Setting of methods for analysis of mucosal antibodies in seminal and vaginal fluids of HIV seropositive subjects from Cambodian and Italian cohorts

Abstract

Background: Genital mucosae play a key role in protection from STD and HIV infection, due to their involvement in both horizontal and vertical disease transmission. High variability of published observations concerning IgA isolation and quantification underlies the strong requirement of specific methods able to maximize investigation on HIV-specific IgA.

Methodology: Genital fluids from 109 subjects, including male and female cohorts from Italy and Cambodia, were collected, aliquoted and processed with different techniques, to assess optimal conditions maximizing mucosal antibody recovery. Three sampling techniques, up to sixteen preservation conditions, six ELISA methods and four purifications protocols were compared.

Principal findings: The optimal method here described took advantage of Weck-Cel sampling of female mucosal fluids. Immediate processing of genital fluids, with the addition of antibiotics and EDTA, improved recovery of vaginal IgA, while the triple addition of EDTA, antibiotics and protease inhibitors provided the highest amount of seminal IgA. Due to low amount of IgA in mucosal fluids, a high sensitive sandwich ELISA assay was set; sensitivity was enhanced by milk-based overcoating buffer and by a two-step biotin-streptavidin signal amplification. Indeed, commercial antisera to detect human immunoglobulins showed weak cross-reactivity to different antibody types. Three-step affinity purification provided reproducible immunoglobulin recovery from genital specimens, while conventional immuno-affinity IgA purification was found poorly manageable. Affinity columns were suitable to isolate mucosal IgA, which are ten-fold less concentrated than IgG in genital specimens, and provided effective separation of IgA monomers, dimers, and J-chains. Jacalin-bound resin successfully separated IgA1 from IgA2 subfraction.

Conclusions/significance: Specific, effective and reliable methods to study local immunity are key items in understanding host mucosal response. The sequence of methods here described is effective and reliable in analysing humoral local responses, and may provide a solid advance to identify and measure the effective mucosal responses to HIV.

Figure 1. Design of the study.

The scheme summarizes the methods tested in each step of immunoglobulins purification and quantitation.

Figure 2. Immunoglobulin concentration in seminal fluids from Italian men.

Box plot summarizes the immunoglobulin concentrations (median and range) observed in seminal fluids from healthy donors, according to the Additives (cryopreservations only, single, double or triple additives) and the Processing Modes (addition of preserving agents made pre- or post-fluid incubation). All specimens were immediately processed after sampling; the values are given in microgram/mL. Panel legends: A. IgG range in pre-incubation samples; B. IgG range in post-incubation samples; C. IgA range in pre-incubation samples; D. IgA range in post-incubation samples. Additives legends: Null, cryopreservation only; EDTA: ethylenediaminetetraacetic acid; IP, protease inhibitors; AB, Antibiotics. Box legends: Box short sides represent the third (Q3) and the first quartile (Q1), respectively, while the asterisk (*) within the box indicates the mean and the horizontal bar (--) shows the median value, respectively. Vertical lines above or below the box indicate the corresponding quartile value (Q3 or Q1) plus or minus 1.5 times the interquartile interval (IQ = Q3−Q1).

Figure 3. Sandwich ELISA assay.

The scheme introduces the six sandwich ELISA protocols that were compared in the study.

Figure 4. Affinity purification of immunoglobulins.

Comparison of the four chromatographic methods for immunoglobulin isolation that were evaluated in the study.

Figure 5. Immunoglobulins purification from vaginal fluids.

Panel legends: A. IgG purification by Protein G column. B. IgA purification by ionic exchange column. C. IgA subtypes isolation by gel filtration.

Figure 6. Three-step vs jacalin affinity purification of mucosal immunoglobulins.

Box plot comparing IgA and IgG immunoglobulin purification by two different methods assayed in the study. Left panel shows IgA and IgG values measured in jacalin-bound and -excluded fractions, respectively; right panel presents IgA and IgG values measured in the two bound fractions. Values are expressed in total micrograms per sample, in log-scale. From the left to the right, respectively: Jacalin–agarose purification (white box): IgA from healthy controls; IgA from HIV-positive subjects; IgG from healthy controls; IgG from HIV-positive subjects. Three-step affinity purification (grey box): IgA from healthy controls; IgA from HIV-positive subjects; IgG from healthy controls; IgG from HIV-positive subjects. Box legends: Box short sides represent the third (Q3) and the first quartile (Q1), respectively, while the horizontal bar (-) shows the median value. Vertical lines above or below the box indicate the corresponding quartile value (Q3 or Q1) plus or minus 1.5 times the interquartile interval (IQ = Q3−Q1).