Blockers Practical Guide

Immunoassay Blocking Reagents Practical Guide ISO Certified 13485:2016

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Recommended Blockers

BLOCKERS FOR INTERFERENCE IN SANDWICH IMMUNOASSAYS

Animal IgG – Passive Blockers • Suited for mixed species assays (e.g. MAb/PAb) • Species of blocker must be the same as the host of the capture or detection antibody

TRU Block ™ – Active HAMA & RF Blocker • Suited for double mouse monoclonal assays • Removes HAMA, HA & RF interference

TRU Block ™ Ready 8001 TRU Block ™ Ultra 8000 TRU Block ™ A66800H TRU Block ™ 2 A66802H TRU Block ™ 3 A66803H

Interfering HA (e.g. HAMA) TRU Block

Mouse IgG A66186M (9-13mg/mL) A66185M (50-55mg/mL) A66189M (45-55mg/mL, No Azide) A66185M-LY (Lyophilized)

TRU Block

IgM RF

Detection Antibody (mouse origin) Capture Antibody (mouse origin) Antigen

Blocking Buffer

Goat IgG A66200H Rat IgG A64391R Rabbit IgG A66100H

Chicken IgY A01302C (Lyophilized) Sheep IgG A66400S (70-77mg/mL, Liquid)

*Formulations differ in their ratio of various proprietary ingredients that confers unique blocking characteristics.

In double mouse monoclonal assays, a specific blocker is required to remove a particular type of HA interference called human anti-mouse antibodies (HAMA) and Rheumatoid Factor (RF). A HAMA blocker contains a specific binder directed against all types of heterophilic interference including HAMA and RF. Once bound to the interfering antibodies, TRU Block prevents further binding of HA to other assay components through steric hindrance. Active blockers can typically be used in lower concentrations than passive blocking reagents, which minimizes the reduction in assay signal commonly associated with passive blockers. RECOMMENDED CONCENTRATION: For best performance, TRU Block should be included as part of the sample or conjugate diluent, at a recommended concentration range: Product Protein Concentration Application TRU Block ™ Ready Single-step dilution with recommended dilution of 1:1000 to 1:10 ELISA & LF

Passive blocking reagents work by preventing interfering antibodies from binding to the capture or detection antibodies by providing alternate binding sites. Animal IgG (e.g. Goat IgG) can only block one type of interference (e.g. human anti-goat antibodies) so typically more than one type must be used, depending on the host of both the capture and detection antibodies. Animal IgG must be added in excess concentration and the effectiveness depends on the affinity of interfering antibody for the animal IgG. RECOMMENDED CONCENTRATION: • 10x the concentration of the MAb/PAb being used in the assay (e.g. if 5μg/mL of Ab/conjugate, add 50μg/ mL Animal IgG). • Can be added to the sample or conjugate diluent but ideally should be in contact with the patient sample before incubation with the assay capture antibody.

TRU Block ™ ULTRA TRU Block ™ TRU Block ™ 2 TRU Block ™ 3

Range: 24-26 mg/mL ELISA, CLIA & LF

Range: 24-26 mg/mL ELISA & CLIA

Range: 24-26 mg/mL

CLIA & LF

24.3 mg/mL

ELISA

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Blockers Practical Guide

BLOCKERS FOR INTERFERENCE IN IGM CAPTURE ASSAYS

IgG Absorbent IgG Absorbent is a purified goat anti-human IgG (GAH IgG Fc) Fc fragment designed for the removal of human IgG and IgG/rheumatoid factor (RF) complexes from serum prior to testing for specific IgM antibodies in ELISA or other immunoassays. Removal of IgG interference has been demonstrated to significantly increase the sensitivity of IgM detection in immunoassays. Meridian’s IgG Absorbent is tested by immunoelectrophoresis to ensure specificity for IgG and no cross-reactivity to IgM or IgA. Goat anti-human IgG (GAH IgG Fc) L15406G. IgM Assay Diluent IgM Assay Diluent is intended for use in qualitative and quantitative assays that detect IgM antibodies. This diluent is formulated to reduce assay interference from a patient’s IgG antibodies, rheumatoid factor, heterophilic antibodies, and other non-specific proteins that can affect the immunoassay results. Blocking proteins within the IgM Assay Diluent are in high excess and will bind to any open binding site on the assay solid phase to prevent binding from interfering factors. When included as a part of a test kit, IgM Assay Diluent can increase the sensitivity of an IgM detection assay.

RECOMMENDED REAGENT: L15406G Goat anti-human IgG Fc (GAH IgG Fc) Dilute prior to adding to patient sample. Recommend diluting 1:10 in PBS.

A dd in a ratio of 1:10 to patient sample and allow to incubate 5-30 minutes.

