Protein Levels in Nonstimulated and Stimulated Tears of Normal Human Subjects

Roderick J. Fullard* and Christopher Snyderj-

Atraumatically collected nonstimulated (<1 /til/min) and stimulated (>50 ^l/min) tears from 30 clinically normal subjects were fractionated by size exclusion high-performance liquid chromatogra- phy (SE-HPLC). Enzyme-linked immunosorbent assay (ELISA) and kinetic assays were applied to relevant HPLC fractions to quantitatively identify 12 tear proteins. Secretory IgA levels were much higher in nonstimulated than in stimulated tears, and a similar disparity was seen also with IgAl and IgA2 in the HPLC fraction containing secretory IgA. IgM levels were also higher in nonstimulated tears. Levels of the primary lacrimal gland proteins, lactoferrin, tear specific prealbumin, and lysozyme were similar in both types of tears. Significantly higher concentrations of the major serum proteins, IgG, transferrin, and serum albumin were measured in nonstimulated tears. Overall, 8 of the 12 proteins assayed were present at significantly higher concentrations in nonstimulated tears. These results show that tear flow rate strongly influences the protein profile obtained. Therefore, to allow valid comparisons of tear protein profiles within and between studies that use atraumatic collection procedures, an indication of flow rate during collection should be reported. Invest Ophthalmol Vis Sci 31:1119-1126,1990

The influence of tear flow rate on tear protein pro- pettes, followed by 300 n\ of additional stimulated files is an important aspect of tear biochemistry, but secretion over the next 5 min. Significant trends in one that is frequently overlooked. In a previous arti- HPLC profiles were evident in these five successive cle, ' size-exclusion high performance liquid chroma- tear samples. The proportion of total protein in tography (SE-HPLC) was used to demonstrate the HPLC peak 1 (high-molecular-weight fraction) pro- effect of tear flow rate on protein profiles of micropi- gressively decreased with each sample and overall by pette-collected tears. SE-HPLC of tear samples a factor of 6.6 (from 25 to 3.8%). Similarly, peak 5 yielded a chromatogram with five main peaks, peak 1 decreased by a factor of 2.9. The proportion of total representing the highest MW protein fraction. Differ- protein in peaks 3 to 5 increased 1.7, 1.9, and 1.4X ences between nonstimulated and stimulated tear respectively. Total protein content decreased from HPLC profiles were most evident in SE-HPLC frac- 9.1 to 6.0 mg/ml over the five samples.1 tions 1 and 5, both making up a considerably larger The importance of using atraumatic collection proportion of total protein in nonstimulated tears. methods was also demonstrated in this article1 by Moreover, differences between successive tear sam- repeating the above experiment for samples 1 to 4, ples collected after the onset of stimulus indicated a but using Schirmer strips for tear collection rather strong dependence of tear protein composition on than polished micropipettes. Total protein content of tear flow rate and total reflex tear volume secreted Schirmer strip-collected samples exceeded that of the after stimulus. Immediately after initiating nasal corresponding micropipette-collected samples 2.2, stimulus of reflex tearing, samples representing 0-5, 2.0, 1.9, and 1.2X respectively. In addition, the trends 5-10, 10-20, and 20-50 /x\ were collected in rapid reported above for the five major SE-HPLC fractions sequence over less than 1 min in separate micropi- in successive micropipette-collected samples all were replaced by considerably more uniform distributions in Schirmer strip-collected samples, indicating an From the University of Alabama at Birmingham *Department of Physiological Optics and fDepartment of Optometry, University overriding contribution by traumatically derived Station, Birmingham, Alabama. proteins to all tear samples.' Supported by National Institutes of Health grants EY-07783 The aim of the current study was to use a com- (RO1), EY-03039 (CORE), and RR05807 (BRSG). bined HPLC-enzyme-linked immunosorbent assay Submitted for publication: September 13, 1989; accepted Oc- (ELISA) procedure to compare nonstimulated and tober 13, 1989. Reprint requests: Roderick J. Fullard, OD, PhD, University of stimulated tear protein profiles of a group of clinically Alabama at Birmingham, Department of Physiological Optics, normal subjects in order to establish normal levels of University Station, Birmingham, AL 35294. 12 specific proteins in each type of tear fluid. HPLC

