EXPT # 13-15: Qualitative Detection of HBsAg, Ab to HCV, Ab to HIV  Dane particle (whole viral part of HBV)

Hepatitis is inflammation of the liver  HBsAg (formerly : Australia antigen)

o First marker to appear in 1-2 weeks in active hepatitis
Hep A request for HAVIgM infection

Associated with ALT/alanine aminotransferase - Associated with hepatocellular carcinoma, AFP at alpha-1 in
electrophoresis, oncofetal antigen high in uterus baby
Detects antigensss! HEP B serologic marker

HEPATITIS B SURFACE ANTIGEN (HBsAg) - 1. HBsAg/ Australia antigen- 1st to appear

1% liver cirrhosis; produces the highest to viral hepatitis - 2. HBeAg- marker of infectivity, if increased titer very infectious

 Hepatitis virus is causative agent of viral hepatitis A, B, C, D, E, G - 3. Anti-HBc- current/recent infection- if positive plus HBsAg= acute
hepatitis infection
o Not self-limiting : hepatitis B & C (patient can have chronic
disorder) - 4. Anti-HBe- means you’re recovering/safe

 Hepatitis C : 85% of infected patients have chronic - 5. Anti-HBs- denotes immunity to the infection
liver disease (higher rate of chronicity) Routes of transmission
o Self-limiting : hepatitis A, D, E, G 1. Exposure to blood
 Hepatitis D : coinfection/ superinfection 2. Blood drinking
 Coinfection is simultaneous infection; superinfection 3. Parenteral/needlestick injury
already acquired hepB + hep D wherein px has 99%
hep b ; recovery is acquired at childhood 4. Sexual

 Patient is infected first with hepatitis B before 5. Needleprick/intravenous route

hepatitis D HEPATITIS C VIRUS (HCV) and dengue are flaviviruses- common factor
 Hepatitis D infection depends on HBsAg of Hepatitis is their asymptomatic character; shows if in abundance 85% chronicity-
B 20% end stage liver cyst

HEPATITIS B VIRUS (HBV) 15% recovers

Acute: less than 6 months Traditional confirmatory: recombinant Immunoblot assay test

Chronic: more than 6 months failure in seroconversion or production of  Causative agent of hepatitis C
antibody towards that certain antigen  RNA virus
 Causative agent of hepatitis B
 Flavivirus
 DNA virus  No vaccine available due to its capability of mutating to escape the
 Hepadna virus- contains only one never both, specialization 2ndary or host’s immune system
latent infection; hepatocarcinoma  Hepatitis C is also known as Non-A Non-B hepatitis
 Antigens : HBsAg (surface), HBeAg (envelope), HBcAg (core) cannot  Most common cause of post-transfusion hepatitis after blood
be detected because HbeAg covers it transfusion
 Antibodies : Anti-HBs, anti-HBe, anti-HBc
  No vaccine because keeps mutating o 3 weeks afteracute
HUMAN IMMUNODEFICIENCY VIRUS (HIV)  Principle : lateral flow immunochromatographic assay/
immunochromatography
CD4-helper cells
o Mobile phase : analyte in question
HTLV I, II, leukemia
 Also known as o Stationary Control and : detects; where ag bind to ab

o Human T-cell Lymphocytic Virus III (HTLV III)- trophic o Burgundy color = (+) reaction

o AIDs-related virus (ARV) Control

Conjugate pad Test band
band
o Lymph Adenopathy Virus (LAV) Goat anti-
HBsA Mouse anti-HBsAg Antibody Non-conjugated
mouse IgG
 Has 2 strains : HIV I (United States) and HIV II (Africa) g conjugated w/ colloid gold HBsAg antibody
Ab
 Hiv I subgroups: M( A-H), N, O Recombinant HCV Antigen
Recombinant Goat anti
HCV conjugated w/ colloidal gold and
 HIV II- A B HCVAg rabbit IgG
rabbit IgG gold colloid
 Tests 1) recombinant HIV-1- Antigen
HIV HIV-1 antigen Goat anti-
2) recombinant HIV-2- Antigen
HIV-2 antigen rabbit IgG
3) rabbit-IgG-gold conjugate
o Screening test : ELISA;
o Screening; sandwich with antibody hiv will combine with ab,  Pipetting scheme
washing to remove excess, add enzyme labeled ab- 1. HBsAg reagent = 100 uL/ 3 drops
conjugation substrate and enzyme
2. Anti-HCV- 10ul/ 1 drop + 1drop diluent
o Confirmatory test : Western Blot- detects proteins
3. Anti-HIV-1 and 2- 100ul/ 3 drops and 1 drop diluent
 Report as positive if (+) in at least two :
-spx is serum
 P24 (always positive)- because it’s the core
antigen, GP120 or GP21, GP160  Read test kit within 15 minutes

o Traditional test : Radio Immunoblot Assay (RIBA)  Control is always positive, if not, reject test kit results

