Serology Assays

Principle of the Direct Method of Immunofluorescence Assay

Detects antigen directly from sample

One reagent

Principle of the Indirect Method of Immunofluorescence Assay

Two-step, two antibody bindings:

Primary antibody binds to target of interest

Secondary antibody-FITC (conjugate) specific to Primary antibody binds next

Antigen and Antibody for Anti-Nuclear Antibody (ANA) IFA

Antigen: Human laryngeal carcinoma epithelial cell line (HEp-2)

Antibody: Auto-antibodies to nuclear materials

Principle of ANA IFA

Indirect Immunofluorescence

Specimen used for ANA IFA

Serum

Clinical Significance of ANA IFA

Screening for auto-immune diseases like SLE

Positive and Negative results for ANA IFA

Positive: Fluorescence pattern (homogeneous, speckled, nucleolar, centromere)

Negative: No Fluorescence

What disease is synonymous with a Centromere pattern?

Scleroderma with CREST Syndrome

Antigen and Antibody for Anti-nDNA (dsDNA)

Antigen: Crithidia luciliae with kinetoplast

Antibody: nDNA antibodies

What is the principle of Anti-nDNA (dsDNA)

Indirect immunofluorescence

Specimen used for Anti-nDNA (dsDNA)

Serum

Clinical Significance of Anti-nDNA (dsDNA)

Supports Active SLE diagnosis and some drug induced lupus syndromes

Positive and Negative results for Anti-nDNA (dsDNA)

Positive: Fluorescence of kinetoplast → large chunky glow (homogeneous)

Negative: no fluorescence

Antibody and Antigen for Anti-ENA Panel (Smith, RNP, SS-A, SS-B, Jo-1, ScI-70)

Antibody: Anti - (Smith, RNP, SS-A, SS-B, Jo-1, ScI-70)

Antigen: Extractable nuclear antigens

Principle of Anti-ENA Panel (Smith, RNP, SS-A, SS-B, Jo-1, ScI-70)

Indirect Immunofluorescence

Specimen used for Anti-ENA Panel (Smith, RNP, SS-A, SS-B, Jo-1, ScI-70)

Serum

Clinical Significance of Anti-ENA Panel (Smith, RNP, SS-A, SS-B, Jo-1, ScI-70)

Smith: SLE

RNP: SLE, MCTD

SS-A (Ro): Sjögrens Syndrome

SS-B (La): Sjögrens Syndrome

Jo-1: PM/DM

ScI-70: PSS, Scleroderma

Positive and Negative results of Anti-ENA Panel (Smith, RNP, SS-A, SS-B, Jo-1, ScI-70)

Positive:

• Smith: Speckled pattern

• RNP: Speckled pattern

• SS-A: Speckled pattern

• SS-B: Speckled pattern

• Jo-1: Cytoplasmic pattern

• ScI-70: Homogeneous, Nucleolar pattern

Negative: no fluorescence

Homogeneous pattern as a follow-up biomarker is synonymous with

Anti-nDNA

Speckled pattern as a follow-up biomarker is synonymous with

Extractable Nuclear Antigen (ENA)

Cytoplasmic pattern as a follow-up biomarker is synonymous with

Anti-mitochondrial/Smooth Muscle

Antibody and antigen for Mitochondrial Antibody (AMA)

Antibody: anti-mitochondrial antibody

Antigen: kidney tubules/stomach parietal cells

Principle of Mitochondrial Antibody (AMA)

Indirect Immunofluorescence

Specimen used for Mitochondrial Antibody (AMA)

Serum

Clinical Significance of Mitochondrial Antibody (AMA)

Detection of Hepatic diseases

• Primary biliary cirrhosis

• Primary sclerosing cholangitis

• Autoimmune hepatitis

• Etc.

Positive and Negative results of Mitochondrial Antibody (AMA)

Positive: Mitochondrial Fluorescence (ring appearance)

Negative: No Fluorescence

Antibody and Antigen Smooth Muscle Antibody (ASMA)

Antibody: Anti-smooth muscle

Antigen: Kidney and stomach muscular layers

Principle of Smooth Muscle Antibody (ASMA)

Indirect Immunofluorescence

Specimen used for Smooth Muscle Antibody (ASMA)

Serum

Clinical Significance of Smooth Muscle Antibody (ASMA)

Detection of Hepatic diseases

• Primary biliary cirrhosis

• Primary sclerosing cholangitis

• Autoimmune hepatitis

• Etc.

