immunological testpreferably homologous, reference or ... [… · immunological test methods (itm)...

5678 ⟨1102⟩ Immunological Test Methods / General Information Second Supplement to USP 35–NF 30

format that is stable. Plans should be in place to update ing its binding to an immunosorbent surface and its subse-archived data so that, as technology changes, archived data quent detection by the use of enzymatic hydrolysis of a re-can still be retrieved. Regulatory agencies require that raw porter substrate, either directly (as with an analyte that hasdata be available for various lengths of time after the com- enzymatic properties or is directly labeled with an enzyme)pletion of a study or regulatory filing. Finally, data must be or indirectly (by means of an enzyme-linked antibody thatsecure from corruption, alteration, or access by unautho- binds to the immunosorbed analyte). Qualitative results pro-rized personnel.■2S (USP35) vide a simple positive or negative result for a sample. Con-

verting quantitative to qualitative results based on a cutoffvalue that separates positive and negative results is commonpractice. Because the performance properties of the assaydepend heavily on the cutoff value, the process used to de-termine the cutoff should be evidence-based and well docu-mented. Quantitative assays determine the quantity of theanalyte based on the interpolation of a standard calibration

Add the following: curve with known analyte concentration, run simultaneouslyin the same assay. This standard should be an appropriate,preferably homologous, reference or calibration material

■⟨1103⟩ IMMUNOLOGICAL TEST that is representative of the analyte(s) of interest. The powerof immunoassays has been demonstrated by the variety ofMETHODS—ENZYME-LINKED procedures that have evolved, including alternative solid sur-faces such as beads of different sorts, various plastics inIMMUNOSORBENT ASSAY (ELISA) plates of different configurations, and alternative detectionmethods, e.g., chemiluminescence and fluorescence. ELISAassays are widely used in the biopharmaceutical industry forvarious applications such as identity, purity, potency, detec-tion or quantitation of antibody or antigen, and otherpurposes.INTRODUCTION

Immunological Test Methods (ITM) utilize binding be- Basic Principlestween an antigen (Ag) and antibody (Ab). (See Appendix 1for a complete list of acronyms used in this chapter.) En- The essential steps of an ELISA can be broken down aszyme-linked immunosorbent assay (ELISA) is one of the follows (see Figure 1):most widely used ITM for characterization, release, and sta- 1. Binding of the capture reagent (generally an antibodybility testing of biotechnology products to help ensure the or antigen), which functions as an immunosorbent forquality of biological drug substances and drug products. capture of the analyte, to a solid surface;The term ELISA is used here in a broader sense and includes 2. Removal of excess, unbound capture reagent followedenzyme immunoassays (EIA), as well as alternative detection by blocking of unoccupied binding sites with a blockingmethods, e.g., chemiluminescence and fluorescence. protein such as albumin, gelatin, casein, or other suitableThis chapter provides analysts with general information material;about principles, procedures, experimental configurations, 3a. Incubation of the analyte (in the test sample or refer-assay development, and validation for solid-phase ITM like ence standard) with the capture reagent to bind theELISA and can be used for the other immunoassay varia- analyte onto the solid surface, followed by the washingtions mentioned above. The chapter also covers reference away of unbound material in the test sample and detec-standard(s) and control(s) used for immunoassays. The infor- tion of the analyte. Direct detection occurs when themation can be adapted to the specific procedures of a mon- analyte has enzymatic activity or has been linked to aograph. This chapter does not cover immunoassays for the detector molecule (e.g., enzyme); ormeasurement of immune responses to product in animals or 3b. Incubation of the analyte (in the test sample or refer-humans (e.g, serological or cellular assays), non-immunoas- ence standard) with the capture reagent to bind thesays (e.g, receptor-ligand interactions), or other related analyte onto the solid surface, followed by the washingapproaches. away of unbound material in the test sample and subse-The chapter is part of a group of general information quent detection of the analyte (Figure 1, step 3a). Indi-chapters for immunological test methods [Immunological rect detection occurs when the analyte is detected byTest Methods—General Considerations ⟨1102⟩, Immunological the addition of a secondary enzyme-labeled reagent (Fig-Test Methods—Immunoblot Analysis ⟨1104⟩ (proposed), and ure 1, step 3b); andImmunological Test Methods—Surface Plasmon Resonance 4. Quantification of the analyte by addition of a sub-⟨1105⟩], and also is related to the general information chap- strate suitable for the detector used (e.g., TMB, 3,3′,5,5′-ters for bioassays [Design and Development of Biological As- tetramethylbenzidine), followed by comparison of thesays ⟨1032⟩, Biological Assay Validation ⟨1033⟩, and Analysis test sample to the reference standard.of Biological Assays ⟨1034⟩].

Definition

ELISA can be defined as a qualitative or quantitative solid-phase immunological method to measure an analyte follow-

Official from December 1, 2012Copyright (c) 2012 The United States Pharmacopeial Convention. All rights reserved.