RECOMMENDED REAGENT: 8120 IgM Assay Diluent

In a separate tube, dilute the patient serum sample in the IgM Assay diluent at a 1:21 dilution or greater (mix well). The diluent must be standardized with the other assay components.

SOLID PHASE BLOCKING BUFFERS

J82100B - ELISA Blocking Buffer (PBS, pH7.4) J82300B - Lateral Flow Blocking Buffer (PBS, pH 7.4) • Non-mammalian based blockers • Blocks non-specific binding to a solid phase subtrate to improve assay sensitivity • Stabilizes bound proteins on microplate/membrane • Increases signal-to-noise ratio • Use at 1x concentration or with further dilution

J16430D - Coating Stabilizer and Blocking Buffer • Improves stability and function of antigens/proteins bound to a solid phase • Ideal for very unstable proteins or for less labile antigens/ proteins. Dilute up to 1:1 in the assay’s current blocker • Buffer pH 7.2 ± 0.2

A64801B - Bovine Serum Albumin (BSA) • Ideal for casein-sensitive antibodies, such as phospho-specific antibodies • Can be used in ELISA, Western blot, and IHC • Use at 1-5% concentration in PBS at pH 7 • Lyopholized

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Recommended Blockers

An isotype control acts as a negative control in flow cytometry and immunohistochemistry assays by differentiating non-specific background signal from specific antibody signal. In addition, Human Immunoglobulin can be used to measure the type of immunoglobulin/ isotype in a patient’s sample, as a standard blocking agent, or as a protein coating agent for applications including immunofluorescence, immunocytochemistry, Western blot, and ELISA. ISOTYPE CONTROLS & OTHER BLOCKERS

RECOMMENDED REAGENT: Human IgG • A50170H, IgG

Mouse IgG • A66186M, IgG

IgM • A32169H, IgM • A99850H, IgM • A50168H, IgM • A50713H, IgM (mu Chain) • A50591H, IgM Fc5u

IgM • A03169M, IgM, kappa

• A50175H, IgG (Fc) • A50177H, IgG (Fab) • A50183H, IgG1, kappa • A50184H, IgG2, kappa • A01384H, IgG3 • A01241H, IgG4, kappa

• A31171H, IgG1, kappa • A01696M, IgG2a, kappa IgA • A03090M, IgA, kappa

IgE • A03900M, IgE

IgE • A50384H, IgE

• A01235H, IgE, kappa • A01268H, IgE, kappa • A75671H, IgE, kappa & lambda

IgA • A50167H, IgA

Rat IgG • A64391R, IgG

Sheep IgG • A66400S, IgG

• A50166H, IgA1, kappa • A50165H, IgA1, lambda • A50555H, IgA2, kappa

Isotype controls are primary antibodies that lack specificity to the target antigen, but match the class and type of the primary antibody used in the application. They act to reduce non-specific binding by the primary/capture antibody through blocking Fc receptor binding or nonspecific antibody interactions with cellular proteins, carbohydrates, and lipids. The isotype control antibody should match the primary/capture antibody’s host species, isotype (including heavy chain (IgA, IgG, IgD, IgE, or IgM) and light chain (kappa or lambda) class) and if relevant, conjugation. In addition, isotype controls should be used at the same concentration as the primary antibody.

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4 | Blockers Practical Guide Blockers r ti l i

Blockers are used in ELISA and LF assays to reduce interference from proteins in patient samples that could produce false results and an incorrect diagnosis. Why Use Blockers?

Choosing the best assay format depends on the intended application of the assay, the type of samples to be analyzed, the availability of reagents and whether the assay is intended for a single analysis in one lab, or is intended to be used in many laboratories by various technicians. Generally, an immunoassay will fall into one of the following categories: •  Sandwich Antigen Detection ELISA - designed to measure the amount of target antigen/analyte •  Antibody Capture ELISA - used to screen for antibodies (e.g: lgG, lgM, lgA & lgE) to a specific target •  Competitive ELISA - detects antigen/analyte present in a sample and is commonly used when the antigen is small with only 1-2 epitopes Several parameters that are critical to assay performance are common among all of these formats: (1) the choice of solid phase (2) the choice of antibodies/antigen and (3) the choice of blocking agents. In order to produce an assay with high sensitivity and specificity, the most critical element is selecting antibodies or antigens that have a highly specific interaction with the target molecule. However, it is possible to improve assay sensitivity and specificity with the use of blocking agents. These blockers work by reducing non-specific binding resulting in an increase in signal-to-noise ratio. Non-specific interactions in an assay can occur (1) between the solid phase and non-target proteins, which can absorb to the surface of the solid substrate and (2) between antibodies within the assay itself and endogenous antibodies present within a patient’s sample. To prevent non-specific binding, blocking buffers are used after the solid-phase coating step to block any remaining open binding sites. Other blocking agents are used in the sample preparation to prevent interfering antibodies from binding to the assay antibody components.

