Guide for Eco-sensible ELISA Developmentkomabiotech.co.kr/www/techniques/pdf/ELISA_guide.pdf · The Guide for Eco-sensible ELISA Development starts with an overview of the differe

Guide for Eco-sensible

ELISA Development

Kem-En-Tec Diagnostics A/S www.kem-en-tec.com

Kem-En-Tec Diagnostics’ Guide for Eco-sensible ELISA

Development provides step by step information for

ELISA optimization in an environmentally sensible way.

It includes hints, tips and troubleshooting for the de-

velopment of assays based on chromogenic detection

of Alkaline Phosphatase (AP) and Horseradish Peroxi-

dase (HRP).

The Guide for Eco-sensible ELISA Development starts

with an overview of the different types of ELISA for-

mats. This is followed by sections describing the steps

and the reagents involved in ELISA development and

the critical factors that need to be addressed for each

step in order to develop a successfully optimized ELISA.

Eco-sensible properties of Kem-En-Tec Diagnostics’

products have green highlights.

1

Table of contents

TABLE OF CONTENTS ................................................................................................................................... 1

INTRODUCTION TO ELISA ............................................................................................................................ 2

1. ELISA DESIGN ...................................................................................................................................... 4

2. COATING ............................................................................................................................................ 9

3. WASHING ......................................................................................................................................... 10

4. BLOCKING AND STABILIZING ELISA PLATES ....................................................................................... 11

5. DILUTION OF SAMPLES, STANDARDS & CONTROLS .......................................................................... 14

6. DILUTION AND STABILIZATION OF THE CONJUGATE ......................................................................... 16

7. ENHANCED CONJUGATES: AMPLIFICATION SYSTEMS ....................................................................... 19

8. SUBSTRATES ..................................................................................................................................... 21

ELISA OPTIMIZATION ................................................................................................................................ 24

EXAMPLE OF ECO-SENSIBLE ASSAY OPTIMIZATION: SANDWICH ELISA ..................................................... 25

DEVELOP YOUR ASSAY STEP-BY-STEP WITH KEM-EN-TEC DIAGNOSTICS A/S ............................................ 26

Look for catalogue numbers in the end of this guide and ask for samples!

Abbreviations

Ab Antibody Abs Absorbance Ag Antigen AP Alkaline Phosphatase ELISA Enzyme Linked Immunosorbent Assay HAMA Human anti-mouse antibodies HRP Horseradish Peroxidase NSB Non Specific Binding OD Optical Density PBS Phosphate-Buffered Saline RT Room Temperature RTU Ready-To-Use SA Streptavidin SNR Signal-to-Noise Ratio TBS Tris-buffered Saline

Legend for images

Antigens

Antibodies

Enzyme

Reacting enzyme

Substrate

Reacting substrate

Blocking agents

Biotin

Streptavidin

2

Introduction to ELISA

ELISAs are used to detect and quantitate peptides, proteins, antibodies and hormones in biologi-cal specimens. Antibodies (Ab) or antigens (Ag) are adsorbed to a solid surface and bind with high affinity their complementary reagents that are typically conjugated to enzymes. The enzymatic reaction triggered by appropriate substrates allows the quantitation of the analyte of interest.

ELISAs are useful tools because they allow for the rapid screening or quantitation of a large num-ber of samples. Other techniques have been developed, but ELISA remains popular because of the ease of performance of the assay, accuracy, and the low cost.

This guide is divided in eight sections that follow the traditional development of an ELISA.

1. ELISA Design This section introduces the most common ELISA formats, pre-senting their advantages and disadvantages. 2. Coating Suggestions for optimizing the coating of the solid phase are listed in this section. 3. Washing Wash out excess reagent to reduce background signal and in-crease the signal-to noise ratio (SNR). Guidelines for optimizing the washing buffer and the washing procedures are suggested. 4. Blocking and stabilizing ELISA plates The critical aspects to consider for preventing non-specific-binding (NSB) are discussed. Kem-En-Tec Diagnostics’ Synthetic Blocking Buffer and WellChampion are ideal reagents for block-ing and stabilizing ELISA plates. 5. Dilution of samples, standards & controls Specimens, standards and controls need to be diluted in specific buffers before adding them in the microwells. Kem-En-Tec Diag-nostics’ SamplePLUS2, Effect Diluents, and Protein-StabilPLUS are the ideal diluents for reducing cross reactions and interfer-ences. The use of Ab’s is also described in this section. 6. Dilution and stabilization of the conjugate Specific diluents are used for achieving the optimal concentra-tion of conjugated Ab’s or Ag’s to be added in the assay. Most of Kem-En-Tec Diagnostics’ diluents also have stabilizing properties to prevent loss of activity. The Alkaline Phosphatase (AP) and Horseradish Peroxidase (HRP) labels are also described. 7. Enhanced conjugates: amplification systems Kem-En-Tec Diagnostics has developed an amplification system based on the biotin-streptavidin binding, which enables 100 fold signal enhancement. 8. Substrates Adding the substrate is the last step in an ELISA. This guide fo-cuses on chromogenic substrates for AP and HRP. The broad range of Kem-En-Tec Diagnostics’ pNPP and TMB substrates is described in details for the highest level of optimization.

Figure 1. A schematic description of the most common type of ELISA, the sandwich ELISA.

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Environmental considerations during assay development

Over the past decade, IVD manufacturers have been working towards more enviromentally con-scious and sustainable manufacturing. Much of this effort has been focused on reducing usage of facility water and energy use, reducing facility waste, increasing facility recycling, and sourcing recycled/recyclable product packaging. Eco-sensible options are now available for the develop-ment and production of diagnostics assays.

Many common components used in ELISAs contain biological and chemical hazards which were traditionally used to increase assay quality and to prolong assay shelf-life. Considering that the average automated immunoassay analyzer in a clinical lab can run 200 tests per hour while pro-cessing samples 24/7, these components can generate a considerable amount of hazardous waste. ELISAs have not traditionally been eco-sensible and several components in this type of assay are usually environmentally harmful:

Many components contain bovine serum albumin (BSA). This is a natural product from bo-vines and can carry disease. It is tightly regulated to avoid this issue. Kem-En-Tec Diagnostics totally avoids using BSA in its products. For instance Kem-En-tec Diagnostics has developed proprietary blockers which are more eco-sensible and block with high efficiency as well.

