Assay Development Protocols

Sample Proposal 1
Monoclonal – Polyclonal Sandwich Assay Development

Required materials and reagents to begin assay development:
1.Analyte.
2.Antibodies.
3.Analyte negative samples.
4.Analyte positive samples.

Step I: Reagent Preparation and Initial Assay Titration
1.Labeling of specific polyclonal antibody (with HRP, for example) for assay signal generation.
2.Selection of compatible antibodies.
3.Determination of a working titration of capture monoclonal antibody and detection polyclonal antibody conjugate.
4.Development and optimization of ELISA plate coating procedures for optimal antigen capture.

Step II: Assay Optimization
1.Selection of proper diluents for sample and conjugate signal generation.
2.Development of special additives to eliminate sample matrix effects such as interference and non-specific binding.
3.Construction of a standard curve to mimic performance characteristics of the sample matrix.
4.Development of a functional assay protocol within the target sensitivity range of the analyte.
5.Elimination or modification of extraction or sample preparation steps.

Step III: Assay Validation
1.Definition and documentation of assay performance characteristics essential for optimal assay utility (such as sensitivity and precision).
2.Documentation of diluent performance parameters to assure proper matrix composition (such as recovery and linearity).
3.Fine-tuning of specific assay components and incubation protocols to meet final performance requirements.

Step IV: Production of 25 Finished Kits
1.Quality control assessment of final components.
2.Final assembly, packaging, and delivery of components for 25 ELISA kits.

Total Assay Development Cost: range of $10,000 – $15,000
Estimated Time: 15-31 weeks

Sample Proposal 2
Antigen-Coated Microtiter Plate Assay Development

Required materials and reagents to begin assay development:
1.Analyte.
2.Antibodies.
3.Analyte negative samples.
4.Analyte positive samples.

Step I: Reagent Preparation and Initial Assay Titration
1.Labeling of specific polyclonal antibody (such as goat IgG anti-human IgE) with HRP) for assay signal generation.
2.Determination of a working titration of capture antigen and detection polyclonal antibody conjugate.
3.Development and optimization of ELISA plate coating procedures for optimal antibody capture.

Step II: Assay Optimization
1.Selection of proper diluents for conjugate signal generation.
2.Development of special additives to eliminate sample matrix effects such as interference and non-specific binding.
3.Construction of a standard curve to mimic performance characteristics of the sample matrix.
4.Development of a functional assay protocol within the target sensitivity range of the target antibody.
5.Elimination or modification of extraction or sample preparation steps.

Step III: Assay Validation
1.Definition and documentation of assay performance characteristics essential for optimal assay utility (such as sensitivity and precision).
2.Documentation of sample performance parameters such as dilution and linearity and nonspecific background signal generation.
3.Fine-tuning of specific assay components and incubation protocols to meet final performance requirements.

Step IV: Production of 25 Finished Kits
1.Quality control assessment of final components.
2.Final assembly, packaging, and delivery of components for 25 ELISA kits.

Total Assay Development Cost: range of $10,000 – $15,000
Estimated Time: 15-31 weeks

Sample Proposal 3
Competitive Inhibition 96-Well Micrometer Plate ELISA Development

Required materials and reagents to begin assay development:
1.Analyte.
2.Antibodies.
3.Analyte negative samples.
4.Analyte positive samples.

Step I: Reagent Preparation and Initial Assay Titration
1.Selection of compatible monoclonal antibodies and labeled analyte.
2.Titration of capture antibody and labeled analyte to working dilutions.
3.Development and optimization of ELISA plate coating procedures for optimal antigen capture and competitive inhibition of labeled antigen binding to the plate-bound capture antibody.

Step II: Assay Optimization
1.Selection of proper diluents for sample and conjugate signal generation.
2.Development of a functional assay protocol within the target sensitivity range of the analyte.
3.Construction of a functional standard curve to mimic performance characteristics of the sample matrix.

Step III: Assay Validation
1.Definition and documentation of assay performance characteristics essential for optimal assay utility (such as sensitivity and precision).
2.Documentation of diluent performance parameters to assure proper matrix composition (such as recovery and linearity).
3.Fine-tuning of specific assay components and incubation protocols to meet final performance requirements.

Step IV: Packaging of Final Test Kit Product
1.Quality control assessment of final kit components.
2.Final assembly, packaging, and delivery of components for 25 immunoassay kits.

Total Assay Development Cost: range of $10,000 – $15,000
Estimated Time: 21-27 weeks

Sample Proposal 4A
One Step Immunoassay Development

Required materials and reagents from Client:
1.Capture antibody and detection antibody pair.
2.Purified analyte.
3.Analyte positive samples.
4.Analyte negative samples.Extraction buffer.
5.Any existing assay kits or strips for comparative studies.

