1. بسم الله الرحمن الرحيم

2. Faculty of Allied Medical Sciences
Clinical Immunology & Serology Practice
(MLIS 201)

3. AUTOANTIBODIES IN THE DIAGNOSIS OF AUTOIMMUNE DISEASES
Prof. Dr. Ezzat M Hassan
Prof. of Immunology
Med Res Inst, Alex Univ

4. Objectives To understand the concept of auto-immunity
To define auto-antibodies
To know the classes of autoimmune diseases
To know how to detect auto-antibodies
To know the antibodies in rheumatic diseases
To define the ANA and their patterns
To describe different methods for detection ANA and how to interpret the results
To know different auto-antibodies in rheumatoid arthritis, methods of detection and their clinical importance
To describe ANCA and anti-phosphlipid antibodies and their clinical significance
To define some-organ specific auto-antibodies
To define the acute phase proteins

5. Introduction
The main function of the immune system is to discreminate between self and non-self antigens
This is maintained by a process called immune tolerance.
The break-down of tolerance will result in an immune response directed at one or more self (auto) antigens
This leads to an abnormal auto-immune response causing autoimmune diseases with the production of various auto immune effector components .
One of these effectors is the production of auto-antibodies.

6. Many autoantibodies are associated with autoimmune diseases
Autoantibodies are immunoglobulins that bind to self antigens (autoantigens)
Autoantigens include self molecules in the nucleus, cytoplasm, and cell surface
Many autoantibodies are associated with autoimmune diseases
Autoantibodies can be detected in healthy persons (at low level) without clinical significance
Mechanisms how normal immunity change to autoimmunity are multiple

7. Autoimmunity Results of loss of autotolerance Results in inflammation
Autoimmune diseases are either:
Organ specific or
Organ non-nespecific (systemic)

9. LABORATORY DETERMINATION OF AUTOANTIBODIES:
Based on immunochemical detection by:
* agglutination
* precipitation: * turbidimetry
* immunobloting
* ELISA
* immunofluorescence
determination of autoantibodies can be:
* qualitative ve/-ve
* quantitative: * titer
* arbitrary units
* concentration (standard)

10. IMMUNOFLUORESCENCE DETERMINATION
OF AUTOANTIBODIES:
Detection of autoantibodies reacting with tissue antigens
by IIF test using :
* Tissue sections from different animal species
* Cell suspensions:
Hep-2 cell line (ANA)
Granulocytes (ANCA)
Crethidia Luciliae (Anti-dsDNA)

11. Autoantibodies and Rheumatologic Diseases: When and How to Use Laboratory tests

12. 1-Antinuclear antibodies
(ANA)

13. Antinuclear antibodies (ANA)
Serologic hallmark of systemic autoimmune connective tissue diseases
React with cell nuclear apparatus; may bind DNA, RNA, nuclear proteins, nucleolar proteins & protein-nucleic acid complexes (RNP)
Their levels may correlate with intensity of inflammation
Healthy persons may have low levels of ANA

14. mitotic
apparatus
centromere
ANA
ENA: extractable nuclear antigens

15. ANA in rheumatologic diseases
SLE (Systemic Lupus Erythematosus) Drug-induced SLE RA (Rhematoid Arthritis)(40%) Polymyositis/dermatomyositis (75%) MCTD (Mixed Connective Tissue Sisease)
ANA in non-rheumatologic diseases (eg. Autoimmune thyroiditis, Autoimmune hepatitis, Hepatitis C)
Normal Population 5%
Higher in women, elderly

16. Detection of ANA 1- BY IMMUNOFLUORESCEINCE Using :
* tissue Sections: Mouse liver & kidney
* cell suspensions: * Hep-2 cell line (ANA)
* Crethidia Luciliae (Anti-dsDNA)
Immunofluorescence is commonly used to look for antibodies binding to cellular components : Anti-Nuclear, and also anti-cytoplasmic Antibodies
Ethanol and methanol fixation may remove Ro/SSA antigens from cells, so the cells are fixed with acetone

