Aggregates upon itself under improper conditions Must generate particles - Not economical or practical to purchase raw particles Optimization - Each protein needs to be optimized for conjugation separately Arista Biologicals Inc

Disperse Dyes Positive Aspects Can choose colors desired - Dyes come in many colors as with latex Negative Aspects Require larger amount of protein to be conjugated - Similar to latex particles Must generate particles - Not commercially available Difficult to reproduce - Due to nature of particle production Relatively large particles can aggregate during assay - Similar to latex Can be difficult to rehydrate - Similar to colloidal gold Arista Biologicals Inc

Conjugation Preparation Concerns and Choices Scale up - Ease and reproducibility Covalent vs. noncovalent methods - Generally covalent methods produce a more stable product. Covalent methods multistep in nature Conjugation ratios - The amount of protein to detector reagent Blocking reagents: Type to be used - Compatibility issues with remainder of reagents and materials in system Buffers - Stability issues and compatibility to final system must be addressed Final concentrations - Stability and usability for application method concerns Liquid Stability - Storage and shipping conditions Arista Biologicals Inc

Latex Particle Conjugation Passive Absorption Removal of Surfactants Dialysis Dilution Chromatographic - ion exchange, gel permeation Diafiltration - stirred cell, tangential filtration Arista Biologicals Inc

Latex Particle Conjugation Cont… Passive Absorption Cont … Optimization of Conjugation conditions pH - Optimal conditions are generally not difficult to determine for a wide range of proteins. Typically optimal coating found at neutral to slightly basic conditions Mass - The amount of protein to conjugate will depend on particle size because of surface area considerations and must be determined experimentally. Typically for antigen assays the general rule is more is better until saturation is observed but in the case of hapten assays the opposite may be true in order to get the proper displacement curves. Coating buffer - Typically phosphate or glycine buffers are acceptable. This is generally not one of the most important variables. Many people use elevated temperatures for coating in order to increase the speed of conjugation and get a smoother preparation Arista Biologicals Inc

Latex Particle Conjugation Cont… Passive Absorption Cont … Optimization of Conjugation conditions Blocking - This can be very critical depending on the other buffers used in preparing other components of the final assay system. Typically proteins or polymers used such as BSA, PEG, PVA, PVP etc. By varying these materials and amounts one can increase sensitivity and decrease nonspecificity. Molecular weight of these materials can also alter their effects. Washing - Generally centrifugation, gel permeation, or tangential filtration method are used Diluent - Critical for stability of reagent and can effect sensitivity and specificity. Typically phosphate and glycine buffers used. Generally coating and diluents can be similar in makeup. Arista Biologicals Inc

Latex Particle Conjugation Cont… Covalent Absorption Choice of Ligands and Chemistry Carboxyl - CDI type reagents are the most common reagents used Primary amine - CDI or glutaraldehyde are commonly used Others Optimization Linker - The type of coupling reagent must be compatible with starting material One step vs. Two step methods - The one step methods are the easiest where conditions are optimized to add the latex, coupling agent, and the protein to be conjugated all add the same time and then just blocking and washing is performed. In the two step method the latex or protein is first activated by the coupling agent and then the excess coupling agent is removed prior to the addition of the final component. This increases coupling efficiency in many cases. This can be difficult to perform with reagents which react with water etc. Mass - This must be optimized as with the passive absorption methods Coupling buffers and conditions - Similar to passive absorption methods. However must stay away from buffers containing similar chemical structures to those being coupled. You must also use conditions to minimize crosslinking of reactants. Washing - Similar to passive absorption methods Blocking Conditions - Similar to passive absorption methods Final Diluents - Similar to passive absorption methods Arista Biologicals Inc

Colloidal Gold Conjugation Passive Absorption Production of Colloidal Gold Particles Determine size of particle desired - Typically 20-40nm particles are used in these membrane based formats. The larger the particle the stronger the color observed. The smaller the particle the more reddish in color. The larger the particle the more purple in color observed. The larger the particle the less stable and more difficult to reproduce. Determine which reducing agent desired - Typically citric, tannic or malic acid are used. Determine pH, temperature, and other conditions required for proper particle production. Arista Biologicals Inc

