Anti TTG Antibodies Test Purpose, Procedure, Principle, Result Interpretation, Report Formate and Clinical Signification

The Anti-tTG antibody assay is an immunological serological test used to detect autoantibodies directed against tissue transglutaminase (tTG), an intracellular enzyme involved in post-translational protein modification. The test is primarily utilized for autoimmune enteropathy assessment, especially gluten-related immune disorders.

The assay may detect IgA and/or IgG class antibodies, depending on the test configuration.

Anti TTG Test Types:

1. Anti-TTG IgA Test:
   • This test measures IgA (immunoglobulin A) antibodies against tissue transglutaminase.
   • IgA antibodies are commonly used because they are found in the mucous membranes of the gastrointestinal tract, which is where celiac disease primarily affects.
   • It is the primary form of the Anti-TTG antibody test and is considered highly specific for celiac disease diagnosis.
   • Results are typically reported in units such as U/mL (units per milliliter) or IU/mL (international units per milliliter).
2. Anti-TTG IgG Test:
   • In some cases, individuals with celiac disease may have IgA deficiency, which can lead to false-negative results on the Anti-TTG IgA test.
   • In such cases, the Anti-TTG IgG test is used to detect IgG antibodies against tissue transglutaminase.
   • This test is often used as an alternative when IgA deficiency is suspected or confirmed.
   • Results are also reported in units such as U/mL or IU/mL.

Principle of the Test

The Anti-tTG assay is based on an antigen–antibody immunological reaction.

Method Type:

• Enzyme-Linked Immunosorbent Assay (ELISA) – most common
• Chemiluminescent Immunoassay (CLIA)
• Fluorescence Enzyme Immunoassay (FEIA)
• Radioimmunoassay (RIA) – obsolete/rare

ELISA Principle

• Microtiter wells are coated with recombinant human tissue transglutaminase antigen
• Patient serum antibodies (IgA or IgG) bind to immobilized tTG
• Enzyme-labeled anti-human Ig conjugate binds to antigen-antibody complexes
• Substrate (e.g., TMB) reacts with enzyme producing a color change
• Color intensity is directly proportional to antibody concentration
• Measured photometrically at 450 nm

Clinical Significance (Technical)

Elevated Anti-tTG Levels

• Indicates autoimmune-mediated mucosal injury
• Reflects loss of immune tolerance to tissue transglutaminase
• Strong association with:
  • Gluten-mediated autoimmune enteropathy
  • Malabsorption syndromes
• High titers correlate with intestinal villous atrophy severity
• IgA Anti-tTG shows highest diagnostic accuracy
• IgG Anti-tTG is significant in IgA-deficient states

Low or Undetectable Levels

• Suggest absence of autoimmune response
• May occur in:
  • Early disease
  • Immunoglobulin deficiencies (e.g., selective IgA deficiency)
• Requires correlation with total serum IgA

Specimen Requirements

Patient Preparation (Technical)

• No fasting required
• Sample should be collected before immunosuppressive therapy
• Ongoing gluten exposure may affect antibody levels
• Avoid repeated freeze-thaw cycles
• Recent blood transfusion may interfere

Reagents & Materials Required

Reagents

• Recombinant human tTG-coated microplates
• Enzyme-labeled anti-human IgA or IgG conjugate
• TMB substrate
• Stop solution (0.5–1N sulfuric acid)
• Wash buffer concentrate

Calibrators

• Multi-level calibrators traceable to manufacturer standard

Controls

• Negative control
• Low positive control
• High positive control

Equipment

• Micropipettes (10–1000 µL)
• ELISA washer
• Microplate reader
• Incubator (37 °C)
• Timer

Wavelength

• Primary: 450 nm
• Reference: 620–630 nm (optional)

Reagent Storage

• 2–8 °C
• Do not freeze coated plates

Test Procedure (Step-by-Step)