1119

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fractionation of tears and subsequent ELISA of rele- volume in each tube and continually monitoring the vant HPLC fractions were shown previously to over- rate of filling, it was possible to obtain a very good come some of the interference problems seen in estimate of tear flow rate at all times throughout the whole-tear ELISA and also allowed all ELISAs to be collection procedure. Inferior tear prism height also conducted on a smaller total tear volume.' was monitored continually during collection, and the subject was instructed to immediately report any ocu- Materials and Methods lar irritation or perception of increased tear flow. Any time that tear flow rate was estimated by the collector Informed consent was obtained from all subjects to be in excess of 1 /zl/min, the sample in that tube after the nature of all procedures had been fully ex- was discarded. It should be noted that several subjects plained to them. who passed the initial clinical selection procedure were ultimately excluded from the study due to the Selection of Clinically Normal Subjects inability to maintain a sufficiently slow tear flow rate. Stimulated tears were collected by 20-/A micropi- Volunteers taking regular medications of any kind pette. Tear flow was initiated by inserting a cotton (including topical ocular medications and oral con- swab (Q-Tip) into the nose and stimulating the sneeze traceptives) or with a history of contact lens wear or reflex. In four cases, irritation of the nasal mucosa by anterior eye disease were excluded from the study. A ammonia vapor was necessary to establish a suffi- battery of clinical tests directed at diagnosis of ocular ciently rapid flow rate. Direct ocular surface irritation surface disease, with particular emphasis on detection was always avoided. After establishing a rapid reflex and characterization of dry eye states, was used. tear flow rate, two 20-yul samples were collected in Briefly, the clinical protocol included: 1) the rapid sequence, usually in less than 30 sec. The initial McMonnies questionnaire to screen for likely dry eye 20-^1 sample was discarded. In order to assure that a patients;2 2) noninvasive tear break-up time measure- 3 flow rate of at least 50 ^1/min was established, the ment using a Xeroscope provided by Tonge; 3) de- maximum time allowed to fill both 20-^1 tubes was tailed slit-lamp examination; 4) assessment of tear 45 sec. If the 45-sec time limit was exceeded before viscosity, tear debris, and inferior tear prism height; collecting both samples, the entire procedure was re- 5) measurement of lacrimation kinetics using poly- peated until it fell into the required time frame. All ethylene-covered, calibrated "Schirmer-Holly" 4 tear samples were transferred to 400-/A polypropyl- strips provided by Holly; and 6) evaluation of fluo- ene tubes and frozen at -20°C for less than 24 hr rescein and Rose Bengal staining. Selection of normal prior to the first stage of analysis. subjects for the current study was based on a negative result for all clinical tests. Thirty subjects (16 female, 14 male) were admitted to the study. The average age HPLC Fractionation of Tear Samples of the subjects was 36.9 yr (10 in the 20-29-yr age All tear samples were separated into 11 fractions on group, 8 from 30-39, 7 from 40-49, 4 from 50-59, a TSK G 3000 SW size exclusion column (300 X 7.5 and 1 over 60 yr). Results of the clinical procedure 5 mm) attached to a Rainin (Woburn, MA) gradient are described elsewhere. HPLC system; Isco (Lincoln, NE) variable wave- length absorbance detector set to monitor 280 nm; Tear Collection and Gilson (Middleton, WI) FC-203 fraction collector. The mobile phase was 0.5 M NaCl/0.1 M phos- All tear samples were collected by flame-polished 1 phate, pH 5, flow rate 0.5 ml/min. Chromatographic micropipette using totally noninvasive collection profiles were analyzed by a Dynamax (Rainin) HPLC procedures. Nonstimulated tears were collected using Method Manager data acquisition system, allowing 10-^1 micropipettes, care being taken to avoid ocular real-time peak area integration. surface contact. Tears were collected from the right eye only, except in three cases where they were col- Protein Assays lected from both eyes and pooled. Average time for collection of 10 /x\ nonstimulated tears was 20-30 ELISA: In developing and running each ELISA, a min. All samples were collected by an investigator data analysis program, ELISANALYSISII6 was used. experienced in tear collection techniques (RJF) and This program facilitated isolation of the linear region extreme care was taken to maintain tear flow rate at of logit-log transformed standard data and sample <1 /il/min. This was done by using six or more mi- data. Parallelism of standard and sample dose-re- cropipettes per subject and filling each tube only to sponse curves could be tested readily using ELISAN- the orange band 1.5 mm from the polished collecting ALYSIS to confirm the validity of each ELISA and end (a volume of 1.67 jul). By collecting such a small the choice of protein standard in each case. Sandwich