17. IIF-ANA test Materials Specimen Collection Substrate slide
Hep-2 (human epithelial cells)
Positive Control
Specific autoantibody activity
Negative Control
Pooled normal human sera
Universal FITC conjugate
Fluorescein conjugated to AHG
Mounting media
polyvinyl alcohol
Phosphate buffered saline
Evans Blue counter stain
Specimen Collection
Serum (recommended)
Avoid grossly hemolyzed or lipemic samples produce increased nonspecific background staining of the subtrate

18. Procedure:
Bring all reagents, materials and patient specimens to ambient temperature. (Approximately 20 min)
Construct a humidity chamber.
Prepare a 1:40 dilution (5µl serum: 200 µl PBS) for each specimen.
Remove slide from foil bag. Place in humidity chamber. Immediately add 25 l controls or diluted test serum to the appropriate wells.
Cover humidity chamber. Incubate ambient temperature for 30 minutes.

19. Procedure:cont. Rinse each slide briefly with a stream of PBS.
Wash slides for 10 minutes in a staining dish filled with PBS. Gently agitate staining dish several times or use a magnetic stirrer during the wash.

20. Procedure:cont.
Remove each slide from staining dish and blot excess PBS from around the wells using blotting strips. (N.B. Do not touch the strip to the substrate wells.)
Place slides in humidity chamber and immediately dispense 25 l of FITC conjugate into each well.
Cover humidity chamber & Incubate for 30 ambient temperature in the dark. Exposure to light will cause quenching of the fluorescent stain.

21. Procedure:cont. Rinse each slide briefly with a stream of PBS
Wash slides for 10 minutes in a staining dish filled with fresh PBS and 5-6 drops of Evan’s blue counterstain. During wash, turn on microscope.
NOTE: This wash step should also be set up in a dark place. Exposure to light will cause quenching of the fluorescent stain.

22. Procedure:cont.
Remove slides, blot, and apply 4 1 drops of mounting media per slide, making sure to cover all wells.
Add coverslip.
View under fluorescent microscope in a dark room.
Evaluate each well for fluorescent staining
using +ve & -ve control wells as
a reference.

23. Y Y DETECTION OF AUTOANTIBODIES BY INDIRECT IMMUNOFLUORESCENCE NO NO
POSITIVE RESULT
(AUTO Ab+)
NEGATIVE RESULT
(AUTO Ab-)
LIGHT EMISSION
(FITC  GREEN)
UV LIGHT
UV LIGHT
NO EMISSION
FLUOROCHROME - CONJUNG.
ANTISERUM AGAINST HUMAN Ig
WASHING
WASHING Y NO
SERUM (AUTO Ab)
WASHING Y
AUTO Ag NO
WASHING
CELLS or Tissue
GLASS slide

24. Interpretation of Results
The test for autoantibodies is NEGATIVE if no specific pattern of fluorescence is observed on the substrate.
The test for autoantibodies is POSITVE when the Hep- 2 substrate shows a specific pattern of fluorescence.

25. Patterns Peripheral or rim = dsDNA Homogenous = dsDNA, histones
Speckled = RNP
Nucleoli = Ku Antigen
Centromeric = Kinetochore
Cytoplasmic = Jo-1 (RNA synthetase)

26. Patterns Peripheral or rim Homogenous Speckled Centromeric Cytoplasmic
Nucleoli

27. Anti-dsDNA (native DNA)
kinetoplast formed
by dsDNA.
Substrate
is protozoon
Crithidia luciliae,
with kinetoplast
formed by
dsDNA. .
antigen: native dsDNA
clinical association: Significantly (95%) associated with SLE
Correlated with disease activity

28. Detection of ANA (cont.)
2- By molecularly characterized antigens
Targets (antigens) are produced by biotechnology
Methods:
ELISA tests
immunobloting

29. METHODS
immunoblot
ELISA
recombinant or purified antigens

30. CLINICAL APPROACH TO ANA TESTING
ANA by IFA
Negative
Positive
Repeat
anti-ds DNA
anti-Sm
SS-A(Ro)
SS-B(La)
U1 RNP
(titer)
ELISA
Negative
(stop)
Positive
Clinical Hx/PE (+) supportive AutoAb results

