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Standard Operating Procedure

COMPETITION ELISA FOR MALARIA 10/198 AS A STANDARD

Table of Contents

Standard Operating Procedure...................................................................................1

Competition ELISA for Malaria 10/198 as a standard.................................................1

1 Purpose...............................................................................................................2

2 Introduction..........................................................................................................2

3 Scope...................................................................................................................2

4 Definitions/abbreviations......................................................................................3

5 Equipment/Antigens/Reagents............................................................................3

6 Procedure............................................................................................................4

7 Data processing and reporting.............................................................................8

8 References .....................................................................................................10


1 PurposeThis SOP describes the procedures to demonstrate the antigen specificities of malaria antibodies in immune serum or plasma by direct competition with different allelic forms of the homologous antigen.

2 IntroductionThe competitive ELISA, cELISA, measures the ability of a solution phase antigen(s) to compete with an immobilised coating antigen for the test antibodies. In this instance the assay measures the cross reactivity of the antibodies elicited by a candidate vaccine against homologous and heterologous alleles of the malarial apical membrane antigen (AMA1).It is expected that the principle of the assay can be applied to a range of antigens / antibodies.

This SOP covers the competition ELISA protocol and incorporates use of the Anti-malaria (Plasmodium falciparum) human serum (1st International Reference Reagent), 10/198, (Bryan D, et al. 2017) as a reference material. 10/198, is a pooled lyophilised serum of the individuals from African individuals infected with malarial parasites.

A brief overview of cELISA is shown in the schematic diagram. cELISA is a 3 day process consists of two parts, a. Serum Dilution determination and b. competition ELISA

a. Serum Dilution Determination (To determine the concentration of antibodies to be used to ensure that competition / reduction in signal is measurable).

Day 1: Coating of 96 well plates and incubating overnight at 40C. Day 2: Perform serum/plasma dilution and analyse.

b. Perform competition ELISA. (Use concentration of antibodies determined in (a) in the presence of immobile antigens as well as varying concentration of competitor antigen in mobile phase)

Day 2: Coating of 96 well plates and incubating overnight at 40C. Day3: Perform cELISA and analysis.

Schematic diagram of competition ELISA (Day 3).

Please read this SOP at its entirety.

3 ScopeAssays listed in this SOP are performed in Category Level 2 laboratories.


4 Definitions/abbreviationsName Abbreviation

Competition ELISA cELISA

First WHO ReferenceReagent for Anti-malaria

(Plasmodium falciparum) Human

serum

Standard 10/198

Room Temperature RT

Antibody Ab

Antigen Ag

5 Equipment/Antigens/Reagents5.1 Equipment

Item Supplier Catalogue number

ELISA plates- immunoplateMaxisorp

Nunc 442404

Plate reader Any AnyPlate Washer and if notavailable use hand washing technique

Any Any

2ml polystyrene screw topvials

Any Any

50ml polystyrene centrifugetubes

Any Any

5.2 Antigens/reagentsItem Supplier Catalogue number

Recombinant antigens (AMA-1, MSP-1, DiCos1,2,3 etc)

Any Any

1 step ultra TMBsubstrate solution

Thermo scientific 34029

2M Sulphuric Acid Any Any

Horseradish peroxidase-conjugated rabbit anti- human IgG. Use 1 in 5000 in Reagent Diluent.

Dako P0214

Standard-First WHOReference Reagent for Anti-malaria (Plasmodium falciparum) Human serum

National Institute ofBiological Standards

and Controls (NIBSC)

www.ni b sc.org/ pro d u c t s .a s px and code 10-198

Negative control - serumor IgG from a non- immunized or unexposed Human

Any- availablecommercially or in

house (Serum/plasma or purified IgG)

Any


5.3 Prepared Solutions in Advance5.3.1 Coating Buffer

1.59 g Na2CO3 + 2.93 g NaHCO3 per litre in distilled water. Carbonate/Bicarbonate solution should be at pH 9.4 - 9.6. Coating buffer can be stored for up to 6 months at 4°C.

