ToRCH IgG/IgM Assay Development Guide

ToRCH IgG/IgM Assay Development

www.MeridianBioscience.com/LifeScience

ToRCH Overview

ToRCH is an acronym for a group of infections that can cause significant birth defects and even fetal death. Meridian Life Science offers a complete range of antigens and other reagents for the detection of IgG and IgM antibodies in various assay formats such as EIA, rapid anti-IgM assays and Immunofluorescence (IFA).

The ToRCH test measures the levels of an infant’s antibodies against five groups of chronic infections: toxoplasmosis, rubella, cytomegalovirus (CMV), herpes simplex virus (HSV) and other infections. The “other infections” usually include syphilis, hepatitis B, coxsackie virus, Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human parvovirus. These infectious diseases are all associated with congenital abnormalities resulting from maternal infection. Although these organisms typically cause only asymptomatic or mild infection in the mother, they can have serious consequences for the fetus. If the infection occurs during the first three months of pregnancy and if it is a primary infection (newly acquired during pregnancy), the risk of congenital abnormalities is much higher as compared to a secondary or reactivated infection. CMV is the most common cause of congenital infectious disease with a much higher rate of transmission (10% vs. 1%) for mothers with a primary infection compared to a reactivation. Consequently it is a very important part of prenatal care to recognize these infections in the first trimester of pregnancy.

IgM vs IgG SEROLOGY TIMELINE

For most ToRCH organisms, the initial screening test is based on the detection of antibodies to the organism. Subsequent screening, if required, is carried out using a monoclonal antibody-based immunofluorescent assay (IFA). Assays are commercially available for the detection of IgG, IgM, or both IgG/IgM antibodies. In most cases, IgG reactivity in the absence of IgM reactivity is indicative of a past infection, while IgM reactivity in the absence of IgG reactivity indicates a current infection. However, for some ToRCH diseases such as toxoplasmosis and CMV infections, IgG avidity has recently been found to be useful for identifying primary infections. An IgG antibody produced in the first few months following an initial infection has a lower avidity than an IgG antibody produced several months or years later; consequently, low-avidity antibody can be used to specifically identify high-risk mothers with a primary infection. To protect a fetus from ToRCH infection, early diagnosis through first trimester screening is critical.

2 | ToRCH REAGENTS - IgG and IgM Assay Development

Meridian ToRCH Antigens & Antibodies

Antigens and Antibodies

Assay Reagents

Toxoplasma gondii

Anti-human IgM

8200

Native Ag

Z01235M MAb to IgM W01258G Goat PAb ( µ chain) W01259G G oat PAb ( µ chain), low

R01573 R01581

Rec. Ag. p30 (SAG1), E. coli

Rec. Ag. p35 (GRA8), E. coli C01523M MAb to T. gondii SAG1 (p30) protein C01589M MAb to T. gondii (38kDa protein)

cross-reactivity to IgA & IgG

Anti-human IgG

Rubella Virus

Z86238M M Ab to IgG (Fc), no cross-reactivity with IgA, IgM or IgE

6075 Native Ag., Grade III, highly pure 6076/6123 Native Ag., Grade IV, highly pure R01491 Rec. Ag., E1 Mosaic, E. coli EV9525 MAb to Rubella, purified EV9526 MAb to Rubella, purified

IgG Absorbent (Required for IgM Assays)

L15406G Goat anti-human IgG (Fc) 8120 IgM Diluent

Cytomegalovirus (CMV)

7511 7600

Native Ag., IgM Concentrate Native Ag, IgG/IgM Concentrate

Solid Phase Blocking Buffers

EV9268 EV7509 R18102 R01561 R01562 R01563

Native Ag., Grade II

J82100B J82300B

ELISA blocking buffer

Native gB Ag.

Lateral Flow blocking Buffer J16403D Coating stabilizer and Blocking Buffer

Rec. gB Ag., E. coli

Rec. Ag pp52 (UL44), E. coli Rec. Ag pp65 (UL83), E. coli

Rec. Ag pp150 (UL32), E. coli C86314M MAb to Early Antigen (65kDa protein) C8A022M MAb to Immediate Early Antigen, pp72

Herpes Simplex Virus (HSV) 1

To order

7305

Native Ag.