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Second Supplement to USP 35–NF 30 General Information / ⟨1103⟩ Immunological Test Methods 5679

Figure 1. Essential steps for performing an ELISA.1

antibody that is being developed as a drug. In this case,ASSAY DESIGNanti-idiotypic or other antibodies specific for the antibodyare used to develop the assays. Five general categories of ELISA are described in Table 1

and in the sections that follow. The assay designs are flexi- 1 Capture reagent binding, blocking, analyte binding, detector antibody bind-ble and, depending on specific needs, can be modified from ing, and analysis are the five basic steps in an ELISA. Capture reagent bind-

ing, blocking, and analyte binding steps are each followed by a washing stepthese procedures. The choice of format depends primarilyto remove unbound reagents before the addition of the next reagent. Beforeon the amounts and purity of reagents and equipment analysis an appropriate substrate is added, followed by measurement of the

available. On some occasions the analyte being character- substrate by appropriate equipment for detection. Quantitation of unknownstakes place by comparison with a standard curve.ized actually is an antibody, as in the case of a monoclonal




Table 1. Representative ELISA Types

ELISA Type Required Reagents Attributes DisadvantagesDirect detection • Capture analytea • Rapid because only one antibody • May modify the conformation of

• Labeled primary antibody spe- is used the analytecific for antigen • Uses less reagent • Sensitive to matrix and adjuvant

• Analyte is immobilized components• Not commonly used• Poor sensitivity

Indirect detection • Capture analytea • Versatile because a variety of pri- • Longer because of more incuba-• Primary antibody specific for mary antibodies can be used with tion and washing stepsantigen the same secondary detector• Labeled secondary detector • Improved sensitivity because ofantibody that binds to the pri- signal amplificationmary antibody • Analyte is immobilized

Competitive • Analytea can be used as a cap- • Good for assessing antigenic • Format difficult to troubleshootture reagent or can be labeled cross-reactivity • Limited dynamic rangewith a detection label • Appropriate for smaller proteins• Antibody specific for analyte with single epitopescan be used for capture or la- • Requires only a single antibodybeled for detection • Analyte in solution competes for• Labeled secondary antibodies binding to primary antibodyto bind to primary antibody ifan indirect format is used

Sandwich • Primary capture antibody spe- • Improved sensitivity • Requires relatively large amountscific for analyte • Good for quantitative assays for of pure or semipure specific an-• Sample solution containing larger multi-epitope molecules tibodyanalytea • Analyte measured in solution • Not suited for smaller proteins• A different primary enzyme- that may have only a single epitopeantibody conjugate specific for or a few closely spaced epitopesanalyte

a This reagent can be either purified or partially purified. The terms “analyte” and “antigen” are used interchangeably when describing ELISAs.

antibody complex is washed away. Substrate is added, andDirect ELISAthe inhibition of the reaction (e.g., colorimetric, elec-trochemiluminescence, fluorescence, or chemiluminescence)Directly Labeled Antibody: In this assay an antigen is is measured relative to the reaction when no competitorcoated onto a solid surface and the remaining unbound re- antigen is added. The amount of inhibition is inversely pro-active sites are blocked (Figure 2A). Then a solution contain- portional to the amount of antigen in the test sample. Com-ing a specific antibody labeled with a detector is added. petitive assays can also measure small molecules by coatingAfter incubation, the unbound antibody is washed away, fol- an antibody to the plate that is specific to the small mole-lowed by the addition of an appropriate substrate for the cule. The small molecule is often biotinylated with a longdetector used. linker that does not interfere with binding between the cap-

Directly Labeled Antigen: This assay is similar to that us- ture antibody on the plate and the small molecule. Antigening a directly labeled antibody except that the antibody is (the small molecule) in the sample then competes with thecoated onto the solid surface and a labeled antigen is used labeled small molecule for binding to the capture antibody.as the detector. After washing, a detection reagent (e.g., streptavidin labeled

with HRP) is added to detect the binding complex.Direct Antigen Competitive ELISA: This assay is similar toIndirect ELISAthe Direct Antibody Competitive ELISA, except that it is usedto detect soluble antibodies. The antigen is conjugated toIn this assay an antigen is coated onto a solid surface andthe detector and the antibody is coated onto the solidthen, after blocking, a solution containing a specific an-surface.tibody is added (Figure 2B). After incubation, the unbound

antibody is washed away, followed by the addition of an Indirect Antibody Competitive ELISA: This assay is similaranti-immunoglobulin (anti-Ig) detector antibody. Anti-Ig de- to the Direct Antibody Competitive ELISA, except that insteadtectors are available commercially for specific Ig classes and of directly labeling the antibody, the test uses a labeled anti-subclasses from a variety of species, which makes this assay Ig reagent for detection.format useful for isotyping of antibodies. In addition, the Indirect Antigen Competitive ELISA: This assay is similaruse of a labeled anti-Ig detector amplifies the signal com- to the Direct Antigen Competitive ELISA, except that insteadpared to a Direct ELISA, thereby increasing assay sensitivity. of directly labeling the antigen, the test uses a labeled sec-

ondary antibody for detection.

