IMMUNOSERO (LABORATORY WORKSHEETS)

Test Principle Type of Test Type of Sample Antibodies Antigen Result Interpret Sample
Required
Rapid Plasma Carbon Non-Treponemal Serum/Plasma Reagin antibodies Carbon suspension Black clumps; 50 uL
Reagin Agglutination; Test • Produced by coated with LIPID Flocculation
infected host COMPLEXES
Note: Heat agains his
inactivation own cell
unnecessary components
• Non-specific
• Seen in
chickenpox,
hepatitis, IM,
leprosy,
tuberculosis,
malaria
FTA-ABS Fluorescence using Treponemal Test
Fluorescence dark field
Treponemal microscopy
Antibody
Adsorption

Serum/EDTA TPHA uses preserved 10 uL

Treponema plasma avian erythrocytes coated
Pallidum with extracted antigens of
Hemagglutinatio T. pallidum
n Test (TPHA) (Nichols strain)

Anti-HBsAg Immunochromatogra Serum/Plasma 15-20 minutes 100 uL

phic Assay
HIV Testing Chromatographic Other Notes: 10 uL serum HIV recombinant 5-20 minutes
Immunoassay ELISA – Or 20 uL Whole proteins
Screening Blood HIV1:
• P24
Note: Sensitive to Western Blot • Gp41
humidity & Heat Cofirmatory
HIV2:
• Gp36
Anti-Streptolysin Latex Agglutination Serum Streptococcal 3 minutes 50 uL
O Streptococcal Abs exoenzymes bound to
(ASO) Do not use Plasma inert latex particles

Rheumatoid Lates Agglutination Distinct agglutination 3 minutes

Arthritis Serum Rheumatoid Factor Human IgG bound to indicates a RF
polystyrene lated particles content of greater than
IgM antibody directed 20IU/mL in the
against IgG undiluted sample

Note:
Heavy bacterial
contamination may
cause false positive

Lipemic sera can cause

non-specific rxns
 STORAGE CONDITIONS
• Store Serum 2-8C for no longer than 72 hrs AFTER collection, if the test cannot be done in the same day
• Specimens may be kept refrigerated for up to 24 hours. Freezing specimens at -20 C is advised for longer storage.

SPECIMEN STORAGE (INSERTS)

HEPATITIS B
• 2-8C for 3 days
• - 20C for long term storage
Note: It is not suitable to test whole blood samples which have been stored at 2-8C for more than 7 days

RPR
• 2-8C for 7 days
• -20 C for 3 months

ASO
• 2-8C for 7 days
• -20C for longer periods

OTHER NOTES
ASO REAGENTS RA Reagents

Reagents: Buffer Concentrate (20x)

ASO latex reagent • Concentrated Glycine Saline Buffer is to be diluted 1:20 withpurified water
• A suspension of polystyrene particles coated with streptococcal
exoenzymes. RF – Latex Reagent
• A suspension of latex particles sensitized with human IgG in Glycine saline
ASO positive control Buffer
• A stabilized human serum reactive with the test reagent. Ready for use; do not
dilute (200IU/mL). Positive Control
• A stabilized prediluted human serum containing RF; reactive with the latex
ASO negative control reagent
• A stabilized human serum non-reactive with the test reagent. Ready for use; do
not dilute. Negative Control
Glycine-Saline Buffer (20x) • A stabilized prediluted human serum; nonreactive in the test system.
• pH = 8.2 +/- 0.1
• A diluent containing 0.1M glycine and 0.15M NaCl.
• Dilute buffer according to instructions on the label.
• All reagents contain 0.1% (w/v) sodium azide as a preservative.
• Store all reagents at 2-8 C. Do not freeze.
 INTRODUCTION PROCEDURES
RPR RPR
1. Reagent Prep Carbon Antigen is labelled with Antigen lot number, expiry, and
Treponema pallidum transfer dates
• Causative agent of syphilis, STI
• “great imitator” Stability is 3 months or until expiry date (whichever comes first)
• Many individuals do not show symptoms