5.3.2 Blocking BufferPBS + 0.05% v/v Tween20 + 3% w/v BSA. It can be stored for up to 1 week at 40C.

5.3.3 Reagent DiluentPBS + 0.05% v/v Tween20 + 0.5% w/v BSA. It can be stored for up to 1 week at 40C.

5.3.4 Wash Buffer: - PBS + 0.05% Tween.

Table 1. Day 1, Serum determination plate layout1 2 3 4 5 6 7 8 9 10 11 12

A HB3

B

C No Antigen

D

E DiCo3

F

G No Antigen

H

Antigens diluted in coating buffer at 2ug/ml final concentration and dispense 50ul/well. No antigen wells receives 50ul of coating buffer only.

6 Procedure6.1 Day 1: Determination of Serum DilutionAn initial titration of serum or plasma is performed to determine the optimal dilution of serum or plasma to use in the subsequent cELISA.

6.1.1 Coat plates with 50μl/well of each recombinant Ag at 2μg/ml final concentration in coating buffer e.g. refer to Table 1: Serum Determination Plate Layout.

6.1.2 For no antigen wells dispense 50μl/well coating buffer.6.1.3 Cover plate with sticky clear tape and incubate overnight at 40C.

6.2 Day 2: Serum determination ELISA and plate coating for cELISA

PREPARATION IN ADVANCE BEFORE STARTING ELISA

Bring all reagents at room temperature such as Coating buffer, Reagent Diluent, Blocking Buffer, one step ultra TMB substrate solution, Serum or Plasma and wash buffer.

Reconstitution of lyophilised 10/198 standard

Reconstitute 1 vial of lyophilised 10/198 standard according to the manufacturer’s protocol. In brief, 1 vial of 10/198 is reconstituted with 1ml of distilled water and leave at RT for 10 minutes, transfer contents to a screw top vial and store at RT until use. For short term storage up to 2 months and long term storage i.e. more than 2 months, it is advisable to store reconstituted 10/198 at 40C and -200C respectively.

Serum determination ELISA

6.2.1 Wash plates 6x with wash buffer using plate washer.


1 2 3 4 5 6 7 8 9 10 11 12

ANeg

control 10/198HB3

BNeg

control 10/198

C No Antigen

D

ENeg

control 10/198DiCo3

FNeg

control 10/198

G No Antigen

H

Serum/Plasma - 2 fold dilution in reagent diluent, 100ul final volume

Standard 10/198, use 1 in 3000 dilution in reagent diluent

6.2.2 Dispense 200ml Blocking Buffer per well and incubate at RT for 1.5hrs.6.2.3 During 1.5hrs of plate incubation, prepare standard 10/198 dilution for e.g.

Table 2, serum determination plate layout, for one plate, use standard 10/198, 1 in 3000 dilution in Reagent Diluent as positive control

Therefore, calculate required amount per plate e.g. for plate layout in Table 2, 100μl (per well) x 4 wells = 400μl + 200μl extra therefore, total 600μl of diluted standard 10/198 is required per plate.. Negative control is diluted according to the manufacturer’s

protocol.

Table 2:- Day 2, Serum Determination Plate Layout

Antigens

6.2.3 Wash plates 6x with wash buffer using plate washer.6.2.4 Titrate serum or plasma in reagent diluent, 2-fold from 1:500 from column 1 or

more concentrated if Ab titres are expected to be low (e.g. in infants). Final volume is 100μl/well.

6.2.5 Add 100μl/well of positive control and negative control in the appropriate wells.

6.2.6 Cover plates and incubate for 2hrs at RT.6.2.7 Last 10 minutes of incubation, prepare Horseradish peroxidase- conjugated

rabbit anti-human IgG in reagent diluent.6.2.8 Wash plates 6x with wash buffer using plate washer.6.2.9 Add 100μl/well of diluted (1 in 5000) Horseradish peroxidase-conjugated

rabbit anti- human IgG from step 6.3.2.7.6.2.10 Cover plates and incubate for 1hr at RT.6.2.11 Wash plates 6x with wash buffer using plate washer.6.2.12 Add 100μl ultra TMB substrate solution.6.2.13 Cover plates with aluminium foil and incubate for 20min at RT.6.2.14 Add 50μl/well of 2M sulphuric acid stop solution.6.2.15 Read plates as soon as possible in plate reader at 450nm absorbance.