TYPES OF IMMUNOASSAY SOLID PHASES

Binding Capacity

Material

Type of Interaction

Nitrocellulose

High High High

Hydrophobic, Hydrophilic

PVDF Nylon

Hydrophobic Hydrophobic

Plates & Tubes Polystyrene

Low Low

Hydrophobic Hydrophobic

Polyvinyl

Derivatized microtiter plates

Covalent, Hydrophobic, Hydrophilic

Low

Beads

Polystyrene Denvatized Polystyrene

Moderate

Hydrophobic

Covalent, Hydrophobic, Hydrophilic Covalent and Hydrophobic

High

Microparticles

High

A solid surface which has a high binding capacity but does not cause damage to the native protein conformation of the immobilized antigen or antibody is ideal.

KEY PERFORMANCE DIFFERENCES BETWEEN MONOCLONAL AND POLYCLONAL ANTIBODIES

In general, a MAb is often chosen as the primary antibody to establish the highest level of specificity in an assay, and a PAb is chosen as the secondary antibody, to amplify the signal via multiple binding events. However, any combination can be used. All candidate antibodies must be tested together with the intended sample type in order to select the best performers.

Monoclonal antibodies (MAb) • Generally produced in mice or recombinantly, and recognize a single epitope. • Since only one antibody molecule can bind to the antigen, the interaction is highly specific but can lack sensitivity, depending on the affinity constant.

Polyclonal antibodies (PAb) • Produced in goats, sheep, chicken, rabbits and other animals. • Polyclonal sera is a heterogeneous composite of antibodies with unique specificities. The concentration of specific antibody (PAb) is typically 50-200mg/mL. • PAbs are able to recognize multiple epitopes on any one antigen which makes them less sensitive to antigen mutational changes. • PAbs are useful when the nature of the antigen is not well known. However, their quantity is limited by the lifespan of the host animal.

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Immunoassay Formats that Require Blockers

Sandwich Antigen Detection Immunoassays

This is a sensitive and robust method which captures the target antigen between two antibodies (capture and detection antibody). The capture antibody is bound to the solid phase. The antigen-containing sample is applied followed by a wash step to remove unbound antigen. A detection antibody is added that binds directly to the antigen. The capture and detection antibodies must bind to non-overlapping epitopes on the antigen. Either monoclonal or affinity-purified polyclonal antibodies can be used as capture and detection. The antigen can be measured with a conjugated detection antibody (direct detection) or a matched set of unlabeled detection and conjugated secondary antibodies (indirect detection).

Detection Antibody Secondary Antibody

Detector Label

Detector Label

Detection Antibody Capture Antibody Antigen

Capture Antibody Antigen

Direct Detection

Indirect Detection

Antibody detection Assay (IgG, IgM & IgA)

The antigen is immobilized on the solid phase by direct absorption. The antibody-containing sample is applied followed by a wash step. Detection of the antibody can then be performed using an conjugated detection antibody (direct detection) or a matched set of unlabeled detection and conjugated secondary antibodies (indirect detection). Direct detection is shown below.

Detector Label

Detection Antibody

Detector Label

Detection Antibody

Patient’s IgG Ab

Patient’s IgM Ab

Antigen

Antigen

IgG Detection

IgM Detection

Competitive Assay

A competitive binding process between the patient’s target analyte and labeled antigen (inhibitor antigen). Antibody specific for the target analyte is coated onto the solid phase. Patient sample and labeled inhibitor antigen are incubated with the pre-coated antibody and compete for binding sites. Unbound labelled antigen/analyte is removed by washing. The more analyte in the sample, the less the labelled inhibitor antigen will bind to the antibody. Therefore, the weaker the assay signal, the higher the concentration of analyte in the patient sample. This is a common method for small antigens that have only 1-2 epitopes.

Detector Label

Patient Analyte

Inhibitor Antigen

Capture antibody

Direct Detection

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Blockers Practical Guide

Solid phase quantitative immunoassays such as ELISA, lateral flow and Western blot all involve the immobilization of antibodies to a surface. Non-specific binding to this surface by other proteins or biomolecules can reduce an assay’s specificity and sensitivity. Solid phase blocking agents are specifically designed to saturate these unoccupied binding sites to prevent non-specific binding, while enhancing assay sensitivity. Blocking the Solid Phase

When developing a new immunoassay, the first step is to optimize the antigen or capture antibody coating conditions on the solid phase in order to maximize the amount of protein coated. After coating, any remaining unoccupied binding sites must be blocked in order to prevent non- specific binding of subsequent reactants. An ideal blocking agent is typically protein-based as it is able to block both hydrophobic and hydrophilic sites on the solid phase. In addition, it can serve as a stabilizing agent and prevent denaturation while proteins react at the surface of the solid phase. The concentration of the blocker and the amount of blocking time must be optimized for each assay. Using inadequate amounts of blocker will result in excessive background and a reduced signal-to-noise ratio. Using excessive concentrations of blocker may mask antibody- antigen interactions causing less sensitivity. ELISA without blocking buffer Unbound debris such as proteins, interfering molecules in the patient sample, and assay reactant bind to the solid phase and compete with the specific antigen-antibody reaction causing background noise and reduction in specific assay signal.

