Our previous results showed higher level of anti-Hib IgG (2.41 g/ml) average geometric mean concentration (GMC) regardless of age and gender, followed by levels of IgM (0.91 g/ml) and IgA (0.34 g/ml), reflecting the prevalence of immunity against Hib. Current study was evaluated the avidity profile for these high anti-Hib concentration. The IgM avidity analysis did not revealed any significant results between male and female values. While, the average of IgA avidity in male samples were significantly ( 0.05) higher than the average of female. Also, The result showed that high percentage of population has moderate to high IgG avidity. The study showed lower avidity percentages of both IgM and IgA comparing to IgG. More studies are suggested to be done on individuals with low avidity considering the secondary immunodeficiency in participant’s history.
Capsular polysaccharide (CPS)-protein conjugate Hib vaccine has become obligatory in the national immunization program in Saudi Arabia in 2000 [2]. Serum anti-CPS antibodies allow immunity against invasive Hib disease ( 0.15 g/ml of these antibodies is a serological indication for short-term immunity protection, while a concentration of 1.0 g/ml represents long-term protection) [12, 26, 27]. Hence, antibody levels measurement and circulation data review in worldwide populations are the key steps for accurate evaluation of vaccine-induced immunity.
In 2000, the immunogenicity in Saudi infants was examined after three doses of Hib (HbOC) vaccine. Anti-Hib antibodies levels after three doses (14.4 g/ ml) were higher than those reported recently from the USA (4.4 g/ml), but similar to data which have been previously reported (16.8 g/ml) [2, 3, 13, 17, 31]. This phenomenon of low antibody level has been observed in countries using Hib vaccine on a wide scale and for all types of Hib vaccines [3]. This could be caused by the total decrease in the overall exposure of infants to Hib infection, and consequently, the absence of the natural boosting effect to the vaccine [3]. This suggests that in the early periods of Hib vaccination on a wide scale, the primary series of three doses will create high levels of antibody, which will not be the case in other cohorts of children vaccinated a few years later.
The term antibody affinity could represent the strength of the binding of a single antibody type (e.g. monoclonal antibody) and a single antigen. The antibody population in human serum is polyclonal (heterogeneous); therefore, it is impossible to measure antibody affinity. Nevertheless, alterations of the affinity notion have been developed, and measurements of the strength and stability of the antigen-antibody contacts in a mixed antibody population have been termed antibody avidity measurements [11, 23]. Also, the antibody avidity can be detected by several techniques and its levels will fluctuate, depending on the technique employed. Each technique has its limitations, and it is difficult to match the readings of specific technique with those of another, given the variety of methods used in experimentation and analysis. The homogeneity of serum antibodies is the main factor that affects the avidity results, since high antibody heterogeneity leads to more challenges in the accurate measurement of antibody avidity [11, 23].
To test antibody avidity, chaotropic agents (e.g. ammonium or sodium thiocyanate) have been used widely; these agents serve as elute (dissociate) molecules or molecules interfering with antigen-antibody binding, and therefore, only antibodies with strong avidities can bind to antigens [6, 18]. Accordingly, thiocyanate concentration obstructs electrostatic interactions that facilitate the formation of antigen-antibody complexes, and concentrations of 0 to 5 M thiocyanate are regularly used. The higher the antibody avidity, the more antigen-antibody complexes are formed, and as a result, a greater concentration of chaotropic agent is expected to disrupt the development of such antigen-antibody connections. In one study, samples showing values of 68.1%, between 68.1 and 75.8%, or 75.8% were stratified as low-, moderate-, or high-avidity sera, respectively [10].
Antibody avidity is used as a marker for the evaluation of memory induced after conjugate vaccination [9]. Avidity affected by the type of Hib vaccine, the mean anti-Hib PS Ab avidity of the serum pool from the infants vaccinated with HbOC was threefold higher than that of the pool from infants vaccinated with Hib-OMP, which demonstrate that the molecular form of the Hib CPS immunogen dictates quality of Ab function [14, 15]. Hib vaccines elicit IgG avidity regardless of IgG concentration [1]. Likewise, patients with X-linked hypogammaglobulinemia and other high-risk individuals for Hib diseases (including splenectomy, malignancies associated with immunosuppression, and sickle-cell anaemia) have been successfully treated by passive immunization with pooled immunoglobulin [30].