Cluster of differentiation  Reporting of results:

Ratio: 2:1 is normal  HBsAg – reactive/ non-reactive (ANTIGEN)

AIDS= 0.5:1 / 1:2  Anti-HCV, Anti-HIV – positive/negative (ANTIBODY)

CD4:CD8 QUALITATIVE DETECTION OF ANTIGEN-ANTIBODIES

Qualitative determination
(if positive)
↓
TEST KIT Rerun the test
 HBsAg is the 1st marker to appear in blood in acute hepatitis (if positive proceed to)
↓
o 1 week - 2 months after exposure Quantitative determination
(if positive)
o 2 weeks – 2 months bfore onset of symptoms
↓
Rerun the test again
(if positive)
↓
HBsAg o 2nd – dengue viral shock
- More than or equal to 2 ng/ml  All serotypes are sense positive RNA viruses
- + burgundy o Serotypes : DENV-1, DENV-2, DENV-3, DENV-4
- Confirm by ELISA  Vector
- Qualitative test only o Mosquito – Aedes aegypti (common in Eastern
- Limitations to all cross reaction with high titers of heterophil countries)/tropical country
antibodies and rheumatoid factor – Aedes albopictus (common in Western
countries)/ cold areas
- Hbv is the common cause of persistent viremia, chronic liver,
hepatocellular carcinoma  After recovery from dengue fever, one develops short term immunity
from all serotypes
- Hepatotropic DNA
 Subsequent infection is more severe
- Core has polymerase, coat has lipids, proteins and
carbohydrates called HbsAg DETECTION OF ANTIGENS
- Chronic carriers: 6-12 months persistence, no seroconversion  NS1 (non-structural protein) Right side of test
HCV- igG,M,A o 1 to 9 days after onset of illness
- Detects antibody  IgG  indicates past infection/ secondary infection
- Qualitative o Primary infection : 14th day after onset of illness
- Because of chronic o Secondary infection : 1st & 2nd day after onset of illness
- Routes: iv drugs, sexual contact  IgM  higher titer in primary infection, indicates current infection
- ssRNA with synthetic or recombinant protein o Primary infection : 5-10 days
HIV-1 and 2- dilute at 1:50 or 1:100 , retest to confirm more o Secondary infection : 4-5 days
Surface hep b: active ANAMNESTIC RESPONSE
E= active infectious  the secondary immune response occurring on subsequent exposure
Igm and e- current to an antigen that has previously been encountered
 memory cells remember which antigens to use against the infection,
thus it can respond quicker in times of dengue virus infection (Memory
Report as non or reactive for hiv cells DO NOT FORGET the antigens)
 predominant antibody is IgG
EXPT # 16: Qualitative Detection of Dengue NS1 Antigen & IgG/ IgM Ab TEST KIT
DENGUE VIRUS (DENV)/dengue fever
 principle: immunochromatography/ lateral flow
 Flavivirus and RNA virus immunochromatographic assay
 Clinical presentation  pipetting scheme
o st
1 – viral hemorrhagic fever 1. NS1 = 100 uL or 3 drops
 2. IgG/ IgM = 10 uL + 4 drops diluent Test pad: mouse monoclonal anti-dengue ns1
 Read test kit within 15 minutes Control: goat anti-mouse IgG
DENGUE IgG, IgM= detects antibodies
Secondary infection is more severe and fatal For rapid, qualitative and differential detection
Malariae lives in polluted waters while dengue lives in clean Control: rabbit anti-dengue IgG
waters
Test pads for IgGand IgM: mouse monoclonal anti-human IgG and IgM –
gives color
Stages: Conjugate pad: recombinant dengue virus envelope protein- gold colloid
1: fever + is purple
2: hemorrhagic fever
3: dengue shock/absence of blood supply Spx EDTA whole blood
Negative results: retest after 3-5 days
In CBC testing, decrease WBC count= because of viral infection Invalid results are due to insufficient antibodies or spx levels, incorrect
indicated by lymphocytes technique
Increase hematocrit= because of escape of body fluid to the level Control line is for procedural control
plasma of vessel will escape
Cross reactivity with FLavivirus group
Increased wbc= bacterial/neutrophil
Dengue virus
Dengue virus introduces holes making holes to the blood vessels the
St. Louis encephalitis
platelet will try to cover up the holes
Japanese encephalitis
West nile
Severe viremia= deplete platelet count px has low plt
Yellow fever virus
Sanofi- vaccine for dengue called DENGVAXIA last December 2015 1 st
country to use is MEXICO
+ result: PURPLE
M- primary
G- secondary or past
Both:
Tests= CBC, virology, IgM, IgG
Coffee like poopings
NS1 left side= early acute dengue, qualitative
Conjugate pad: detects antigen
Mouse monoclonal anti-dengue ns1 with gold colloid