Email: orders@meridianlifescience.com Phone: +1 901-382-8716 Fax: +1 901-333-8223 Meridian Life Science, Inc. 5171 Wilfong Rd. / Memphis, TN 38134 www.MeridianBioscience.com/LifeScience ISO Certified Company

VTI520 Rec. Ag. glycoprotein G 1 (S. cerevisiae) C05014MA MAb to Nucleocapsid protein (155kDa) C66150M MAb to Glycoprotein G 1

Herpes Simplex Virus (HSV) 2

7705

Native Ag.

VTI530

Rec. Ag. glycoprotein G 2 (S. cerevisiae)

www.MeridianBioscience.com/LifeScience | 3

Common Types of ToRCH Assays

ToRCH IgG & IgM Capture Assays

A ToRCH serologic test detects IgM and IgG antibodies to the ToRCH panel of infectious diseases (Toxo, Rubella, CMV and HSV). IgM is the immediate antibody that is produced once a human is exposed to a bacteria, virus or a toxin and disappears within 2-3 weeks. It is then replaced by IgG which lasts for life and provides lasting immunity. Meridian’s ToRCH antigens are suitable for IgG and IgM detection. They can be used in a range of immunoassay formats including, but not limited to, ELISA, LF, CLIA, rapid assays, and bead-based assays.

Detectable product

GENERAL ASSAY PRINCIPLE 1. Solid-phase (assay plate, beads, etc) is coated with the antigen 2. Blocking buffer is added to block the remaining binding site 3. Sample is added and patient’s IgG or IgM antibody binds to the antigen 4. Detection can either be by direct or indirect methods

INDIRECT

Substate

Secondary Antibodies

Enzyme

Blocking Reagent

Antigen Patient’s IgG or IgM Ab

INDIRECT DETECTION: uses a labeled secondary antibody for detection. The secondary antibody has specificity for human IgG or IgM.

ToRCH Rapid Anti-IgM Assays

Rapid anti-IgM assays are particularly sensitive in demonstrating IgM responses early in the illness. These assays work by binding IgM-specific antibodies in the patient’s specimen to a solid phase coated with an anti-IgM capture antibody. Soluble antigen is added in excess allowing the specific IgM antibody-antigen reaction to occur in the absence of competing immunoglobulin isotypes. Finally, a labelled detection antibody is added which has specific reactivity against the antigen. Assay sensitivity can be highly dependent on the purity of the antigen used. ELISA capture for IgM determination minimizes interference of rheumatoid factor.

GENERAL ASSAY PRINCIPLE 1. Solid-phase is coated with anti-human IgM (MAb or PAb Blocking buffer is added to block the remaining binding site) 2. IgM-specific antibodies in the patient’s sample bind to the anti-human IgM 3. Antigen (e.g. Rubella, toxo) is added in excess and a antibody- antigen-antibody complex forms 4. Detection can either be by direct or indirect methods

Substate

Detectable product

Detectable product

Enzyme

Anti-ToRCH Antigen

IgM captured from patient sample

ToRCH Antigen

Anti-human IgM antibody Recommended:

Cat# Z01235M Cat# W01258G Cat# W01259G

DIRECT DETECTION

INDIRECT DETECTION

DIRECT DETECTION: uses a labeled antigen that reacts directly with the antibody.

INDIRECT DETECTION: uses a labeled secondary antibody for detection.

4 | ToRCH REAGENTS - IgG and IgM Assay Development

How to Increase Assay Sensitivity

Use a solid phase blocking buffer

Solid phase blocking buffers are designed to efficiently prevent non-specific binding, reduce background noise, and stabilize coated proteins to enable more sensitive immunoassays. They work by blocking unoccupied spaces on the surface to prevent non-specific binding to this surface by other proteins or biomolecules.

Recommended Reagents:

Blocking Buffer for ELISA

J82100B J82300B

Blocking Buffer for Lateral Flow, PBS Based

J16430D Coating Stabilizer and Blocking Buffer

Use IgG absorbant to remove IgG and RF

Recommended Reagents:

The sensitivity and specificity of IgM detection can be compromised by the presence of IgG in the patient sample. There are two major mechanisms by which IgG can interfere with assays for IgM and cause a false result: • by competing with specific IgM for substrate binding sites • by forming immune complexes with Rheumatoid Factor (RF) which can compete with specific IgM for substrate binding sites Removal of IgG and RF-IgM can be accomplished by pre-treating the patient specimen with goat anti-human IgG.