In Brazil, nearly all children (97.56%) vaccinated with Hib-combined vaccine (manufactured completely in Brazil after technological allocation of Hib vaccine production from Glaxo SmithKline, Belgium) acquired an effective anti-CPS IgG antibody ( 1.0 g/mL), whereas an anti-CPS IgM was detected in 64.24% of children ( 0.15 g/mL). Only 18.88% of infants with elevated CPS antibody IgG levels ( 1.0 g/mL) acquired a high IgM antibody response. Anti-CPS IgG antibody concentrations were considerably greater than those of anti-CPS IgM antibody. These data prove the prevalence of IgG antibodies over IgM antibodies in response to CPS (ratio 17:1). IgG antibodies were mainly of the IgG1 subclass. A rise in IgG avidity was also seen throughout the program of immunization [16].
As we previously demonstrated that higher level of anti-Hib IgG (2.41 g/ml) regardless of age and gender, followed by levels of IgM (0.91 g/ml) and IgA (0.34 g/ml), which reflecting the prevalence of immunity against Hib within Jeddah population, Saudi Arabia. The current study was evaluated those anti-Hib antibodies affinity for Hib CPS [4, 32, 33].
Materials and methods
Participants and samples
One thousand three (1003) venous blood samples (2–5 mL) were obtained by venepuncture with informed consent from consecutive healthy individuals attending the local medical facility in Jeddah, Saudi Arabia, for routine medical check-up, from the period of February 2014 to January 2016. Volunteers with immune disorders or who had receiving immunomodulators were excluded based on medical history data and participants’ answers. The participants include 490 males vs. 513 females with ages ranged from one year to 91 years old for the both. The average age for all participants is 42.4 years, includes the average 42.8 years for male participants and the average 42 years for female participants. Most of participants are Saudi nationals 964 with 486 males and 478 females. Other nationalities (39 samples) include 1 American female (Sample# 398), 2 Egyptian males (Sample# 6 and 208), 9 Egyptian females (Sample# 15, 49, 115, 186, 323, 427, 462, 498 and 1599), 1 French female (Sample# 555), 1 Indian male (Sample# 130), 2 Indian females (Sample# 472 and 608), 1 Indonesian female (Sample# 85), 1 Jordanian female (Sample# 143), 2 Malaysian females (Sample# 22 and 598), 1 Macedonian female (Sample# 1091), 1 Moroccan male (Sample# 197), 7 Moroccan females (Sample# 75, 131, 151, 210, 292, 549 and 1244), 6 Pakistani females (Sample# 8, 596, 601, 640, 1121 and 1642), 1 Palestinian female (Sample# 511), and 3 Pilipino females (Sample# 86, 602 and 611). Samples have been age and gender-stratified as shown in results section.
Samples selection for IgG avidity
Sex
Age
1 g/mL samples
1 g/mL samples
Total
Male
1–10
4
4
8
Female
1–10
3
5
8
Male
11–20
1
3
4
Female
11–20
2
2
4
Male
21–30
2
2
4
Female
21–30
2
2
4
Male
31–40
2
2
4
Female
31–40
1
3
4
Male
41–50
2
2
4
Female
41–50
2
2
4
Male
51–60
3
2
5
Female
51–60
2
2
4
Male
61–70
3
2
5
Female
61–70
3
2
5
Male
71–80
1
2
3
Female
71–80
2
2
4
Male
81–90
1
0
1
Female
81–90
1
0
1
Total
37
39
76
Samples selection for IgM avidity
Sex
Age
1 g/mL samples
1 g/mL samples
Total
Male
1–10
4
3
7
Female
1–10
2
3
5
Male
11–20
1
2
3
Female
11–20
1
2
3
Male
21–30
1
1
2
Female
21–30
2
2
4
Male
31–40
1
1
2
Female
31–40
1
1
2
Male
41–50
1
1
2
Female
41–50
1
1
2
Male
51–60
2
1
3
Female
51–60
3
1
4
Male
61–70
1
1
2
Female
61–70
2
1
3
Male
71–80
3
1
4
Female
71–80
3
1
4
Male
81–90
0
0
0
Female
81–90
0
0
0
Total
29
23
52
All sera were separated from clotted whole-blood samples by centrifugation at room temperature, and kept frozen to 20C in 1.5 mL Safe-Lock Eppendorf tubes. All samples were diluted to 1:100 in phosphate buffer saline (PBS).