Manifestations:
1. Chancre – primary
2. Condylomata lata – secondary
3. Gummas – tertiary

Chancre – endothelial cell thickening, aggregation of lymphocytes,

macrophages, & plasma cells
2. Qualitative
Test in 50 uL of sample
Microtiter 1 drop of control
Plates Rotator 100 rpm for 8 minutes

Read MACROSCOPICALLY over light box or under high intensity

incandescent lamp
3. Results Medium – Large Clumps
Interpretation (Reactive)
Small Clumps
(Weakly Reactove)

No Clumping or Roughness
(Non-Reactive)

INTRODUCTION PROCEDURES
TP-PHA
TPHA Test Kit Components: TPHA Test Kit Components:
1. Test cells: avian erythrocytes coated with antigens of T. pallidum
1. Test cells: avian erythrocytes coated with antigens of T. 2. Control cells: avian erythrocytes
pallidum 3. Sample diluent: Saline solution containing absorbents
2. Control cells: avian erythrocytes 4. Positive control: rabbit antiserum (titre 1:1280)
5. Negative control: normal rabbit serum
3. Sample diluent: Saline solution containing absorbents
4. Positive control: rabbit antiserum (titre 1:1280)
5. Negative control: normal rabbit serum
PROCEDURE
1. Label the wells as follows: S, PC, NC, T, and C.
a. Legend: S (sample), PC (positive control), NC (negative control), T
(test), C
(control)
2. Into the S well, add 190 uL of diluent. Add 10 uL of sample and mix
thoroughly.
3. Add 25 uL of positive control and negative control to designated wells.
4. Transfer 25 ul of diluted sample from step 2 to the T and C wells.
5. Resuspend the Test and Control cells thoroughly.
 6. Add 75 uL of Test Cells to PC and NC wells.
7. Add 75 uL of Test Cells to T well, and 75 uL of Control Cells to C well.
8. Mix wells thoroughly and incubate at 15-30°C on a vibration-free surface
for 45-60
minutes.
9. Read the agglutination patterns. Record the results

INTRODUCTION PROCEDURES
Anti-HbsAg Test
1. 100 uL of serum or plasma
Hepatitis B 2. Interpret Results
• Double stranded DNA virus composed of 27mm nucleocapsid core At 15-20 minutes
surrounded by HbsAg (Hepatits B Surface Antigen)
Note: Do not interpret AFTER 30 MINUTES
HbsAg – initial marker to appear after weeks of exposur; good indicator of
disease progression
• Peak during ACUTE STAGE; declines as patient recovers
• Formerly known as Australia Antigen(Au) antigen or Hepatitis
Associated Antigen (HAA)
Results POSITIVE
Interpretation Presence of two bands in Test & Control Window

NEGATIVE
• Contol Line Only

INVALID
• No visible lines in control & Test line
• No visible line in control
Limitations Negative results does not mean there is no presence of HbsAg antibody

INTRODUCTION PROCEDURES
Anti-HbsAg Test
1. Test devices and specimens should be stored in room temperature 15-30
Hepatitis B minutes before
• Double stranded DNA virus composed of 27mm nucleocapsid testing.
core surrounded by HbsAg (Hepatits B Surface Antigen) 2. Remove the test device from sealed pouch, and place it on a level and
clean surface.
3. Transfer 100ul of serum or plasma into the sample well.
HbsAg – initial marker to appear after weeks of exposur; good indicator of 4. Wait for the purple or red line in the test region to appear after the test
disease progression begins to work.
• Peak during ACUTE STAGE; declines as patient recovers 5. Interpret test results at 15-20 minutes. Do not interpret test result after 30
• Formerly known as Australia Antigen(Au) antigen or Hepatitis minutes.
Associated Antigen (HAA)

INTRODUCTION PROCEDURES
HIV
Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired 1. Prepare all test strips, sealed bottle and specimens in room
Immunodeficiency Syndrome (AIDS). temperature before testing.