L15406G Goat anti-human IgG FC (GAHG)

Dilute prior to adding to patient sample. Recommend diluting 1:10 in PBS. Add in a ratio of 1:10 to patient sample and allow to incubate 5-30 minutes. IgM 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.

8120

How to use Blocking Buffer and GAH IgG

General Protocol

STEP 2 Add blocking buffer A. Add the blocking solution directly to the wells, blotting membrane or nitrocellulose membrane depending on the assay type being used. Use at 1X concentration or with further dilution ELISA Blocking Buffer: Cat# J82100B Lateral Flow Blocking Buffer: Cat# J82300B Coating Stabilizer & Blocking Buffer: Cat# J16430D B. Incubate at room temperature for 30 minutes to 2 hours C. Continue with

STEP 4 Add patient sample mixture to the reaction well A. Incubate patient sample with the antigen B. Perform wash steps: remove non-bound reagents

STEP 1 Optimize the plate/nitrocellulose-coating conditions for the antigen A. Coat the plate with antigen: 2-10 μg/mL solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline (pH 7.4) or carbonate- bicarbonate buffer (pH 9.4) B. Incubate plate for several hours to overnight at 4-37°C C. Remove coating solution, perform wash steps

STEP 3 Pre-incubate GAHG with the patient’s serum A. Dilute GAH IgG 1:10 in PBS B. Add diluted GAH IgG to patient sample 1:10 and mix C. Incubate for 3-5 min and proceed with sample testing

If patient IgM is present it binds to the antigen.

Goat anti-human IgG (GAHG)

Patient’s serum with both IgG and IgM antibodies

Patient sample

your process and reagents according to the assay protocol

Patient’s serum and GAHG are pre-incubated prior to adding to plate

The GAHG complexes

with the patient’s IgG

STEP 5 Detection by direct or indirect methods

Antigen is coated to the plate

www.MeridianBioscience.com/LifeScience | 5

Commercial Assays & Recommended Products

Toxoplasma IgM

Detection System

Recommended Products

Assay Type

Solid Phase

Antigen

Mouse anti-human IgM, F(ab’)2 to Toxo & Rec. p30 antigen

Z01235M, C01523M and R01573 Z01235M, C01523M and 8200 Z01235M, C01523M and 8200 Z01235M, C01523M and 8200 W01259G, C01523M and 8200

Abbott ARCHITECT

CMIA IgM-capture sandwich method

Paramagnetic microparticles

Direct, CL

Abbott AxSYM

MEIA IgM-capture sandwich method

Microparticles Mouse anti-human IgM, MAb to Toxo p30 & native lysate Indirect, MUB

Siemens IMMULITE

CLIA IgM-capture sandwich method

Paramagnetic particles

Mouse anti-human IgM, MAb to Toxo & p30 antigen

Direct, CL

DiaSorin LIAISON

CLIA IgM-capture sandwich method

Paramagnetic particles

Mouse anti-human IgM, MAb to Toxo & native antigen Goat anti-human IgM, MAb to Toxo p30 & native antigen (RH Sabin strain)

Indirect, CL

BioMérieux VIDAS

ELFA IgM-capture sandwich method

Solid phase receptacles

Direct, MUB

Toxoplasma IgG

Detection System

Recommended Antigens

Assay Type

Solid Phase

Antigen

Abbott ARCHITECT ARCHITECT Avidity

Paramagnetic microparticles Paramagnetic microparticles

Rec. antigens to 30 and p35 (GRA8), anti-human IgG Rec. antigens to 30 and p35 (GRA8), anti-human IgG

CMIA

Indirect, CL R01573, R01581 and Z86238M

CMIA 2 assays with and without blocking buffer to dissociate low-avidity antibodies