Selecting samples for avidity
We tried to select very high and very low antibody concentration as far as we can (Tables 1–3).
Hib polysaccharide
Pure capsular polysaccharide (CPS) of Hib was obtained from FUNDAÇÃO BUTANTAN (Prof. Joaquin Cabrera-Crespo, Instituto Butantan, Centro de Biotechnologia Lab. Bioprocessos I Av. Vital Brasil 1500 05503-900 Sao Paulo, SP Brazil). CovaLink NH Microwell Plates (Treated microwell plates), Thermoscientific (Cat No. NUNC-478042) were used to chemically conjugate the Hib CPS to NH group of the plates in a indirect ELISA.
Anti-human antibodies
All antibodies (first or secondary antibodies) used as well as substrates were as described previously [32, 33].
ELISA assay to evaluate IgG avidity using elution with increasing concentration of ammonium thiocyanate
Samples selection for IgA avidity
Sex
Age
1 g/mL samples
1 g/mL samples
Total
Male
1–10
4
4
8
Female
1–10
4
4
8
Male
11–20
2
1
3
Female
11–20
3
2
5
Male
21–30
1
1
2
Female
21–30
2
1
3
Male
31–40
1
1
2
Female
31–40
2
1
3
Male
41–50
2
1
3
Female
41–50
2
1
3
Male
51–60
1
1
2
Female
51–60
2
1
3
Male
61–70
2
1
3
Female
61–70
2
2
4
Male
71–80
2
1
3
Female
71–80
1
1
2
Male
81–90
0
0
0
Female
81–90
1
1
2
Total
34
25
59
ELISA assay to evaluate IgG avidity using elution with increasing concentration of ammonium thiocyanate were designed as described by Romero-Steiner et al. [29].
Antigen coating and washing
Purified PRP antigens at a concentration of 17 g/mL were dissolved in 1% (-(3-dimethylaminopropyl)--ethyl-carbodiimide hydrochloride (EDC) and dispensed into CovaLink NH microwell plates, 50 l/well and incubated overnight at 37C. PBS was prepared by reconstitute of 1 tablet of the product in 200 mL of water, the resulting solution is 1 PBS. Two g (1%) of gelatin and 20 l (0.05%) of Tween 20 were added to make washing solution [28, 29].
First washing procedure and blocking step
After overnight incubation, plates contents were discarded and plates washed three times by washer machine with 50 l of washing solution. Blocking solution was prepared by adding 4 g (2%) of gelatin to 200 mL washing solution. Plates blocked by the addition of 100 l/well of blocking solution and incubated for 60 min at 37C [21, 22, 28].
Sampling and washing
Each selected serum (50 l) was tested in duplicate and added to microtiter plates wells and incubated for 60 minutes at 37C. Wells washed 4 times and patted dry on fresh paper towels. Plates contents were discarded and plates washed three times by washer machine with 50 l of washing solution.
Adding chaotrope agent and washing
Increasing concentrations (0, 0.005, 0.016, 0.050, 0.150, 0.440, 1.330, and 4.000 M) of ammonium thiocyanate were used as the chaotropic agent. 50 l/well added and incubated for 15 min. in room temperature. Plates contents were discarded and plates washed two times by washer machine with 50 l of washing solution [28, 29].
Adding anti-IgG human antibody and washing
Anti-IgG antibody were prepared based on 1:50,000 dilution in washing solution. Fifty l of anti-IgG antibody was added to each well, incubated for 30 minutes at 37C. All plates contents discarded and plates washed 3 times and patted dry on fresh paper towels [8, 19, 24].