Reagents can also contain toxic preservatives such as mercury, azide and other toxic compo-nents which can be environmentally harmful. Kem-En-Tec Diagnostics avoids using any toxic chemicals as preservatives, but still maintains long product stability.

In order to maintain stability, substrates can be solvent based. Kem-En-Tec Diagnostics sub-strates are all aqueous based and contain no harmful organic solvent, but still maintain supe-rior activity and stability.

The acid used to stop the ELISA reaction can be hazardous (ex. greater than 0.5M H2SO4). Kem-En-Tec Diagnostics TMB is formulated to require a non-hazardous concentration of sul-phuric acid for stable stop stability, less than 0.3 M H2SO4.

During the execution of ELISAs, water is wasted in the washing steps. Kem-En-Tec’s Well Champion alleviates the need for 2 to 3 washing steps in the normal assay procedure allow-ing for less water wasting.

Kem-En-Tec Diagnostics’ eco-sensible options do not compromise assay stability or quality, and can reduce production costs. In some cases, non-hazardous alternatives can enhance perfor-mance, simplify assay production, and facilitate product transportation and distribution. The de-velopment and use of eco-sensible approaches enable individual IVD manufacturers to achieve more sustainable practices and allows other members of the IVD ecosystem (including the healthcare consumer) to improve their environmental sustainability as well, so that in the end, everyone benefits.

4

1. ELISA Design

ELISA procedures currently used for diagnostic testing are developed as combinations and modifi-cations of a number of basic assay formats.

Figure 2. A representative diagram showing the different formats used for making an ELISA

Type of antigen immobilization

The immobilization of the antigen of interest can be accomplished by direct adsorption to the assay plate. This normally occurs through passive hydrophobic interactions between the plate and non-polar protein residues of the antigen. Alternatively a capture antibody, i.e. a primary anti-body that recognizes an epitope of the antigen or an anti-Ig (or protein A/G), is adsorbed to the plate and can be used to immobilize the antigen of interest. Using a capture antibody is normally the preferred method when the antigen concentration is low and when the antigen does not ad-here properly to the solid phase.

Figure 3. Schematics of the two types of analyte immobilization. Left) An example of direct adsorption: the antigen is adsorbed to the microwell surface; Right) An example of immobilization via a capture antibody: the capture antibody is adsorbed on the microwell surface and binds to the antigen to be detected.

ELISA

Antigen Immobilization

Direct Adsorption

Capture Antibody

Assay

Type

Competititve Non -

Competitive

Detection Method

Direct Indirect

5

Competitive vs. Noncompetitive Format

ELISAs can be designed in a competitive or a noncompetitive format. The difference is based on the presence of excess (noncompetitive) or limiting (competitive) antibody binding sites.

Competitive Noncompetitive

Plate adsorption

Figure 4. Competitive assays can be performed either using direct adsorption or capture Ab.

Figure 5. Noncompetitive assays can be based either on direct adsorption or capture Ab.

Format descritpion

When the reference Ag is bound to the solid phase, the sample Ag competes with it for a limited amount of labeled Ab in solution (Figure 4, Direct adsorption). When the Ab is adsorbed on the solid phase, the labeled reference Ag in solution competes with sample Ag for binding to a limited amount of Ab (Figure 4, Capture Ab). It is essential for the Ab to be in limited quanti-ties in the assay. This allows for the reference Ag and the sample Ag to compete for the limited Ab’s binding sites. If there is a saturat-ing amount of Ab, a small amount of Ag might show no detectable change.

The Ab binding sites exceed the number of Ag being detected (Figure 5). This property makes the noncompetitive format the most sensitive.

Advantages Better for quantitation

Preferred in case of small Ag size or when the Ag has only one antibody-binding site.

Can be highly specific, even in the presence of cross-reactivity

More sensitive assay

Can be used to assay multi-epitope mole-cules

Disadvantages Only a limited amount of Ab can be used Not recommended when the Ag is small and has only one epitope or Ab-binding site

Quantitation A decrease in signal indicates the presence of the Ag in the sample when compared to wells without sample. This happens because the sample Ag and the reference Ag compete for the same Ab binding site. Quantitation is ob-tained by generating a standard curve of con-centration of added reference Ag vs activity. The activity in the standard curve is inversely related to the Ag concentration in the sample.

Quantitation is obtained by comparing the activity obtained with a sample to the activity measured from a standard curve made with reference Ag. Since the measured signal is proportional to the concentration of Ag pre-sent, this assay is often referred to as a pro-portional ELISA. The activity in the standard curve is directly proportional to the Ag con-centration in the sample.

6

Direct vs Indirect Detection

The antigen can be detected directly by means of a labeled primary antibody or indirectly by means of a labeled secondary antibody that binds to the unlabeled primary antibody.

Direct detection Indirect detection

Plate adsorption

Figure 6. Direct detection in an assay based on direct adsorption of the Ag: the primary Ab is conjugated to the enzyme and binds to the Ag to be detected.

Figure 7. Indirect detection in an assay based on direct adsorption of the Ag: a secondary Ab is conjugated with the enzyme and binds to the primary Ab that had specifically reacted with the Ag to be detected.

Format descritpion

In a direct ELISA the enzyme is coupled to the primary/detecting Ab that binds directly to the Ag. It is not frequently used in ELISAs.

In an indirect ELISA the enzyme is conjugated to a secondary Ab that is specific for the de-tection of primary Ab only, and not for the eventual capture Ab. In order to avoid interferences, capture and primary antibodies should be from different host species.

Advantages Only one Ab is used

No cross-reactivity of secondary Ab

Simple, quick assay procedure

The same Ab-conjugate can be used to analyze different samples if all the primary Ab’s are from the same species

There is a large choice of labeled secondary Ab’s available in the marketplace

maximum immunoreactivity of the primary Ab is retained because it is not labeled

Signal amplification can be obtained as a consequence of the several epitopes availa-ble on each primary Ab that can be bound by the labeled secondary Ab

Possibility to use different detection mark-ers with the same primary Ab

Disadvantages Potential matrix effects (interferences of an unknown cause from a biological sample)

Labeling of the primary Ab can affect its immunoreactivity

Each new ELISA requires the labeling of a primary Ab (a time-consuming and expen-sive process)

Limited signal amplification

Requires an additional incubation step

More non-specific binding issues due to the cross-reactivity of the secondary Ab.

