Association of Low Concentrations of Serum Mannose-Binding Protein with Recurrent Infections in Adults

Patients.Patients’ specimens were remnants of serum samples sent to Specialty Laboratories for routine clinical testing and were from two different groups. Group I consisted of 47 adult patients (33 females, ages 29 to 86, and 14 males, ages 24 to 72) with non-human immunodeficiency virus (HIV)-related recurrent infections and a history of repeated hospitalization. Group II consisted of 73 pediatric patients (30 females and 43 males, ages <19 years) and 56 adult patients (35 females and 21 males, ages >19 years) with recurrent infections whose sera had been sent for a humoral immune response survey following pneumococcal vaccination.

Control subjects.Group III (the control group) included 23 healthy pediatric subjects and 50 healthy adult subjects. Sera from the healthy pediatric subjects (ages <19 years) were remnant samples from a previous study on Bartonella-HIV encephalopathy conducted in collaboration with Benjamin Estrada, New Orleans, La. Sera from the adult subjects were obtained from volunteers who were employed at Specialty Laboratories. All the adult serum donors were remunerated.

Purification of human MBP.Human MBP was purified from pooled human AB serum (American Qualex, San Clemente, Calif.) as described earlier (13). Briefly, 500 ml of pooled human AB serum was dialyzed extensively against loading buffer containing 20 mM Tris (pH 7.4), 0.15 M NaCl, 1.0 mM CaCl₂, and 0.05% NaN₃. Following dialysis, the serum was loaded on a mannan-agarose column (Sigma Chemical Co., St. Louis, Mo.) which was equilibrated with loading buffer. The loaded column was washed extensively with loading buffer, and the bound protein was eluted with a solution of 20 mM Tris (pH 7.4), 0.15 M NaCl, 0.01 M EDTA, and 0.05% NaN₃. The fractions with higher absorbances (280 nm) were pooled, and the serum amyloid P component was removed by CaCl₂ precipitation. Following concentration by ultrafiltration (filter from Amicon), the presence of MBPs was confirmed by Western blotting with mouse monoclonal antibodies to human MBP (Harlan Bioproducts for Science, Inc., Indianapolis, Ind.). Protein concentrations were determined with the Bio-Rad protein assay reagent with bovine serum albumin as the standard.

Generation of rabbit antibodies to human MBP.One milligram of purified human MBP was mixed with 1 ml of Titermax (Sigma Chemical Co.) and injected subcutaneously into three rabbits (Universal Animal Care, Bloomington, Calif.). Three weeks following immunization, the rabbits were bled for sera. The sera were tested for MBP-specific antibodies by Western blotting. The immunoglobulin G fraction of the MBP-specific antibodies was purified from the serum with a high titer by using a protein A-Sepharose column (13).

Assay for MBP.Serum MBP concentrations were determined by a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) as described earlier (13). Briefly, Immulon 2 ELISA plates (Dynatech Laboratories, Inc., Chantilly, Va.) were coated overnight at 4°C with 100 μl of purified rabbit anti-human MBP immunoglobulin G per well at a concentration of 1 μg/ml in carbonate buffer (50 mM Na₂CO₃, pH 9.6). The following morning, the plates were washed three times with washing solution (phosphate-buffered saline with 0.05% Tween 20 [PBS-T]) and blocked with 1% bovine serum albumin in PBS-T for 1 h at room temperature (RT). One-hundred-microliter aliquots of serum samples or standards per well were added to the antibody-coated plates and incubated overnight at 4°C. Different concentrations of MBP were used as standards in twofold dilutions. Following incubation, the plates were washed as before and 100 μl of biotinylated anti-MBP mouse monoclonal antibody (0.5 μg/ml) was added per well. The plates were incubated at RT for 2 h. At the end of the incubation, the plates were washed, 100 μl of 1:2,000-diluted alkaline phosphatase-conjugated streptavidin (Boehringer Mannheim Corporation, Indianapolis, Ind.) was added to each well, and the plates were incubated at RT for 1 h. Following incubation, the plates were washed and 150 μl of the substrate,p-nitrophenyl phosphate (1 mg/ml) (Sigma Chemical Co.), was added to all the wells. The plates were kept in the dark for 30 min and then read in an ELISA reader. Data were analyzed by using a four-parameter standard curve (Softmax software; Molecular Devices, Sunnyvale, Calif.).

Reference range.The cutoff value of 5 ng of serum MBP per ml was chosen on the basis of the earlier report that individuals with homozygosity of abnormal MBP alleles had <10 ng of serum MBP per ml (10) and that homozygosity for MBP mutant alleles predisposes individuals to recurrent infections (2).

Statistical analysis.Student’s t test and Fisher’s exact test were employed to evaluate differences between control and patient groups.