Adding antibody conjugate and washing
Streptavidin-peroxidase were prepared based on 1:50,000 dilution in washing solution. Fifty l of Streptavidin-peroxidase was added to each well, incubated for 30 minutes at 37C. All plates contents discarded and plates washed 4 times and patted dry on fresh paper towels [20].
IgG avidity using elution with increasing concentration of ammonium thiocyanate
Sample-Molarity
Mean g/mL
SD
1 - 0.0 M
0.246
0.020
1 - 0.005 M
0.253
0.023
1 - 0.016 M
0.252
0.019
1 - 0.05 M
0.234
0.019
1 - 0.15 M
0.191
0.013
1 - 0.44 M
0.175
0.020
1 - 1.33 M
0.142
0.001
1 - 4 M
0.110
0.003
2 - 0.0 M
0.151
0.008
2 - 0.005 M
0.157
0.006
2 - 0.016 M
0.150
0.003
2 - 0.05 M
0.149
0.004
2 - 0.15 M
0.140
0.006
2 - 0.44 M
0.125
0.005
2 - 1.33 M
0.101
0.004
2 - 4 M
0.097
0.030
3 - 0.0 M
0.314
0.003
3 - 0.005 M
0.303
0.002
3 - 0.016 M
0.301
0.008
3 - 0.05 M
0.302
0.018
3 - 0.15 M
0.272
0.004
3 - 0.44 M
0.218
0.005
3 - 1.33 M
0.138
0.010
3 - 4 M
0.118
0.004
4 - 0.0 M
0.793
0.028
4 - 0.005 M
0.796
0.047
4 - 0.016 M
0.770
0.008
4 - 0.05 M
0.782
0.041
4 - 0.15 M
0.708
0.020
4 - 0.44 M
0.506
0.016
4 - 1.33 M
0.233
0.009
4 - 4 M
0.177
0.054
Average
0.294
0.014
IgG avidity using elution with increasing concentration of ammonium thiocyanate.
Substrate incubation, stop, and reading
Fifty l of TMB substrate was added to each well, incubated for 15 minutes in the dark. The liquid in the wells turn blue. Fifty l was of 1% sulphuric acid was added to each well, tapped gently to mix, the enzyme reaction stopped and liquid in the wells turned yellow. Absorbance of the entire plates was read on reader machine at 450 nm using a single wavelength within 10 minutes after stop solution addition [25].
ELISA assay to evaluate IgG, IgM, and IgA avidity using single elution 4 M concentration of ammonium thiocyanate
ELISA assay to evaluate avidity using single elution 4 M dilution of ammonium thiocyanate were designed as described by Romero-Steiner et al. [29]. All steps were the same as Section 3.2, except for step 3.2.5, where only 4 M concentration was added. For anti-IgM and anti-IgA, 1:1,500 and 1:50,000 respectively were used.
Selecting ammonium thiocyanate concentration to use in avidity testing
Four samples were selected to evaluate IgG elution by increasing concentrations (0.005, 0.016, 0.050, 0.15, 0.44, 1.33, and 4 M) of ammonium thiocyanate. The effect of chaotrope started at 0.15 M concentration for all four samples and the concentration of 1.33 M showed effect of approximately 50%, while all four samples still showed reading at concentration of 4 M. Thus, the concentration of 4 M was selected to evaluate IgG, IgM, and IgA avidity using single concentration of ammonium thiocyanate. Table 4 and Fig. 1 show the result of IgG avidity using elution with increasing concentration of ammonium thiocyanate.