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ELISA7 of relevant HPLC fractions1 was used in all Prior to adding color reagent, plates were washed cases. Checkerboard titrations were run initially .to four times in PBS-Tween 20, pH 7.2. Two Hundred optimize antisera dilutions and to determine the use- microliters color reagent, consisting of 0.55 mM 2,2'- ful working range for each standard curve. Coating azino-di-(3-ethylbenzathiazoline sulfonate) (ABTS) antisera were always applied in excess (2.0-10.0 and 0.3 mM H2O2 in 55 mM citrate buffer, pH 4.0 8 9 /v.p?/ml) to minimize the effects of surface desorption was then added to each well. After 30-60 min (de- on the linear range of the logit-log dose-response pending on the assay), plates were read on a com- curve. A similar procedure was used for all assays. puter-interfaced Biotek (Winooski, VT) EL-312 ki- Ninety-six-well Nunc (Roskilde, Denmark) Maxi- netic microplate reader. Standard curves were fitted sorp immunoplates were coated overnight at 4°C and estimates of sample concentrations were calcu- with 100 ix\ per well of antibody at a concentration of lated using ELISANALYSIS II.6 Details of the between 2.0 and 10.0 Mg/ml (depending on the assay) antisera used and assay designs are summarized in in 50 mM carbonate buffer, pH 9.6. They were next Table 1. washed three times with phosphate-buffered saline Kinetic Assays: Peroxidase and lysozyme assays (PBS), pH 7.2 in a Dynatech (Chantilly, VA) Ultra- were both run in Nunc 96-well microplates using the wash II automatic microplate washer, and 100 ti\ Biotek EL-312 Kinetic Microplate Reader. The PBS-0.5% gelatin, pH 7.2 was added per well to block ABTS assay was used to measure peroxidase activity, unreacted sites. Plates were blocked for 1 hr at 37°C. with bovine milk lactoperoxidase as the standard. They then were washed three times with PBS-Tween Substrate concentrations in the final assay solution 20, pH 7.2 and 100 ^tl per well of samples and stan- were 0.32 mM ABTS and 0.16 mM H2O2 in 0.1 M dards in PBS-Tween 20, 0.1% gelatin, pH 7.2 were citrate buffer, pH 4.3. To initiate the reaction, tear applied and incubated for 2 hr at room temperature SE-HPLC fraction 5 (Fig. 1) was added to the sub- on an orbital shaker. HPLC-fractionated tear samples strate and the reaction monitored at 405 nm every 15 were run in two previously optimized dilutions, both sec for 2.5 min. The rate of change of absorbance for in duplicate. After washing three times with PBS- the initial minute of the reaction was used for tear Tween, pH 7.2, 100 n\ per well of antibody (indirect HPLC fractions, and for generation of a standard ELISA) or conjugate (direct ELISA) in PBS-Tween, curve with serial dilutions of lactoperoxidase stan- 0.1% gelatin, pH 7.2 was added and incubated for 90 dards. Tear peroxidase levels were calculated from min at room temperature on an orbital shaker. For the lactoperoxidase standard curve. indirect ELISA, the same procedure was used for all Lysozyme activity was measured by the Micro- subsequent antibody layers. coccus lysodeikticus assay. The rate of clearing of a