Competitive ELISAsSandwich ELISA

Direct Antibody Competitive ELISA: This assay is used todetect or quantitate soluble antigens (Figure 2C). It requires Direct Sandwich ELISA: In this assay an antibody is immo-an antigen-specific antibody that has been conjugated to an bilized onto a solid surface and blocked, and then a solutionappropriate detector, e.g., horseradish peroxidase, alkaline containing a specific antigen is added (Figure 2D). After anphosphatase, ruthenium, or fluorescein. It also requires a incubation step, the unbound material is washed away, andpurified or partially purified antigen for coating. The antigen a labeled detector antibody is added. This assay format re-is coated onto a solid surface, followed by a blocking step. quires two antibodies, each of which binds to differentThe antibody–conjugate is incubated with the test solution epitopes on the surface of the large and complex molecule.containing soluble antigen. The mixture is then added to The two antibodies are specific for the antigen, and the an-the immobilized antigen, incubated, and unbound antigen-




Figure 2. Schematic representations of direct, indirect, competitive, sandwich, and bridging ELISAs.2 (Ab = antibody; Ag =antigen (or analyte); Preincub = preincubation)

tigen should be sufficiently large and complex to accommo- a laboratory repeatedly engineers a particular epitope intodate the binding of two antibodies. multiple fusion proteins. In this case, the laboratory can use

certain common qualified reagents (e.g, an antibody to aIndirect Sandwich ELISA: Alternatively, instead of directlyglutathione S-transferase region in multiple fusion proteins),labeling the detector antibody, an anti-Ig antibody detectorfacilitating rapid sandwich immunoassay development. Smallcan be used. Indirect sandwich immunoassay formats canantigens with a limited number of epitopes available for an-be considered only if each binding reagent is from a uniquetibody binding restrict ELISA format choices. If there is onlyspecies (e.g., a sandwich assay using two mouse monoclo-one binding epitope, then ELISA methods that use the sand-nal antibodies for capture and detector could not be de-wich/two-site binding or other bridging formats cannot betected indirectly because the resulting signal may becomeused because they require at least two available epitopes forindependent of the antigen concentration). antibody binding. In addition, small molecules are not usu-Bridging ELISA: This subset of Sandwich ELISA assays often ally used as a capture reagent on a plate because the pro-uses a single antibody for both capture and detection (Fig- cess may interfere with binding to the detection reagent.ure 2E). If a monoclonal antibody is used, it requires that Examples of such small molecules are some peptides, oligo-the target antigen have at least two identical epitopes that saccharides, nucleotides, and antibacterials. Analysts usuallyare adequately spaced to prevent steric hindrance so that adopt a competitive assay format for such small analytes.one epitope binds to the capture antibody and the other Different assays and formats may demonstrate differentepitope binds to the detector antibody. Alternatively, a properties and characteristics, e.g., specificity, precision, ac-polyclonal antibody can be used but still requires that the curacy, sensitivity, dynamic range, dose-response ratio, sam-target antigen be large enough to accommodate the bind- ple throughput, sensitivity to interference, and simplicity oring of two antibody molecules. With respect to specificity efficiency for automation. Ease of validation also may varyand sensitivity, bridging assays usually are suitable for most between different assay protocols and formats. Assay de-large molecules. signs with replicates in adjacent wells could be biased ifthere are location effects; hence, in this case, replicatesshould not be in adjacent wells. Assay designs that are con-CHOICE OF ASSAYvenient to perform on 96-well plates, using relatively fewsingle-channel pipet actions and more multi-channel pipetDeciding which ELISA procedure or format to use oftenactions, are usually easier to adapt to automation. Assaysdepends on individual choice and availability of reagents,with steep dose-response curves are generally better able toinstruments, and other equipment. For example, sometimesdeliver high precision estimates; however, some assays with

2 The type of ELISA format depends on the availability of reagents, the in- steep dose-response curves are imprecise in the EC50 andtended purpose of the assay, and the physicochemical characteristics of the require a wider dose range.analyte of interest. For a Bridging ELISA, the capture and detector antibodiesrecognize the same epitope, and therefore the target antigen must have atleast two epitopes available for binding.




temperature. Although different combinations of coatingPROCEDUREStimes and temperatures often result in the same coating effi-ciency, the stability of the capture reagent (which should bedetermined during method development) influences which

Solid Phase conditions to select. Analysts must assess the impact of vary-ing the coating time in order to determine the robustness of