Plate coating for cELISA

This step can be performed any time during Day 2. For each serum or plasma sample, whole plate is coated with each Ag of interest and competed with the same Ag and other desired Ag to be competed with the immobilised Ag. Please refer to the Introduction section of cELISA.

6.2.16 Each plate is coated with 50μl/well of one antigen at 2μg/ml final volume.6.2.17 Cover plate with sticky clear tape and incubate overnight at

40C.


6.3 Day 2: Analysis of Serum determinationTwo analysis can be performed from serum determination ELISA in order to choose correct serum dilution for use in cELISA on Day 3.

6.3.1 Analysis based on OD450 nm 1-1.5This analysis is performed mainly when individual laboratory is performing cELISA on serum or plasma.

6.3.1.1 For each Ag, OD’s of serum or plasma is plotted against serial dilution using four parameter logistic function.

6.3.1.2 Based on the curve, choose serum or plasma dilution that results in between thelinear part of the curve and OD450 nm above 1.

(Example: For HB3 coated Ag wells, 1 in 800 dilution of serum or plasma antibodies fits in the linear part of the curve which resulted in OD450 1.5 . Therefore, 1 in 800 serum dilutions will be selected for HB3 Ag in use for cELISA on Day 3. Similarly, DiCo3 coated Ag wells, serum / plasma dilution will be choose based on the linear fit on the curve and OD450 nm

above 1)

6.3.2 Analysis based on serum dilutionThis analysis is performed mainly when 2 or more laboratories are involved in performing cELISA with the use of same serum or plasma across all laboratories.

6.3.2.1 For each Ag, OD’s of serum or plasma is plotted against serial dilution using four – parameter logistic function.

6.3.2.2 Based on the curve, choose one serum dilution that fits in the linear curve from all the curves generated at individual laboratory.

(Example: For serum dilution 1 in 800, Laboratory A results in OD450 nm 1.2, Laboratory B results in OD450 nm 2, Laboratory C results in OD450 nm 1.5 etc. Regardless of the OD450 nm, it is advisable to choose same dilution of serum or plasma for subsequent use in cELISA on Day 3)

Chosen serum dilution will be used in the Day 3 cELISA.

6.4 Day 3: cELISAPREPARATION IN ADVANCE BEFORE STARTING cELISA

Bring all reagents at room temperature such as Coating buffer, Reagent Diluent, Blocking Buffer, one step ultra TMB substrate solution, Serum or Plasma, antigens, and wash buffer.

Table 3. Day 3, cELISA Plate Layout

1 2 3 4 5 6 7 8 9 10 11 12

Compe titor Ag HB3 μg/ml

Compe titor Ag 3D7 μg/ml

Compe titor Ag DiCo3 μg/ml

Compe titor Ag DiCo1 μg/ml

Compe titor Ag FVO μg/ml

Se rum Standard - 100ul/we ll

A 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 250 250

B 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 750 750

C 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 2250 2250

D 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 6750 6750

E 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 20250 20250

F 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 60750 60750

G 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 182250 182250

H no competitor Ag no competitor Ag no competitor Ag no competitor Ag no competitor Ag Blank

6.4.1 Wash plates 6x with wash buffer using plate washer.6.4.2 Dispense 200ml Blocking Buffer per well and incubate at RT for 1.5hrs.6.4.3 During 1.5hrs of plate incubation, prepare following reagents


6.4.3.1 Competitor antigen preparation 60μg/mlMake up all antigens at 60μg/ml in reagent diluent and store at RT until its use.

6.4.3.2 Standard preparationMake up standard 10/198 at 1 in 250 dilution in reagent diluent from neat and store at

RT until its use.

6.4.3.3 Serum or plasma dilution for each Antigen

Serum or plasma dilutions were chosen from point 6.3 Day 2: Analysis of Serum determination. For each Ag coated plate, serum or plasma dilution may be different or same and therefore, it may require preparing serum or plasma dilution in advance

for each Ag. For e.g. Plasma A results in 1 in 800 dilution for HB3 Ag, 1 in 200 dilution

for DiCo3. Plasma B results in 1 in 100 dilution for HB3 Ag, 1 in 2000 dilution for DiCo3.