IDEAL BLOCKING AGENTS HAVE THE FOLLOWING CHARACTERISTICS: • Effectively block nonspecific binding of assay reactants to the surface of the well • Do not disrupt the binding of assay components that have been adsorbed to the well • Act as a stabilizer (prevent denaturation) of assay reactants on the solid phase • Do not cross-react with other assay reactants • Do not possess enzymatic activity that might contribute to signal generation of the substrate or degradation of the reactants • Perform consistently across various lots

ELISA with blocking buffer Blocking buffer is designed to bind to open sites on the solid phase, preventing unbound debris from non-specifically binding.

Detector Label

Detector Label

Detection Antibody Capture Antibody Antigen

Detection Antibody Capture Antibody Antigen

Unbound Debris

Blocking Buffer

GENERAL BLOCKING PROTOCOL

1. After coating the solid-phase with primary antibody, add the blocking solution directly to the wells, beads, blotting membrane or nitrocellulose membrane. 2. Determine the best concentration of blocker for your assay. Blockers can be used at 1x concentration or diluted. 3. Determine the optimum incubation temperature and time for proper absorption of the blockers. Longer times and higher temperature increase the rate of blocking. Typical temperatures are 25°C - 30°C incubated for 30 minutes to 2 hours. 4. Proceed to wash steps.

NOTE: In addition to blocking, it is essential to perform thorough washes between each step. Washing steps are necessary to remove unbound reagents and decrease background noise. Insufficient washing will allow high background. A common technique is to use a diluted solution of the blocking buffer along with some added high-purity detergent.

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Blocking Assay Interference

Immunoassay interference is a general term for substances that can change the outcome of an assay by causing a false positive or false negative test result. Examples of potentially interfering particles include endogenous antibodies or other binding proteins present in a patient sample, polyreactive antibodies or autoantibodies (heterophiles), and human anti-animal antibodies. BACKGROUND INFORMATION ON IMMUNOASSAY INTERFERENCE

TRUE Positive Result

Interfering substances can have non-specific reactions that disrupt the reaction between the analyte and reagent antibodies in an immunoassay by either: (1) out-competing the analyte of interest for binding to the assay antibodies (false negative) or (2) simulta- neously binding to the assay capture and detection antibodies in the absence of any analyte (false positive). Assays that are inherently vulnerable to interference include double- sandwich antigen detection assays, competitive assays and lgM capture assays. Specific examples which have been reported in the literature are assays for ToRCH, CEA, CA-125, CK-MB, LH, FSH, prolactin, TSH, AFP, cardiac troponin I (cTnl) and hCG. The most common type of interference in sandwich and competitive assays is heterophilic antibodies (HA) which are naturally occurring human antibodies with low affinities that can react with immunoglobulins from different species, including mouse, goat, rabbit, sheep and chicken. In diagnostic assays, HAs are able to bind to multiple and seemingly unrelated epitopes to disrupt the assay’s specific antigen-antibody interaction. Human anti-mouse antibodies (HAMA) is one type of HA interference that specifically binds to mouse antibodies. Due to the high use of mouse monoclonal antibodies in commercial diagnostic immunoassays, HAMA interference is the most widely experienced type. Rheumatoid factor (RF), an autoantibody that reacts with the patient’s own immunoglobulin (lg), may also cross-react with animal lg resulting in “RF interference”, which is similar to HA/HAMA interference. Generally, isotype (Fc region)-specific interfering HA is more common than idiotype (Fab or binding site)-specific HA. lgM capture assays generally experience two types of interference. The first is from high levels of patient lgG antibodies that can compete with lgM for antigen binding sites on the solid phase. Since lgG antibodies are highly abundant, representing approximately 75% of serum antibodies in humans, they can outcompete lgM due to their sheer quantity. The second type of interference is caused by lgM RF which can produce false-positive signals by reacting with the Fc fragment on immunoglobulins. RF are found in 1% to 4% of the general population and in 75% of adult patients over 65 years of age.

Detection Antibody Capture Antibody Antigen

Blocking Buffer

FALSE Negative Result

Blocking Buffer

Interfering antibody blocks the binding site on the capture or detection antibody. Occurs in both sandwich and competitive assays.

FALSE Positive Result

Interfering Antibody (HA interference)

Blocking Buffer

Interfering antibody binds to both the capture and detection antibody even in the absence of the antigen. Occurs in sandwich assays only.

