2cycle gia with pldh assay

2-cycle GIA with pLDH assay

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Aim
The aim of this 2-cycle Growth Inhibition Assay is to quantify inhibition of invasion
or growth of P. falciparum into erythrocytes. The inhibition is thought to be mediated
by P. falciparum specific antibody in the presence of purified IgG fraction from
animals immunized with P. falciparum antigens. Allowing the parasites to grow for
two cycle facilitates the detection of the biological effects of antibodies that act later
in the life cycle (growth inhibition rather than invasion inhibition). The read out
system is detection of pLDH (plasmodium Lactate DeHydrogenase), this enzyme is
produced by live parasites and well detectable when they are in late trophozoiet and
schizont phase.
Principle
3-Acetylpyridine Adenine Dinucleotide (APAD) and Lactate (present in substrate
buffer) are converted by pLDH to APADH and Pyruvate. APADH reduces the
chromogenic substrate Nitro Blue Tetrazolium (NBT) using the enzyme diaphorase.
This results in the formation of Nitro Blue Formazan (NBF), a deep purple soluble
stain that can be measured at a wavelength of 650 nm.
Schematic representation:

Materials
❏
Positive control: BPRC 98 rabbit standard at 6 mg/ml total Ig. Negative control: Negative rabbit, rhesus or human IgG fraction, purified similarly as the samples. 96-well flat-bottom culture plates with individual lids: Greiner Bio-one #655180 Centrifuge for culturing and spinning down RBC’s: Beckman Coulter Allegra X-22R Centrifuge for harvesting of GIA-plates: Beckman Spinchron R Centrifuge Eppendorf centrifuge: Eppendorf centrifuge 5424 2cycle GIA with pLDH assayb.doc Page 1 of 4 Eppendorf tubes: Eppendorf Safe-lock 1.5 ml #0030 120.086 Filters: 0.45 µm: Whatman FP30/0,45 CA-S #10462100 Flatbed shaker: Edmund Bühler TiMix CONTROL Humidified box: plastic box with wet tissues or water at the bottom Humidified incubator: Sanyo O2/CO2 incubator Platereader: Bio-Rad Model 680 Microplate Reader 100% Methanol: Merck #1.06009.2500; CH3OH, M=32.04 RPMI1640 + 20% Human serum + 30 µg/ml Gentamycin To prepare 200 ml 2xCmed: Mix 160 ml RPMI1640 + 40 ml Human serum + 120 µl Gentamycin. Store at 4 °C. APAD: Sigma #A5251; C22H28N6O14P2, M=662.44 3-Acetylpyridine Adenine Dinucleotide To prepare 10 ml stock solution of 10 mg/ml: dissolve 100 mg of APAD in 10 ml distilled water. Make 50 µl aliquots and store at -20 °C. Diaphorase from Clostridium kluyveri: Sigma #D5540 To prepare 30 ml stock solution of 50 units/ml: dissolve 1.500 units Diaphorase in 30 ml distilled water. Make 200 µl aliquots and store at -20 °C. EDTA: Merck #1.00944.1000; C10H16N2O8, M=292.25 Ethylenediamine tetraacetic acid To prepare an 8 mM EDTA solution: Dissolve 116.9 mg in 40 ml RPMI1640, adjust to pH 7.5, fill up to 50 ml with RPMI1640 and sterilise through a 0.45 µm filter, store at 4 °C. Gentamycin: Gibco #15750-037; stock = 50 mg/ml Giemsa buffer: Merck #1.09468.0100; Na2HPO4*2H2O KH2PO4 Buffer tablets pH 7.2 To prepare 1 litre Giemsa buffer: dissolve 1 buffer tablet in 1 liter distilled water. To prepare fresh Giemsa stain: dilute 1 part Giemsa stain in 4 parts Giemsa buffer, filter through a 0.45 µm filter, use immediately. Heat-inactivated Human Serum A+: Bloedbank Leidsenhage, store at -20 °C. Human RBC O+ (multipe donors): Bloedbank Rotterdam, store at 4 °C. LDH-buffer: Sigma #L7022; C3H5NaO3, M=112.06 Sodium L-lactate To prepare 500 ml of the buffer, mix 50 ml of 1M TRIS-HCl (pH 8.0) and 450 ml distilled water. Add 2.8 g Sodium L-Lactate and 1.25 ml Triton X-100. Mix on a magnetic stirrer at room temparature for at least 30 minutes. Make 50 ml aliquots and store at -20 °C. NBT: Sigma #N6639; C40H30N10O6*2Cl, M=817.64 2cycle GIA with pLDH assayb.doc Page 2 of 4 Method
Timing of 2-cycle experiment
Timing is essential in a 2-cycle GIA, which depends on the stage the synchronised
parasites are in. There are two time schedules you can follow:
Development stage:
ripe schiz.
medium addition
late throps
late trophs
Development stage:
medium addition
schizonts
late trophs