Solid phases are available in a variety of forms (e.g, mem- the assay procedure.brane, plate, or bead) and chemistries (e.g, nylon, nitrocel- Coating Temperature: Coating temperature and time arelulose, polyvinylidine fluoride (PVDF), polyvinyl, polystyrene, closely related assay parameters. The coating temperatureor a chemically derivatized surface). The selection of the depends on the binding kinetics and stability of the antigen.solid phase determines the most likely binding mechanism, Higher temperatures can increase the rate of adsorption andi.e., hydrophobic, hydrophilic, or covalent interactions. In may shorten the coating time, but they are likely to affectgeneral, compared to plates, beads offer higher capacity interaction sites and to reduce antigen-antibody affinity.and are more commonly used in clinical assays whereas Typical combinations of time and temperature are 1–4 h atplates are more commonly used to test biotechnology prod- ambient temperature, 15 min to 2 h at 37°, or overnight atucts. Additional information on plates is provided below. 4°. Analysts should determine the effects of variations inCoating the Solid Phase—Immobilization of Capture Rea- temperature in order to assess the robustness of the assaygent: Capture reagents are coated onto a solid phase by procedure.adding a solution containing the capture reagent to the sur- Buffers: Buffers used for diluents, coating, blocking, andface. The most commonly used solid-phase materials for washing plates can affect overall assay performance. Buffercapture reagent immobilization are plastic 96-well microtiter components can interact with the test sample and inhibitplates. Those with flat-bottom wells are recommended for binding. They also can cause low antigen sensitivity or highspectrophotometric readings, and round-bottom well plates nonspecific background activity.are useful for visual assessment of a dye’s color develop- Diluent—Buffers [e.g., phosphate-buffered saline (PBS) or im-ment. The degree of coating is influenced by the concentra- idazole-buffered saline] with polysorbate 20 (0.01%–0.1%)tion of capture reagent, temperature during coating, dura- are used commonly for different ELISA steps as a diluent andtion of capture reagent adsorption, the surface properties of washing buffer.the solid-phase material, and the nature of the buffer of the

Coating Buffers—Coating buffers should maximize assay con-capture reagent solution. Although the optimum coatingsistency and promote binding of the capture reagent to theconcentration must be determined for each capture reagent,solid phase. Commonly used coating buffers include 50 mMconcentrations of 1–10 µg/well are most commonly used.carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; and 10 mMThe volume of capture reagent added to each well usuallyPBS, pH 7.2. The choice of coating buffer depends on thecorresponds to the sample volume that will be analyzed,nature of the individual antigens and should be determinedi.e., 50–100 µL. Coating duration, temperature, and buffersempirically.are discussed separately below. During the coating proce-Blocking Agents and Buffers—A blocking agent is a com-dure analysts should avoid introducing bubbles. Proteinspound (e.g., protein or detergent) that should saturate thethat bind to plastic can be denatured, which alters antige-remaining immunosorbent binding sites following capturenicity. In such cases, a capture antibody or an intermediaryreagent (antibody or antigen) binding. This reduces nonspe-protein such as Protein A or Protein G can be used. In addi-cific binding of analyte and nonanalyte components to thetion, streptavidin can be used if the reagent is biotinylated.immunosorbent matrix and/or the absorbed reagent. Non-The pH of the coating buffer should be optimized based onspecific binding occurs when protein in the test samplethe isoelectric point of the capture reagent and the surfacebinds to the plastic of the microtiter plate or absorbed rea-properties of the assay plate chosen.gent instead of specifically binding to the capture reagent ofMicrotiter Plates: The composition and commercial sourceinterest. Nonspecific binding can be reduced by addingof the microtiter plate can influence binding of the captureblocking reagent to the wells and by the addition of an-reagent during coating. Several microtiter plates from differ-other protein such as bovine serum albumin (BSA) to theent suppliers should be compared using a single coatingdilution buffer. The choice of blocking agent should be gov-procedure to select those that provide high specificity forerned by the nature of the capture reagent, plate, coatingthe capture reagent of interest and low nonspecific back-buffer, test sample diluent, and related factors. If any ofground. Comparisons of different grades of plates from athese parameters changes, a change in blocking agent maysingle supplier also may be needed. Clear plates typically arebe needed. Commonly used blocking agents include BSA,used for colorimetric ELISA, and opaque plates often arenonfat milk, gelatin, casein, normal horse serum, fetal bo-used for chemiluminescent and fluorometric ELISA. Acidicvine serum, polysorbate 20, and others. Several grades ofcapture reagents may require a lower pH solution to neu-BSA are available commercially, and the optimal gradetralize repulsive forces between the protein and solid phase.should be empirically determined for each assay. In addi-Peptides often require optimization of buffer pH based ontion, many commercial blocking and assay diluent reagentstheir charge for optimal coating conditions during assay de-are available for ITMs.velopment. Polysaccharides, lipopolysaccharides, or glyco-

proteins may be difficult to coat directly to the plate andmay require a capture antibody or a buffer that contains Adding Samples and Reagentslysine or glutaraldehyde. Coating with an antibody can beenhanced by precoating the microtiter plate with Protein A Samples and reagents generally are pipetted into theor Protein G or a combination of the two, which allows ELISA plate wells. Care should be taken to avoid cross-con-binding to the Fc region so that the Fab portion can bind to tamination, frothing, or bubbles. Labor-saving equipmentthe analyte of interest. However, care must be taken to en- such as electronic pipets, automated liquid handlers, platesure that subsequent secondary antibodies do not react with washers, and robotic pipets also can be used to improvethe Protein A- or Protein G-coated wells. In this case, for precision, reduce analyst-to-analyst variability, and increaseexample, chicken IgY or another appropriate antibody class throughput.could be used. Microtiter plate formats other than the 96-