31. To summarize… Screen for ANAs using IIF on slides with HEp-2 cells
If it’s positive look for the specific antigen that the antibody is reacting by using ELISA or immunoblot
Checking for anti-dsDNA antibodies only when the symptoms are suspicious of SLE AND the ANA is positive
Guidelines suggest that the only antibodies that need to be quantified are dsDNA (to predict a flare, and nephritis risk)

32. 2- Rheumatoid Factors (RF) & Related antibodies

33. Rheumatoid Arthritis
It is a chronic inflammatory disease affecting mainly the joints and may affect other connective tissues.
It is characterized by the presence of various circulating auto-antibodies including:
Rheumatoid factors (RFs)
Anti-keratin antibody (AKA)
Anti-preinuclear factor
Anti-cyclic citrulinated peptide (Anti-CCP) antibodies.
ANA (low titer)

34. 1-Rheumatoid Factor (RF)
RFs are auto-antibodies against Fc fragment of self-IgG
Most RFs are of IgM but IgG & IgA RFs are also observed
It is present in 70-90% of RA patients
High titer RF is assocciated with extra-articular complications and systemic vasculitis
It is an important diagnostic and prognostic parameter
Negative RF: No rule out RA

35. MOLECULAR TARGETS FOR RHEUMATOID FACTORS
V domains
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
-S-S-
C1
-S-S-
-S-S-
binding
sites
for RF
Agal IgG
C2
Asn 297
C domains
-S-S-
-S-S-
C3
His 435
-S-S-
-S-S-
GA – specific RF

37. IMMUNOPATHOGENESIS OF RHEUMATOID ARTHRITIS
binding of RF to IgG
deposition of immunocomplexes
to joint synovia
IgG RF

38. 1-Rheumatoid Factor (RF) Cont.
Although RF can exist as IgG, IgM, IgA & IgE isotypes, the IgM-class RF is the main class identified by clinically available diagnostic tests.
RF is detected by :
1- Qualitative & Semi Quantitative Tests:
Rose-Waller hemagglutination (RBCs coated with human IgG)
Latex agglutination (Latex particles coated with human IgG)
2- Quantitative Tests:
ELISA, Turbidimetry and Nephelometry
Measurement of IgM or IgG RF By ELISA does not give significantly more clinical information than traditional tests such as the Rose-Waaler or latex agglutination tests.

39. RF Latex Agglutination Test
It is the most common screening serological test for RA
Sample collection:
Allow 2 ml blood to clot at room temp (Formation of clot must be complete)
Separate serum and store at 2-8 °C if used within 2-3 days or at -20 oC if used later

40. Procedure If +ve Semi-quantitative test (Serial Dilutions)
Bring all reagents to room temp.
Prepare 1:20 dilution of test serum (50 μl of serum to 1 ml of glycine buffer)
Place 50 μl of diluted serum on to the test slide
Add 1 drop of well shaken latex reagent
Mix the two drops together with a clean stirrer and spread out to the edge of the test area
Rock the slide gently and observe for macro-agglutination
Read at 2 minutes under a direct light source
A definite clumping is reported as reactive (R).
No clumping is reported as non-reactive (N).
If +ve Semi-quantitative test (Serial Dilutions)

41. Incidence of RF in Rheumatic Diseases
Rheumatoid Arthritis
Sjogrens,s syndrome
Systemic lupus erythematosus
Dermatomyositis
Mixed connective tissue disease
Cranial arteritis
Polymyalgia rtheumatica
Polyarthritis
Scleroderma
Juvenile rheumatoid arthritis
70-90 58 18 16 13 10 7 6

42. 2-AKA Highly specific for RA, occur in 40% of RA patients
Present in 1/3 of RF negative patients with RA
Detected at early stages, even before joint symptoms
Associated with more active &/or sever forms of RA
AKA, by IIF, on rat oesophagus

43. 3- Anti-perinuclear factor
Not used now due to low sensitivity and in-consistency of the substrate
4- Anti-CCP (By ELISA)
IgG antibody against Cyclic Citrullinated Peptide (CCP)
Highly specific (96%) and moderately sensitive (78%) for RA.
Not found in other diseases (contrast to RF)
Detected in 70% of early RA.
Detected in 30-46% of sero-negative RA.
Should be a one time test, does not need to be repeated or followed
It is predictive of erosive disease.