Positive and Negative result of Smooth Muscle Antibody (ASMA)

Positive: Fluorescence in kidney and stomach muscular layers

Negative: No Fluorescence

Antigen and Antibody of Fungal Antibodies (Histoplasma, Blastomyces, Coccidioides, Aspergillus)

Antibody: Fungal Antibodies (Histoplasma, Blastomyces, Coccidioides, Aspergillus)

Antigen: Fungal Antigens

Principle of Fungal Antibodies (Histoplasma, Blastomyces, Coccidioides, Aspergillus)

Immunodiffusion/precipitation in gel matrix

Specimen type for Fungal Antibodies (Histoplasma, Blastomyces, Coccidioides, Aspergillus)

Serum

Clinical Significance of Fungal Antibodies (Histoplasma, Blastomyces, Coccidioides, Aspergillus)

Histoplasma: Past/current infection

Blastomyces: Active/recent infection

Coccidioides: Active/recent infection

Aspergillus: Infection/colonization/allergy

Negative results do not exclude infection

Results for Fungal Antibodies (Histoplasma, Blastomyces, Coccidioides, Aspergillus)

Identity, Partial Identity, Non-Identity, No reaction

Histoplasma: 2 bands (M and H) → M band more common

Blastomyces: One band

Coccidioides: At least one band (may have two)

Aspergillus: Must produce >/= 2 bands

What does an Identity Reaction look like

Smooth curve

What does a Partial Identity Reaction look like

Smooth curve with a single spur

What does a Non-Identity Reaction look like

Two precipitate lines which cross to form an “X”

What does a No Reaction look like

No visible precipitate line

Antigen and Antibody of Geenius HIV ½ Supplemental Assay

Antigen: Recombinant proteins specific to HIV-1 & HIV-2

Antibody: Antibody to HIV-1 or HIV-2

Principle of Geenius HIV ½ Supplemental Assay

Immunochromatographic/Lateral Flow

Specimen used in Geenius HIV ½ Supplemental Assay

Serum

Clinical Significance of Geenius HIV ½ Supplemental Assay

Confirmation for HIV-1 or HIV-2

Results of Geenius HIV ½ Supplemental Assay

Positive: Control and test line appears colored

Negative: Control line appears colored

Antigen and Antibody of Syphilis RPR test

Antigen: Cardiolipin-Lecithin-Cholesterol with Carbon particles; Stabilized with EDTA and choline chloride

Antibody: Reagin (Nonspecific AB: Anti-Cardiolipin)

Principle of Syphilis RPR test

Flocculation (Precipitation) (Non-Treponemal Test)

Specimen of Syphilis RPR test

Serum or Plasma

Clinical Significance of Syphilis RPR test

Suggests infection with Treponemal pallidum

Quantitate and confirm with Treponemal test (Bioplex or TP-PA)

False positives: IM, SLE, Pregnancy, etc.

Results of Syphilis RPR test

Positive: Black Aggregates with white background

Negative: No Aggregates

Antigen and Antibody of BioPlex Syphilis Assay (Total IgM + IgG)

Antigen: 3 cloned T. pallidum proteins (Colored beads)

Antibody: Treponemal Total IgM + IgG

NO SORBENT USED

Principle of BioPlex Syphilis Assay (Total IgM + IgG)

Enzyme Immunoassay with Fluorescent Detection

Specimen used for BioPlex Syphilis Assay (Total IgM + IgG)

Serum

Clinical Significance of BioPlex Syphilis Assay (Total IgM + IgG)

Screening tool for Reverse Sequence Syphilis Algorithm

What is the Reverse Sequence Syphilis Algorithm

Test on BioPlex initially → Positive → RPR test → Positive → Quantitate

If RPR test is negative → alternant Treponemal test (TP-PA) → positive = Syphilis

TP-PA Negative → BioPlex result was a false positive

Results of BioPlex Syphilis Assay (Total IgM + IgG)

Positive: Bead IDed + Fluorescence

• Continue with RPR

Negative: No Bead IDed + No Fluorescence

Antigen and Antibody of Treponema pallidum Particle Agglutination (TP/PA)

Antigen: SORBENT of Reiter treponemes and Gelatin Particles sensitized with T. pallidum AG

Antibody: Treponema pallidum AB

Principle of Treponema pallidum Particle Agglutination (TP/PA)

Passive Agglutination

Specimen of Treponema pallidum Particle Agglutination (TP/PA)

Serum or Plasma

Clinical significance of Treponema pallidum Particle Agglutination (TP/PA)

Used in Reverse Sequence when BioPlex Treponemal IgG + IgM is positive and RPR is non reactive (unless prior syphilis) for CONFIRMATION of Syphilis (Rules out False Positive)

Results of Treponema pallidum Particle Agglutination (TP/PA)

Positive: Smooth mat of agglutinated particles on the bottom of microtiter well

Negative: Compact button of non-agglutinated particles on the bottom of microtiter well

Antigen and Antibody of VDRL CSF Syphilis test

Antigen: Cardiolipin-Lecithin-Cholesterol

Antibody: Reagin (Nonspecific AB)

Principle of VDRL CSF Syphilis test

Flocculation (Precipitation) (Non-Treponemal Test)

Specimen of VDRL CSF Syphilis test

CSF

Clinical Significance of VDRL CSF Syphilis test

Suggests Neurosyphilis

Quantitation required

Results of VDRL CSF Syphilis test

Positive: Black Aggregates with white background

Negative: No Aggregates

Antigen and Antibody of Lyme Disease Assay EIA

Antigen: SPR coated with recombinant chimeric proteins (B. burgdorferi)

Antibody: AB to B. burgdorferi

Principle of Lyme Disease Assay EIA

Enzyme-Linked Immunofluorescence Assay (ELFA) via Solid Phase Receptacle (SPR)

