An immunoglobulin G1 monoclonal antibody highly specific to the wall of Cryptosporidium oocysts

Abstract

The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG(1) monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.

FIG. 1

Comparison of the fluorescence intensities of C. parvum oocysts stained with sera of three dilutions (1:1,000, 1:10,000, and 1:100,000) from the soluble-extract, oocyst wall, or whole-oocyst control groups that were then stained with an anti-IgG or an anti-IgM fluorescently labeled antibody. Data are calculated by subtracting the fluorescent value (arb) obtained from mouse serum samples after two immunizations from the preimmune serum samples from serum samples from individual mice. MFI data obtained from each group were then averaged, and the standard deviations were calculated.

FIG. 2

Comparison of antibodies for staining oocysts in water samples. The axes show side-scatter (x axis) versus green-fluorescence (y axis) data. The MFI oocyst population was also recorded. Control 1 (A) is oocysts only, and control 2 (B) is unstained oocyst in a water concentrate. Control 3 (C) is Cry104 with no oocysts in a water concentrate, and the negative control (D) is stained with an anti-Giardia antibody. Note the differences in the separation between the oocysts and the debris particles obtained with different antibodies. Cry26 (E) and 15H10 (F) data are also presented. (G) Cry104 shows the greatest separation of the oocyst population from debris in water samples, with the highest MFI of the oocyst population.

FIG. 2

Comparison of antibodies for staining oocysts in water samples. The axes show side-scatter (x axis) versus green-fluorescence (y axis) data. The MFI oocyst population was also recorded. Control 1 (A) is oocysts only, and control 2 (B) is unstained oocyst in a water concentrate. Control 3 (C) is Cry104 with no oocysts in a water concentrate, and the negative control (D) is stained with an anti-Giardia antibody. Note the differences in the separation between the oocysts and the debris particles obtained with different antibodies. Cry26 (E) and 15H10 (F) data are also presented. (G) Cry104 shows the greatest separation of the oocyst population from debris in water samples, with the highest MFI of the oocyst population.

FIG. 2

Comparison of antibodies for staining oocysts in water samples. The axes show side-scatter (x axis) versus green-fluorescence (y axis) data. The MFI oocyst population was also recorded. Control 1 (A) is oocysts only, and control 2 (B) is unstained oocyst in a water concentrate. Control 3 (C) is Cry104 with no oocysts in a water concentrate, and the negative control (D) is stained with an anti-Giardia antibody. Note the differences in the separation between the oocysts and the debris particles obtained with different antibodies. Cry26 (E) and 15H10 (F) data are also presented. (G) Cry104 shows the greatest separation of the oocyst population from debris in water samples, with the highest MFI of the oocyst population.

FIG. 2

Comparison of antibodies for staining oocysts in water samples. The axes show side-scatter (x axis) versus green-fluorescence (y axis) data. The MFI oocyst population was also recorded. Control 1 (A) is oocysts only, and control 2 (B) is unstained oocyst in a water concentrate. Control 3 (C) is Cry104 with no oocysts in a water concentrate, and the negative control (D) is stained with an anti-Giardia antibody. Note the differences in the separation between the oocysts and the debris particles obtained with different antibodies. Cry26 (E) and 15H10 (F) data are also presented. (G) Cry104 shows the greatest separation of the oocyst population from debris in water samples, with the highest MFI of the oocyst population.

FIG. 2

Comparison of antibodies for staining oocysts in water samples. The axes show side-scatter (x axis) versus green-fluorescence (y axis) data. The MFI oocyst population was also recorded. Control 1 (A) is oocysts only, and control 2 (B) is unstained oocyst in a water concentrate. Control 3 (C) is Cry104 with no oocysts in a water concentrate, and the negative control (D) is stained with an anti-Giardia antibody. Note the differences in the separation between the oocysts and the debris particles obtained with different antibodies. Cry26 (E) and 15H10 (F) data are also presented. (G) Cry104 shows the greatest separation of the oocyst population from debris in water samples, with the highest MFI of the oocyst population.

FIG. 2

Comparison of antibodies for staining oocysts in water samples. The axes show side-scatter (x axis) versus green-fluorescence (y axis) data. The MFI oocyst population was also recorded. Control 1 (A) is oocysts only, and control 2 (B) is unstained oocyst in a water concentrate. Control 3 (C) is Cry104 with no oocysts in a water concentrate, and the negative control (D) is stained with an anti-Giardia antibody. Note the differences in the separation between the oocysts and the debris particles obtained with different antibodies. Cry26 (E) and 15H10 (F) data are also presented. (G) Cry104 shows the greatest separation of the oocyst population from debris in water samples, with the highest MFI of the oocyst population.

FIG. 2

Comparison of antibodies for staining oocysts in water samples. The axes show side-scatter (x axis) versus green-fluorescence (y axis) data. The MFI oocyst population was also recorded. Control 1 (A) is oocysts only, and control 2 (B) is unstained oocyst in a water concentrate. Control 3 (C) is Cry104 with no oocysts in a water concentrate, and the negative control (D) is stained with an anti-Giardia antibody. Note the differences in the separation between the oocysts and the debris particles obtained with different antibodies. Cry26 (E) and 15H10 (F) data are also presented. (G) Cry104 shows the greatest separation of the oocyst population from debris in water samples, with the highest MFI of the oocyst population.

FIG. 3

Western blot of C. parvum protein extracts probed with mouse serum from the following mouse groups: E, soluble-extract mice; W, oocyst wall mice; and C, oocyst control group (whole oocysts). Results obtained with MAbs are presented as follows: M1, Cry26 (IgM); M2, Cry104 (IgG1); M3, Cry212 (IgM); and M4, Crypto-a-Glo (IgM). Cry26, Cry104, and Cry212 were from Macquarie University Centre for Analytical Biotechnology, and Crypto-a-Glo was from Waterborne, New Orleans, La.

FIG. 4

Binding curves of the three FITC-labeled MAbs Cry26, Cry104, and Immucell. The oocyst concentration of 10⁷/ml was constant for every antibody dilution. Relative fluorescence intensity values for each of the 20 serial dilution were recorded and plotted. The value for 50% maximal binding was then calculated for each MAb by reading the value off the curve.