Development and validation of the universal competitive ELISA for Trichinella-specific

antibody detection in humans and animals

Ljiljana Sofronic-MilosavljevicReference Laboratory for Trichinellosis,

Institute for the Application of Nuclear Energy - INEP, University of Belgrade, Belgrade , Serbia

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

Since the mid-1970s, the indirect enzyme-linked immunosorbent assay(i-ELISA) has been the most commonly used serological method fordiagnosis of Trichinella-infection.Trichinella Antigens used in ELISA:

Crude worm extracts CWE in i-ELISA75-90% test sensitivity, cross-reactions with antibodies against otherparasites (Ascaris suum, Toxocara canis, Trichuris suis andNippostrongylus brasiliensis)

Excretory-secretory antigens (ES L1 Ag) in i-ELISA90-100% test sensitivity for detection of Trichinella-specific antibodies inhumans, and 90-99.6% in swine.

Synthetic (β-tyvelose) or recombinant antigens (p53 antigen ofT. spiralis)- These antigens only increased test specificity, but significantly reducedtest sensitivity.

ELISA in diagnosis of Trichinella-infection

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

The use of monoclonal antibodies specific against some components of ES L1 in ELISA

For antigen preparation in i-ELISA or As competing antibodies in competitive ELISA (c-ELISA) Manly provided an excellent results in terms of test sensitivity andspecificity, but none of these systems has yet been developed andvalidated for detection of Trichinella-specific antibodies in differenthost species.

The US4 ELISA was used to detect specific antibodies inhumans (Escalante et al., 2004)

The 7C2C5 ELISA was developed and validated for application in swine (Gamble and Graham, 1984).

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

The shortcomings of commercially available ELISA tests

Low level of specificity of tests, especially in case of infection detection in pigs and game.

Low level of sensitivity of the test in the initial stages of the infection.

Each commercially available kit relates to detection of one type of antibody (IgG) in one type of host - non-universality.

There are no commercially available tests for a number of potential hosts (from the game group)

Research goal: Development and validation of the universalELISA test for the detection of Trichinella-specific antibodies inhumans and different animals using the same components andtest parameters, improved performance compared to existingELISA tests.

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

The Trichinella c-ELISA is based on usages of monoclonal antibodies 7C2C5 - specific foran epitope unique to the muscle larvae of the entire Trichinella genus. It occurs on the45-, 49- and 53-kDa protein components of T. spiralis ES L1 antigens.

In a competitive ELISA model, the enzyme-labeled mAb-7C2C5 is in competition withTrichinella-specific serum antibodies for the same binding site on ES L1 antigen that isbound to a solid phase.

The measurable signal in the c-ELISA is inversely proportional to the concentration ofspecific antibodies present in the examined sera and derives exclusively from theinteraction of mAb 7C2C5 and the target epitope.

7C2C5 mAt - HRP

Trichinella-specific serum antibody

Principle of the NRLT INEP Trichinella c-ELISA

ES L1 Ag

A monoclonal antibody is considered to be a suitable candidate for use in c-ELISA, if it is specific for the strictly conserved epitope

that is also recognized by the specific antibodies raised during the infection.

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

WB analysis: Reactivity of ES L1 Ag blotted on a nitrocellulose membrane with 7C2C5 mAt(Line 1) or highly positive human sera (Line 2) at Fig A and Fig B.Inhibition test: Pre-incubation of ES L1 Ag with 7C2C5 mAt inhibits the reactivity ofTrichinella spiralis-specific antibodies from human (Line 3), horse (Lane 4) and pig serum(Line 5) as presented at Fig. A and reactivity with T. britovi-specific antibodies (Lane 3, Fig B)present in human sera.

Reactivity 7C2C5 mAb with T. spiralis ES L1 antigen and its ability to inhibit binding of anti-Trichinella antibodies present in sera of different

host species and infected with T. spiralis or T. britovi.

Fig A Fig B

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

Defining reference sera for test development Production and characterization of ES L1 Ag parasite

- In-vitro cultivation of Trichinella muscle larvae- Isolation of antigen from the medium

Production and characterization of 7C2C5 mAb- The cultivation of 7C2C5 hybridoma cells in in-vitro conditions, their application in inbred

mice and isolation of the ascites that contains mAt in a high concentration suitable for further work.- Testing the immunoreactivity of 7C2C5 mAb from ascites by Western blot technique.- Purification of monoclonal antibodies from ascites- Quality check ie. testing of the immunoreactivity of isolated antibodies in the indirect

ELISA system (with ES L1 antigens on the solid-phase and polyclonal antibodies labeled with HRP as asecondary reagent).

The labeling of 7C2C5 mAb with horseradish peroxidase (HRP)Examination of optimal conditions for binding of T. spiralis ES L1 antigens for several types

of unmodified polystyrene micro titer plates,Determination of optimal dilution of serum and conjugate and their volume relationships.

Development of the Trichinella c-ELISA / methodology

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

Code number Elisa index (%) Interpretation of results(cut-off > 11.8%)

1 31.7 Positive2 84.9 Positive3 98.2 Positive4 91.6 Positive5 98.1 Positive6 101.2 Positive7 12.2 Positive8 98.1 Positive9 7.8 Negative

10 7.9 Negative

Eight human serums of different levels of positivity and two negative humansera were defined as Serbian NRLT reference material, upon verification ofthe results in the European Union Reference Laboratory for Parasites,Istituto Superiore di Sanita`, Rome, Italy.