Pipets: Single, multichannel, and robotic pipets with set orwell variety, such as half volume 96-well or 384-well plates,fixed volumes are available. The type and accuracy of pipetscan be used to increase throughput and/or conserveshould be evaluated for each application. Regular mainte-reagents.nance and professional calibration of pipets should be per-Coating Time: Coating time depends on binding kinetics, formed and documented.stability, concentration of capture reagent, and incubation




Pipet Tips: A variety of pipet tips are available, some of other assay reagents should be considered; for example, en-which are specific to the type of pipet. The type and accu- dogenous biotin is found in milk and serum, and serum mayracy of the pipet tip, particularly related to the viscosity and contain antibody to viral or bacterial proteins. Therefore,nonspecific binding of the materials, should be evaluated for screening of serum lots may be necessary. The volume ofeach application. blocking solution added to the well should be greater than

the maximum reaction volume used for later steps so thatall of the potential surface area that may interfere with the

Washing binding reaction is blocked.In addition, Ig in the test materials can be removed by

Wash steps are included throughout the ELISA procedure using buffers that inhibit antibody conformation or aggre-to remove the unbound coating antigen, sample, and de- gate the heterophilic antibodies, by blocking with nonim-tection reagents. Washing is critical for assay performance, mune serum, or by removing Fc regions in critical antibodycan be a source of assay failure, and is important to evaluate reagents, thereby reducing or eliminating undesired immu-during method development. Multiple approaches can be nological interactions that cannot be addressed by theused for washing. Manual procedures include using a blocking reagents described above. Negative control wellssqueeze bottle, dipping the microtiter plate in wash buffer, can be included to monitor nonspecific reactions. The na-and adding wash buffer with a multichannel pipet or hand- ture of the negative control wells depends on the assay butheld multi-channel (8- or 12-pin) manifolds. Analysts should can include blocked wells without coating antigen, eliminat-wash carefully to avoid cross-well contamination. Automatic ing the primary or secondary antibody, or using buffer inmicroplate washers generally provide more washing consis- place of sample. Control wells also can be useful as part oftency. Strip-well and multiwell washers are available. Most system suitability testing.automatic washers can be programmed for different dis-pensing volumes and speeds, number of washes, speed ofbuffer aspiration, and amount of residual buffer left in the Pretreatment of Sampleswell. Incorrectly programmed or maintained as well as in-completely cleaned automatic washers can cause assay vari- Although ELISA methods are designed to measure anation and elevated assay background. analyte in complex mixtures, the presence of other materials

can prove problematic if they interfere with analyte detec-tion. In order to ensure assay specificity, the specific proce-

Incubation dure to treat samples to remove nonspecific interfering sub-stances (e.g., reducing agents or precipitates) can be

ELISAs are incubated following the addition of samples determined empirically during method development andand reagents. The optimal time, conditions, and tempera- then can be incorporated into the validated assay. Any sam-ture of each incubation step should be determined during ple-processing step should be evaluated against the poten-method development. Incubation times vary from minutes tial that the treatment will alter the test article’s propertiesto overnight. Commonly used incubation temperatures are and/or introduce further variability that results in biasedambient temperature, 4°, and 37°. ELISA plates commonly measurements. Samples, standards, and controls should beare sealed or placed in a secondary container to avoid evap- prepared and handled in processes as similar to each otheroration or contamination during incubation. Atmospheric as possible. Analysts should verify that sample pretreatmentsconditions such as dry or humidified incubation should be have not damaged the sample so much that it can noevaluated during method development. Rocking, shaking, or longer be measured (e.g., by spiking experiments).rotating the microtiter plates may be necessary or desirabledepending on the kinetics of binding.

Detector AntibodiesBlocking Conditions and Nonspecific Reactions Depending on ELISA format, detector antibodies labeled

with enzyme or other labels can be used as primary or sec-After immobilization and removal of the unbound antigen ondary reagents to enable detection of the immobilized

or antibody, unoccupied binding sites are blocked to ensure analyte. In a direct or competitive ELISA (Figure 2A and Fig-that the measured analyte in the test article or subsequent ure 2C), after the analyte is bound to the immunosorbent(detection) reagents does not bind nonspecifically to the surface, excess analyte is washed away and the immobilizedsolid surface or to the coated antigen or antibody. If nonanalyte is detected using a detector antibody that is consid-specific binding occurs, any reported signal could bias the ered to be the primary antibody. In other ELISA formatsmeasurement and may reduce the sensitivity and dynamic (Figure 2B, 2D, and 2E), the analyte-specific Ig (nonconju-range of the assay. Blocking is critical to ensure the sensitiv- gated primary antibody) is allowed to bind to the immobi-ity and/or specificity of the assay. Sources of nonspecific lized analyte, and any excess antibody is washed awaybinding fall into two general categories: before the addition of a detector antibody, which is termed