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Blockers Practical Guide

INTERFERENCE IN SANDWICH IMMUNOASSAYS

A sandwich immunoassay uses two antibodies (either monoclonal or polyclonal) that bind to different sites on the antigen or analyte of interest. The capture antibody binding affinity for the antigen is usually the main determinant of immunoassay sensitivity. However, human anti-animal immunoglobulin antibodies (HA antibodies) can interfere with this interaction and reduce assay sensitivity and specificity.

Mixed Species Assays

Double Mouse Monoclonal Assays

e.g. goat, sheep, rabbit, rat, chicken, mouse

Detection Ab

Mouse MAb Antigen

Antigen

Blocking Buffer

Blocking Buffer

e.g. mouse, chicken, rabbit, rabbit, goat

Mouse MAb

Capture Ab

For mixed species sandwich assays, the IgG blocker used must be the same as the host of the capture and detection antibodies. More than one species of IgG is required when two different antibody species are used in a sandwich assay.

For double mouse monoclonal antibody sandwich assays, mouse IgG in addition to an active HAMA blocker (TRU Block) should be used to remove a specific type of HA interference called human anti-mouse antibodies (HAMA) and Rheumatoid Factor (RF).

INTERFERENCE IN IgM CAPTURE ASSAYS

IgM antibodies are the first type of antibodies produced by the immune system in response to an infection. Consequently, lgM detection assays have proven to be valuable diagnostic tools that assist in identifying early and recent infections. However, lgM antibodies only comprise 5% to 10% of all the antibodies in the body. In contrast, lgG antibodies are the most abundant immunoglobulin and comprise about 75% to 80%. In order to ensure an lgM assay is both sensitive and specific, it is necessary to reduce assay interference, especially from the more plentiful lgG antibodies and other non-specific proteins, such as rheumatoid factors (RF).

Goat Anti-human IgG or IgM Diluent

Detection Antibodies

Patient IgG

Detector Label

Patient’s IgM

Antigen

IgM Capture Assay

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Product List ABBREVIATIONS

Ab

IgM IgY

Antibody

Immunoglobulin M Immunoglobulin Y

Aff. Pur.

Affinity Purified

Block

LF

Tested as a blocking agent Bovine Serum Albumin Enzyme Immunoassay Fragment crystallizable region of an antibody Heavy and light chain in IgG

Lateral flow

BSA

MAb Monoclonal antibody Monospecific Monospecific when tested by immunoelectrophoresis Neat

EIA, ELISA

Fc

Whole, unpurified, undiluted antisera

H&L

Neph

Nephelometry

HA

PAb PBS

Heterophillic antibodies

Polyclonal antibody

HAMA

Human anti-mouse antibodies

Phosphate buffered saline

HRP IEP IgA IgE IgG

Purified

Horseradish peroxidase Immunoelectrophoresis

Refer to the product COA regarding the extent of purification and/or process used

Immunoglobulin A Immunoglobulin E Immunoglobulin G

TIA Tris

Turbidimetry

T ris (hydroxymethyl) aminomethane based-buffer

Animal Serums Animal serum contains a diverse mixture of proteins which can absorb heterophile interfering agents in various assays. Animal serum can be used in place of or in addition to animal IgG. The species of animal serum must be the same as the antibody reagent in the assay. Specificity Cat Number Host / Source Format Bovine Serum, Sterile Filtered N21010B Bovine Neat Canine Plasma, Pooled/Mixed Gender, Unfiltered, Non-Sterile (Heparin) N01262C Canine Plasma Fish Serum (cell culture grade) N82800F Fish Neat Goat Serum, Sterile Filtered N66001G Goat Neat Goat Serum, Sterile Filtered and Heat Inactivated N66111G Goat Neat Horse Serum, Sterile Filtered N21010H Horse Neat Mouse Serum, Filtered N14010M Mouse Neat Porcine Serum, Sterile Filtered N64250P Pig Neat Rabbit Serum N66010R Rabbit Neat Rabbit Serum, Sterile Filtered and Heat Inactivated N01267R Rabbit Neat Rat Serum, Filtered N64110A Rat Neat Sheep Serum, Sterile Filtered N21010S Sheep Neat

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Blockers Practical Guide

Chicken Immunoglobulins Chicken Igs should be used in sandwich or competitive ELISAs or lateral flow assays that use a chicken antibody. Any human anti-chicken antibodies in the patient sample that could potentially interfere with the assay signal can bind to the chicken IgY, preventing non-specific binding that could interfere with assay results.

Specificity IgY, Chicken IgY, Chicken IgY, Chicken

Cat Number

Host / Source Chicken Eggs

Format Purified

A01366C A01460C A01302C

Chicken Serum Purified Chicken Serum Purified

Goat Immunoglobulins Goat Igs should be used in sandwich or competitive ELISAs or lateral flow assays that use a goat antibody. Any human anti-goat antibodies in the patient sample that could potentially interfere with the assay signal can bind to the goat Ig, preventing non-specific binding that could interfere with the assay results.