7

Sandwich ELISA

The most common type of diagnostic ELISA is the sandwich assay. The sandwich ELISA is a combi-nation of different formats that results in a powerful detection tool for diagnostic markers. It is a typically non-competitive method where the immobilization of the antigen is accomplished via a capture antibody, so that the antigen is bound between two antibodies (from here the name “sandwich”). The detection can be direct or indirect.

Figure 8. A representative diagram showing the formats used to design a Sandwich ELISA

The label on the secondary antibody can vary, but most commonly is the horseradish peroxidase (HRP) or the alkaline phosphatase enzyme (AP). Detection and quantitation of the bound labeled secondary antibody is accomplished by assessing its enzymatic activity in the presence of a sub-strate.

Figure 9. Schematic of a direct sandwich ELISA and an indirect sandwich ELISA: the analyte is immobilized using a cap-ture Ab adsorbed to a microwell plate. In a direct sandwich ELISA a labeled secondary Ab is used to “sandwich” the analyte. In an indirect sandwich ELISA a secondary Ab “sandwich” the analyte and a labeled Ab specifically binds the secondary Ab. Washing occurs between each step of the process, allowing for only analyte-specific complexes to re-main bound to the plate.

Sandwich ELISA

Antigen Immobilization

Capture Antibody

Assay

Format

Non -Competitive

Detection Method

Direct Indirect

8

An overview of procedures for the most common ELISA formats

9

2. Coating

The coating step refers to the passive adsorption of the Ab or Ag to the assay solid phase through hydrophobic interactions, van der Waals forces, hydrogen bonding and ionic interactions. Optimal coating depends on a number of conditions such as the affinity of the biomolecule to the solid phase surface, the coating buffer and its pH, the coating time and temperature.

The solid phase

The market offers a variety of microtiter plates made in polystyrene or derivatives of polystyrene.

Coating of hydrophobic molecules is recom-mended on what are traditionally called “low binding” plates (bind up to 100-200 ng of IgG/cm2), while coating of hydrophilic mole-cules is recommended on “high binding” plates (bind up to 400-500 ng of IgG/cm2). “Medium binding” plates are generally pre-ferred when the background signal needs to be reduced.

Different plates should be tested while opti-mizing an assay in order to reach the desired binding capacity and to aim to a CV ≤ 5% over the plate.

Coating buffers and pH

Coating solutions are prepared by diluting the coating protein in an alkaline buffer at the desired concentration. Some proteins may require specific conditions or pretreatment for optimal binding to occur.

Optimal adsorption occurs at pH 7-9 in a salt concentration appropriate for maintaining the solubility and the native conformation of the biomolecule. Detergents should be avoided in the coating buffers, since they often compete for binding and cause low, uneven binding of the coated protein.

Coating time and temperature

In order to have a thorough adsorption and minimize well to well variations, the plate should be incubated overnight (16-18 hours) at 4° C after the coating solution is applied.

Hints and Tips

Optimize the protein concentration

For Noncompetitive assays:

Use a coating solution with protein con-centration of 2-10 μg/mL

Use more protein than can be bound during the assay

For Competitive assays:

Lower the coating concentration

Ensure that the Ab is the limiting factor

Avoid “Hooking” (background) In case of “hook” effects, use concentra-tions lower than the plate's maximum bind-ing capacity.

Kem-En-Tec Diagnostics recommends

All plates and reagents should reach RT or reaction temperature before coating.

Do not pile the plates, since this may cause “edge effects”.

Seal plates during incubation to avoid evaporation from the wells.

Choose the coating buffer

Buffer for most biomolecules:

100 mM K2HPO4, pH 7.2

Cheap and easy-to-make

Buffer for Ab’s on “high binding” plates

10 mM PBS, pH 7.2

Buffer for antigens

50 mM carbonate buffer, pH 9.6


Increasing the adsorption’s speed

Incubating at RT for 2-3 hours, ensuring even heating across the plate

Putting the plate on a shaking table


10

3. Washing

Washing is necessary between each step of the ELISA to remove excess non-bound reagents and thus reduce background signal and increase the SNR. Washing is performed by filling the wells of the microtiter plate entirely with water-based buffers.

Insufficient washing may result in high background, while excessive washing may determine a decrease in the assay sensitivity.

Washing buffer

The washing buffer is a physiologic solution that must avoid denaturation of the binding partners in an ELISA step and preserve en-zyme activity.

As long as the pH of the washing buffer is at about 7-8 and the ion strength is kept low (e.g. 0.1 M), a variety of buffers will work depending on the assay under development.

Buffers such as Tris-buffered saline (TBS), phosphate-buffered saline (PBS), Imidazole-buffered saline or K2HPO4-NaCl are usually used. To aid in decreasing and avoiding non-specific binding, a detergent such as 0.1%-0.05% Tween20, Triton X-100 or a milk pro-tein such as casein is typically included in the washing buffer.

Washing procedure

Wash should be performed 3 - 5 times after each incubation step in order to disrupt low affinity nonspecific interactions.

Manual washing Allow a few minutes soak with washing buffer at each washing step. Flip the plate upside down on dry paper towels and tap out the excess washing buffer. Repeat the washing and tapping cycles without letting the plate dry between the wash steps. Drying can cause reduction of activity.

Automatic washing Program the plate washer to repeat at least 3 cycles of washing and aspiration.

Hints and Tips

With background issues add extra detergent or protein to the washing buffer.

Add a final washing step using a buffer without detergent if detergent affects the activity of the enzyme.

If background issues persist, use a diluted solution of the blocking buffer along with some added detergent. For best results, use detergents to prevent contamination with enzyme inhibitors or peroxides.

Optimize the washing buffer

For issues with AP

Never use a phosphate buffer

Use a Tris buffer with high pH (8.4 – 8.5)

For issues with HRP

Never use sodium azide (NaN3) as pre-servative

o It is toxic o It denatures HRP

For all enzymes

Use an imidazole buffers (can increase the HRP activity)


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4. Blocking and stabilizing ELISA plates

Blocking

After coating, the unoccupied sites on the microplate’s wells surface must be blocked in order to reduce/prevent non-specific binding in the next steps of the ELISA. If blocking fails it will result in high background, low specificity and low sensitivity, i.e. low signal-to-noise ratio (SNR).