44. 3- Anti-Neutrophil Cytoplasmic Antigen (ANCA) antibodies

45. Anti-Neutrophil Cytoplasma Antigens
(ANCA) ANTIBODIES
Diagnostic for various systemic autoimmune vasculitis.
*2 types By IIF
Cytoplasmic (C-ANCA), & Peri-nuclear (P-ANCA)
* substrate are ethanole or formalin fixed human granulocytes

46. ANCA
ELISA
proteinase-3
myeloperoxidase

47. Diagnostic Specificity of (ANCA)
Disease % Positive
Wegener,s granulomatosis (c-ANCA)
Generalized
Active
Full remission
Localized
Polyarteritis group (p-ANCA)
Polyateritis nodule
Churg-Strauss syndrome
Polyangitis overlap syndrome
Idiopathic crescentic glomerlonephritis
Inflammatory Bowl Diseases (Atypical)
Normal Indiveduals 96 41 67 32 50 75
100
70-82
1-2

48. 4- Antiphospholipid antibodies
(APLA)

49. Anti-Phospholipids Antibodies (APLA)
Heterogeneous antibodies, against phospholipids
Cardiolipin and its co-factor, β2-glycoprotiens are primary antigens of phospholipids.
Anti-cardiolipin antibodies (ACA) are raised in various diseases especially SLE complicated with thrombotic manifestations.
They are measured in serum by ELISA
SLE patients who make anti-phospholipid antibodies will make VDRL test look like it’s positive because the VDRL particles are coated with phospholipids.

50. Disease Association of Anti-Cardiolipin
Antibodies
Condition
% Incidence
Recurrent Venous Thrombosis
Recurrent Fetal Loss
Hemolytic Anemia
Thrombocytopenia
Arterial Occlusions
Pulmonary Hypertension
28-71
28-64 38
27-33
25-31
20-40

51. 4- ORGAN-SPECIFIC AUTOANTIBODIES

52. ANTI- AGAINST GASTRIC PARIETAL CELLS
(GPC) Abs
antigen: antigen H+/K+/ATP-asa
clinical association: pernicious anemia (80-90%),
autoimmune endocrine diseases,

53. Anti-Liver-Kidney Microsomal
(LKM-1)
Anti Mitochondrial Abs
antigen: cytochrome mono-oxygenase mitochondrial antigen
clinical association: markers of autoimmune hepatitis

54. ANTI-RETICULIN Abs (R1)
antigen: reticulin R1
clinical association: Coeliac disease

55. ANTI-ENDOMYSIUM Abs (EMA)
antigen: tissue transglutaminase
clinical association: Coeliac disease

56. ANTI-SMOOTH MUSCLE Abs(ASMA)
antigen: cytoskeletal antigens
clinical asociation: marker of autoimmune hepatitis I, inflammatory diseases

57. Common Auto-antibodies in some autoimmune diseases
Specfic Autoantibody
Disease
Anti-ds DNA & Anti-Sm
SLE
Anti Histone
drug-induced lupus
Anti-Jo-1
polymyositis
Anti-U1-RNP
Mixed Connective Tissue Disease
Anti-Smooth Muscle, Anti-LKM
autoimmune hepatitis
Anti-CCP
Rheumatoid Arthritis
Anti-Scl-70
Scleroderma
ANCA
Autoimmune vasculitis

58. THE PRESENCE OF AUTOANTIBODIES HAS TO BE INTERPRETED
SUMMARY:
THE PRESENCE
OF AUTOANTIBODIES
HAS TO BE INTERPRETED
IN CLINICAL CONTEXT

59. Acute phase proteins Inflammatory markers
Produced mostly by hepatocytes
Acute phase response: is the increase of serum proteins in response to cytokine release (esp. Il-1& IL-6) from monocytes and macrophages in inflammatory states
Examples:
CRP:
Precise function unknown; activates complement, but also anti-inflammatory
Rapidly fluctuates in response to inflammation
Others: serum amyloid A (SAA), ferritin

60. Study question
Mention the methods used to determine Anti-nuclear antibodies

61. Assignment
Write shortly on the methods used to determine Anti- nuclear antibodies (ANA).

62. Thanks