Indirect, CL R01573, R01581 and Z86238M

Siemens AVIDA Centaur

Paramagnetic particles

CLIA

Native p30 antigen

Direct, CL

8200

Siemens IMMULITE 2000

Paramagnetic particles Paramagnetic microparticles Paramagnetic microparticles

Partially purified antigen, mouse anti-human IgG

CLIA

Indirect, CL 8200

DiaSorin LIAISON DiaSorin LIAISON Avidity

Native antigen, anti-human IgG Native antigen, anti-human IgG

CLIA

Indirect, CL 8200 and Z86238M

CLIA 2 assays with and without a 6M urea elution step to dissociate low-avidity antibodies

Indirect, CL 8200 and Z86238M

BioMérieux VIDAS BioMérieux VIDAS Avidity

Solid phase receptacles Solid phase receptacles

ELFA

Native antigen

Direct, MUB 8200

ELFA 2 assays (reference and test) with and without a disassociation wash step to remove low-avidity

Native antigen (RH Sabin strain), anti-human IgG

Direct, CL

8200 and Z86238M

ELFA: Enzyme-linked fluorescence assay CLIA: Chemiluminescent Immunoassay CMIA: Chemiluminescent Microparticle Immunoassay MUB: Methyl-umbellifery CL: Chemiluminescence MEIA: Microparticle Enzyme Immunoassay ECLIA: Electrochemiluminescence Immunoassay

6 | ToRCH REAGENTS - IgG and IgM Assay Development

Rubella IgM

Detection System

Recommended Products

Assay Type

Solid Phase

Antigen

Abbott ARCHITECT i2000SR Roche ELECSYS Siemens IMMULITE 2000 Siemens AVIDA Centaur XP

Paramagnetic microparticles

Viral lysate (strain HPV77) and mouse anti-human IgM MAb Indirect, CL 6076/6123

CMIA

Recombinant rubella like particles and rubella-specific antibodies

CLIA IgM-capture sandwich method Magnetic beads

Indirect, CL R01491 and EV9525 or EV9526

CLIA IgM-capture sandwich method Polystyrene beads

Viral lysate (strain HPV77)

Indirect, CL 6076/6123

Viral lysate and rubella specific antibody

CLIA IgM-capture sandwich method Paramagnetic particles

Indirect, CL 6076/6123 and EV9525 or EV9526

Rubella IgG

Detection System

Recommended Products

Assay Type

Solid Phase

Antigen

Abbott ARCHITECT i200SR

Partially purified viral lysate (strain HPV77), mouse anti-human IgG MAb

Paramagnetic microparticles

CMIA

Indirect, CL 6075 and Z86238M

Abbott AxSYM

MEIA

Microparticles Purified viral lysate and anti-human IgG

Indirect, MUB 6075 and Z86238M

Siemens AVIDA Centaur XP Siemens IMMULITE 2000 Beckman ACCESS 2 Roche ELECSYS BioMérieux VIDAS Ortho VITROS Eci

Purified viral lysate (strain HPV77) and mouse anti- human IgG FC

Paramagnetic particles

CLIA moderate complexity

Indirect, CL 6075 and Z86238M

Paramagnetic particles Paramagnetic particles

CLIA moderate complexity

Viral lysate

Indirect, CL 6075

EIA

Viral lysate

Indirect, CL 6075

Recombinant E1 antigen, rubella like particles and rubella specific antibodies Viral lysate (strain MR383) and anti-human IgG Viral lysate and mouse-anti- human IgG

CLIA, double antigen sandwich with an IgG-capture method ELFA, sandwich immunoassay method

Magnetic beads

Indirect, CL R01491 and EV9525 or EV9526

Solid phase receptacles

Indirect, MUB 6075 and Z86238M

Indirect, Luminescence 6075 and Z86238M

CLIA high complexity

Plastic wells

CMV IgM

Detection System

Recommended Products

Assay Type

Solid Phase

Antigen

Viral lysate (strain AD169), rec. antigen pp150 and pp52, and anti-human IgM Rec. antigen CMV pp150, pp52, pp65, pp38, and anti-human IgM Purified antigen (strain AD169), Goat anti-human IgG, goat anti-human IgM

Abbott ARCHITECT

Paramagnetic microparticles

CMIA

Indirect, CL R01561, R01563 and W01259G

R01561, R01562, R01563, R01567 and W01259G 7600 or 7511 and W01259G and L15406G R01561, R01563 and Z01235M 7600 or 7511 and Z01235M