1. Ionic or hydrophobic interactions occur when binding is the secondary antibody.mediated by nonspecific ionic or hydrophobic interac- To facilitate detection, in all ELISA formats that use en-tions between assay reagents and the solid surface or zyme-conjugated antibodies, a substrate specific for the con-another assay reagent. jugated enzyme is introduced into the assay system. An en-

2. Immunological interactions occur when binding is me- zymatic reaction ensues, converting a substrate into adiated by unintended antigen-antibody interactions. soluble product that can be measured using appropriateThis occurs when antibody preparations used in the wavelengths and a suitable reader.assay interact with other assay reagents. For example, ELISA sensitivity depends on the quality of the reagentsif an ELISA was designed to test a serum-derived and the detection system, including the label and substrate.analyte using murine capture and detection antibod- If multiple differently conjugated antibodies are available,ies, antibodies in the test article with reactivity to mu- analysts should select one appropriate for the assay. Duringrine Ig (also known as heterophilic antibodies) could this evaluation, the dilution of each conjugate that yieldsbe nonspecifically detected in the assay. desirable sensitivity and specificity should be determined us-

The choice of blocking agent (examples are found in the ing appropriate controls.Blocking Agents and Buffers section above) is determined em- The most commonly used labeling enzymes for conjugat-pirically, and the balance between the reduction in nonspe- ing to antibodies include alkaline phosphatase (AP), horse-cific binding and the impact on assay sensitivity should be radish peroxidase (HRP), and galactosidase. These enzymesassessed during method development. Cross-reactivity with are highly specific, sensitive, and stable in catalyzing chro-




mogenic, luminescent, or fluorescent reactions. para-Ni- analyte capture and detection. Any changes of critical bio-trophenyl phosphate (pNPP) is a commonly used substrate logical reagents should be evaluated (see, for example,for AP. Commonly used substrates for HRP include TMB, guidance contained in Design and Development of BiologicalOPD (o-phenylenediamine dihydrochloride), and ABTS [2,2′- Assays ⟨1032⟩.)azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diam- Source: The availability and quality of the starting materialmonium salt] (see Table 2). The substrates for AP and HRP should be controlled so that manufacturing of the (purified)are chromogenic and result in the formation of a colorimet- reagent can be reproducibly and consistently performed,ric product that can be measured using a spectrophotome- potentially over several decades. Because critical reagentster. Chemiluminescent and fluorescent substrates for AP and are biological molecules, sources can range from chemicalHRP also are available, and in many cases they are available synthesis (e.g., peptides) to complex biological matricesas commercial kits. Disodium 3-(4-methoxyspiro{1,2-diox- (e.g., antibodies prepared from serum, monoclonal antibodyetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl from ascites/cell culture, or fermentation/cell culture prod-phosphate (CSPD) is a known chemiluminescent substrate ucts). When appropriate for the intended use of the assay, afor AP (see Table 2). Well-known fluorescent substrates for single lot of a critical reagent can be manufactured to estab-galactosidase include MG (4-methylumbelliferyl galactoside) lish a substantial supply and to prevent lot-to-lot variability.and NG (nitrophenyl galactoside). If a chemiluminescent In other instances it may be appropriate to include in thesubstrate is used, then a luminometer is required to quanti- validation multiple lots or multiple suppliers in order totate the formed product. A fluorometer is needed if a fluo- demonstrate that the assay is sufficiently robust for its in-rescent substrate is used in the ELISA. tended use.

Table 2 also provides a summary of the advantages and Purity: In general, the purity of critical reagents should bedisadvantages of different types of ELISA substrates. Colori- assessed to ensure the removal of impurities and manufac-metric substrates have been prevalent since the origin of turing process residuals that can influence reagent perfor-ELISAs and may yield robust assays that generally are more mance and/or stability.cost efficient than assays that use chemiluminescent andSpecificity: The specificity of a critical reagent refers to itsfluorescent substrates. Nevertheless, chemiluminescent andability to capture or detect only the analyte of interest. Thefluorescent ELISA methods may yield more rapid and sensi-reagent must be specific to the analyte and should showtive assays with a wider dynamic range than assays that uselittle nonspecific binding or no cross-binding to off-targeta colorimetric readout. The final choice of readout shouldmolecules in complex test materials.be governed by the assay’s purpose and the requirements ofStability: The stability of critical reagents should be empiri-the assay.cally determined to ensure assay performance over time (is-sues include accuracy, precision, reproducibility, and assay

ASSAY DEVELOPMENT AND VALIDATION drift). Long-term (months to years) stability of critical re-agents under required storage conditions (e.g., with definedPLANtemperatures and containers) should be determined so thatappropriate expiry dating can be assigned. Short-term (min-utes to days) stability (and freeze/thaw and room tempera-

Critical Reagent Development ture stability for frozen critical reagents) also is required toensure day-to-day assay accuracy, precision, and