A. Manual ELISA Method

1. Bring reagents and samples to room temperature
2. Add 100 µL calibrators, controls, and samples to wells
3. Incubate at 37 °C for 30 minutes
4. Wash wells 3–5 times
5. Add 100 µL enzyme conjugate
6. Incubate at 37 °C for 30 minutes
7. Wash thoroughly
8. Add 100 µL TMB substrate
9. Incubate 10–15 minutes in dark
10. Add 100 µL stop solution
11. Read absorbance at 450 nm

B. Automated Method

• Load samples, reagents, and calibrators
• Program assay protocol
• Perform calibration as per analyzer instructions
• Analyzer performs incubation, washing, detection
• On-board reagent stability: 7–14 days (method dependent)

Calculation Formula

Manual ELISA Calculation

$$\text{Anti-tTG (IU/mL)} = \frac{\text{Sample Absorbance}}{\text{Calibrator Absorbance}} \times \text{Calibrator Value}$$Anti-tTG (IU/mL)=Calibrator AbsorbanceSample Absorbance​×Calibrator Value

Units: IU/mL

Example:
Sample OD = 1.20
Calibrator OD = 0.80
Calibrator = 10 IU/mL
Result = (1.20 / 0.80) × 10 = 15 IU/mL

Reference Ranges

Note: Reference ranges must be validated by each laboratory.

Interpretation (Technical / Professional)

High Levels

• Autoimmune antibody production
• Active mucosal immune injury
• Correlates with:
  • Anti-endomysial antibodies
  • Elevated inflammatory markers
  • Malabsorption biochemical profile

Low Levels

• Absence of autoimmune activity
• Possible IgA deficiency
• Early disease or remission state

Interfering Factors / Sources of Error

• Hemolysis → false elevation
• Lipemia → optical interference
• Icterus → absorbance distortion
• High bilirubin
• Immunosuppressive drugs ↓ levels
• Improper washing
• Incorrect anticoagulant
• Temperature fluctuations
• Instrument drift
• Microbial contamination

Quality Control Requirements

• QC Levels: Normal, Low Positive, High Positive
• Frequency: Daily or per batch
• Rules: Westgard 1-2s, 2-2s, R-4s
• Charts: Levey–Jennings mandatory
• Corrective Action: Recalibrate, repeat run, check reagents

Calibration Requirements

• Multi-level calibration
• With new reagent lot
• After analyzer maintenance
• Linearity typically up to 200 IU/mL
• Recalibrate if QC fails

Instrument Maintenance Notes

• Daily probe washing
• Weekly optical cleaning
• Monthly deep wash cycles
• Check temperature calibration
• Prevent carryover contamination

Reporting Format

Test: Anti-tTG Antibodies (IgA)
Result: 18 IU/mL
Reference Range: <10 IU/mL
Flag: H
Method: ELISA
Technical Note: Results should be interpreted with total IgA levels.

Critical (Panic) Values

• 100 IU/mL (method-dependent)

Follow institutional policy for notification.

Troubleshooting Guide

Safety Precautions

• Use PPE (gloves, lab coat)
• Treat all samples as biohazard
• Dispose reagents per MSDS
• Avoid aerosol formation
• Decontaminate spills immediately

Test Limitations

• False negatives in IgA deficiency
• Cross-reactivity with other autoimmune antibodies
• Not suitable for monitoring acute changes
• Method-dependent sensitivity variations

Notes for Lab Students

• Always check total IgA status
• Strict washing steps are critical
• QC validation is mandatory
• Never report without calibration verification

Viva / MCQs

1. Anti-tTG detects antibodies against which enzyme?
2. Primary wavelength used in ELISA?
3. Most common specimen type?
4. Main cause of false low results?
5. Which QC rule detects random error?

Answers:

1. Tissue transglutaminase
2. 450 nm
3. Serum
4. IgA deficiency
5. R-4s rule

FAQs:

References (Technical)

• CLSI Immunoassay Guidelines
• IFCC Autoimmune Diagnostics Manual
• WHO Laboratory Quality Standards
• Manufacturer ELISA Inserts
• Peer-reviewed Immunology Journals

Possible References Used