Therefore, all the above dilutions are prepared 2x concentrated. Examples listed in Table 4.

6.4.4 After 1.5 hours of blocking buffer incubation. Wash plates 6x with wash buffer using plate washer.

6.4.5 Add 50μl reagent diluent to all wells except row A wells and all wells in column 11 and12.

Table 4. Examples of Plasma samples for preparaing 2x dilution for use in cELISA

Sample Dilutions obtained from Day 2 of serum determination

Dilutions to be prepared on Day 3 for cELISA

HB3 DiCO3 HB3 DiCO3

Plasma A 1 in 800 1 in 200 1 in 400 1 in 100

Plasma B 1 in 100 1 in 2000 1 in 50 1 in 1000


Table 5. Titration of competitior Ag wells and Standard 10/198

Competitor Ag HB3 μg/ml Competitor Ag 3D7 μg/ml Competitor Ag DiCo3 μg/ml Competitor Ag DiCo1 μg/ml Competitor Ag FVO μg/ml Standard 10/198

A 75ul of HB3 @ 60ug/ml

75ul of HB3 @ 60ug/ml

75ul of 3D7 @ 60ug/ml

75ul of 3D7 @ 60ug/ml

75ul of DiCo3 @ 60ug/ml




75ul of FVO @ 60ug/ml

75ul of FVO @ 60ug/ml

150 ul of serum

150 ul of serum

B 25ul from well A + 50 ul RD

25ul from well A + 50 ul RD











C 25ul from well B + 50 ul RD

25ul from well B + 50 ul RD











D 25ul from well C+ 50 ul RD

25ul from well C+ 50 ul RD









50ul from well C + 100 ul RD

50ul from well C + 100 ul RD

E 25ul from well D + 50 ul RD

25ul from well D + 50 ul RD











F 25ul from well E + 50 ul RD

25ul from well E + 50 ul RD











G25ul from well F +50 ul RD, mix well and discard 25 ul

25ul from well F + 50ul RD, mix well

and discard 25


and discard 25

25ul from well F +50 ul RD, mix well and discard



and discard 25


and discard 25


and discard 25


and discard 25


50ul from well F +100 ul RD, mix

well and discard

50ul from well F +100 ul RD, mix

well and discard

H no competitor Ag and only 50 ul of RD no competitor Ag and only 50 ul of RD no competitor Ag and only 50 ul of RD no competitor Ag and only 50 ul of RD no competitor Ag and only 50 ul of RD 100 ul RD 100 ul RD

6.4.6 Add 75μl of relevant Ag (competitor Ag) at 60μg/ml to row A. Refer to Table 56.4.7 Pipette out 25μl of competitor Ag from row A and perform 3 fold dilution down the

plate from Row A- Row G. Refer to Table 5.6.4.8 At row G, mix well contents and discard 25μl as to maintain 50μl final volume in row

G. Note: Do not titrate in Row H. Refer to Table 5

6.4.9 For standard 10/198 wells which are columns 11 and 12: Add 100μl of reagent diluent from row B to row H.

6.4.10 Add 150μl of standard 10/198 pre-diluted 1 in 250 to row A. Refer to standard preparation listed in “6.4 Day 3: cELISA”.

6.4.11 Pipette out 50μl from row A and perform 3 fold dilution down the plate till Row G.6.4.12 At row G mix well contents and discard 50μl as to maintain 100μl final volume in row

G. Note: Do not titrate in Row H (Blank wells). Refer to Table 5

6.4.13 Finally, Add 50μl of 2x serum prepared for each Ag coated plate to all wells (Table 4) except all wells in column 11 and 12.

6.4.14 Incubate plates for 2hrs at RT.6.4.15 Wash plates 6x with wash buffer using plate washer.6.4.16 Last 10 minutes of incubation, prepare Horseradish peroxidase- conjugated rabbit

anti-human IgG in reagent diluent.6.4.17 Wash plates 6x with wash buffer using plate washer.6.4.18 Add 100μl/well of diluted (1 in 5000) Horseradish peroxidase-conjugated rabbit anti-

human IgG.6.4.19 Cover plates and incubate for 1hr at RT.6.4.20 Wash plates 6x with wash buffer using plate washer.6.4.21 Add 100μl ultra TMB substrate solution.6.4.22 Cover plates with aluminium foil and incubate for 20min at RT.6.4.23 Add 50μl/well of 2M sulphuric acid stop solution.6.4.24 Read plates as soon as possible in plate reader at 450nm absorbance.