Key considerations for critical reagents are source, purity, reproducibility.specificity, and stability. For quality measurements, ITMsuse reference standards along with critical reagents for

Table 2. Enzyme Conjugates and Substrates

Principle of theReadout Enzymatic Reaction Enzyme Substrate Reader Advantages Disadvantages

Colorimetric Produces a colored AP, HRP pNPP TMB Spectrophotometer • Robust • Lessproduct that yields OPD • Economical sensitiveabsorbance values di- ABTS • Reagent availabili-rectly proportional to tyanalyte concen-tration

Chemilumines- Produces a light emis- AP CSPD Luminometer • Wide assay • Requires specialcent sion that is directly dynamic range plates

proportional to • Lower coating • Costlyanalyte concentration concentrations

• More sensitive• Rapid signal gen-eration

Fluorescent Produces excitation-in- Galactos- MG Fluorometer • Rapid • Requires specialduced light emission idase NG • Sensitive platesthat is directly propor- • Costlytional to analyte con- • Interference bycentration excipients




should take care in the selection of an appropriate curve-Feasibility/Pilot Studiesfitting algorithm. In other cases, ELISA assays are used forqualitative purposes to determine whether a sample is posi-The steps of the process by which an ELISA method istive or negative based on a sensitivity threshold.developed, validated, and used in routine sample analysis

are described below:• Generate or purchase critical reagents to measure the Basic Statistical Analysisanalyte. Determine storage conditions and stability.• Understand the performance goals for the assay system. Basic statistical methods are not detailed here. General in-• Develop the assay to the point that there is a detecta- formation chapter Analytical Data—Interpretation and Treat-ble concentration response curve. ment ⟨1010⟩ addresses important fundamentals, including• Perform method development/robustness testing. data handling; computation of means, standard deviations,• Prepare the reference/calibration standard and control and standard errors; detection of and methods to addressand assess stability. nonconstant or nonnormal variation; detection of and man-• Establish assay procedures, appropriate controls and agement of outliers; and procedures for and interpretationlimits, assay and sample acceptance criteria, and of statistical tests and confidence intervals. The concepts be-instrumentation. hind validation, goals, designs, analysis, and practical meth-• Determine method performance, and qualify method ods for validation are described in general information chap-for accuracy, specificity, precision, and robustness, in- ters Analytical Data—Interpretation and Treatment ⟨1010⟩,cluding qualification of all applicable sample types to be Validation of Compendial Procedures ⟨1225⟩, and Biological As-analyzed. say Validation ⟨1033⟩. General test chapter Design and Analy-• Validate the assay. sis of Biological Assays ⟨111⟩ contains guidance on combin-• Implement the method (technology transfer) in the ing results from independent assays.testing laboratory, including training and qualification

of analysts.• Monitor assay performance. Nonlinear Statistical AnalysisDuring assay development, the critical parameters and re-

agents that are required for the assay should be assessed Nonlinear calibration for immunoassays draws on manyand set at levels that yield desired assay performance. In sources for statistical design and analysis. These includemany instances several parameters may be evaluated, and methods for assessing and addressing nonconstant variance,well-designed experiments can accelerate assay develop- designs and analysis methods for experiments with complexment, particularly for assessing the potential interaction of structures, and validation. The concepts behind linear cali-several inputs. bration design, analysis, and inverse regression apply in

Many ELISA procedures are product specific, and external nonlinear calibration, and professional statisticians can helpreference/calibration standards may not be available. The apply these appropriately.preparation and stability of reference/calibration standardsshould be considered early in assay development.

Reporting ResultsAssay Validation Reported estimates of concentration should be under-

stood as having an associated confidence interval based onAssay validation is executed according to guidances from the results of the validation. The reported value or estimate

appropriate regulatory bodies (e.g., ICH Q2) to demonstrate used to describe a sample can be based on a combinedthat the particular test used for an analyte is appropriate for result from multiple assays.its intended use. More information about assay validationcan be found in the general information chapter Validationof Compendial Procedures ⟨1225⟩ or in general information APPENDIX 1chapter Biological Assay Validation ⟨1033⟩ if the ELISA is usedas a surrogate potency assay. See Appendix 2 for additionalinformation.

Abbreviations

DATA ANALYSIS Ab—AntibodyAg—Antigen

The analysis of ELISA data can be simple (e.g., a linear ABTS—2,2′-Azino-bis[3-ethyl-benzothiazoline-6-sulfoniccalibration with inverse regression) or complex (e.g., a non- acid]diammonium saltlinear calibration curve with inverse regression). The type Anti-Ig—anti-immunoglobulinand rigor of data analysis depend largely on the assay sys- AP—Alkaline phosphatasetem and the intended uses of the assay. For example, data BSA—Bovine serum albuminreduction may estimate a concentration (e.g., ng/mL) of an CSPD—Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-unknown sample using a calibration curve. Other ap- (5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenylproaches include estimation of the half-maximal inhibitory phosphateconcentration (IC50) or effective concentration (EC50), esti- EIA—Enzyme immunoassaymation of the amount of a sample that yields the same ELISA—Enzyme-linked immunosorbent assayresponse as the EC50 (or IC50) on a standard curve, and an HRP—Horseradish peroxidaseestimate of the relative activity of a test sample compared to Ig—Immunoglobulina reference/calibration standard. More extensive guidance ITM—Immunological test methodsabout statistical methods for potency analysis are given in MG—4-Methylumbelliferyl galactosidegeneral information chapters Design and Development of Bio- NG—Nitrophenyl galactosidelogical Assays ⟨1032⟩ and Analysis of Biological Assays ⟨1034⟩. OPD—o-Phenylenediamine dihydrochloride