Blocking buffer The blocking buffer is a solution of compounds that passively adsorbs to unoccupied binding sites on the surface of the plate. The ideal blocking buffer improves the SNR by:

Blocking all potential binding sites of nonspecific interaction

Reducing background signal therefore improving the assay sensitivity

Not altering or obscuring the epitope for Ab binding

When preparing a blocking solution, optimization of the blocking step is required. Improper opti-mization may results in excessive background when the concentration of blocking agent is too low; or in enzyme inhibition and masking of the Ab-Ag interactions when the concentration of blocking agent is too high.

Categories of blocking agents Traditionally there are two categories of blocking agents: detergents and proteins.

Most of the detergents used as blockers are non-ionic compounds that disrupt non-specific pro-tein interactions. Nevertheless detergents may disrupts hydrophobic interactions, may leave hy-drophilic sites unblocked or may interfere with the enzyme activity. Moreover detergents can be washed away by washing buffers that contain detergents.

Proteins block both the hydrophilic and hydrophobic sites on the solid phase but enhance the risk of cross-reactions, tend to be inconsistent from batch-to-batch and unstable in solution. A com-monly used blocking agent is bovine serum albumin (BSA), which is relatively inexpensive, effi-cient and biochemically inactive. However, BSA is heavily regulated worldwide because of the risk of contamination by the prion responsible for the bovine spongiform encephalopathy.

Synthetic Blocking Buffer for ELISA (# 4520)

Properties

Ready-to-use

BSA-free and protein-free

Superior quality

Advantages

Compatible with a wide variety of Ab’s

Highly effective and stable

High blocking capacity

Improves signal-to-noise ratio

Reduces the risk of false positive cross reactions

Instructions for use After the coating step, add the Synthetic Blocking Buffer and incubate the plate for 5-10 min. at RT. Remove the solution from the wells by aspiration or dumping.

Stability 4 years stored at 2-8°C


12

Plate stabilization

When storage of coated plates is required, a stabilization buffer is necessary in order to prevent loss of activity of the adsorbed biomolecules. This procedure traditionally did require few addi-tional steps in the assay development.

Drying conditions should be optimized for a particular molecules being adsorbed before full scale processing begins. This can be done by coating, drying and storing a set of plates for at least 1 - 2 days and comparing the activity to freshly made plates using the same batch of coating reagent. Traditional stabilization methods require the protecting buffer to be easy to remove with a wash-ing step prior the use of the stored plate: uneasy removal can cause interference with the assay signal (background).

Figure 10. A visual comparison between steps required for blocking as compared with the steps traditionally required for stabilization.

The ELISA production process generates a significant amount of water waste, caused mostly by the consumption of water during the wash steps. Water waste can be limited by reducing the number of washing steps in the assay and by combining ELISA steps wherever possible.

A new generation of novel dual-purpose reagents that combines the blocking step with the plate protection step in one single operation, can remove two washing steps from the ELISA procedure (Figure 10 and Figure 11). This dramatically speeds up both the coating and the stabilization pro-cesses and can completely eliminate the need for washing during pre-coated plate production.

13

WellChampion (# 4900)

One step blocking and stabilization that works with both hydrophobic and hydrophilic plates.

Figure 11. Traditional blocking and stabilization versus blocking and stabilization using WellChampion. A dual-purpose reagent as WellChampion combine the features of a blocking agent and a stabilizer for plates, eliminates two washing steps, and enhance the assay sensitivity and precision.

Properties

BSA-free and biologically inactive

Free of azide, mercury or other toxic preservatives

Stabilizes and preserves microwell plates coated with protein or other biomolecules

Efficiently blocks any free binding sites on the microwell surface without creating any inter-ference

Advantages

Eliminates intermediate washing steps; hence it dramatically speeds up plate coating and stabilization, and ultimately reduces water waste

Blocking and stabilization can be done in one step

Prevents degradation, denaturation and leaching

Improves assay sensitivity (OD) and precision over the plate

Instructions for use 1. After the coating incubation, DO NOT EMPTY WELLS. Fill the wells to the top with Well-

Champion. When using automatic equipment adjust the pump speed to ensure maximum mixing of the reagents.

2. Incubate for 10-15 minutes at RT. Precision (CV% values) can be improved by gently stirring. 3. Empty the plate by inversion or aspiration: DO NOT WASH. 4. Dry in darkness overnight at RT or for 3 hours at 37°C. 5. Pack the stabilized plate in an airtight bag with a desiccant. 6. It is recommended to evaluate whether it is necessary to prewash the plates prior to use.

Stability 4 years stored at 2-8°C. Avoid direct exposure to light.

Orange WellChampion (# 4901)

WellChampion is also available in an orange version. A colored plate supplied in an ELISA kit will indicate a uniform and effective pre-coated plate. Pre-staining does not affect the product performance; it will however show nice circles proving a uniformly coated plate.


14

5. Dilution of samples, standards & controls

Biological samples such as plasma, urine, saliva, serum, milk, cells and tissues extracts, contain the analyte to be quantified. The target antigen must also be present in known amounts in standard and control solutions.

Sample dilution

Samples tested in an immunoassay can require dilution for several reasons. Among others, these reasons include:

The Hook Effect can occur when an overwhelming amount of Ag is present in an assay and the sample reads as a false-negative. It is thought that the excess Ag binds to the receptor sites, preventing the conjugate antigen from being bound. Assaying a diluted serum will usually unmask this phenomenon and allow for an accurate measurement.

Complex sample matrices, such as serum and plasma, may contain interfering factors that af-fect the ability of the assay to accurately quantify the target analyte. Strong interferences are often caused by rheumatoid factors and HAMA’s. This matrix effect can cause high back-ground in the negative control or false negatives in the sample measurement. To reduce this effect the sample should be diluted, i.e. to show assay linearity.

The samples tested in an immunoassay might require dilution to be read within the dynamic range of the assay, i.e. to ensure that the signal response is within the standard curve.

It is important that buffers designed for sample dilution also reduces matrix effects and minimize nonspecific binding interactions in order to optimize the assay without affecting the correct bind-ing of the Ab or Ag.