Table 1. ELISA designs

Antigen Coating antibody Layer 3 Conjugate Negative control

igM 2.5 /ig/ml 31 ng/ml 125 ng/ml IgA-SC," IgA,b E,c a a d GAH-M* MAH-M-MAbf GAM-Ig-HRP* serum-IgM IgA-SC 2.5 n%Jm\ 1.0 Mg/ml IgM/G/E, A,lact,b GAH-a*a GAH-SC-HRF serum-IgAd IgAl 2.0 /ug/ml 50 ng/ml IgA2,h M, G, E, lact, MAH-IgAl-MAbf GAH-Fab-HRP*8 serum-IgA IgA2 2.0 Mg/ml 250 ng/ml IgAl,hM, G, E, lact, MAH-IgA2-MAbf GAH-Fab-HRP serum-IgA IgA 2.5 /ug/ml 625 ng/ml 83.3 ng/ml IgM, G, E, lact, serum- GAH-a MAH-a-MAbf GAM-Ig-HRP IgA IgG 2.5 fig/m\ 16 ng/ml IgA-SC, A, M, E, alb,b GAH-7*a GAH-Fab-HRP serum-IgGd Lactoferrin 5 Mg/m1 65 ng/ml IgA-SC, A, G, tran,b alb RAH-Lact*b RAH-lact-HRP*b Albumin 5 Mg/ml 250 ng/ml Tran, lact, IgG, A GAH-Albe GAH-Alb-HRF Transferrin 3.125 ^g/ml 625 ng/ml Lact, alb, IgG, A SAH-Tranj SAH-Tran-HRPj Lysozyme 10 Mg/ml 50 ng/ml 50 ng/ml IgA-SC, G, A, alb, tran, RAH-Lyso1 RAH-Lyso-Biotink Strep-HRP*a RBPj

* Affinity purified. (University of Alabama at Birmingham); 'Dako (Carpinteria, CA); jBiode- t Purified antibody. sign (Kennebunkport, ME); kcustom preparation from Southern Biotechnol- Immunochemica! suppliers: "Southern Biotechnology Associates (Bir- ogy Associates (Birmingham, AL). mingham, AL); bPel-Freez (Rogers, AR); cChemicon (Temecula, CA); GAH, goat anti-human; MAH, mouse anti-human; serum-IgM, serum "Sigma (Saint Louis, MO); 'Nordic (Capistrano Beach, CA); fBehring Diag- minus IgM; lact, lactoferrin; tran, transferrin; alb, serum albumin; strep, nostics (La Jolla, CA); "Jackson (West Grove, PA); "gift from Dr. J. Mestecky streptavidin; RBP, retinol binding protein.

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SE-HPLC 10 ul Non-stimulated tears SE-HPLC 20 ul Stimulated tears 0.075-,

0.10-

0.00 0.00-1 10.0 20.0 10.0

ELUTION TIME (mins) ELUTION TIME (mins) Fig. 1. SE-HPLC profiles of (A) 10 ^1 nonstimulated tears and (B) 20 nl stimulated tears from a typical study participant. Numbers above each chromatogram indicate elution positions of the 11 HPLC fractions collected for each tear sample. Separation conditions: TSK G 3000 SW column, mobile phase 0.5 M NaCl/0.1 M sodium phosphate, pH 5, flow rate 0.5 ml/min.

