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).
ToRCH IgG/IgM Assay Development
www.MeridianLifeScience.com
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).
IgM vs IgG SEROLOGY TIMELINE
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.
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 R01797 R01803
Rec. Ag. p30 (SAG1), E. coli Rec. Ag. p35 (GRA8), E. coli Rec. Ag. p30 (SAG1), P. pastoris
cross-reactivity to IgA & IgG
Rec. Ag. P29 (GRA7), E. coli C01523M MAb to T. gondii SAG1 (p30) protein C01589M MAb to T. gondii (38kDa protein)
IgG Absorbent (Required for IgM Assays)
L15406G Goat anti-human IgG (Fc) 8120 IgM Diluent
Rubella Virus
6075 Native Ag., Grade III, highly pure 6076/6123 Native Ag., Grade IV, highly pure EV9525 MAb to Rubella, purified EV9526 MAb to Rubella, purified
Solid Phase Blocking Buffers
J82100B J82300B
ELISA blocking buffer
Lateral Flow blocking Buffer J16403D Coating stabilizer and Blocking Buffer
Cytomegalovirus (CMV)
7511 7600
Native Ag., IgM Concentrate Native Ag, IgG/IgM Concentrate
EV9268 EV7509 R18102 R01561
Native Ag., Grade II
Native gB Ag.
Rec. gB Ag., E. coli
Rec. Ag pp52 (UL44), E. coli
R01563 Rec. Ag pp150 (UL32), E. coli C8A022M MAb to Immediate Early Antigen, pp72
To order
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.MeridianLifeScience.com ISO Certified 13485:2016
Herpes Simplex Virus (HSV) 1
7305
Native Ag.
VTI520
Rec. Ag. glycoprotein G 1 (S. cerevisiae)
Herpes Simplex Virus (HSV) 2
7705
Native Ag.
VTI530
Rec. Ag. glycoprotein G 2 (S. cerevisiae)
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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
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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 and R01581
CMIA 2 assays with and without blocking buffer to dissociate low-avidity antibodies
Indirect, CL R01573 and R01581
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
CLIA 2 assays with and without a 6M urea elution step to dissociate low-avidity antibodies
Indirect, CL 8200
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
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 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
Abbott AxSYM
MEIA
Microparticles Purified viral lysate and anti-human IgG
Indirect, MUB 6075
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
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 EV9525 or EV9526
Solid phase receptacles
Indirect, MUB 6075
Indirect, Luminescence 6075
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, 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
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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
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
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
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
Magnetic particles
Rec. HSV-2 gG 1 and mouse anti-human IgG
CLIA, two steps
Indirect, CL VTI530
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
EIA multiplex flow three steps
VTI520 and VTI530
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 and 7705
Life Science Division 5171 Wilfong Road | Memphis, TN 38134 +1 901-382-8716 www.MeridianLifeScience.com
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