Immuno lab practical 1

What it detects or measures: Antibodies against HIV proteins

HIV indirect ELISA

Principle or reaction: Enzyme-linked color reaction

HIV indirect ELISA

What it detects or measures: Reagin antibodies associated with syphilis screening

RPR

Principle or reaction: Flocculation

RPR

What it detects or measures: Human chorionic gonadotropin

hCG rapid test

Principle or reaction: Lateral-flow sandwich immunoassay

hCG rapid test

What it detects or measures: Heterophile antibodies associated with infectious mononucleosis

Mono test

Principle or reaction: Agglutination of treated horse red blood cells

Mono test

What it detects or measures: Antibodies against streptolysin O

ASO

Principle or reaction: Antigen–antibody reaction

ASO

What it detects or measures: Rheumatoid factor, typically IgM against the Fc portion of IgG

RF rapid test

Principle or reaction: Agglutination of IgG-coated particles

RF rapid test

What it detects or measures: C-reactive protein

CRP rapid test

Principle or reaction: Reverse passive latex agglutination

CRP rapid test

Organism: HIV, an RNA retrovirus that targets CD4+ immune cells

Important viral proteins: gp120, gp41, and p24

HIV indirect ELISA

Procedures:

1) Coated antigen

2) Primary antibody

3) HRP-linked secondary antibody

4) ABTS substrate (TURNS GREEN)

HIV indirect ELISA

Practical interpretation: The positive control should turn green and the negative control should remain clear. If the controls fail, do not interpret patient samples. This teaching assay detects antibodies only; a negative result cannot exclude very recent ______

HIV indirect ELISA

Test group: Non-treponemal

Detects: Reagin antibodies

Antigen: Cardiolipin, lecithin and cholesterol in a modified antigen preparation

Reaction: Flocculation

Reading: Macroscopic, aided by charcoal

Serum: Unheated

Rotation in the manual: 100 rpm for 8 minutes

RPR

Test group: Non-treponemal

Detects: Reagin antibodies

Antigen: Cardiolipin, lecithin and cholesterol

Reaction: Flocculation

Reading: Microscopic

Serum: Heated at 56°C for 30 minutes

Rotation in the manual: 180 rpm for 4 minutes

VDRL

Organism: Treponema pallidum, a spirochete

Reagin: A nonspecific antibody produced in response to lipids released during tissue invasion; it is not directed specifically against T. pallidum

RPR, VDRL, and febrile agglutinins

Widal test, Salmonella O → Somatic (cell-wall) antigen; compact, granular clumps

Febrile agglutinins

Widal test, Salmonella H → Flagellar antigen; loose, flocculent clumps

Febrile agglutinins

Proteus antigens used in febrile agglutination testing

Proteus OX19, OXK, OX2

Used in testing for brucellosis

Brucella abortus antigen

Used in testing for tularemia

Francisella tularensis antigen

Analyte: Human chorionic gonadotropin (hCG)

Specificity: The β subunit provides greater immunologic specificity for hCG

Test format: Qualitative lateral-flow sandwich immunoassay

hCG pregnancy testing

hCG: C and T lines

Positive: hCG detected above the assay cutoff

hCG: C line only

Negative: hCG not detected at or above the cutoff

hCG: No C line

Invalid, regardless of the T line

Reaction sequence: Specimen migrates by capillary action → hCG binds labeled anti-hCG antibody → complex is captured by immobilized anti-hCG antibody at T → labeled conjugate is captured at C

hCG pregnancy test

Organism: Epstein–Barr virus (EBV), a member of the herpesvirus family

Target detected: Heterophile antibodies, described in the manual as IgM antibodies

Reaction: Agglutination of chemically treated horse erythrocytes

Mono test

Positive reaction: Dark agglutination against a blue-green background.

Negative reaction: No visible clumping; smooth suspension against a green-brown background

Mono test

Reagent: Color-enhanced, treated horse red blood cells

Positive control: Diluted positive human serum

Negative control: Nonreactive diluted human serum

Confirmatory serology: EBV-specific VCA-IgM, VCA-IgG, and EBNA

Mono test

Organism: Group A Streptococcus (Streptococcus pyogenes)

Bacterial target: Streptolysin O, a hemolytic toxin

Patient antibody: Antistreptolysin O (ASO)

Test method: Latex agglutination

ASO

Positive reaction: Agglutination when the target is present at a detectable level.

Clinical association: Evidence of prior streptococcal infection, including in evaluations of rheumatic fever and post-streptococcal glomerulonephritis (PSGN)

ASO

Anti-DNase B

Anti-NADase

Anti-hyaluronidase

Anti-streptokinase

Anti-M protein

what test is it associated with?

ASO

Test target: Rheumatoid factor, an autoantibody typically of the IgM class.

Antibody target: Fc portion of IgG

RF

Latex test: Uses particles coated with human IgG in the described latex method.

Positive reaction: Visible agglutination.

Negative reaction: Smooth, homogeneous suspension.

RF

Clinical association: Rheumatoid arthritis, but a positive result is not definitive because RF may occur in other conditions.

RF

Analyte: C-reactive protein, an acute-phase protein produced by the liver.

Test method: Reverse passive latex agglutination.

Reagent: Latex particles coated with anti-CRP

CRP

Antigen in the reaction: CRP in the patient's serum.

Positive reaction: Visible agglutination.

Negative reaction: Smooth, homogeneous suspension.

CRP

Key distinction: In CRP testing, the latex carries anti-CRP. The CRP in the patient sample is the antigen being detected. The manual also notes that postzone reactions can occur, so diluted and undiluted specimens should be considered when suspected.

CRP

Smooth mat of cells in microtiter wells

MHA-TP

Fluorescent anti-human globulin

FTA-ABS

Agglutination — visible clumping

RF, CRP, ASO, Mono, and febrile agglutinin tests

Flocculation — fine particles or flakes

RPR

Color change in an ELISA well

HIV antibody indirect ELISA

Lateral-flow lines

rapid hCG testing

Dilution Factor

A whole number, such as 5

EX.

1 : DF

Reported Dilution

Written as a ratio, such as 1:5

Sample volume

Use when preparing a stated dilution

Diluent volume

What must be added to the sample