0.3 mg/ml suspension of M. lysodeikticus in 50 mM found by the 12 specific assays to contain no identi- sodium phosphate, pH 6.3 was measured from 30 sec fiable protein other than a trace amount of secre- to 2.5 min at 450 nm.10 Serial dilutions of human tory IgA. urinary lysozyme (Alpha Therapeutic, Los Angeles, HPLC integrator output included area percentage CA) were used to generate a standard curve. Tear of each peak, as well as an absolute measure of peak lysozyme activity was measured in SE-HPLC frac- area (absorbance units X time). This allowed a direct tions 10 and 11 (Fig. 1). This was then converted to comparison of protein content in corresponding tear lysozyme concentration by standard curve inter- HPLC fractions between nonstimulated and stimu- polation. lated tears as well as a direct comparison of total tear Tear TSP Levels: Tear specific prealbumin levels protein content. A volume-corrected nonstimulated/ were quantitated using a Bradford microassay11 on stimulated tear total protein ratio of 1.71 (n = 30, P the SE-HPLC fraction containing tear-specific preal- < 0.005 by t-test) was determined using this method. bumin (TSP). Identity of the TSP-containing SE- A comparison of the percentage of total protein made HPLC fraction has been confirmed previously by up by the six major SE-HPLC fractions between SDS-PAGE12 and isoelectric focusing.1 These earlier nonstimulated and stimulated tears is shown in Ta- studies also revealed the absence of non-TSP contam- ble 3. inants at levels beyond trace quantities. Therefore, Details of the ten ELISAs used for determination of the Bradford assay result for the TSP-containing tear protein concentrations are shown in Table 4. fraction was considered to be a reliable estimate of Levels of the 12 proteins assayed in SE-HPLC frac- tear TSP concentration. Both bovine serum albumin tions are given in Table 5. Since lysozyme was as- and IgG were used to calibrate the Bradford assay, sayed by two different methods (ELISA and M. lyso- with the mean value for the two standards used to deikticus assay), tear lysozyme levels listed in Table 5 determine tear TSP levels. represent the mean value obtained from the two

Results Table 2. Proteins assayed in SE-HPLC fractions Typical size exclusion (SE) chromatograms of 10 ^1 nonstimulated tears and 20 n\ stimulated tears from HPLC the current study are shown in Fig. 1. Eleven SE frac- fraction ELISA Other assay tions were collected from each tear sample. In a pre- 2 IgM, IgA-SC, IgAl,IgA2 vious article,1 the main tear proteins were found to be 3 IgA, IgAl,IgA2 — 4 IgG — present in the equivalent SE-HPLC fractions to cur- 5 — Peroxidase: ABTS assay rent fractions 6 (lactoferrin), 8 (tear specific prealbu- 6 Lactoferrin, transferrin, — min) and 10 (lysozyme). Preliminary ELISAs and ki- albumin 8 — TSP: Bradford assay netic assays were used in the current study to deter- 10 Lysozyme Lysozyme: mine which SE fraction contained each of the 12 M. lysodeikticus assay proteins of interest; this data is summarized in Table 11 Lysozyme Lysozyme: 2. SE-HPLC fraction 1 is the void volume and was M. lysodeikticus assay

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Table 3. Nonstimulated and stimulated tear Discussion SE-HPLC profile comparison The greatest difference between nonstimulated and Nonstimulated Stimulated SE-HPLC peak area peak area stimulated tears was seen in the levels of secretory IgA fraction in the high-molecular-weight SE-HPLC fraction (fraction 2). This also was reflected in the IgAl and 1 1.58 ± 1.18f 1.02 ±0.80 <0.05 2 11.15 ±4.92 3.42 ±2.01 <0.005 IgA2 assay results for this fraction. Several possible 6 21.47 ±2.79 25.21 ±2.52 <0.005 mechanisms may be involved: 1) The rate of secre- 8 7.97 ± 4.84 10.78 ±5.51 tion of secretory IgA by the main lacrimal gland may 10 39.82 ± 6.68 51.03 ±7.27 <0.005 11 16.65 ± 6.08 7.62 ± 4.26 <0.005 fall behind the fluid secretion rate with increasing flow. According to Dartt,13 this is the case for "con- n = 30. stitutive" proteins,14 the rate of secretion of which is * Percentage of total area of integrated peaks. t Mean ± standard deviation. determined by their rate of synthesis, and not by the % Not significant. rate of stimulation. Sullivan et al15 demonstrated that the secretory component is a constitutively secreted lacrimal gland protein in the rat. The current study assays. For all proteins assayed, levels were either indicates that the same is true of humans, IgA-SC higher in nonstimulated tears or approximately equal levels decreasing in stimulated tears as the protein is in nonstimulated and stimulated tears. Consistent not being stimulated. 2) The accessory lacrimal with HPLC profiles, nonstimulated tears contained glands may secrete IgA-SC at a higher concentration 16 significantly higher levels of all proteins assayed in SE than does the main lacrimal gland. Gillette et al fraction 2 (high-molecular-weight fraction). On a have shown that both main and accessory lacrimal total protein basis, the high-molecular-weight frac- gland tissue stained for IgA and secretory component. tion of nonstimulated tears contained 5.6 times more 3) Additional sources to the main and accessory lacri- protein than the corresponding stimulated tear frac- mal glands may be contributing to IgA-SC levels at 17 tion (based on volume-corrected HPLC peak area). low tear flow rates. Franklin et al found IgA-stain- The serum-dominant proteins, IgG, albumin, and ing plasma cells in rabbit conjunctival stroma along transferrin also were present in significantly greater with secretory component in conjunctival epithe- 18 concentrations in nonstimulated tears. None of the lium. However, Cohen and Allansmith were unable serum proteins, however, was present in overwhelm- to find specific secretory component staining in ing amounts. This was taken as evidence that the human conjunctival epithelium and suggested that methods used for collection of both nonstimulated the secretory component staining seen by Franklin et 16 and stimulated tears were truly noninvasive. The al may have been an artifact resulting from cryo- main lacrimal gland proteins, lactoferrin, tear specific preservation of their tissue. prealbumin, and lysozyme all were present in similar If sources other than the main lacrimal gland are quantities in both types of tears. contributing to tear IgA-SC levels, then with increas- SE-HPLC fraction 2 contained IgA, predominantly ing tear flow, lacrimal gland fluid would progressively in the form of secretory IgA. Subclass ELISAs re- dilute the nonlacrimal gland contribution. Reports of vealed a distribution biased slightly towards IgA2, total tear IgA levels are much more commonly found more so in stimulated than in nonstimulated tears. in the literature than are reports of IgA-SC or IgA