IgG avidity (g/mL)
Male
Female
Gender/Age/S.#
Total IgG
IgG avidity
Avidity %
SD
Gender/Age/S.#
Total IgG
IgG avidity
Avidity %
SD
Male
4.64
0.54
44.76
6.24
Female
4.00
0.44
43.62
3.95
1–10
3.97
0.65
37.73
6.23
1–10
3.37
0.41
30.32
2.85
159
7.03
0.39
5.51
0.12
62
2.03
0.32
15.81
0.00
363
2.23
0.19
8.30
0.38
67
1.53
0.24
15.54
1.10
662
0.93
0.25
26.84
7.21
147
2.43
0.39
16.16
0.69
694
0.10
0.09
92.61
8.66
341
0.64
0.28
44.03
11.89
707
0.64
0.21
33.76
10.57
344
0.53
0.23
44.15
7.98
714
0.23
0.23
98.57
21.94
471
0.49
0.45
92.78
0.00
914
10.96
2.81
25.66
0.54
1421
9.35
0.65
6.94
0.27
1447
9.61
1.02
10.58
0.44
1477
9.99
0.71
7.15
0.84
11–20
7.59
0.75
31.48
3.02
11–20
4.91
0.54
50.27
2.95
281
7.29
0.46
6.29
2.19
58
8.50
0.76
8.89
1.09
655
0.27
0.27
99.73
9.37
527
0.39
0.36
92.22
6.40
857
10.96
1.28
11.67
0.38
589
0.21
0.20
91.89
3.92
949
11.86
0.98
8.23
0.14
950
10.5
0.85
8.06
0.40
21–30
4.59
0.46
48.29
4.59
21–30
3.54
0.68
40.32
1.50
103
7.15
0.40
5.58
0.35
176
6.07
1.12
18.53
2.08
368
0.32
0.28
88.69
7.98
196
7.07
1.03
14.56
0.36
406
0.45
0.41
91.58
9.37
601
0.71
0.30
43.02
3.57
1492
10.43
0.76
7.31
0.65
608
0.31
0.26
85.15
0.00
31–40
3.59
0.41
47.31
4.93
31–40
4.39
0.43
31.64
3.71
262
6.10
0.30
4.98
0.97
143
7.25
0.35
4.85
0.35
339
0.47
0.36
76.96
5.34
151
2.68
0.46
17.31
1.88
424
0.19
0.19
96.95
13.21
327
0.34
0.33
96.58
12.39
1394
7.61
0.79
10.33
0.22
1244
7.29
0.57
7.84
0.23
41–50
3.53
0.79
50.39
8.08
41–50
4.20
0.39
49.85
9.97
3
0.36
0.35
96.89
11.59
84
8.65
0.73
8.40
0.97
104
6.43
0.43
6.76
1.18
168
7.61
0.32
4.23
0.44
304
6.76
1.99
29.39
1.99
372
0.39
0.35
89.47
17.41
391
0.57
0.39
68.52
17.58
549
0.16
0.16
97.31
21.07
51–60
4.82
0.49
45.17
2.04
51–60
4.80
0.36
40.20
0.58
251
10.29
0.55
5.38
0.90
259
9.53
0.32
3.31
1.15
645
0.10
0.08
86.49
0.00
287
9.05
0.75
8.28
0.93
663
0.39
0.11
27.91
8.71
381
0.07
0.06
91.26
0.24
692
0.21
0.20
94.67
0.00
700
0.53
0.31
57.96
0.00
1295
13.11
1.49
11.39
0.58
61–70
4.53
0.31
50.35
4.91
61–70
4.00
0.34
56.76
11.59
408
8.94
0.46
5.19
1.50
249
7.52
0.46
6.18
1.79
530
0.04
0.03
71.88
19.81
583
0.19
0.19
96.95
4.40
709
0.32
0.29
90.22
2.60
658
0.07
0.06
91.43
51.05
743
0.25
0.20
80.07
0.00
661
0.25
0.20
82.62
0.00
1246
13.0
0.57
4.37
0.64
1238
11.99
0.80
6.65
0.70
71–80
3.68
0.57
54.12
7.00
71–80
7.59
0.41
35.46
12.08
140
4.35
0.42
9.73
0.58
303
9.81
0.37
3.77
2.57
720
0.22
0.21
96.69
7.57
635
0.05
0.05
96.20
33.46
726
0.18
0.17
95.42
19.19
1304
12.92
0.83
6.40
0.20
1714
9.99
1.46
14.65
0.67
81–90
0.31
0.23
73.38
0.27
81–90
0.11
0.11
97.56
0.00
370