In general, ELISA assay curves are characterized by a non- PBS—Phosphate-buffered salinelinear relationship between the concentration of the analyte PVDF—Polyvinylidene fluorideof interest and the calculated mean response. Typically, this pNPP—para-Nitrophenyl phosphateresponse curve is defined by a sigmoidal relationship of re- TMB—3,3′,5,5′-Tetramethylbenzidinesponse to concentration. A wide range of mathematicalmodels can fit standard/calibration curves, and analysts




APPENDIX 2

Additional Sources of Information about Specific Topics in Validation and Data Analysis

Analytical Data—Inter- Design and Analysis Validation of Com-pretation and Treatment of Biological Assays pendial Procedures Biological Assay Chapters

⟨1010⟩ ⟨111⟩ ⟨1225⟩ ⟨1032⟩, ⟨1033⟩, and ⟨1034⟩Means XStandard deviations XStandard errors XNon-normality X XNonconstantvariance X X

Outliers X XTests XConfidence intervals X XValidation X XCombining resultsfrom multiple as-says X X

■2S (USP35) degradation products). Stability considerations should in-clude not only the specific compendial requirements, butalso changes in physical appearance of the product thatwould warn users that the product’s continued integrity isquestionable.

Stability studies on active substances and packaged dos-age forms are conducted by means of “real-time,” long-term tests at specific temperatures and relative humidities

Delete the following: representing storage conditions experienced in the distribu-tion chain of the climatic zone(s) of the country or region ofthe world concerned. Labeling of the packaged active sub-

■⟨1150⟩ PHARMACEUTICAL stance or dosage form should reflect the effects of tempera-ture, relative humidity, air, and light on its stability. LabelSTABILITY temperature storage warnings will both reflect the results ofthe real-time storage tests and allow for expected seasonalexcursions of temperature.

The term “stability,” with respect to a drug dosage form,refers to the chemical and physical integrity of the dosage Controlled Room Temperatureunit and, when appropriate, the ability of the dosage unit tomaintain protection against microbiological contamination. Controlled room temperature (see Storage TemperatureThe shelf life of the dosage form is the time lapse from and Humidity in Preservation, Packaging, Storage, and Label-initial preparation to the specified expiration date. The mon- ing under General Notices and Requirements) delineates theograph specifications of identity, strength, quality, and pu- allowable tolerance in storage circumstances at any locationrity apply throughout the shelf life of the product. in the chain of distribution (e.g., pharmacies, hospitals, and

The stability parameters of a drug dosage form can be warehouses). This terminology also allows patients or con-influenced by environmental conditions of storage (tempera- sumers to be counseled as to appropriate storage for theture, light, air, and humidity), as well as the package com- product. Products may be labeled either to store at “Con-ponents. Pharmacopeial articles should include required trolled room temperature” or to store at temperatures “upstorage conditions on their labeling. These are the condi- to 25°” where labeling is supported by long-term stabilitytions under which the expiration date shall apply. The stor- studies at the designated storage condition of 25°. Con-age requirements specified in the labeling for the article trolled room temperature limits the permissible excursions tomust be observed throughout the distribution of the article those consistent with the maintenance of a mean kinetic(i.e., beyond the time it leaves the manufacturer up to and temperature calculated to be not more than 25°. See Meanincluding its handling by the dispenser or seller of the article Kinetic Temperature. The common international guideline forto the consumer). Although labeling for the consumer long-term stability studies specifies 25 ± 2° at 60 ± 5% rela-should indicate proper storage conditions, it is recognized tive humidity. Accelerated studies are specified at 40 ± 2°that control beyond the dispenser or seller is difficult. The and at 75 ± 5% relative humidity. Accelerated studies alsobeyond-use date shall be placed on the container label. allow the interpretation of data and information on short-

term spikes in storage conditions in addition to the excur-sions allowed by controlled room temperature.Stability Protocols The term “room temperature” is used in different ways indifferent countries, and for products to be shipped outsideStability of manufactured dosage forms must be demon- the continental U.S. it is usually preferable for product label-strated by the manufacturer, using methods adequate for ing to refer to a maximum storage temperature or tempera-the purpose. Monograph assays may be used for stability ture range in degrees Celsius.testing if they are stability-indicating (i.e., if they accurately

differentiate between the intact drug molecules and their



immunological testpreferably homologous, reference or ... [… · immunological test methods (itm)...

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