Hints and Tips

SamplePLUS2 (# 4781)

Properties

Phosphate-free


Can be used for dilution of serum, plas-ma, as well as standards & controls

Advantages

Minimizes cross-reactions and non-specific binding without inhibiting specific binding

Contains proprietary synthetic, active and stabilizing components

Stability

2 years when stored at 2-8°C

Effect Diluent (# 5070, 5080, 5090)

Properties

Ready-to-use buffer for optimal discrimi-nation between specific and non-specific reactions

Proprietary buffer formulation with a 7.2 pH

Advantages

Offered in three versions with “Low”, “Medium” and “High” strengths for minimizing interferences from HAMA’s, heterophilic Ab’s and rheumatoid factors

Stability

2½ years when stored at 2-8°C


If dilution is greater than 1/1,000, make the dilution in two steps.

If 20 µl or less is required, make the dilution in a greater volume to assure accuracy.

Avoid making the dilutions in the wells, if possible. The foam created by mixing in the wells allows for protein degradation and inaccurate pipetting.

15

Standards & Controls

Standard curves are generated when an analyte concentration needs to be determined. A stand-ard curve is created by assaying increasing concentrations of a known analyte and plotting the activity generated (OD reading) versus the concentration.

In noncompetitive assays the measured signal is proportional to the concentration of the analyte while in competitive assays the measured activity decreases with the increase of analyte concen-tration. Quantitation of the sample’s analyte is obtained by interpolating the measured activity to the OD-values on the standard curve.

Diagnostics kits require both positive and negative controls. Assay controls are used to verify the assay performance and to ensure that the OD readings are expressed only by the nature and presence of the analyte. The negative control is used to distinguish the background reading from the matrix.

Protein stabilizers Stabilizers are available for preparing standards and controls in a pre-diluted, ready-to-use for-mat. This minimizes dilution errors in dissolving lyophilized standards used to determining the concentration of unknown specimens. It is highly important that a stabilization buffer for stand-ards and controls effectively protects a variety of proteins in solution.

Antibodies

The antibodies are the key actors in the development of an ELISA. In a sandwich ELISA it is im-portant that the antigen binding sites on the two antibodies are non-overlapping. A capture anti-body and a detection antibody that can bind to the same antigen simultaneously without inter-ference are considered to be a matched pair and can be used to successfully develop a sandwich ELISA. Both monoclonal and polyclonal antibodies may be used.

Type of antibody

Monoclonal antibodies Polyclonal antibodies

Specificity Single epitope Multiple epitopes of a single Ag

Advantages Increases the specificity of an assay

Allows for small changes in quanti-tation

Are used to gather as much antigen as possible

Protein-StabilPLUS (# 4720)

Properties

BSA-free

free of azide, mercury or other toxic preservatives

Tris-based buffer with proprietary stabilization components

Advantages

Stabilizes proteins, antibodies and biomolecules in solution

Allows for superior stabilization of pre-diluted standard and controls

Stability



16

6. Dilution and stabilization of the conjugate

Enzymatic detection method

There are several different types of detection technologies that can be used: radiolabeling, fluo-rescent labeling and enzymatic labeling. The most commonly used enzymes in ELISAs are Horse-radish peroxidase (HRP) and Alkaline phosphatase (AP). In order to be used in the assay, HRP and AP molecules must first be conjugated to the detection Ab or Ag. Consequently, the choice of several reagents depends on the enzyme.

Horseradish peroxidase HRP is derived from the root extracts of the horseradish plant. HRP uses H2O2 to oxidize both or-ganic and inorganic compounds. 3,3′,5,5′-tetramethylbenzidine (TMB) is one of the most popular chemical substrates used with HRP because of its high sensitivity and liquid formulation. As the TMB is oxidized by HRP, it turns from a clear color to blue. The intensity of this reaction can be quantitated by reading the absorbance at 650 nm. The kinetic reaction can be stably terminated by the addition of a stop-solution, which is commonly acid-based. Acid stop-solutions change the pH in the microplate’s wells and give a more stable yellow color for analyte measurement, ulti-mately increasing the color intensity by 2-3 fold. The reaction can then be quantified by the ab-sorbance reading at 450 nm.

Alkaline phosphatase AP is a zinc metaloenzyme which catalyzes the hydrolysis of an orthophosporic monoester to yield an alcohol and an orthophosphate. The main colorimetric ELISA substrate for AP is p-nitrophenyl phosphate (pNPP) which yields a yellow reaction product whose absorbance can be quantified by reading the OD at 405 nm. The kinetic reaction can be stably terminated by the addition of a stop-solution and quantified by measuring the absorbance.

Kem-En-Tec Diagnostics’ HRP is purified in large scale through the use of benzhydroxamic acid agarose by repeated affinity chromatography of root extracts from horseradish. The result is an enzyme of high purity and high specific activity.

Kem-En-Tec Diagnostics’s AP is purified from calf intestine by affinity chromatography through a method developed in Kem-En-Tec Diagnostics’ laboratories.

The antibodies or antigens conjugated with HRP or AP can be stored at 2-8°C either as a concen-trated stock solution or diluted in an appropriate buffer to the desired assay dilution range.

Horseradish Peroxidase (# 4120)

Properties

BSA-free


Lyophilized

The product consists mainly of the basic isoenzymes

Activity > 250 U/mg (25°C, Guaiacol as hydrogen donor)

Purity number of (A403/A275) = RZ > 3.0

Stability

4 years stored dry at -20°C

Alkaline Phosphatase (# 4700)

Properties

BSA-free


Contained in buffer: 5 mM Tris, 5 mM MgCl2, 0.1 mM ZnCl2, 50% Glycerol, pH 7

Activity > 1000 U/mg enzyme protein (25°C, 4-nitrophenyl-phosphate, glycin as buffer)

Gel filtration purified: 1 peak

Stability

18 months at 2-8°C


17

Diluents and stabilizers for the conjugates

When diluted biomolecules or conjugates need to be stored for future use, the buffer should also have stabilizing properties to maintain the molecular conformation and prevent loss of activity over time, finally providing ready-to-use working dilutions.

Universal stabilizers are available for the stabilization of both AP- and HRP-conjugates, and for the dilution of polymeric HRP and AP conjugates.