0.31
0.23
73.38
0.27
636
0.11
0.11
97.56
0.00
IgM avidity (g/mL)
Male
Female
Gender/Age/S.#
Total IgM
IgM avidity
Avidity %
SD
Gender/Age/S.#
Total IgM
IgM avidity
Avidity %
SD
Male
1.24
0.17
38.81
2.84
Female
1.60
0.18
31.27
5.29
1–10
0.87
0.15
45.65
3.41
1–10
1.80
0.26
42.55
3.61
159
0.01
0.01
84.99
7.41
451
0.01
0.01
84.99
7.41
899
1.54
0.38
24.68
1.66
471
0.01
0.01
84.99
7.41
1037
0.09
0.01
13.55
1.19
947
3.39
0.37
11.05
0.50
1043
0.01
0.01
84.99
7.41
1177
2.56
0.31
12.27
1.33
1261
0.01
0.01
79.75
0.00
1627
3.01
0.59
19.47
1.42
1457
3.01
0.30
9.85
1.98
11–20
3.24
0.21
14.67
1.11
1495
1.42
0.31
21.79
4.22
205
0.08
0.02
31.62
1.39
11–20
1.98
0.28
37.59
3.16
907
4.92
0.31
6.39
0.69
2
3.10
0.48
15.56
0.55
1586
4.73
0.28
6.01
1.26
195
0.01
0.01
84.99
7.41
21–30
2.58
0.28
22.18
3.82
1395
2.82
0.34
12.23
1.51
13
4.92
0.46
9.33
1.04
21–30
1.10
0.28
16.01
0.39
160
0.01
0.00
5.97
0.00
197
0.01
0.00
5.97
0.00
412
0.45
0.30
66.42
13.38
1629
2.18
0.57
26.04
0.78
1583
4.92
0.34
7.00
0.87
31–40
1.06
0.20
49.15
0.20
31–40
1.73
0.15
7.27
0.12
298
0.01
0.01
79.75
0.00
115
0.01
0.00
5.97
0.00
1715
2.12
0.39
18.56
0.40
1091
3.46
0.30
8.57
0.25
41–50
1.13
0.21
51.78
4.66
41–50
0.85
0.22
55.14
5.47
83
2.24
0.42
18.58
1.90
5
1.70
0.43
25.30
3.52
207
0.01
0.01
84.99
7.41
201
0.01
0.01
84.99
7.41
51–60
0.80
0.10
8.06
0.00
51–60
0.69
0.12
56.05
15.13
501
0.01
0.00
5.97
0.00
10
2.73
0.45
16.61
0.94
629
0.01
0.00
5.97
0.00
123
0.01
0.01
84.99
7.41
1084
2.37
0.29
12.24
0.00
381
0.01
0.01
42.86
52.17
61–70
3.97
0.18
42.13
0.38
747
0.01
0.01
79.75
0.00
998
7.92
0.36
4.50
0.75
61–70
1.06
0.14
13.37
8.12
1638
0.01
0.01
79.75
0.00
775
0.04
0.01
17.82
22.42
71–80
0.52
0.09
48.89
6.61
1029
0.13
0.01
9.02
0.79
116
0.08
0.01
8.89
11.18
1059
3.01
0.40
13.25
1.13
255
0.01
0.01
84.99
7.41
71–80
0.78
0.08
27.44
2.50
643
0.01
0.01
84.99
7.41
140
0.01
0.01
84.99
7.41
1106
1.99
0.33
16.72
0.43
695
0.14
0.01
8.14
1.42
739
0.01
0.00
5.97
0.00
1195
2.95
0.31
10.67
1.16
Average
1.43
0.18
34.90
4.11
IgA avidity (g/mL)
Male
Female
Gender/Age/S.#
Total IgA
IgA avidity
Avidity %
SD
Gender/Age/S.#
Total IgA
IgA avidity
Avidity %
SD
Male
2.10
0.40
14.46
2.39
Female
2.15
0.31
11.14
2.72
1—10
2.69
0.48
13.45
2.36
1–10
1.51
0.12
7.12
2.18
1622
1.95
0.57
29.14
3.66
1688
2.09
0.57
27.18
3.41
914
7.03
1.53
21.80
0.40
1693
0.78
0.07
9.53
1.83
6
8.40
1.40
16.63
1.10
1476
0.82
0.05
5.96
0.87
1509
0.79
0.09
11.32
0.90
1593
1.95
0.10
5.10
1.10
1617
0.80
0.07
9.28
3.57
1696
0.72
0.02
2.99
0.99
1097
0.68
0.05
7.94
4.19
1477
1.70
0.05
2.88