In 2009, WHO estimates about 33.3 million individuals are infected with HIV 2. Transfer 10ul of serum, plasma or 20ul of whole blood into the sample
with 2.6 million new infection. well.
• The HIV-1 and HIV-2 are the two types of HIV that infect humans, but 3. Remove test strip from container, and dip it into sample well
HIV-1 is more common. maintaining it vertically. Do not submerse the strip below the ‘stop
• The HIV targets CD4+ T-Lymphocytes cell or also known as T-helper line’.
cells. This T-cell is crucial in the immune response of the human body 4. Drop 3 drops (about 100ul) of assay diluents into other sample well,
against diseases. and then dip the strip into the well and absorb the assay diluents.
• The transmission of HIV is primarily by four major routes:
(1) Unprotected sexual contact; 5. Wait for the red or purple line in the test region to appear after the test
(2) exposure to infected blood or other body fluids; begins to work
(3) being born to an infectedmother (
4) injection drug use. 6. Interpret test results at 5-20 minutes. Do not interpret test result after
20 minutes.
The majority of HIV infection cases is caused by unprotected sex, particularly,
homosexual contact. However, unprotected anal sex is
riskier than unprotected vaginal sex. And among those who have sex with
other men, unprotected receptive anal sex is riskier than unprotected insertive
anal sex.

There are various serological methods employed to detect the presence of

HIV virus:

• Enzyme Linked Immunosorbent Assay (ELISA) is the screening test

for HIV, while
• Western Blot, is the confirmatory assay.

INTRODUCTION PROCEDURES
ASO
ASO- lysed RBCs 1. 50 uL of PC; 50 uL of ASO NC. 50 uL of Sample
2. Rock for 3 minutes
Group A (Strep pyogenes)
• Produces streptolysin O and S Qualitative Test:
• DNAse A,B,C,D 1. Place all reagents and specimens at room temperature before testing.
• Streptokinase 2. Place one drop (50ul) of ASO positive control on the field of the reaction
• NADase, slide. Place one
• Hyaluronidase drop (50ul) of the ASO negative control on another field. Use pipette/stir
• Diphosphopyridine nucleotidase stick to deliver 1
 • Pyrogenic exotoxin drop (50ul) of undiluted test sample to field #3. Continue likewise with
additional
Diagnostically important Abs unknowns. Retain pipette/stir sticks for mixing step.
• Anti ASO 3. Gently resuspend the ASO latex reagent and add one drop to each test
• Anti-DNAse field.
• Anti-hyaluronidase 4. Mix well using flat end of the pipette. Gently rock the slide for three (3)
minutes and read
immediately under direct light.

INTRODUCTION PROCEDURES
RA
Rheumatoid arthritis is an autoimmune condition affecting multiple joints of Limitations Limitations:
the body.
Reactions that take longer than four (4) minutes may result into an
• The cause of this disease is unknown, but is believed to be the apparent false positive reaction due to drying effect.
consequence of faulty immune response.
Contominated or markedly lipemic sera can result to false positive
• It is frequently manifested by swelling and pain in the joints and by reactions.
degenerative and inflammatory
• process involving cartilage synovial membrane or muscle tissue. Agglutination intensity in the qualitative is not indicative of RF
concentrations. Screening
• There is no cure for rheumatic reactions, therefore, should not be graded.
• arthritis (RA), but recent discovered drugs are found to be effective in
the treatment and prevention Healthy individuals may have rheumatoid factors (approximately 2%)
• of joint deformity. detectable by
agglutination test.
• Other than medication, various factors are also considered in the
treatment of RF found in low titers in these individuals. Incidence of positive
reaction increases with age.
• RA, good self-management including exercise is known to reduce
pain and disability.
Rheumatoid factor may be present with Scleroderma, Still’s disease,
Sjorgren’s syndrome, and Systemic Lupus Erythematosus.
• Rheumatoid factor is a group of antibodies that react with both human
and animal IgG.

• Scientific evidences show that rheumatoid factors participate in the

development of rheumatoid rthritis through formation of antigen-
antibody complexes. Most patients who are found to be
positive with rheumatoid factor have more severe rheumatoid
arthritis than those with negative tests.