1. Optimization of the acute toxoplasmosis immunodiagnostic during the pregnancy . Juan Gabriel Costa1; Jésica Perotti; Pablo Faccendini2; Claudia Lagier2; Iván Marcipar1. 1Labortorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs. Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina 2Departamento de Química Analítica, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina e.mail:
Background and Objectives: The toxoplasmosis is an infection carried out by Toxoplasma gondii protozoan. When women become infected during the pregnancy, this parasite may be transmitted to fetus causing serious health consequences including aborts, malformations or live threatening illness in the child. As an early treatment decrease the risk of congenital transmission, an accurate diagnosis is highly desirable during the course of pregnancy. In this work we investigated the use of acute phase antigens and some fractions thereof to improve the accuracy of immunochemical diagnostic tools to discriminate acute from chronic infections.
Selected Antigen
P35
P22
P30
Different sequences of acute phase antigens
were selected, cloned and induced.
500 pb
97.4
66.2 45 31
20.5
P22c
27 AA
173 AA
Figure 8: Agarose gel electroforesis of P30c digested sequences from pGem-T easy cloning vector (a), vector without insert (b) and molecular marker.
Figure 9: PAGE of P30c protein induced in E. coli BL 21 DE3 (a), TRX protein induced in E. coli BL 21 DE3 (b) and molecular marker.
P22
97.4
66.2 45 31
20.5 14 a b a b b b
P22L
1 AA
188 AA a N C
P22
1 AA
188 AA
Figure 2: Agarose gel electroforesis of P35A. Nucleotide sequence cloned in pGem-T easy cloning vectors (in different concentrations) .
Figure 3: PAGE of purified P35B protein and molecular marker.
Figure 4: Assessed aminoacidic
regions of P22.
Figure 8
Figure 9
Figure 2
Figure 3
500 pb a
97.4
66.2 45 31
20.5 14 a b
Figure 5: Agarose gel electophoresis of P22c PCR product and molecular marker .
Figure 6: PAGE of P22c protein induced in E. coli BL 21 DE3 (a), basal expressed proteins in E. coli BL 21 DE3 (b) and molecular marker.
P30L
76 AA
336 AA 11 N C
P35B
P35A
P35
1 AA
268 AA
1 A
95 AA
145 AA
48 AA
P30c
171 AA
337 AA
P30
Figure 6
Figure 5
Figure 7: Assessed aminoacidic regions of P30.
Figure 1: Assessed aminoacidic regions of P35.
Modelling and epitopes prediction of the selected acute phase antigens
Figure 13: P22 model structure based in computational prediction. Main program used: Modeller 9v3.
Figure 10: P35 model structure based in computational prediction and published experimental data. Main program used: Modeller 9v3. N C C N
Figure 16: Reported crystal structure of a P30 protein highly homologous with the P30 used in this work (identity sequence: %). This structure will be used to model our protein.
Figure 14: Predicted lineal epitopes location within P22 using online program: AAppred.
Figure 11: Predicted lineal epitopes location within P35 sequence using online program AAppred. N C N C
Figure 15: Predicted structural epitopes location withing P22 using online program: Discotope.
Figure 12: Predicted structural epitopes location within P35 sequence using online program Discotope.
Immune Assay
Partial Conclusions:
From the antigens assessed up to now, using indirect ELISA to determine acute toxoplasmosis, P35B was the most promising one. This region allowed the best performance when specific IgG antibodies were determined, followed by IgA. Specific IgM antibodies against P35A or B did not allow discrimination enough between acute and chronic infection.
In P35 protein, experimental results agree with B epitopes prediction; P35B is the most antigenic region in the protein. In P22 antigen, predicted epitopes are mainly placed in P22c although we have not yet experimentally confirmed them.
Future work:
In a next step, specific IgG, A and M antibodies against the different P22 and P30 fragments will be assessed in indirect ELISA in order to determine the usefulness of these assays to discriminate acute from chronic infections. Avidity of specific IgG antibodies against these antigens will also be evaluated for this purposed. Furthermore, tridimensional structure of P30 protein will be modeled and its linear and conformational epitopes predicted. Finally, as a result of our study we will be able to select the fittest regions from these acute phase antigens to optimize the immunodiagnosis during the pregnancy.
IgG
IgM
IgA
P35A
P35B
Amount of sera
SIA 68 44 57
SIC 67 27
SIN 55 61
Average signal
2.36
2.12
2.59
1.75
2.30
1.22
1.39
0.91
2.66
1.45
2.11
0.80
1.07
0.61
2.55
2.08
0.82
Area under curve
SIA vs SIC
0.81
0.85
0.53
0.60
0.73
SIA vs SIC +SIN
0.84
0.90
0.51
0.52
SIP vs SIN
0.76
0.79
0.50
0.56
0.57
Optimal sensibility (SIA vs SIC) (%)
67.60
79.40
50.00
63.60
73.70
70.20
Optimal specifity (SIA vs SIC) (%)
89.60
83.30
63.00
59.30
47.40
68.40
Table 1: Indirect ELISA performed to detect IgG, IgA and IgM, using P35A and P35B antigen. SIP, sera from individuals infected with toxoplasmosis (Acute + Chronic); SIC, sera from individuals with chronic infection; SIA, sera from individuals with acute infection; SIN, sera from individuals without infection.