3.77
1279
0.65
0.04
6.78
2.20
15
3.38
0.07
2.04
2.32
1529
1.24
0.06
4.74
2.86
1487
0.68
0.01
1.27
3.14
11–20
1.64
0.28
11.10
2.48
11–20
2.56
0.33
10.02
3.18
927
3.53
0.74
21.08
2.62
929
4.08
0.83
20.45
1.22
1553
0.78
0.08
10.83
3.67
1143
0.78
0.10
13.42
1.83
1349
0.62
0.01
1.38
1.14
28
6.74
0.68
10.05
10.55
21–30
1.95
0.54
22.51
1.49
909
0.60
0.02
3.98
0.00
368
3.12
0.98
31.61
1.14
1506
0.62
0.01
2.18
2.28
228
0.78
0.10
13.42
1.83
21–30
2.23
0.47
14.07
4.31
31–40
2.06
0.35
12.98
3.23
145
5.17
1.29
24.87
0.41
31
3.34
0.66
19.67
5.53
86
0.80
0.08
9.91
2.67
1715
0.78
0.05
6.29
0.92
640
0.72
0.05
7.45
9.83
41–50
1.82
0.30
13.84
2.47
31–40
1.76
0.33
12.89
3.07
43
3.83
0.71
18.48
4.83
239
3.64
0.86
23.76
1.37
104
0.73
0.10
13.53
0.97
68
0.83
0.08
9.52
2.57
1700
0.88
0.08
9.50
1.61
634
0.81
0.04
5.40
5.26
51–60
1.90
0.49
21.12
1.17
41–50
1.74
0.36
14.61
1.36
30
3.02
0.89
29.46
1.41
27
3.66
0.93
25.55
1.36
91
0.78
0.10
12.77
0.92
99
0.80
0.07
9.28
1.78
61–70
1.88
0.33
12.05
2.33
63
0.77
0.07
9.01
0.93
33
4.10
0.88
21.56
0.52
51–60
2.01
0.45
14.56
0.67
1205
0.78
0.08
10.18
2.75
19
4.49
1.22
27.08
0.16
117
0.77
0.03
4.41
3.72
109
0.77
0.07
9.01
0.93
71–80
1.74
0.38
14.73
3.23
1679
0.78
0.06
7.59
0.92
1012
3.69
1.02
27.63
2.31
61–70
3.65
0.60
16.28
2.63
1363
0.78
0.07
9.53
3.67
860
4.20
1.11
26.47
3.05
1376
0.77
0.05
7.04
3.72
93
0.77
0.14
18.87
5.58
25
8.87
1.08
12.18
0.96
87
0.78
0.06
7.59
0.92
71–80
2.01
0.08
5.50
3.45
1292
0.75
0.06
7.81
4.71
789
3.26
0.10
3.19
2.18
81–90
2.14
0.13
7.98
5.39
1317
0.78
0.08
10.83
3.67
736
3.50
0.18
5.13
7.11
Average
2.13
0.35
12.60
2.57
Statistical analysis
-test has been used to compare different data throughout the study, while regression test has been used to test relationship between parameters.
Results and discussion
To evaluate avidity using elution by single 4 M concentration of Ammonium Thiocyanate (4 M), the concentration of 4 M of ammonium thiocyanate has been selected and used based on the pilot study cited in Material and methods section (Table 4, Fig. 1). According to the lowest and highest results of total IgG concentration in 10 years’ interval age categories, 76 samples were selected and used to evaluate the IgG avidity. Statistically, there are no differences between male and female participants in percentage of avidity (Table 5, Fig. 2). While, the regression analysis shows a positive relationship was detected between IgG avidity and IgG concentration ( 0.4431) as seen in Fig. 3.
IgG, IgA and IgM avidity throughout both female and male.
