Exercise 12 Erese, Alexane Noreen D.

ANTISTREPTOLYSIN O DETERMINATION LATEX SLIDE TEST (RAPITEX SLIDE) 3CMT-1L

PROCEDURES Using the flattened end of the appropriate plastic pipette as

a stirrer (step 2), thoroughly mix each sample with reagent
within the full area of the circle.
Bring all test reagents and sample to room temperature.

Discard the disposable pipette.

Using a disposable pipette to draw up and place one free-

Slowly tilt the slide forward and backwards for exactly 2
falling drop of each undiluted sample into its identified circle
minutes and observe for agglutination under a high intensity
of the slide. Retain each pipette for mixing in step 5.
of light.

Deliver one free-falling drop of positive and negative control Record results. Re-wash the glass slides for future use.
into its identified circle.

Result: A test sample is considered to contain ASO

antibodies in excess of 200 IU/ml when agglutination
Mix ASO latex reagent by gently shaking. Add one free- (clumping) is observed when compared to the result of the
falling drop of reagent to each control and sample. negative control.
Exercise 13 Erese, Alexane Noreen D.
FEBRILE ANTIGENS (RAPID SLIDE TEST) 3CMT-1L

PROCEDURES Interpretation of Results:

POSITIVE – Presence of agglutination

NEGATIVE – Absence of agglutination
Dispense 80 ul of undiluted serum in the row of 3cm
diameter circles.

Mix the reagent bottle well and add 1 drop of antigen

suspension to the serum.

Mix well using applicator stick and rotate the slide

for 1 minute.
Exercise 13 Erese, Alexane Noreen D.
NON TREPONEMAL TEST FOR SYPHILIS – RAPID PLASMA REAGIN 3CMT-1L

PROCEDURES Interpretation of Results:

REACTIVE – with flocculation

Dispense 1 drop of undiluted serum sample on circle of a NONREACTIVE – without flocculation
test card.

Using flat end of the stirrer, spread the serum to cover

the entire diameter of the test circle.

Attach the dispensing needle to the plastic bottle.

Withdraw sufficient RPR carbon antigen for the number
of test performed. Maintaining the needle in a vertical
position, allow 1 drop (16 ul) to fall to the center of the
serum sample. DO NOT RE-STIR.

Rotate the RPR test card for 8 minutes at 100 rpm

Exercise 14 Erese, Alexane Noreen D.
TREPONEMA PALLIDUM PARTICLE AGGLUTINATION ASSAY (TPPA) 3CMT-1L

PROCEDURES Mix the contents of the wells thoroughly (for approximately

30 seconds) using a plate mixer (automatic vibratory
shaker). DO NOT USE [Link] cover the plate and let
Place 4 drops (100μL) of Sample Diluent in well #1 and 1 drop it stand at room temperature (+15 to +30°C) for 2 hours
(25μL) in wells #2 through #4 using calibrated pipette before reading. The incubation can be extended to
dropper. overnight without any perceptible difference in patterns.

Add 25μL of specimen to well #1 using micropipette.

Fill the micropipette or a diluted with 25 μl of the diluted

solution well #1 and mix well. Transfer 25 ul of the mixture of
specimen and sample diluent into well #2. Then mix well and
repeat this procedure again with wells #2, #3, and #4 to
obtain serial dilutions.

Place 1 drop (25μL) of unsensitized particles in well #3, 1

drop (25μL) of sensitized particles in well #4 using droppers
supplied in the kit.
Exercise 15 Erese, Alexane Noreen D.
HEPATITIS B SURFACE ANTIGEN USING ENZYME IMMUNOASSAY 3CMT-1L
TECHNIQUE (MONALISA HEPATITIS B SURFACE ANTIGEN ULTRA)

PROCEDURES Dispense 100 ul of mixture in each well incubate 25-30

minutes at +37°C with cover.

Prepare your worksheets and map the one (1) positive

control and three (3) negative controls and samples. Label Add 100 ul of stopping solution.
the wells if it is possible.

Titer reading of the wells in the spectrophotometer.

Add 100 ul of negative control, positive control and samples.
Then, Add 50 ul of conjugate (R7) to all negative control,
positive control and samples. Record the absorbance of controls and samples.

Check the assay validity:

Incubate the sample wells for 90 minutes in dark
NEGATIVE CONTROL : ≤ 0.080
area/environment.
POSITIVE CONTROL : ≥ 1.000

Wash and soak with 30 seconds interval in every wash and

soak.