Specimen of Lyme Disease Assay EIA

Serum

Clinical Significance of Lyme Disease Assay EIA

If AB detected there was a prior exposure to B. burgdorferi, requires Western Blot or Secondary EIA for confirmation of Lyme Disease

IgG: Immune status

IgM: Active infection

Results of Lyme Disease Assay EIA

Positive: Fluorescence

Negative: No Fluorescence

Antigen and Antibody of Cryptococcus Antigen Lateral Flow Immunochromatography

Antigen: Cryptococcal AG

Antibody: Anti-CrAg as Monoclonal AB

Principle of Cryptococcus Antigen Lateral Flow Immunochromatography

Lateral Flow Immunochromatography

Specimen of Cryptococcus Antigen Lateral Flow Immunochromatography

Serum or CSF

Clinical significance of Cryptococcus Antigen Lateral Flow Immunochromatography

Diagnosis of Crytococcosis

Most commonly infecting AIDS/HIV/Impaired cell-mediated immunity

Results of Cryptococcus Antigen Lateral Flow Immunochromatography

Positive: 2 lines (control and test lines)

Negative: 1 line (control line)

Antigen and Antibody of Mono Slide Test (Remel Color Slide II)

Antigen: Guinea pig kidney AG and horse erythrocytes

Antibody: Heterophile AB (IgM)

Principle of Mono Slide Test (Remel Color Slide II)

Direct Hemagglutination

Specimen of Mono Slide Test (Remel Color Slide II)

Serum or Plasma

Clinical Significance of Mono Slide Test (Remel Color Slide II)

Screen test for EBV infection

Symptoms: Sore throat, fever, lymphadenopathy

If Negative and symptoms persist, perform specific EBV AB

Results of Mono Slide Test (Remel Color Slide II)

Positive: Agglutination with blue background

Negative: No Agglutination with brown background

Antigen and Antibody of Fecal Calprotectin (Diasorin Liaison)

Antigen: Calprotectin protein

Antibody: Anti-calprotectin

Principle of Fecal Calprotectin (Diasorin Liaison)

EIA with Chemiluminescence

Specimen of Fecal Calprotectin (Diasorin Liaison)

Stool

Clinical Significance of Fecal Calprotectin (Diasorin Liaison)

Differentiate IBS vs IBD

Results of Fecal Calprotectin (Diasorin Liaison)

Positive: Elevated levels

Negative: Normal levels

What assays does the Vidas System include

CMV IgG EIA, CMV IgM EIA, Toxoplasma IgG EIA, Toxoplasma IgM EIA, Lyme IgG and Lyme IgM. (As alternant method if needed:  Measles, Mumps and Varicella IgG)

Antigen and Antibody of the Vidas System

Antigen: disease agent coating SPR (CMV IgG EIA, CMV IgM EIA, Toxoplasma IgG EIA, Toxoplasma IgM EIA, Lyme IgG and Lyme IgM. (As alternant method if needed:  Measles, Mumps and Varicella IgG))

Antibody: Anti- CMV IgG EIA, CMV IgM EIA, Toxoplasma IgG EIA, Toxoplasma IgM EIA, Lyme IgG and Lyme IgM. (As alternant method if needed:  Measles, Mumps and Varicella IgG)

Principle of the Vidas System

Enzyme-Linked Immunofluorescence Assay (ELFA) via Solid Phase Receptacle (SPR)

Specimen of the Vidas System

Serum

Clinical Significance of the Vidas System

Presumptive identification of disease (CMV IgG EIA, CMV IgM EIA, Toxoplasma IgG EIA, Toxoplasma IgM EIA, Lyme IgG and Lyme IgM. (As alternant method if needed:  Measles, Mumps and Varicella IgG))

Results of the Vidas System

Positive: Fluorescence

Negative: No Fluorescence

Antigen and Antibody of Rubella test

Antigen: Rubella AG

Antibody: Rubella AB

Principle of Rubella test

Passive Agglutination (or EIA)

Specimen used for Rubella test

Serum

Clinical Significance of Rubella test

Detect Acquired (2 samples; rash vs 2 weeks later), Congenital (2 samples; birth vs 6 months), or Immune Status (1 sample; random)

Pregnancy (1 sample; within 10 days of exposure)

Results of Rubella test

Acquired:

• Positive: >/= 4 fold increase

• Negative: No difference b/w samples

Congenital:

• Positive: Increase or same titer

• Negative: decrease in titer

Immune status:

• Positive: Antibody present

• Negative: Antibody absent

Pregnancy:

• Positive: Antibody present (immune)

• Negative: Antibody absent (able to pass to baby)

Antigen and Antibody of BioPlex Bead System Panels (Autoimmune, EBV, Rash, Syphilis Panels)

Antigen: colored beads

Antibody: Specific AB for Autoimmune, EBV, Rash, and Syphilis

Principle of BioPlex Bead System Panels (Autoimmune, EBV, Rash, Syphilis Panels)

Enzyme Immunoassay with Fluorescent Detection

Specimen of BioPlex Bead System Panels (Autoimmune, EBV, Rash, Syphilis Panels)

Serum