Fifty-two samples were selected according to the lowest and highest results of total IgM concentration in 10 years’ interval age categories. The results harvested did not revealed any significant differences between male and female participants in percentage of avidity (Table 6, Fig. 2). By using the regression analysis, a positive relationship was detected between IgM avidity and IgM concentration ( 0.6104) as seen in Fig. 3.
Fifty-nine samples were selected according to the lowest and highest results of total IgA concentration in 10 years’ interval age categories. The average of IgA avidity in male participants samples were significantly ( 0.05) higher than the average of female (Table 7, Fig. 2) and by using regression analysis, positive relationship was detected between IgA avidity and IgA concentration ( 0.758) as seen in Fig. 3.
The relative IgG avidity results ranged between 3.31 and 100%. Avidity affected by the type of Hib vaccine and the molecular form of the Hib CPS immunogen [14, 15], which may explain the wide range of avidity data in recent study (3.31%–100%). Therefore, our avidity results possibly directed by the types of Hib vaccines that used since the introduction of Hib vaccination in Saudi Arabia, and the diversity of molecular structure of different Hib strains that colonized the community, or may be affected by bacterial antigens cross-reactivity [5]. Due to high concentration of ammonium thiocyanate which is used in current study comparing to study of Romero-Steiner et al. and study of Gomez-de-Leon et al. [10, 29], samples showing relative avidity values of 10%, from 10 to 30%, or 30% were categorized as low-, moderate-, or high-avidity sera, respectively. The average IgG avidity is considered high (44.8%) in both gender, 44.76% in male and 44.78% in female. Twenty-six participants (34.2%) from 76 participants show low avidity, 15 participants (19.7%) show moderate avidity, and 35 participants (46%) show high avidity. The high percentage of moderate and high avidity of IgG (65.7%) in addition to the high avidity average (44.8%) may reflects the strength of immunity in Saudi community, since the strong avidity is associated with protective efficacy and the impossibility of Hib vaccination failure [6]. Low avidity is distributed between male (50%) and female (50%), which may reflect the relation-less between gender and low avidity, only one participant is younger than 5-year-old (exactly 4-year-old) with 8.2% avidity. The low avidity may be resulted from unrecorded underlying diseases due to poor registration system, such as sickle cell disease and congenital asplenia [6]. Positive relationship ( 0.44) has been detected between total IgG concentration and avidity regardless of age and gender. The age category 1–5 year shows high level of avidity (33.7%) which is comparable to the findings of Agbarakwe et al., when IgG avidity is elicited by Hib vaccination [1]. The relative IgM avidity results ranged between 0.53 and 66.42%. The average IgM avidity is 12.51%, the male average is 10.49% and female average is 14.38%, with no statistically difference. From 52 participants, there was 32 (61.53%) showed low avidity, 17 (32.69%) showed moderate avidity, while only 3 females (5.77%) showed high avidity. Positive relationship has been detected between total IgM concentration and avidity ( 0.6104). The relative IgA avidity results ranged between 0 and 29.46%. The average IgA avidity is 12.5%, the male average is 14.46% and female average is 10.96%. From 59 participants, 31 (52.54%) show low avidity, 27 (45.76%) show moderate avidity, and 1 (1.69%) show high avidity. Positive relationship ( 0.758) has been detected between total IgA concentration and avidity regardless of age and gender. The lower avidity percentages of IgM and IgA comparing to IgG may reflects the main role of IgG in anti-Hib immunity over IgM and IgM. To the best of our knowledge, there were no previous studies that focusing on the avidity of anti-Hib IgM and IgA, so our study considers the first in this point. The incidence of invasive Hib disease in Saudi Arabia remains low, which may correlated to circulating antibody concentration/avidity. Although, anti-PRP antibody concentrations wane rapidly after the 12-month booster [7].
Avidity regression analysis of IgG, IgA, and IgM.
Conclusion
This study was examined IgG, IgM, and IgA antibody avidity within Jeddah population vaccinated with Hib. The majority of examined samples demonstrated a high IgG avidity. So, this herd immunity circulating in Jeddah population against Hib was offer by moderate to high IgG affinity against CPS of Hib, and less extend by lower avidity of IgM and IgA when comparing with IgG. Forthmore, studies may need to figure out the relationship between avidity and aging.
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