Specificity

Cat Number

Host / Source

Format Purified

IgG, Goat-Differential ppt

A66200H

Goat Serum

Human Immunoglobulins Human Igs can be used in flow cytometry, IHC, sandwich or competitive ELISAs or lateral flow assays to block non-specific binding. The selected antibody should match the primary/capture antibody’s host species, isotype (including heavy chain (IgA, IgG, IgD, IgE, or IgM) and light chain (kappa or lambda class) and if relevant, conjugation.

Specificity IgE, kappa IgE, lambda

Cat Number

Host / Source

Format

A01268H A01720H A75671H A50183H A50184H A01241H A32169H A50591H A50166H A50165H A50555H A50167H A50170H A50177H A50175H A01384H A50713H A50168H

Human Myeloma Neat Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified Human Myeloma Purified

IgE, kappa and lambda

IgG1, kappa IgG2, kappa IgG4, kappa

IgM >95% Pure

IgM, Fc5U IgA1, kappa IgA1, lambda IgA2, kappa

IgA IgG

Human Plasma Human Plasma Human Plasma Human Plasma Human Plasma Human Plasma Human Plasma

Purified Purified Purified Purified Purified Purified Purified

IgG (Fab) IgG (Fc)

IgG3

IgM (mu Chain) IgM >95% Pure

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Product List

Mouse Immunoglobulins Mouse Igs should be used in sandwich or competitive ELISAs or lateral flow assays that use a mouse antibody. Any human anti-mouse antibodies (HAMA) present in the patient sample that could potentially interfere with the assay signal can bind to mouse Ig, preventing non-specific binding that could interfere with the assay results.

Specificity IgE, Mouse

Cat Number

Host / Source

Format

A03900M A01696M A03090M A31171H A03169M

Mouse Serum Aff.Pur. Mouse Myeloma Purified Mouse Myeloma Aff.Pur. Mouse Myeloma Purified Mouse Myeloma Purified Mouse Serum Purified Mouse Serum Purified Mouse Serum Purified Mouse Serum Purified Mouse Serum Purified Mouse Serum Purified

IgG2a, kappa, Mouse IgA, kappa, Mouse IgG1, kappa, Mouse IgM, kappa, Mouse

IgG, Mouse Protein A (9-13mg/ml) Dialyzed Azide Free

A66186M-NA

IgG, Mouse-Caprylic Acid Cut

A66187M A66186M A66181M A66189M A66185M

IgG, Mouse-Protein A (9-13 mg/mL) IgG, Mouse-Protein A (22-28 mg/mL)

IgG, Mouse Protein A (45-55mg/mL) No Azide

IgG, Mouse-Protein A (50-55 mg/mL)

IgG, Mouse-Protein A (50-55mg/ml)

A66185M-LY

Mouse Serum Purified Lyopholized

Rabbit Immunoglobulins Rabbit Igs should be used in sandwich or competitive ELISAs or lateral flow assays that use a rabbit antibody. Any human anti-rabbit antibodies in the patient sample that could potentially interfere with the assay signal can bind to rabbit Ig, preventing non-specific binding that could interfere with the assay results.

Specificity

Cat Number

Host / Source

Format

IgG, Rabbit-Differential ppt IgG, Rabbit-Differential ppt

A66100H A66105R

Rabbit Serum Purified Rabbit Serum Purified

Rat Immunoglobulins Rat IgG should be used in sandwich or competitive ELISAs or lateral flow assays that use a rat antibody. Any human anti-rat antibodies in the patient sample that could potentially interfere with the assay signal can bind to rat IgG, preventing non-specific binding that could interfere with the assay results.

Specificity

Cat Number

Host / Source

Format Purified

IgG, Rat

A64391R

Rat Serum

Sheep Immunoglobulins Sheep Igs should be used in sandwich or competitive ELISAs or lateral flow assays that use a sheep antibody. Any human anti-sheep antibodies in the patient sample that could potentially interfere with the assay signal can bind to sheep Ig, preventing non-specific binding that could interfere with the assay results.

Specificity

Cat Number

Host / Source

Format

IgG Sheep, >=70mg/mL

A66400S

Sheep Serum Purified

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Blockers Practical Guide

Solid Phase Blockers Solid phase blocking agents are designed to saturate unoccupied binding sites on the solid phase to prevent non-specific binding. Typically blocking buffers are able to block both hydrophobic and hydrophilic sites on the solid phase. In addition, they can serve as stabilizing agents and prevent denaturation as proteins react at the surface of the solid phase.