HRP-StabilPLUS (# 4530)

Properties

BSA free


Ready-to-use

Based on a Tris-based buffer with stabi-lizing components

Advantages

Provides superior stabilization of HRP conjugates at high and low dilutions

Cost effective

Allows for the conjugate to be stored at least 4 years at 2-8°C

Minimizes assay errors related to daily dilution variation

Stability


AP-StabilPLUS (# 4540)

Properties

BSA free


Ready-to-use AP stabilization buffer

Advantages

Provide superior stabilization of AP con-jugates at high and low dilutions

Cost effective



Stability



UNI-StabilPLUS (# 5230 - to come)

Properties

BSA free


Ready-to-use

Suitable for the stabilization of both HRP- and AP-conjugates

Advantages

Provides superior stabilization of HRP- as well as AP conjugates at high and low dilutions

Cost effective



Recommended for dilution of Ab’s directed against rabbit immunoglobulins

Stability



18

Stabilizers that are also able to enhance the binding capacity of the antibody allow further dilution of the conjugates, thus resulting in substantial savings on production costs, reduced background and increased SNR.

Hints and tips: choose the right buffer

The same diluent or stabilizer can be used to target different biomolecules and for different pur-poses. The following table presents the purposes that each Kem-En-Tec Diagnostics’ buffer is suit-able for and will guide you to the optimal choice.

Application

Buffer cat. No.

Standards & Controls

Samples HRP-

conjugates AP-

conjugates Antibodies

SamplePLUS2 4781 D D D D

Effect Diluent 5095 D D D D

Protein-StabilPLUS 4720 D/S D/S

HRP-StabilPLUS* 4530 D/S

AP-StabilPLUS 4540 D/S

UNI-StabilPLUS 5230 D/S D/S

Antibody Enhancer* 4660 D/S D/S D/S

Table 1. Applications of Kem-En-Tec Diagnostics’ buffers. The letter “D” indicates that the product can be used as a diluent for the corresponding application in the table heading; the letter “S” indicates that the product can also be used as a stabilizer. The buffers marked with the asterisk “*” cannot be used with antibodies directed against rabbit immu-noglobulin’s.

Antibody Enhancer (# 4660)

Properties

BSA free


Ready-to-use

Signal enhancing buffer

Advantages

Provides the highest SNR by increasing 40-80% the specific Ab activity without increasing the non-specific binding. Enhancement is observed only at high dilution (at least 1:2000).


Cost effective


Less conjugate can be used in the assay

Stability



19

7. Enhanced conjugates: amplification systems

In some applications the primary antibody is labeled with a molecule of biotin. Proteins with high affinity for the vitamin biotin, such as avidin and streptavidin (SA), strongly bind to biotin and can be labeled with enzymes in order to enable the colorimetric detection. Since the biotin molecule is easily conjugated to antibodies and enzymes, avidin-biotin methods and SA-biotin methods are a valid alternative:

when a secondary Ab specific for the primary Ab is not available

when signal amplification is required.

Streptavidin

Streptavidin (SA) is a protein produced by Streptomyces avidinii and isolated by purification from fermentation broth. Streptavidin consists of 4 identical subunits, each bearing an active binding site for the vitamin biotin, therefore it binds 4 moles of biotin per mole of protein with a very high affinity. The high affinity of the biotin-streptavidin interaction results in a very quick reaction with a high signal to noise ratio. The protein is used in combination with biotinylated antibodies in order to obtain amplified signals detecting sub-nanogram amounts of antigen.

Streptavidin can also be coated directly on the solid phase in the first step of an ELISA and used as a generic capture system for biotinylated antibodies with poor coating properties. Having plates pre-coated with streptavidin reduces the need for developing new coating methods.

In biotin-SA methods, primary or secondary antibodies are conjugated to molecules of biotin and finally link the antigen to labeled streptavidin molecules (Figure 12). The use of Streptavidin and SA based systems has many advantages over direct coupling of the antibody to the enzyme:

A single primary Ab can be associated with multiple HRP or AP molecules, with considerable increase in sensitivity.

Streptavidin conjugates are very stable.

Labeled SA systems are flexible and not specific to a single assay because they can be used with a variety of biotinylated Ab, without the need of searching for different secondary Ab specific for the primary antibody used in the ELISA.

Because of the high affinity between SA and biotin, the complex between the SA and the bio-tinylated molecule is rapidly formed and stable.

Streptavidin (# 4610)

Properties

BSA-free


Lyophilized in 50 mM NaCl (pH 9.0)

Activity > 13 U/mg (one unit binds 1 μg of biotin)

Purity: SDS – 1 band (non-reduced)

Molecular weight: 55.000 Dalton

Ip: slightly acidic, 5-6

Stability

No detectable decomposition within 18 months when stored dry at -20°C

Stable over a wide range of pH and decompose only in the presence of SDS > 60°C


20

Enhanced conjugates

Conventional biotin-SA systems are limited to few detection molecules per protein. Kem-En-Tec Diagnostics has developed conjugates specific for biotin with an enhanced molar ratio of enzymes covalently coupled to a polymer backbone. These enhanced conjugates enable low detection lim-its in immunoassays when only minimal amounts of specific Ab/Ag (pg/fg) are available, thus re-placing the conventional SA-HRP and SA-AP conjugates.

Figure 12. Conventional vs. Enhanced biotin-Streptavidin system: in the enhanced biotin-SA system, one of the SA mol-ecules linked to the polymeric backbone binds to the biotin molecule labeled to the primary Ab, which is detected by the amplified reaction of the multitude of enzymes linked to the polymeric backbone.

The Enhanced Streptavidin-AP also overcomes the conjugation and stability issues typical of AP, that are related to the large size of this molecule, which otherwise limits the number of AP en-zymes that can be labeled to an Ab or SA.

Hints and Tips

Enhanced Streptavidin-HRP (# 4740)

Properties

Streptavidin and HRP covalently coupled to a hydrophilic polymer backbone

Specificity: biotinylated probes

1 mg HRP/mL stabilized in TRIS buffer

Recommended dilutions: 1:1.000 to 1:100.000

Advantages

Signal amplification

Ultrasensitive detection

Faster assay time

Can be used with virtually any biotin labeled probe

Stability 2 years when stored at 2-8°C

Enhanced Streptavidin-AP (# 5150)

Properties

Streptavidin and AP covalently coupled to a hydrophilic polymer backbone

Specificity: biotinylated probes

1 mg conjugate/mL

Recommended dilutions: 1:1.000 to 1:100.000

Advantages

Signal amplification

Ultrasensitive detection

Faster assay time

Can be used with virtually any biotin labeled probe

Stability 2 years when stored at 2-8°C


Avoid intra-batch and inter-assay variations by preparing a pre-diluted stock solution of conjugate in the range of 1:1000 and 1:5000.

For optimal results, dilute the enhanced conjugates in UNI-StabilPLUS.