Colloidal Gold Conjugation Cont… Passive Absorption Cont... Optimization of Conjugation Conditions Covalent vs. noncovalent conjugation pH - Optimal conditions are generally more difficult than latex to determine for a range of proteins. Must be determined experimentally for each new protein. Mass - The amount of protein used to conjugate will depend on particle size because of surface area conditions and must be determined experimentally. Typically for antigen assays the general rule is more is better until saturation is observed but in the case of hapten assays the opposite may be true in order to get the proper displacement curves. Coating Buffer - Typically carbonate and other soft buffers used Blocking Buffer - Proteins such as BSA or large molecular polymers such as PVA, PEG, PVP, etc. are generally utilized Washing - Centrifugation or ultrafiltration are the most common methods used to remove free protein. Diluent - Soft buffers such as phosphate, glycine, etc. used. Must retain both immunological and colloidal stability Arista Biologicals Inc

Types of Assays Utilizing the Membrane Formats Antigen Detection Sandwich Assays Polyclonal-Monoclonal Pairs Monoclonal - Monoclonal Pairs Antibody Detection Assays Antigen - Antibody Pairs Antigen - Antigen Pairs Competitive Hapten Assays Antigen - Antibody Pairs Arista Biologicals Inc

Monoclonal or Polyclonal Antibodies? Monoclonal Antibodies These antibodies are the most reproducible and in most cases are the highest in activity. They are relatively more expensive than polyclonal antibodies and are not always available or the best material. Polyclonal Antibodies These antibodies are usually more available and less expensive than the monoclonal antibodies. They have a wider range of specificity which can be a good or bad thing depending on the situation. Special care must be be taken to ensure reproducibility. There is always a danger when changing lots or sources. Affinity Purification of Polyclonal Antibodies In most cases it is necessary to affinity purify polyclonal antibodies to increase sensitivity and decrease nonspecificity in these types of systems. These systems require approximately 100 to 1000 fold more activity than ELISA and RIA type system. Arista Biologicals Inc

Recombinant Versus Native Antigens Recombinant Antigens These type of antigens are generally very easy to reproduce and in many cases give superior sensitivity and specificity over native antigens. Depending on the antigen it may be more or less expensive than native material. Variation can be observed lot to lot. Native Antigens In many cases are easier to find a good source for. In some cases give superior specificity when a recombinant does not contain all variants of an antigen. In some cases can be less expensive. Some variation from lot to lot. Synthetic Peptides These materials are almost always the least expensive choice if available. However they may lack the immunological activity of the above choices and may need to be used in mixtures in order to achieve the specificity requirements. These materials usually are required to be conjugated prior to immobilization. This is the fastest growing source for antigens used in rapid membrane diagnostics. Arista Biologicals Inc

Purity and Conjugation of Proteins Utilized for Immobilization Purity Controls specificity in the case where a contaminant can cause false positive results. Can also cause sensitivity problems since in many cases we are straining for sensitivity in these formats and impurities compete for sites on the membrane or conjugate. Conjugation Prior to Immobilization In many cases this increases immunological reactivity acting as a spacer between antigen and the solid support providing better access for antibody binding. Arista Biologicals Inc

Nonspecificity Reactions and Imcompatibility Issues Antibody-Antibody Interactions In many cases nonspecific interactions occur between two different antibodies in these assays. In most cases it is an attraction or nonspecific interaction due to heterophile type reactions or rheumatoid factor type interactions especially when two or more species are involved. Using additives such as free IgG, aggregated IgG, mouse blocking reagents, and even surfactants can reduce these interactions. Fragmentation of antibodies also is used to remove these interactions since Fc portions of these antibodies cause these interactions in many cases. Arista Biologicals Inc

Nonspecificity Reactions and Imcompatibility Issues Antibody-Antigen Interactions In most cases this is caused by contamination of antigens with other material such as E. coli in some recombinant antigens used in antibody assays. Increased purity or addition of serum, surfactants, blocking reagents can reduce these type of interactions. Conjugate-Membrane Interactions In many cases materials nonspecifically bind to conjugates or cause conjugates to aggregate and bind or get stuck to the membrane. By varying the blocking reagents used such as polymers, surfactants, and proteins it is generally easy to reduce these interactions. In other cases it is usually an antibody or antigen interaction which is actually being observed. Arista Biologicals Inc

Cost and Reproducibility Issues Cost Since we use approximately times more reagents in these assays, cost of reagents is more critical than in EIA and RIA systems. You must identify inexpensive but reliable sources for material. Before you test a material for acceptability it should be kept in mind whether it is reasonable to use the material for production Reproducibility The largest problem once you have developed the test and begin producing in production quantities multiple times is getting a source capable of supplying the material in large orders time after time. In many cases for large production type assays it is not unusual to require grams of antibodies and antigens or liters of conjugate. Arista Biologicals Inc