Internal reference material of NRLT

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

Sera samples used for the Trichinella c-ELISA development and validation:Human sera – positive (T. spiralis) 80Human sera – positive (T. spiralis)Human sera - negative (blood donors)

20100

Human sera – negative (from outbreaks) 40

Human sera potentially cross-reactive with ESL1 antigens of T.spiralis 170

Swine sera - positive 45

Swine sera - negative 70

Swine sera – from experimental infection 45

Swine sera potentially cross-reactive with ES L1antigens of T.spiralis

10

Horse sera - negative 55Horse sera - from experimental infection 42

Total: 677

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

Protocol for the Trichinella c-ELISA preparation

Equal volumes of the serum to be tested(diluted 1:50, 50 µl/well) and mAb 7C2C5-HRP(diluted 1:3000, 50 µl/well), prepared in blockingbuffer are dispensed at the same time intorelevant wells.

Incubation takes place at 37 ºC for 30 minutes. After incubation, all unbound materials are

removed with washing buffer (PBS-T) Color is allowed to develop for 10 min with

chromogenic solution (3,3`,5,5`-tetramethylbenzidine hydrogen peroxide – TMBsubstrate). After stopping the reaction with 2MH2SO4, optical densities (OD) are measured at450 nm on an ELISA reader.

% inhibition (PI) = 100 – (OD of test sample/ OD of monoclonal control × 100)

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

The developed c-ELISA was validated according to the

World Organization for Animal Health Recommendations

(OIE, 2012b)Determination of:

Cut-off points (ROC curve analyses)

Analytical specificity

Diagnostic sensitivity and specificity,

Repeatability

The Trichinella c-ELISA validation

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

Cut-off value determination (ROC curve analyses)

Human sera Swine sera

According to ROC analysis, the best cut-off for human sera is 19.4%, swine sera is 19.3%for PI. With these values, sensitivity and specificity were 100% in both cases (the AUC was 1),which suggests that the defined cut-off points are suitable for interpreting ELISA results.

It can be concluded that when c-ELISA is performed under the same conditions, a single cut-off value set at 19.4% can be used for determination of sera status regarding

Trichinella infection for both humans and swine.

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

None of the examined sera from human and swine exhibited cross-reaction giving afalse positive result in the Trichinella c-ELISA. The highest observed level of mAb bindinginhibition was 17%, a value below the 19.4% threshold established for samples containingTrichinella-specific antibodies. In this way, the c-ELISA correctly classified all potentiallycross-reactive samples as negative.

Analytical specificity of the Trichinella c-ELISA testNumber of

subjects

Number of positive test

result

Maximal PI in category (%)

Hum

an s

era

Other parsitic infection

Toxoplama gondii 50 - 7Echinococcusgranulosus

45 - 8

Toxocara canis 10 - 13

Autoimmune disease with presence of:

Antimitochondrial type 2antibodies

20 - 11

Rheumatoid factor 10 - 9

Antinuclear antibodies 20 - 17

Antismoothmuscleantibodies

5 - 14

Antythiroid microsomal antibodies 10 - 11

Swin

e se

ra

Other parsitoses

Ascaris suum 2 - 17Hyostrongylus sp. 1 - 12Trichuris sp. 4 - 14

Oesophagostomum sp. 1 - 7

Trichuris sp, Oesophagostomum sp, Eimeria sp.

2 - 12

Diagnostic sensitivity and specificity of the Trichinella C-ELISA

c-ELISA result

SerumNumber of

Trichinella infected subjects

AbsentNumber of

Trichinella free subjects

Total

Positive True positive 150 False positive 0 145

Negative False negative 0 True negative 385 350

Total 150 385 535

The diagnostic specificity of the c-ELISA was 100%, calculated as TN/(TN+FP), and the sensitivity was

100%, calculated as TP/(TP+FN). The positive predictive value was 100%, calculated as TP/(TP+FP), and

the negative predictive value was 100% calculated as TN/(TN+FN), where TP is the number of true

positives, FN the number of false negative, TN the number of true negatives and FP the number of false

positive results.

Classification of test results into true or false positive and negative categories with the knownstatus of each sample in a two-way (2x2) table. The cut-off value used for tests resultsclassifications was 19.4 % PI.

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

CONCLUSION Trichinella c-ELISA, based on 7C2C5 mAb, was developed to detect

Trichinella-specific antibodies in sera from humans and swine infected with T.

spiralis, with potential use for sera from other animal hosts (confirmed for

horses) and other species of Trichinella (confirmed for T. britovi).

The test employs a single antibody, mAb 7C2C5 (HRP labeled), as both the

competing and detecting reagent, which allows detection of specific

antibodies irrespective of their isotype or host origin.

This novel c-ELISA exhibited 100% specificity and sensitivity, as

confirmed by a Western blot test.

The assay is easy-to-use (one incubation step), and the 45 minute time

required for the procedure is shorter than in any other ELISA format.

This test could be useful for both detection and surveillance of Trichinella

infections.Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

Sofronic, 13th Workshop of National Reference Laboratories for Parasites, 24 -25 May 2018, EURLP, ISS, Rome, It

THANK YOU FOR YOUR ATTENTION

Keeping memory of Dr Edoardo Pozios̕ visit to INEP at 2007. – Retirement Farewell Fond regards an very best wishes: Alisa, Ljilja, Natasa and Mija