Prepare 1:10 Buffer Mixture, 9 parts of substrate buffer and 1 part

substrate chromogen depending on the number of samples and
controls in tests. Important is to deliver 100 ul of each mixture in
all wells.
Exercise 16 Erese, Alexane Noreen D.
ANTI-HCV USING ENZYME IMMUNOASSAY TECHNIQUE (INTEC 3CMT-1L
ADVANCED HCV ELISA)

PROCEDURES Dispense 50 uL of Color A into each well. Then, dispense 50

uL of Color B into each well. Gently tap the plate to
thoroughly mix the liquid in the wells.
Prepare Washing Solution. Dilute concentrated wash buffer
in Distilled water using a 1:20 diultion.
Cover the plate with adhesive film. Incubate for 10 minutes
at 37'C.
Prepare and label the microtiter plate. Dispense 100 uL of
sample diluent into each well.
Dispense 50 uL of Stopping solution into each well. Gently
tap the plate after.
Dispense:
10uL of Negative control in Well A1, B1, and C1
10uL of HCV Positive control in Well D1 and E1 Read the optical density at 450/ 620 nm using a plate
10uL of sample in their corresponding wells reader.

Gently tap the plate to thoroughly mix the the liquid. Then, Check the assay validity:
cover the plate with adhesive film. Incubate for 25 minutes NC < 0.100 = VALID
at 37'C. NC > 0.100 = INVALID

Wash the plate with a minimum of 330 uL of washing PC > 0.800 = VALID
solution with a soak time of at least 30 seconds. Repeat this PC < 0.800 = INVALID
procedure 5 times. Blot on absorbent.
Exercise 17 Erese, Alexane Noreen D.
HIV AG-AB USING ENZYME IMMUNOASSAY TECHNIQUE (GENSCREEN
3CMT-1L
ULTRA HIV AG/ AB)

PROCEDURES Prepare 1:10 Buffer Mixture, 9 parts of substrate buffer and 1

part substrate chromogen depending on the number of
samples and controls in tests. Important is to deliver 100 ul
Prepare the worksheet and map the two (2) positive antigen of each mixture in all wells.
control, two (2) positive antibody and three (3) negative
controls and samples. Label the wells if it is possible.
Dispense 100ul of mixture in each well incubate 30 minutes
with cover.
Add 75 ul of negative controls, positive controls and
samples. Then, add 25 ul of conjugate 1 in all wells.
Add 100 ul of stopping solution

Incubate for 1 hour ± 4 minutes at +37°C

Titer Reading at 420/620 to 700 nm using a plate
reader/spectrophotometer.
Wash and soak for four (4) times with 30 seconds interval in
each washing.
Check the assay validity:
Negative control: < 0.170
Add 100 ul of conjugate 2 in all wells. Then, incubate for 30 Antigen control: > 0.900
seconds ± 4 minutes at +37°C. Antibody control: < 0.900

Wash and soak for four (4) times with 30 seconds interval in
each washing.
Exercise 18 Erese, Alexane Noreen D.
TESTING FOR THE HETEROPHILE ANTIBODY OF INFECTIOUS 3CMT-1L
MONONUCLEOSIS BY THE CLEARVIEW MONOR TEST

PROCEDURES For whole blood (venipuncture) samples: Hold the dropper

upright and add two drops of whole blood (about 50 L) to the
sample well (S) of the test device. Then add one drop of
Allow the test device, sample, buffer, and controls to reach sample buffer to the sample well. Start the timer.
room temperature (15oC to 30oC) before testing.

For whole blood (fingerstick) samples: Add one capillary

Remove the test device from the foil pouch and use it as tube of blood (about 50 L) to the sample well (S) of the test
soon as possible. For best results, perform the test device. Then add one drop of sample buffer to the sample
immediately after opening the foil pouch. well. Start the timer.

For serum or plasma samples: Hold the dropped upright and

Place the test device on a clean and level surface.
add one drop of serum or plasma (about 25 L) to the sample
well (S) of the test device. Then add one drop of sample
Wait for the red line(s) to appear. The result should be read buffer to the sample well. Start the timer. Avoid trapping air
at 5 minutes. The background should be clear before the bubbles in the sample well.
result is read.
Exercise 19 Erese, Alexane Noreen D.
TESTING FOR THE HETEROPHILE ANTIBODY OF INFECTIOUS
3CMT-1L
MONONUCLEOSIS BY PAUL-BUNNEL TEST

PROCEDURES Pipette 0.1 mL of positive control serum into tube 1 of the control
row. Then, mix and serially transfer 0.25 mL through tube 9,
discarding 0.25 mL from tube 9. Tube 10 of this row contains no
Inactivate complement in sera to be tested by heating in a serum and serves as a negative control.
56oC water bath for 30 minutes.