Table 4. ELISA details Standard linear Logit-log Antigen working range (ng/ml) curve slope Slope SE (%) R Background IgM 16-1024 -1.10 1.31 -0.999 0.100 IgA-SC 5-1250 -0.998 3.93 -0.991 0.134 IgA 2.5-2000 -1.25 3.59 -0.992 0.051 IgAl 1-200 -0.960 2.89 -0.989 0.073 IgA2 6-2800 -0.838 3.76 -0.992 0.058 IgG 0.5-270 -1.042 1.25 -0.999 0.049 Lactoferrin 2-128 -1.124 1.73 -0.998 0.072 Albumin 0.2-110 -0.999 1.38 -0.999 0.049 Transferrin 1.2-27 -1.013 4.66 -0.989 0.056 Lysozyme 0.05-6.5 -0.855 1.19 -0.993 0.070

SE, standard error; R, correlation coefficient.

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Table 5. Protein levels in SE-HPLC fractions of nonstimulated and stimulated tears

Tear protein level (fig/ml; mean ± SE) Ratio HPLC fraction* Protein Nonstimulated Stimulated NS/Si P 2 IgM 2.94 ± 0.60 0.56 ± 0.09 5.25 0.005 2 IgA-SC 793.1 ± 139.3 94.77 ± 16.61 8.37 0.005 2 IgAl 422.1 ±87.8 44.5 ± 9.0 9.49 0.005 2 IgA2 444.3 ± 100.8 72.9 ± 5.9 6.09 0.005 3 IgA 12.15 ±0.88 3.30 ± 0.70 3.68 0.005 3 IgAl 8.50 ± 2.27 2.47 ± 0.43 3.44 0.05 3 IgA2 NRD§ NRD — — 4 IgG 1.50 ±0.24 0.30 ± 0.09 5.03 0.005 5 Peroxidase 6.71 ±0.38 3.91 ±0.23 .72 0.005 6 Lactoferrin 1,741.4 ± 193.0 1,587.6 ±441.3 .10 NS* 6 Albumin 42.02 ± 11.71 12.16 ±3.76 .J.46 0.05 6 Transferrin 0.99 ±0.11. 0.50 ± 0.09 1.98 0.005 8 TSP 1,677.4 ± 188.8 1,245.3 ± 103.4 1.35 NS 10 Lysozyme" 3,297 ± 429 3,162 ±376 1.04 NS 11 Lysozyme11 273 ± 158 265 ± 146 1.03 NS