Specificity

Cat Number

Host / Source

Format

Blocking Buffer for ELISA

J82100B J82300B J16430D J16200D A64801B A01245B A51300B

n/a n/a n/a n/a

n/a

Blocking Buffer for Lateral Flow Assays, PBS based

Liquid Liquid Liquid

Coating Stabilizer & Blocking Buffer Conjugate Stabilizer Diluent, HRP

BSA, Standard Powder Gamma Globulin, Bovine Gamma Globulin, Bovine Bovine Serum Albumin (BSA) Bovine Serum Albumin (BSA)

Bovine Bovine

Purified Purified Purified Aff.Pur. Aff.Pur.

Bovine Serum

C5BP01-188 Chicken C5BP01-296 Chicken

IgG Absorbents IgG absorbents are designed for the removal of human IgG and Ig / rheumatoid factor (RF) complexes from serum and plasma prior to testing for specific IgM antibodies in ELISA or other immunoassays. Removal of IgG interference has been demonstrated to significantly increase the sensitivity of IgM detection.

Specificity

Cat Number

Host / Source

Format Purified

IgM Assay Diluent

8120

Goat Goat Goat Goat

IgG (Fc) IgG (Fc) IgG (Fc)

L15406G

Monospecific

G5G16-048

Aff. Pur. Purified

L62540G

TRU Block ™ - HAMA & RF Blocker In double mouse monoclonal sandwich assays, an active blocker against human anti-mouse antibodies (HAMA) and rheumatoid factor (RF) should be used to prevent non-specific interference leading to false assay results. TRU Block is a proprietary active blocker that can significantly reduce assay interference and improve assay results. It can be used at a lower concentration than passive blocking reagents such as mouse IgG.

Specificity

Cat Number

Host / Source

Format Purified Purified Purified Purified Purified

TRU Block™, Ready Heterophilic Antibody Interference, Active Blocker TRU Block™ 2, Heterophilic Antibody Interference, Active Blocker TRU Block™ 3, Heterophilic Antibody Interference, Active Blocker TRU Block™, Heterophilic Antibody Interference, Active Blocker TRU Block™ Ultra, Heterophilic Antibody Interference, Active Blocker

8001

Confidential

A66802H A66803H A66800H

Mouse & Confidential Mouse & Confidential Mouse & Confidential

8000

Confidential

HAMA & RF Reagents HAMA (human anti-mouse antibodies) or rheumatoid factor positive patient samples that can be used to test the effectiveness of a blocking agent in any type of assay.

Specificity

Cat Number

Host / Source Human Plasma Human Plasma Human Plasma

Format

Human Anti Mouse Antibody (HAMA) Plasma Rheumatoid Factor (RF) Control Serum Rheumatoid Factor (RF) Control Serum

A01431H A01270H A12916H

Neat Neat Neat

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Product List

Secondary antibodies are used for indirect detection and they can provide an increase in sensitivity through signal amplification, and greater flexibility for labeling and detection. The secondary antibody should not be from the same host species as the primary antibody, but it should have the species specificity for the primary antibody. For example, if the primary antibody is sourced from rabbits, the secondary antibody should be anti-rabbit but it should be raised in a host species other than rabbit (e.g. goat anti-rabbit secondary). SECONDARY ANTIBODIES

Mouse Immunoglobulins

Polyclonal Sheep

Specificity

Cat Number Format Isotype Z01253M Purified IgG2b

Specificity IgA, Human

Cat Number W90010F R5G10-048

Format

IgA (Fc), Human IgA (Fc), Human

FITC

Z01253P

HRP IgG2b

IgG (H&L), Human

Aff.Pur.

IgA (Secretory Component), Human

Z42750M Purified IgG1

Polyclonal Rabbit

IgA, Human

Z01250M Purified IgG1 Z86104M Purified IgG2a MAGG16-264 Purified IgG2b,k Z01247M Purified IgG Z54101M Purified IgG1

Specificity IgA, Human

Cat Number W90010F W99114C W99165C W99122P R5G10-048 W01256R

Format

IgE (Fc), Human IgG (Fc), Goat IgG (Fc), Human IgG (Fc), Human IgG (Fc), Human IgG (Fc), Human IgG (Fc), Human IgG (Fc), Rabbit

Biotin

IgG (Fc), Canine IgG (Fc), Human

Aff.Pur. Aff.Pur.

IgG (Fc) (min x w/Hu), Goat

HRP

Z86101P

HRP IgG

IgG (H&L), Human IgG (H&L), Mouse IgG (H&L), Sheep IgG (H&L), Sheep

Aff.Pur. Aff.Pur.

Z86213M Purified IgG Z86238M Purified IgG1

R5SG10-048 Aff.Pur.