Abbott AxSYM

MEIA

Microparticles

Indirect, MUB

Siemens IMMULITE 2000 Roche ELECSYS BioMérieux VIDAS

CLIA 3 step assay, IgM-capture sandwich method ECLIA IgM-capture sandwich method

Paramagnetic particles Paramagnetic microparticles

Direct, CL

Rec. antigens CMV pp150 and pp52, and anti-human IgM Direct, CL Viral lysate (strain AD169), mouse anti-human IgM Indirect, MUB

ELFA IgM-capture sandwich method Solid phase receptacles

www.MeridianBioscience.com/LifeScience | 7

Commercial Assays & Recommended Products continued

CMV IgG

Detection System

Recommended Antigens

Assay Type

Solid Phase

Antigen

Abbott ARCHITECT i2000SR Abbott ARCHITECT Avidity

Paramagnetic microparticles Viral lysate

CMIA

Indirect, CL 7600 or 7511

CMIA, two assays with and without liquid CMV antigen to neutralize high-avidity CMV antibodies

Paramagnetic microparticles Viral lysate, anti-human IgG Indirect, CL

7600 or 7511 and Z86238M

Abbott AxSYM MEIA

Microparticles Viral lysate

Indirect, MUB 7600 or 7511

Siemens IMMULITE 2000 Roche ELECSYS Roche ELECSYS Avidity BioMérieux VIDAS

Paramagnetic particles Paramagnetic microparticles Paramagnetic microparticles

CLIA

Purified Antigen

Indirect, CL 7600 or 7511

ECLIA, one step double sandwich method

Rec. antigens CMV pp150, pp52, pp28, pp38 Rec. antigens CMV pp150, pp52, pp28, pp38

Direct, CL R01561 or R01563

ECLIA, two assays with and without chaotropic conditions to dissociate low-avidity antibodies

Direct, CL R01561 or R01563

ELFA two step sandwich method Solid phase receptacles

Viral lysate, anti-human IgG Direct, MUB 7600 or 7511 and Z86238M

ELFA two assays with and without 6 M urea to dissociate low-avidity antibodies

BioMérieux VIDAS Avidity

Solid phase receptacles

Viral lysate, anti-human IgG Direct, MUB 7600 or 7511 and Z86238M

HSV-1 & HSV-2 IgG/IgM

Detection System

Recommended Antigens

Assay Type

Solid Phase

Antigen

DiaSorin LIAISON HSV-1 Type Specific DiaSorin LIAISON HSV-2 Type Specific

Magnetic particles

Rec. HSV-1 gG 1 and mouse anti-human IgG

CLIA, two steps

Indirect, CL VTI520, Z86238M

Magnetic particles

Rec. HSV-2 gG 1 and mouse anti-human IgG

CLIA, two steps

Indirect, CL VTI530, Z86238M

Focus PLEXUS HerpesSelect® 1 and 2 IgG Biorad BIOPLEX 2200 HSV-1 & HSV-2 IgG

Rec. HSV-1 gG 1, HSV-2 gG 1 and goat anti-human IgG Rec. gG1 and synthetic peptide p8C:BSA derived from gG2 sequence (patented) and mouse anti-human IgG and mouse anti-human FXIII

Indirect, Fluorescent

VTI520, VTI530 and L15406G

EIA multiplex flow three steps

Beads

Carboxy-coated dyed beads

Direct, Fluorescent

VTI520, VTI530, and Z86238M

EIA multiplex flow three steps

Roche ELECSYS HSV-1 IgG

ECLIA, two step antigen sandwich

Paramagnetic microparticles Rec. HSV-1, HSV-1 viral lysate Direct, CL VTI520 and 7305

Siemens Immunlite 2000 Herpes 1/2 IgG

HSV-1 (strain MacIntyre), HSV-2 (strain G) viral lysate and anti-human IgG

Paramagnetic particles

CLIA

Indirect, CL 7305, 7705 and Z86238M

Life Science Division 5171 Wilfong Road | Memphis, TN 38134 901.382.8716 • 800.327.6299 www.MeridianBioscience.com/LifeScience

06/18

8

Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8

meridianlifescience.com

Powered by