For further signal amplification, dilute the enhanced conjugates in the Antibody Enhancer.

21

8. Substrates

AP- and HRP-conjugates can be detected and quantitated using chromogenic substrates that re-sult in soluble, colored products when development takes place. The levels of the enzymatic reac-tion, and consequently the analyte’s concentration, are determined by monitoring the signal de-velopment at specific wavelengths. The most commonly used chromogenic ELISA substrates are 3,3',5,5'-Tetramethylbenzidine (TMB) for HRP and p-NitroPhenyl Phosphate (pNPP) for AP.

Substrate type Specificity Reaction

color Reaction read at

Stopped reaction

Stopped reaction read at

pNPP AP Yellow 405 nm Yellow 405 nm

TMB HRP Blue 620-655 nm Yellow 450 nm

p-NitroPhenyl Phosphate (pNPP)

Kem-En-Tec Diagnostics’s pNPP substrates are liquid buffer solutions containing stabilized pNPP. In the presence of AP, pNPP is hydrolyzed rapidly to p-nitrophenol and inorganic phosphate. The reaction results in a water-soluble yellow end product (p-nitrophenol) that absorbs light at 405 nm. The reaction can be stopped by adding an equal amount of 1 M sodium hydroxide (NaOH) solution and the absorbance read at 405 nm. The intensity of the color is directly proportional to the concentration of the reactant.

Figure 13. Stability data and Signal-to-Noise Ratio of Kem-En-Tec Diagnostics’ pNPP and pNPP ONE in a 2-layer ELISA.

0

10

20

30

40

50

Dako Rb a-Hu IgG/AP

SNR

Signal-to-Noise Ratio at day 1

0%

20%

40%

60%

80%

100%

120%

0 12 24 36

Re

mai

nin

g ac

tivi

ty

Months at 2-8°C

pNPP

pNPP ONE

Stability and relative sensitivity

pNPP (# 4400)

Properties

Free of harmful organic solvents

Development time: 15-30 minutes

Advantages

Ready-to-use, mono-component

Negligible batch-to-batch variations

Superior sensitivity (Figure 13)

Stability


pNPP ONE (# 4401)

Properties



Advantages

Ready-to-use, mono-component


Superior background characteristics that determine a higher SNR (Figure 13)

More robust and recommended in hot climate countries

Stability



22

Tetramethylbenzidine

Kem-En-Tec Diagnostics’ TMB solutions combine TMB, buffer and H2O2 for a ready-to-use, chro-mogenic system that yields a blue color whose absorbance is read at 620-655 nm. The reaction can be stopped with sulphuric acid resulting in a yellow color with an absorbance read at 450 nm and a 2-3 fold increase in sensitivity compared to the non-stopped absorbance reading. The stop-solutions recommended below guarantee an extended stop stability of the measured absorbance, with an OD decreases after 1 hour lower than 8%.

Prestained versions All TMB substrates are also available as unique color-coded products for traceable pipetting. The red indicator makes manual pipetting traceable, while in automatic analyzers, it offers a conven-ient tracking system for reagent supply. The color disappears after stopping and does not inter-fere with the assay.

TMB ONE (# 4380) and Prestained TMB ONE (# 4430)

Properties



Stop solution 0.2 M H2SO4

Advantages

Ready-to-use

Mono-component


Extremely stable and robust

Stability


1 year when stored at RT

TMB PLUS2 (# 4395) and Prestained TMB PLUS2 (# 4445)

Properties




Advantages

Ready-to-use

Mono-component


Cost effective

Improved version of the TMB PLUS

Stability




TMB X-tra (# 4800) and Prestained TMB X-tra (# 4810)

Properties




Advantages

Ready-to-use

Mono-component


High signal for a fast assay reaction

Resistant to raised temperatures

Stability



TMB SENS (# 4850) and Prestained TMB SENS (# 4860)

Properties




Advantages

Ready-to-use

Mono-component


Highly superior sensitivity

Fast assay development

Stability

2½ years when stored at 2-8°C

5 months when stored at RT

23

When selecting a substrate for ELISAs, a number of factors should be considered, including the level of sensitivity, the kinetics of the reaction, the shelf life, the temperature of storage and the content of harmful solvents. Kem-En-Tec Diagnostics’ TMB substrates cover a wide range of sensi-tivity levels in order to find the optimal TMB substrate for individual applications.

Figure 14. Compared sensitivity and kinetic curves of Kem-En-Tec Diagnostics’ TMB substrates. Maximum difference between TMB SENS and the other substrates is observed already after 10 minutes of development.

A trademark of Kem-En-Tec Diagnostics’ TMB substrates is the long shelf life both when stored refrigerated and when stored at room temperature (RT), thus allowing transportation at RT.

Figure 15. Kem-En-Tec Diagnostics’ TMB substrates can reach a 4 years stability at 2-8°C and a 1 year stability at RT.

No harmful components All Kem-En-Tec Diagnostics’ TMBs are aqueous based and contain NO harmful organic solvent. Non-hazardous acid concentration (<0.5 M H2SO4) recommended for stopping the TMB reaction do not require harmful labeling, thus avoiding issues in worldwide shipping of the RTU ELISA kits.

Adjustable to Immunoassay All Kem-En-Tec Diagnostics’ TMB products have corresponding diluents for achieving a specific activity level. The diluted TMB solutions maintain the same stability as the undiluted products.

Hints and Tips

0%

50%

100%

150%

200%

250%

Relative sensitivity

TMB ONE TMB PLUS2 TMB X-tra TMB Sens

0,00

0,20

0,40

0,60

0,80

1,00

0 10 20 30

OD

630

Minutes

Kinetic Curves - TMB Substrates

0 1 2 3 4

Years

Guaranteed shelf life at 2-8°C

TMB ONE

TMB PLUS2

TMB X-tra

TMB SENS

0%

20%

40%

60%

80%

100%

0 10 20 30 40 50

Weeks at RT

Stability at room temperature (RT)

TMB SensTMB X-traTMB PLUS2TMB ONE

If the final colored reaction product of the substrate precipitates at high OD values, use a less sensitive substrate and reduce the enzyme concentration by optimizing your assay.

Store substrates in darkness and avoid prolonged exposure to light.

24

ELISA Optimization

Kem-En-Tec Diagnostics suggests ideal combinations of products that can be used as guidelines specific for meeting your main requirement when developing your assay, e.g. for maximized sen-sitivity, for minimized background and for maximized stability.