MARG16-125 Purified IgG2b,k IgG (Heavy Chains), Human Z20142M Purified IgG1,k IgG, Human Z01236M Purified IgG1,k IgG, Sheep P86502M Purified IgG1 IgG1 (Fc), Human Z86102M Purified IgG1 IgG2 (Fc) gamma-2, Human Z86002M Purified IgG2 IgG3 (hinge-region), Human Z86130M Purified IgG1 IgG4 (Fc), Human Z20154M Purified IgG1,k IgG4 (Fc), Human Z86140M Purified IgG1 IgM (Fab-region), Human Z86215M Purified IgG2b IgM (Fc), Human Z86113M Purified IgG1 IgM (Fc-region), Human Z01234M Purified IgG1 IgM (Heavy Chains), Human Z86110M Purified IgG1 IgM (Heavy Chains), Human Z86114M Purified IgG1 IgM, Human Z01235M Purified IgG1,k IgM, Human Z45190M Purified IgG1 IgM, Sheep P86501M Purified IgG1

W99245P W99020C

HRP

IgM, Mouse

Aff.Pur.

Monoclonal Rat

Specificity

Cat Number Format Isotype

IgG2a, Mouse

P87106M Aff.Pur.

IgG1

14

Blockers Practical Guide

Polyclonal Goat Anti-Human

Polyclonal Goat Anti-Mouse

Specificity IgG (H+L)

Cat Number Format W01262FL FITC

Specificity

Cat Number

Format

IgE (epsilon chain)

W03700G HRP G5MG16-048 Aff.Pur. W41202G HRP G5MG20-0482 Aff.Pur. W99062G Aff.Pur. G5MG10-048 Aff.Pur. G5MG10-0482 Purified G5MG10-0484 Aff.Pur. W41250G TRITC W41502G FITC G5MG10-766 Aff.Pur W03910G HRP

IgA

L66605G Monospecific

IgG (Fc) IgG (Fc)

IgE (epsilon)

L62507G Purified G5G41-048 Aff.Pur. G5G41-048 Aff.Pur. K10262G Aff.Pur.

IgE (Fc) IgE (Fc)

IgG (gamma)

IgG (H&L) (min x w/Hu Bov Hrs)

IgE (NHS Adsorbed)

IgG (H&L) IgG (H&L) IgG (H&L) IgG (H&L) IgG (H&L)

IgG (Fc) (min x w/Hu Bov Hrs Ms) W99036P HRP IgG (Fc) (min x w/Hu Bov Hrs Ms Rab) W01252G Aff.Pur. IgG (Fc) G5G16-0482 HRP IgG (Fc) W66040G Aff.Pur. IgG (Fc) W99008A Alk.Phos. IgG (Fc) W99008P HRP IgG (H&L) (min x w/Bov Hrs Ms) W01239F FITC IgG (H&L) W01260AF Alexa Fluor 488 IgG (H&L) W41808G Aff.Pur. IgG (H&L) W99003P HRP IgM (Fcu) L66607G Monospecific IgM (Fcu) W99154A Alk.Phos IgM (Fcu) W99154G Aff.Pur. IgM (mu) (min x w/Bov Gt Ms Rab) W01251G Aff.Pur. IgM (mu) (min w/IgA and IgG) W01259G Aff.Pur. IgM (mu) G5G54-048 Aff.Pur. IgM (mu) G5G54-0482 HRP IgM (mu) G5G56-048 Aff.Pur. IgM (mu) L04354G Monospecific IgM (mu) L62650G Purified IgM (mu) W01258G Aff.Pur. IgM (mu) W66210G Aff.Pur. IgM (mu) W66310G Aff.Pur. Kappa Light Chain (Free & Bound) L15805G Purified Kappa Light Chain (Free & Bound) L62805G Monospecific Lambda Light Chain (Free & Bound) K04365G Purified Lambda Light Chain (Free & Bound) L15905G Purified Lambda Light Chain (Free & Bound) L62905G Monospecific

IgG (H&L, Goat)

IgG1

Polyclonal Goat Anti-Rabbit

Specificity

Cat Number

Format

IgG (Fc) IgG (Fc)

G5G16-766 Aff.Pur. W41613G Aff.Pur. W41600G Aff.Pur. G5RG10-048 Aff.Pur. G5RG10-0482 HRP G5RG14-0483 FITC S5RG10-0481 Alk.Phos. W41604G Aff.Pur.

IgG (H&L) (min x w/Hu Ms Bov)

IgG (H&L) IgG (H&L) IgG (H&L) IgG (H&L) IgG (H&L) IgG (H&L)

W99035P

HRP

Polyclonal Goat Anti-Bovine

Specificity IgG (H&L) IgG (H&L) IgG (H&L)

Cat Number

Format

W01254G Aff.Pur. W99101B Biotin W99101G Aff.Pur.

Polyclonal Goat Anti-Canine

Specificity

Cat Number

Format

IgG (Fc)

W99114C Aff.Pur.

15

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May 2020

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