Step I: Reagent Preparation
1.Purification of analyte-specific monoclonal or polyclonal IgG.
2.Affinity purification of analyte-specific polyclonal IgG.
3.Conjugation of analyte-specific monoclonal IgG (if available) or polyclonal IgG to colloidal gold.
4.Preparation of blocking buffers.
5.Preparation of conjugate diluents.
6.Preparation of extraction buffers.

Step II: Selection of Materials and Assay Optimization
1.Selection of solid step membrane for best lateral flow results.
2.Selection of absorbent media for proper sample handling and flow-rate.
3.Selection of conjugate release media for efficient release of colloidal gold-antibody conjugates.
4.Selection of proper backing material for required rigidity.
5.Titration of capture antibodies and colloidal gold-antibody conjugates to obtain required sensitivity levels for each analyte.
6.Determination of proper blocking buffer to eliminate nonspecific background signals.
7.Development of user-friendly test protocol for field use.

Step III: Assay Validation
1.Definition and documentation of assay performance characteristics essential for optimal assay utility (such as sensitivity and precision).
2.Comparison of results to any current standard ELISA results.
3.Provide 100 test units to client for evaluation.

Total Cost: range of $10,000 – $15,000
Total Time: 8-14 weeks

Required materials and reagents from Client:
1.Several different affinity purified PoAb.
2.Purified natural analyte.
3.Purchased analyte for use in colloidal gold conjugations.
4.Positive samples.
5.Negative samples.
6.Any current tests for comparison.

Step I: Feasibility Study, Reagent Preparation
Each polyclonal will be assessed for its ability to bind analyte-colloidal gold conjugates. Upon completion of Step I, all reagents will be prepared.
1.Conjugation of the analyte to colloidal gold in 3 different sizes (3-6 nm, 8-12 nm, and 17-23 nm gold particles).
2.Preparation of blocking buffers.
3.Preparation of conjugate diluents.
4.Assessment of each PoAb to bind the analyte-colloidal gold.

Step II: Feasibility Study, Selection of Rapid Test Materials and Assay Optimization
All antibodies that successfully bind the analyte-colloidal gold conjugate will be used during this step. The antibodies will be examined individually and in combinations. Step III will begin when the required sensitivity level is reached.
1.Selection of solid phase membrane for best lateral flow results.
2.Selection of absorbent media for proper sample handling and flow-rate.
3.Selection of conjugate release media for efficient release of conjugate
4.Selection of proper backing material for required rigidity.
5.Titration of capture antibodies and analyte-colloidal gold conjugates to obtain required sensitivity levels.
6.Selection of proper blocking buffer to eliminate nonspecific background signals.
7.Development of a user-friendly test protocol for field use.

Step III: Assay Optimization and Validation
Upon completion of Step III, the assay and assay protocol will be complete. All documentation will be in place for manufacturing and the assay will be in its final form.
1.Definition and documentation of assay performance characteristics essential for optimal assay utility (such as sensitivity and precision).
2.Determination of cross-reactivity to other molecules, as recommended by the client.
3.Comparison of results to existing test methods.
4.Final adjustments to the assay protocol.

Step IV: Production and Delivery of 10,000 Tests
1.Begin pilot scale manufacturing.
2.Quality control assessment of final components.
3.Final assembly, packaging, and delivery of components for 10,000 rapid test kits.

Total Cost: range of $10,000 – $15,000
Total Time: 15-24 weeks

Typical Components of an Sandwich Immunoassy

Antagene Inc is a custom service laboratory specializing in antibody production and immunoassay development. Once developed, we will ship the finished test to you so it can be used on-site by your staff, or in the field by your customers. We will then manufacture the components of the test whenever you need more. A typical 96-well microtiter plate sandwich immunoassay kit may include the following components (there are several different formats of an immunoassay; only 1 is presented here), Click to view the components:

Pre-Coated, Stabilized 96-well Microtiter Plate:For microtiter plate assays, the plates are provided ready-to-use. They are pre-coated with the capture antibody, blocked, stabilized, and packaged in a resealable foil pouch with a desiccant packet. Using our methods, the plates may have a real time stability of 1 year when stored at 2^o – 8^o C. With a standard curve (1 blank and 7 standards) and 3 controls, a 96-well microtiter plate format can test 21 samples in triplicate and 37 samples in duplicate.

Sample Diluent:
The sample diluent is used to ensure that the analyte in the sample matrix is measured accurately. It is used to dilute the sample within the target range of the assay.