TO MAXIMIZE ASSAY SENSITIVITY

Enzyme Plate Coat with Block&Stabilize

Plate with Dilute/Stabilize

conjugate in Develop with

HRP High

binding Carbonate

(pH 9.6) WellChampion Antibody Enhancer TMB SENS

AP High

binding Carbonate

(pH 9.6) WellChampion Antibody Enhancer pNPP

General recommendations:

For maximum sensitivity use Kem-En-Tec Diagnostics’ Enhanced Streptavidin-HRP and Enhanced Streptavidin-AP.

TO MINIMIZE BACKGROUND




HRP Low

binding K2HPO4 (pH 7.2)

WellChampion UNI-StabilPLUS TMB ONE

TMB PLUS2

AP Low

binding K2HPO4 (pH 7.2)

WellChampion AP-StabilPLUS pNPP ONE


For minimum background use Kem-En-Tec Diagnostics’ Effect Diluent “High”.

Use coating concentrations lower than the plate's maximum binding capacity, in case of background issues due to “hook” effects.

Block the plate using Kem-En-Tec Diagnostics’s BSA-free Synthetic Blocking Buffer or WellChampion

Add extra detergent or protein (e.g. casein) to the washing buffer.

Use a dilute solution of the blocking buffer along with some added detergent as washing buffer. For best results, use detergents to prevent contamination with enzyme inhibitors or peroxides.

TO MAXIMIZE ASSAY’S SHELF LIFE / STABILITY




HRP Any Any WellChampion HRP-StabilPLUS TMB ONE

TMB PLUS2 TMB X-tra

AP Any Any WellChampion AP-StabilPLUS pNPP ONE

TO IMPROVE THE ASSAY REPRODUCIBILITY


Use the WellChampion to prepare precoated plates to be stored.

Prepare a RTU bulk solution of prediluted conjugates to avoid batch-to-batch and day-to-day varia-tions.

Choose high quality microwell plates.

Prepare and use prediluted standards and controls.

25

Example of eco-sensible assay optimization: sandwich ELISA

26

Develop your assay step-by-step with Kem-En-Tec Diagnostics A/S

ELISA kit components

Kem-En-Tec Diagnostics’ products Standard Sizes* for HRP based ELISA for AP based ELISA

Enzymes HRP (#4120) AP (#4700) 10 mg 25 mg

100 mg bulk

Biotin binding molecule

Streptavidin (#4610) Streptavidin (#4610) 10 mg 100 mg

bulk

Blocking buffer Synthetic Blocking Buffer (#4520)

Synthetic Blocking Buffer (#4520)

100 mL 500 mL

1 L bulk

Plate blocker and stabilizer

WellChampion (#4900) WellChampion (#4900) 1 L bulk

Diluent and Stabilizer for Standards & Controls

Protein-StabilPLUS (#4720) Protein-StabilPLUS (#4720) 100 mL 1 L

bulk

Sample dilution buffers

SamplePLUS2 (#4781)

SamplePLUS2 (#4781)

100 mL 500 mL

1 L bulk

Effect Diluent Low (#5070) Effect Diluent Medium (#5080)

Effect Diluent “High” (#5090)

Effect Diluent Low (#5070) Effect Diluent Medium (#5080)

Effect Diluent “High” (#5090) 500 mL

Effect Diluent kit (#5095): - 100 mL Low - 100 mL Medium - 100 mL High

Effect Diluent kit (#5095): - 100 mL Low - 100 mL Medium - 100 mL High

3x100 mL

Conjugates Enhanced Streptavidin-HRP (#4740)

Enhanced Streptavidin-AP (#5150)

1 mg bulk

Diluent and stabilizers for conjugates

HRP-StabilPLUS (#4530) Antibody Enhancer (#4660) UNI-StabilPLUS (#5230)

AP-StabilPLUS (#4540) Antibody Enhancer (#4660) UNI-StabilPLUS (#5230)

100 mL 500 mL

1 L bulk

Substrates TMB ONE (#4380) TMB PLUS2 (#4395) TMB X-tra (#4800) TMB SENS (#4850)

pNPP (#4400)

pNPP ONE (#4401) 100 mL 500 mL

1 L bulk

* Samples are available on request.

27

Bottling & Packaging

Kem-En-Tec Diagnostics can customize products as well as packaging in our state-of-the art facility to meet different dispensing needs (8 mL – 10 L). Our specialists can assist you with customized packaging and label design. Total batch volumes are up to 1200 L.

We monitor every step of the manufacturing process to ensure that our dispensed product have the same quality as our batch source. Automated liquid filling guarantees specific batch toleranc-es by precision weighing.

Manufacturing & Quality Assurance

Every lot released by Kem-En-Tec Diagnostics has undergone extensive Quality Control at every significant step, assuring our customers a high level of product validation. As an ongoing process we identify and manage critical process parameters.

Our ISO based QA System supports:

Change Control Notification

Batch Sample Storage

Document Control

Audit Availability

Batch Record Availability

Non-conforming Action Procedures Kem-En-Tec Diagnostics can handle our customer’s needs as they grow, ensuring:

Experience in process scale up and validation

Extensive refrigeration space

Filling lines that can accommodate large scale demands

Contact Kem-En-Tec Diagnostics A/S

To order or for Technical Support, please contact Kem-En-Tec Diagnostics A/S at:

Support: [email protected]

Sales: [email protected]

Orders: [email protected]

USA customers: [email protected]

For more information about Kem-En-Tec Diagnostics A/S, visit www.kem-en-tec.com or email us at [email protected].

Kem-En-Tec Diagnostics A/S Kuldyssen 10

DK-2630 Taastrup Denmark

Phone: +45 3927 1771 Fax: +45 3920 0178

www.kem-en-tec.com

US Contact:

83 Maple Avenue, Windsor, Hartford,

CT 06095, USA Phone: 860 298 0234

Fax 860 298 8586 [email protected]

©Copyright 2013, Kem-En-Tec Diagnostics A/S, All right reserved. ELISA GUIDE 13-02

mailto:[email protected]




http://www.kem-en-tec.com/


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Documents

Guide for Eco-sensible ELISA Developmentkomabiotech.co.kr/www/techniques/pdf/ELISA_guide.pdf · The Guide for Eco-sensible ELISA Development starts with an overview of the differe