* See figure 1. § Not reliably detected. t Nonstimulated/stimulated. 11 Mean lysozyme level by M. lysodeikticus assay and ELISA (results of the % Not significant. two assays were not significantly different).

subclass levels. Horwitz et al19 collected tears at a rate samples and standards.24 Differences in the antigen of about 1-2 /il/min by micropipette, obtaining a capture characteristics8 of the coating antisera used in diurnal average total IgA level of 700 ^g/ml using the two types of assays—goat anti-human a and radial immunodifFusion. Other reports, in which the mouse anti-human a{ and a2—may be factors. Any degree of stimulus was less clearly denned but tear subclass preference by the goat anti-human a poly- flow rate must have been well above minimum clonal capture antibody could lead to an underesti- (based on volumes collected), gave total IgA levels mation of total IgA-SC if the subclass for which it had from 186 to 505 Mg/ml.20-22 All of these values fall greater affinity were present in a higher proportion in between the nonstimulated and stimulated tear IgA- the standard versus test sample. This type of problem SC levels determined in the current study. Since ex- would not be expected of the coating antisera in the tremes of flow rate were deliberately used in this subclass-specific ELISAs. However, the goat anti- study (<1 /xl/min for nonstimulated and >50 /zl/min human Fab conjugate may have a light-chain prefer- for stimulated tears), the intermediate values found ence (K or X), which could lead to inaccuracies if the by others together with the current findings support light-chain distribution differed between standards the possibility that tear IgA concentration falls along and test samples.8 It should be pointed out, however, a continuum, decreasing with increasing tear flow that antibody affinity does not generally influence the rate. accuracy of antigen estimation in sandwich ELISA,8 In SE-HPLC fraction 2, an approximately equal this being one of the reasons that a sandwich design proportion of IgA 1 and IgA2 was found in nonstim- was used for all ELISAs in the current study in prefer- ulated tears. The distribution was biased slightly to- ence to including any competitive ELISAs. wards IgA2 in stimulated tear fluid. In agreement Levels of monomeric IgA (HPLC fraction 3) were with the current study, Russell et al23 and Ladjeva et very low in both types of tears. Only IgA 1 was reliably al24 found an approximately equal IgA subclass dis- detected in the IgA subclass assays of fraction 3, indi- tribution in secretions. It is noteworthy that the com- cating a serumlike subclass distribution.23'24 IgM bined IgA 1 plus IgA2 level in tear SE-HPLC fraction levels (SE fraction 2) were considerably higher in 2 exceeded that of IgA-SC. Two possible factors may nonstimulated tears, with the nonstimulated to stim- explain this finding: 1) The IgA subclass assays will ulated ratio greater than 5. Similar mechanisms could detect the a chains of both secretory and polymeric be postulated for the tear IgM ratio as for IgA-SC in (nonsecretory) IgA. This would indicate the presence terms of additional sources to the lacrimal gland. of a moderate amount of polymeric (nonsecretory) Some IgM may also reach the tear fluid via ocular IgA in human tears. 2) The discrepancy between surface vessels,17 with tear levels decreasing with the IgA-SC and IgA subclass levels may alternatively be diluting effect of increased tear flow rate. IgM has due to differences in the assay designs or subtle dif- been reported previously to be consistently present in ferences in the nature of the antigen between test stimulated tears, although Coyle's and Sibbony's22