Assay Diluent:In addition to a specimen diluent, certain matrixes require the use of a special assay diluent (which is applied to the plate just prior to adding the samples, standards, and controls). Assay diluents are often paired with a specific type of sample (such as serum, or cell culture media) to eliminate interference and non-specific binding generated from the matrix of the sample itself. These interferences are especially noticeable when running neat samples. An assay diluent may not be required for every assay.

Calibrator Diluent:
A calibrator diluent is used to ensure that the standards and controls will be measured accurately. This diluent must compliment the target analyte, capture antibody, and resemble the matrix of the sample.

Standards and controls:
A known amount of the analyte (such as 1 ng) is included with a kit and run on every plate. The standard is often lyophilized. Once reconstituted, it is diluted several times to prepare a range of known values with which to compare and properly quantitate the unknown amount of analyte in a sample. Controls are used to confirm the readings of the standard and to compare readings from different plates. Because they contain a known amount of analyte, they should always read within a certain optical density (OD) value based on the standard.

Conjugated Detection Antibody:
For some assays, the detection or ï¿½topï¿½ antibody is often an affinity purified polyclonal antibody conjugated to HRP. The enzyme-antibody conjugate can often be supplied ready-to-use in its working concentration in a special conjugate diluent. The conjugate diluent stabilizes the conjugate and minimizes nonspecific binding of the conjugate onto the blocked plate or matrix residue. The working conjugate will often have a real-time stability of 1 year when stored at 2^o-8^o C .

10X Wash Solution:This specially formulated buffer is used to rinse the plate after the sample and conjugate incubation periods, just prior to the addition of the next reagent. It minimizes matrix residue and non-specific binding interferences of the samples and conjugate.

Single Component Substrate:This reagent reacts with HRP to generate a colored signal product. It can come in many formulations (powdered, 2-component liquid, etc.). Antagene recommends a low-background, high-signal-generating ABTS or TMB that needs no preparation prior to use.

Stop Solution:
An appropriate stop solution is added to the plate with the ABTS or TMB substrate and stops its reaction with HRP. By stopping this reaction after 20 minutes, the plates can equilibrate before reading, which increases the accuracy and sensitivity of the assay.

Typical Immunoassay Protocol

Preparation
     In this example, 1 blank (which is simply the sample diluent), 7 standards, 3 controls, and 37 unknown samples are being tested in duplicate in 1 microtiter plate MoAb-PoAb sandwich immunoassay.

Standards
     As each kit typically comes with only 1 vial of a high-concentrate standard which is lyophilized (freeze dried into a powder), it must be reconstituted, and then further diluted with sample diluent. If the lyophilized standard was at 5000pg, when reconstituted with 5mL, it would have a concentration of 5000pg/5mL or 1000pg/mL. To generate a standard curve, it should be serially diluted 1:2 (500uL standard with 500uL sample diluent) to create standards at 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, and 15.13pg/mL.

Controls
     The controls may or may not come with a kit, but are often lyophilized and must also be reconstituted, typically with 2mL sample diluent. Controls are usually not diluted further.

Unknown Samples
     Any samples thought to have a value higher than the reconstituted standard (at 1000pg/mL) should be diluted with the sample diluent so that it falls between the high standard (at 1000pg/mL) and the low standard (at 15.13 pg/mL).   If they do not need to be diluted, samples often can be run neat. However, if the samples have some sort of interfering substance, such as rheumatoid factors, or complement, they may need to be treated prior to running in the assay. If you do not know about your samples, simply run them in the assay once and see where they fall (neat and diluted samples can be run at the same time). If the values are too high, just dilute, and/or treat the samples and run the assay again. Each investigator must determine their own protocols for sample dilutions and pre-assay treatments.
Immunoassay Procedure

Step	Discription
1	Add 50 uL per well of assay diluent into every well of the plate.
2	Add 200 uL per well of your blanks, standards, controls, or samples onto the plate. Each item should be tested in duplicate (in 2 wells).
3	Cover with plate sealer and incubate 2 hours at room temperature.
4	Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
5	Add 200uL per well of PoAb-HRP conjugate solution into every well of the plate.
6	Cover with plate sealer and incubate 2 hours at room temperature.
7	Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4 cycles. Blot on paper towels.
8	Add 200uL per well of TMB substrate solution into every well of the plate.
9	Incubate 20 minutes at room temperature.
10	Add 50 uL per well 2N HCl stop solution into every well of the plate
11	Read plate at 450nm while subtracting a reference wavelength of 540nm.
12	Calculate data based on OD values of the unknown samples compared to the known values of the standard curve.