Obtain a sample of patient serum to be tested. Pipette 0.1 mL of

patient serum into tube 1 of the patient row.
Place sheep red blood cells into a tube and wash the cells
three times in saline.
Mix and serially transfer 0.25 mL through tube 10, discarding
0.25 mL from tube 10. Then, add 0.1 mL of the 2 percent sheep
Prepare 5 mL of a 2 percent sheep red blood cell red blood cell suspension (prepared in step 3 above) to each tube
suspensionby pipetting 0.1 mL of washed, packed red blood in the control and patient rows.
cells into 4.9 mL of saline.

Shake the tubes to obtain an even mixture. Cover the tubes with
Set up and label two rows of 10 test tubes in a rack. One row parafilm and incubate at room temperature for at least 15
is for a positive-control serum and the other for the patient minutes. A more accurate reading may be obtained by allowing
serum to be tested. Label the control tubes C1 through C10, the tubes to incubate for 2 hours.
and label the patient tubes P1 through P10.

Following the incubation period, read each tube individually for

Pipette 0.4 mL of saline in the first tube of each row and 0.25 macroscopic agglutination, as follows: gently shake the tube to
mL of saline in each remaining tube. resuspend the red blood cells, tilt the tube, and hold up to light.
Compare each tube to the negative control tube and record
results.
Exercise 20 Erese, Alexane Noreen D.
DENGUE NS1 ANTIGEN AND ANTIBODY DETECTION (DENGUE DUO) 3CMT-1L

PROCEDURES Interpretation of Results:

POSITIVE – Presence of agglutination

NEGATIVE – Absence of agglutination
Dispense 80 ul of undiluted serum in the row of 3cm
diameter circles.

Mix the reagent bottle well and add 1 drop of antigen

suspension to the serum.

Mix well using applicator stick and rotate the slide

for 1 minute.
Exercise 21 Erese, Alexane Noreen D.
RUBELLA SEROLOGY 3CMT-1L

PROCEDURES
Set up timer. Result should be read in 10 minutes. Positive
results may be visible in as soon as 1 minute.
Allow test device, specimen, and/or controls to equilibrate
to room temperature (15-30°C) prior to testing.

Remove the cassette from the sealed pouch and use it as

soon as possible. Place the cassette on a clean, flat surface.
Be sure to label the device with specimen‟s ID number.

Fill the capillary tube with specimen not exceeding the

specimen. The volume of specimen is approx. 10 μL. For
better precision, use a pipette capable of delivering 10 μL.

Holding the capillary tube vertically, dispense the entire

specimen into the center of the sample well (S) making sure
that there are no air bubbles. Immediately add 2 drops
(about 60-80 μL) of Sample Diluent to the sample well with
bottle positioned vertically.
Exercise 22 Erese, Alexane Noreen D.
CHIKUNGUNYA SEROLOGY 3CMT-1L

PROCEDURES INTERPRETATION OF TEST RESULTS

1. Negative result: Only band ("C" Control line) within the result
window indicates a negative result.
Carefully read the instruction for using the STANDARD Q 2. IgM Positive result: Two colored bands ("C" Control line and
Chikungunya IgM/IgG Test. Look at the expiry date at the back of "M" Test line) within the result window indicate Chikungunya
the cassette package. Use another lot, if expiry date has passed. IgM positive.
3. IgG Positive result : Two colored bands ("C" Control line and
"G" Test line) within the result window indicate Chikungunya
IgG positive.
Open the cassette package and check the cassette and the
4. IgM & IgG positive result : Three colored bands ("C" Control
color indicator silica gel in cassette package. line, "M" Test line & "G" Test line) within the result window
indicate Chikungunya IgM & IgG positive.
5. Invalid result: If the control band ("C" control line) is not
Using a micropipette or specimen transfer device(to the visible within the result window, the result is considered
black line), collect the serum, plasma or whole blood (10ul). invalid. The directions may not have been followed correctly
or the test may have deteriorated. Re-test with a new
cassette
Add the collected serum, plasma or whole blood to the
specimen well of the cassette.

Add 3 drops (90ul) of buffer into the buffer well of the

cassette.

Read the test result after 15 minutes. The test result can be
read up to 30 minutes.
Exercise 23 Erese, Alexane Noreen D.
MALARIA PF/ PV 3CMT-1L

PROCEDURES Manner of Reporting:

POSITIVE - distinct colored on both the test and control
lines.
Prepare the test kit. Let it stand at room NEGATIVE – distinct colored band on the control line only.
temperature prior to testing. INVALID – control band fails to appear on the result window.

Dispense a drop of blood on the sample hole of the

cassette.

Dispense three drops of the lysing agent in the

buffer hole. The buffer carries the blood along the
length of the RDT.

Allow the test to stand for 20 minutes. Then, observe

for the formation of the test hand.