Preparation of uninfected RBC for use in GIA
1. Use the same batch of RBC used for the culturing of parasites in the last few days.
2. Make a RBC suspension of 1% hematocrit in 2xCmed.
Preparation of parasites
1. Count the parasitaemia in a smear stained with Giemsa, making sure all parasites
are in the late thorps or schizont stage. 2. Transfer the parasite-culture to a 50 ml tube.
3. Spin for 5 minutes at 2000rpm.
4. Remove supernatant.
5. Dilute enough of the parasite-pellet in 2xCmed for 0.1% parasitaemia.
6. Add RBC 50% HT to get a 1% HT suspension.
Assay set-up
1. Use 96-well flat-bottom culture plates with individual lids.
2. Label all plates with the parasite-line used, date, initials and plate-number
3. Pipette 25 µl IgG’s in triplicates (50 µl if you make a serial dilution of the IgG’s, than also pipette 25 µl of plain RPMI in the other wells for serial dilution). 4. Pipette 25 µl positive IgG in triplicate. 5. Pipette 25 µl negative IgG in triplicate. 6. Pipette 25 µl plain RPMI for the schizont controls in triplicates. 7. Pipette 25 µl plain RPMI for the RBC-control. 8. Pipette 25 µl plain RPMI for the monitor-wells. 9. Add to all wells, except the RBC-controls, 25 µl parasite-suspension. 10. Add to the RBC-control-wells 25 µl RBC-suspension. 11. Pipette 25 µl 8 mM EDTA for the EDTA-control. 12. Put the plates in a humidified box (wet tissues or water at the bottom of the box), and place the lid on the box, but don’t close it completely. 13. Put the boxes with the plates in a humidifed incubator at 37 °C, containing 5% 2cycle GIA with pLDH assayb.doc Page 3 of 4 14. Add 50 ul fresh medium to each well at the appropiate time (see time schedule) 15. Make a smear of 5-6 monitor-wells of one of the plates at appropiate times and stain with Giemsa (See protocol 3.6 Giemsa staining) to monitor parasite-stage.

Harvest of assay plates
1. Make a smear of 5-6 monitor-wells of all plates and stain with Giemsa (See
protocol 3.6 Giemsa staining) to check parasite-stage. Harvest at late throps or
schizont stage.
2. Fill new 96-well flat-bottom culture plates with 200 µl/well of cold PBS (one 3. Mix the contents of the wells in the assay plate thoroughly using a multichannel and transfer 100 µl to the PBS-plate (in the same format). Use same tips when possible (i.e. for each sample). 4. Spin all plates (both the original plates and the new plates) for 10 minutes at 5. Remove 240 µl of supernatant without disturbing the pellet. Use same tips where 6. Freeze the plates overnight at -20 °C (or until ready for pLDH-assay) to lyse
LDH assay
NOTE: NBT is light sensitive, so avoid direct light and keep solutions and plates
with substrate in the dark (cover with aluminium foil)
1. Thaw the plates and LDH-buffer and warm up to room temperature (10 ml LDH-
2. Dissolve NBT in LDH-buffer at a concentration of 2mg/10ml. Mix gently and 3. Add 50 µl APAD stock (10 mg/ml) to every 10 ml substrate. 4. Add 200 µl Diaphorase stock (50 units/ml) to every 10 ml substrate. 5. Use substrate immediately. 6. Add 100 µl substrate per well of the harvested plates. One plate every minute. 7. Cover with aluminium-foil and place on a flatbed shaker at 400 rpm at room 8. Incubate for 30 minutes. 9. Measure OD after 30 minutes at wavelength 655 nm (A655). One plate every
Results
OD-values measured by the platereader should be exported to a csv-file.
% inhibitions can be calculated using the following formula:
(A65 5 Sz con trol – A6 55 RBC control) 2cycle GIA with pLDH assayb.doc Page 4 of 4

Source: http://www.optimalvac.eu/files/u2/2cycle_GIA_with_pLDH%20assay_BPRC.pdf