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value of 5.6 ixg/ml for IgM levels in apparently stimu- study may indicate a subtle decline in protein secre- lated tears exceeds the present and previous1 findings tion rate with higher tear flow rate. However, since a of the current authors by one order of magnitude. total protein assay was used on the TSP-containing Somewhat surprisingly, only moderate nonstimu- SE-HPLC fraction, rather than a specific assay for lated/stimulated ratios of the major serum proteins TSP, the difference has not been shown conclusively IgG, transferrin, and albumin were seen. It is gener- to be a function solely of TSP levels. The finding of ally accepted that these proteins are derived from relatively little difference in levels of lactoferrin, TSP, the ocular surface vessels and that their presence in and lysozyme between nonstimulated and stimulated the tears of normals is indicative of collection tears is consistent with previous reports that these are 13 14 trauma.2526 This is based on the wide variation in "regulated" proteins, ' their rate of secretion being their reported tear concentrations—significantly controlled by the rate of stimulation. higher tear levels with collection methods (such as the Results of the current study for the main lacrimal Schirmer strip method) that are known to be trau- gland proteins agree with the findings of Berta,31 who matic—and on the fact that they are present at very found a subtle drop in the levels of the three proteins high concentrations in serum. IgG levels ranging with increasing tear flow rate. Stuchell et al32 reported from undetectable to 32 /ug/ml are typically reported significantly higher levels of lactoferrin and lysozyme for stimulated tears collected by micropipette.20'2228 in stimulated tears. However, they used two different In the case of albumin, 12vr=-54 tig/ml is a typical types of absorbent collection strip to obtain nonstim- range reported for stimulated tears.2028'29 Stuchell et ulated and stimulated tears. In contrast, Selsted and al28 concluded that transferrin levels in micropipette- Martinez33 found a 50% higher lysozyme level in collected stimulated tears must be less than 1 /tig/ml nonstimulated tears collected with cellulose sponges after they failed to detect the protein by crossed im- than in stimulated tears collected by either sponges or munoelectrophoresis. Similarly, Broekhuyse30 was by plastic tubing. Overall, the range of tear lactoferrin unable to detect transferrin in micropipette-collected levels reported for normals is quite high, with typical nonstimulated tears using immunoelectrophoresis. values ranging from 1.3 to 3.1 mg/ml.20'28'33'34 This These results do not contradict the current study; the places the current results for lactoferrin at the lower transferrin levels in both types of tears were found by end of the range. Literature reports for tear lysozyme the more sensitive sandwich ELISA technique to be levels also vary considerably, from about 1 to 3.1 less than 1 /ig/ml. mg/ml.20'28'32'33 Part of the variability in the case of The relatively low levels of IgG and transferrin lysozyme may be explained by the findings of a diur- found in the current study, combined with moderate nal variation study19 of nonstimulated tear lysozyme nonstimulated/stimulated ratios, indicate that collec- levels. According to that study, the nonstimulated tion trauma was not contributing significantly to ei- tear lysozyme concentration gradually decreased ther type of tear sample. Furthermore, the facts that from a high of 2.0 mg/ml at 12:00 PM to a low of 0.6 the nonstimulated/stimulated ratios of IgG, albumin, mg/ml at 3:00 AM.19 Higher than average tear lyso- and transferrin varied from 2 to 5 and that all three zyme values were found in the current study. This proteins were consistently present in measurable may be due in part to the use of a human urinary quantities in stimulated tear samples, indicate that lysozyme standard for both the ELISA and the M. the presence of these proteins in tear fluid is not due lysodeikticus lysozyme assay. entirely to ocular surface trauma. Therefore, the use A slight, but significant, decrease in tear peroxidase of tear albumin, IgG, and transferrin levels as indica- was seen in stimulated tears relative to nonstimulated tors of collection trauma should be viewed with cau- tears. Van Haeringen and Thorig,35 on the other tion. In the current study, albumin and IgG levels hand, found a slight increase in stimulated tear per- were found to vary greatly among individuals, but oxidase levels over nonstimulated tears, but con- higher nonstimulated tear levels were generally asso- cluded that the level probably remained relatively ciated with higher stimulated tear levels for a given constant at all tear flow rates. The findings of a slight subject. decrease in peroxidase levels in stimulated tears in the No significant differences were found between current study and a slight increase in another study35 nonstimulated and stimulated tear levels of the three together support the possibility that peroxidase is a main lacrimal gland proteins lactoferrin, TSP and regulated protein secreted primarily by the main lac- lysozyme. Very similar lactoferrin and lysozyme rimal gland.13 In the current study, no attempt was levels found in both types of tears indicate that the made to identify alternative sources of tear proteins secretion rate of these proteins varies little at higher and so the possibility that peroxidase may be derived tear flow rates. The slight drop in stimulated tear TSP from sources in addition to the main lacrimal gland levels relative to nonstimulated tears in the current was not addressed.

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