Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 16, Num. 3, 1999

Rolando Ochoa Azze, Mayte Nerey Olivares, Juan C Martínez Rodríguez
DACTA. Instituto Finlay.

Ave 27 No. 19805. AP 16017, CP 11600. La Lisa, Ciudad de La Habana, Cuba.
Fax: (53-7) 33 6754; E-mail: ochoa@finlay.edu.cu

Code Number: BA99030

Abstract

Precoating with poly-L-lysine was analyzed in an ELISA used for the quantitative determination of antibodies against serogroup C Neisseria meningitidis capsular polysaccharide. Different coatings were analyzed: purified capsular polysaccharide of serogroup C N. meningitidis did not attach well to polystyrene, but precoating with poly-L-lysine and the use of methylated human serum albumin attached to polysaccharide had a good performance. Cross reactivity to poly-L-lysine was not detected in 48 adults immunized with a divalent (serogroups B and C) meningococcal vaccine (VA-MENGOC-BC^®). Seroconversion and seroresponse were detected in 89.58 and 97.92% of the vaccinated individuals, respectively.

Keywords: ELISA for anti-polysaccharide C antibodies, poly-L-lysine, poly-L-lysine precoating, serogroup C Neisseria meningitidis

Se evaluó el prerecubrimiento con poli-L-lisina en un ELISA para la cuantificación de anticuerpos contra el polisacárido capsular de Neisseria meningitidis serogrupo C. Se analizaron diferentes recubrimientos: el polisacárido C no se unió bien al poliestireno, pero se alcanzaron buenos resultados recubriendo previamente con poli-L-lisina, así como con seroalbúmina humana metilada unida al polisacárido C. No se detectó reactividad cruzada a la poli-L-lisina en 48 adultos inmunizados con una vacuna anti-meningocóccica bivalente contra los serogrupos B y C (VA-MENGOC-BC^®). Se detectó seroconversión en 89,58% de los vacunados y serorrespondió 97,92%.

Palabras claves: ELISA para anticuerpos anti-polisacárido C, Neisseria meningitidis serogrupo C, poli-L-lisina, prerrecubrimiento con poli-L-lisina

Although ELISA procedure has been commonly used for measuring antibodies to protein antigens, its application for polysaccharides has not been successful. Negatively charged polysaccharides do not attach easily to polystyrene in ELISA [1-3]. In order to solve this problem, various methods have been developed.

The uses of capture antibodies [3] are limited because of the extreme difficulty to produce specific immunological reagents. Covalent binding to proteins [1] is tedious and introduces the risk of altering the immunological reactivity. The avidin-biotin method has been used [3-5], but careful methods must be employed to avoid conformational alteration. Methylated human serum albumin (mHSA) has been successfully used to coat with serogroup A and C capsular polysaccharides of Neisseria meningitidis [6, 7], but it is not commercially available and it is difficult to produce. Precoating with poly-L-lysine (PLL) [8-10] has been criticized because it generates a low signal to noise ratio and non-specifically reacts with some sera [3, 5].

We examined the performance of an ELISA using precoating with PLL to measure the antibody response to the serogroup C polysaccharide in subjects vaccinated with a divalent (serogroups B and C) meningococcal vaccine.

The capsular polysaccharide (CPS) antigen of serogroup C N. meningitidis was provided by the "Finlay" Institute, Havana, Cuba. The polysaccharide contained less than 1% of protein and nucleic acids.

Fourty-eight volunteer adults over 40 years old were immunized with VA-MENGOC-BC^® (divalent [serogroups B and C] meningococcal vaccine). The vaccination scheme consisted in two doses of 0.5 mL, with an interval of 8 weeks. Known vaccinations with VA-MENGOC-BC^® or other meningococcal vaccine in the last 10 years, and antecedents of meningitis were exclusion criteria. Venepuncture was performed immediately prior to the first dose (T0) and four weeks after the second dose (T2). Sera were stored at -20 ºC until simultaneous processing.

High-binding ELISA plates (Costar, Cambridge, Ma) were precoated by incubation with 100 µL of PLL per well (3 µg/mL) (Sigma Chemical Co, St. Louis, MO) in 0.15 M phosphate buffered saline (PBS), pH 7.4 for 30 min at 20-25 ºC. After the plates were washed three times with 300 µL of PBS per well, 100 µL of serogroup C N. meningitidis CPS (2.5 µg/mL) in PBS was added to each well. The plates were incubated overnight at 2-8 ºC and then washed three times with PBS containing 0.05% Tween 20 (PBS-T). The standard curve was constructed by performing six two-fold serial dilutions of the "Finlay" standard serum (10 000 U/mL) (starting dilution 1:100) in PBS-T containing 3% skim milk (PBS-TM) (Merck, Germany). PBS-TM was used as zero-standard. The test sera and the control serum were diluted 1:100 in PBS-TM. Of each solution, 100 µL were added in duplicate to the wells for 1 h at 37 ºC. After washing four times with 300 µL of PBS-T, 100 µL of conjugate solution (goat anti-human IgG:alkaline phosphatase) (Sigma, St. Louis, MO) in PBS-TM were added to each well and the plates were incubated for 1 h at 37 ºC. The plates were washed again, and 100 µL of p-nitrophenylphosphate (1 mg/mL) (Sigma, St. Louis, MO) in 0.92 M diethanolamine buffer pH 9.8 were added to each well. The plates were left at 20-25 ºC for 30 min and then the absorbances were determined at 405 nm (Anthos reader 2001, Labtec Instruments, Germany) [9]. Validation, quantitative determination and print out were performed using the ELISA software package from the Center for Disease Control (CDC), Atlanta, USA [11].