7 Data processing and reportingDay 3: cELISA Analysis

When plates are read at wavelength 450nm in plate reader, raw data obtained as optical density are transported to ADAMSEL Software for evaluating residual binding. The residual binding data are plotted against concentrations of competitor Ag.

7.1 Conversion of raw data to obtain concentration from standard curve.The cELISA used was based on coating plates with one antigen of PfAMA1 and mixing test antibody samples (Serum or plasma) with the same or other PfAMA1 Ag to determine the degree to which antibody binding to the coating material was inhibited.


Optical Density raw data from 96 wells are converted into units from standard 10/198 (Reconstituted 10/198 is 100units) curve using 4 parameter logistic function. Software has been developed for this function called ADAMSEL software and alongside instructions for software use can be downloaded free of charge from EURIPRED website. Analysis example as a Fig 1 has been shown below from Kusi et al. 2009 paper publication. Contact EURIPRED website for further advice on analysis or use of ADAMSEL software.

Fig 1: Competition ELISA with protein A-purified antibodies and antibody pools made from single allele immunisations.

A) Assay on 3D7 AMA1-coated plates with anti-3D7 AMA1, anti-Combi (mixed allele immunisation) and anti-FVO/HB3/3D7 antibody pool. B) Assay on FVO AMA1-coated plates

with anti- FVO AMA1, anti-Combi and anti-FVO/HB3/3D7 antibody pool. C) Assay on HB3 AMA1-coated plates with anti- HB3 AMA1, anti-Combi and anti-FVO/HB3/3D7 antibody pool.

IgG pools were made from antibodies raised in single allele immunisations with FVO, HB3

and 3D7 AMA1. All assays were performed with FVO, HB3, 3D7 and CAMP AMA1 proteins as competitor antigens. All IgGs were used at 2 times the pre-determined antibody titre. Plots are representative of at least 2 assay repeats using IgGs from one rabbit per group since the depletion patterns were similar for both rabbits in each immunisation group.


7.2 cELISA IC50 CalculationAfter the concentrations from the cELISA have been calculated using ADAMSEL, the cELISA results files can be merged (per assay plate) using ADAMSEL merge. The resulting Excel

sheet named “Samples All” holds the compiled results data. The amount of Ab remaining bound where competitor is added can be calculated by dividing the concentration in the well

by the concentration in the well (well number 8) without competitor. This can also be done using R statistics software for which a script is provided on the EURIPRED website. For correct

execution of the script it is of mandatory that every sample has a unique name (see also example files on EURIPRED website). The R script then calculates the IC50 values for all samples tested.

ADAMSEL software can be downloaded from https://web.vboxx.nl/shares/file/1a64d3e6684fe7/

8 References1. The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum. Donna Bryan, Nilupa Silva, Peter Rigsby, Thomas Doμgall, Patrick Corran, Paμl W. Bowyer, Mei Mei Ho, and the Collaborative study group. Malaria Journal, 2017; 16: 314.

2. Humoral immune response to mixed PfAMA1 alleles; multivalent PfAMA1 vaccines induce broad specificity. Kusi KA, Faber BW, Thomas AW, Remarque EJ. PLoS One. 2009; 4(12): e8110.

For technical help with cELISA experiment and analysis, please contact : remar q ue@b p rc.nl

and/or : bh ag wati.k h atr i @ n ibsc.org

https://urldefense.proofpoint.com/v2/url?u=https-3A__web.vboxx.nl_shares_file_1a64d3e6684fe7_&d=DwMFaQ&c=bXyEFqpHx20PVepeYtwgeyo6Hxa8iNFcGZACCQj1uNM&r=yjJ_t2zEBIevljqIEO3zEuUjXxQO1vG-0G-4CJzUYME&m=F6HSDl_3U3YTOA1vkHUQzFrJL_6hjqWbJONBZOTKPL0&s=rqtyW1tknRYj94uA5ZStT1uxQfnavCIpN-LuEviGo7g&e=

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