Detection limit was calculated after the blank reagent PBS-TM was tested 80 times in three performances. The mean plus twice the standard deviation was taken as an estimate of the detection limit [12]. The absorbance values were homogenized and transformed to U/mL by the ELISA software package from the CDC [11] using a reference curve. Inter-assay average was calculated.

Half the value of the detection limit was assigned to samples with concentrations below the detection limit.

Wells of columns 1-4 of high-binding ELISA plates were sequentially coated with PLL and serogroup C N. meningitidis CPS as it was described above. Columns 5-8 were only coated with PLL for 30 min at 20-25 ºC, and after washing, wells were soaked in PBS. PBS was also added to wells of the remaining columns, i.e. wells without coating. After incubation overnight at 2-8 ºC, the plates were washed three times with PBS-T. The serum samples collected prior to the first dose, which were above the detection limit for anti-polysaccharide antibodies, were diluted 1:100 in PBS-TM. Of each sample, 100 µL were added in duplicate to coated and non-coated wells, and the plates were incubated for 1 h at 37 ºC. The next steps were performed as previously described.

Sequential coating with PLL and CPS was used as described above in columns 1-4 of a plate. Columns 5-8 were coated with an equal mixture of 5 µg of CPS/mL and mHSA/CPS (gently provided by CDC) [6]. Columns 9-12 were only coated with CPS (10 µg/mL). The next steps were as described. The standard curve was used to analyze the coatings.

Statistical analysis was carried out using appropriate tests in the Statgraphic software [13] on an IBM compatible computer.

Linear regression analysis between standard curves was used to analyze different coatings with respect to mHSA/CPS. Analysis of variance for checking the linear model and the Cochran's C test for homogeneity of variances were performed.

Geometric means and 95% confidence intervals were calculated for anti-polysaccharide C antibody distribution in T0 and T2. Comparison of means was carried out by Student's t test for matched samples after Chi-square test for normality was performed.

The T2/T0 relation was calculated for each pair of samples. The relation ³ 4 was defined as seroconversion and ³ 2 as seroresponse [10].

Meningococci are divided into a number of serogroups on the basis of structural differences in the CPS. The serogroups B, C, Y and W-135 have sialic acid in their CPS. Sialic acid-containing polysaccharides confer resistance to host complement-mediated attack mechanisms. The purified CPS vaccine against serogroup C was among the first chemically pure bacterial vaccines [14, 15]. This vaccine is immunogenic, safe and effective.

The antibody response has shown to correlate well with the bactericidal assay [6, 10, 16, 17], but the measurement by ELISA of antibodies to purified cell surface carbohydrates is often hindered by the failure of lipid-free or protein-free polysaccharides to efficiently and reproducibly bind to a solid phase [1, 2, 5]. On the other hand, the acidic polysaccharides attach well to the basic PLL, so we used it.

Precoating with PLL, which has reactive epsilon-amino groups, resulted in high antibody binding with low background. The standard curve for PLL+CPS and mHSA/CPS was similar. The coating with CPS showed the smallest sensitivity (Table 1). Sensitivity is defined for quantitative immunoassays as the ability to detect small differences in concentration, it is, the ratio of the change in response of the method to the change in concentration of the analyte, or the slope of the analytical calibration curve [12].

Table 1. Analysis of different coatings used in an ELISA for the detection of antibodies against serogroup C N. meningitidis capsular polysaccharide.

The detection limit is the concentration distinguishable from the zero-standard. It was 367 U/mL. Only seven serum samples collected in T0 were above the detection limit. Cross reactivity to PLL was not detected in any sample (Figure). On the other hand, PLL showed a blocking role on the polystyrene surface.

Specific reaction to CPS before vaccination could be explained by a long-lasting protective immunity elicited by VA-MENGOC-BC^®. Cross reactivity with Escherichia coli K92 CPS is less probable because strains of this pathogen have been infrequently found in healthy individuals. Cross reactivity with other CPS has not been demonstrated yet, and serogroup C meningococci are rarely found among healthy individuals [13, 18]. Certainly, vaccination with a serogroup C meningococcal vaccine can not be disregarded, therefore, specific reaction to CPS should be carefully studied to reach a conclusion.

Even though certain sera could react non-specifically against PLL, this ELISA is useful to evaluate the immune response against the serogroup C polysaccharide-based vaccine, and probably for other acidic polysaccharide-based vaccines. The mean of samples collected after vaccination was significantly larger than the mean of samples collected before vaccination (Table 2), and seroconversion (T2/T0 ³ 4) was detected in 43 vaccinated individuals (89.58%), including those with initial reactivity to CPS. Seroresponse (T2/T0 ³ 2) was found in 47 individuals (97.92%). Other investigations showing a high seroconversion and serorresponse [10, 15] support this method. Nevertheless, the background reactivity to PLL of each sample could be substracted if necessary [19].

Table 2. Results obtained in serum samples tested for anti-polysaccharide C antibodies before and after vaccination.

In conclusion, our results support that precoating with PLL is a suitable method for the ELISA used to detect antibodies against serogroup C N. meningitidis CPS.

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