Human recombinant lacritin and lacritin truncation mutants lacking 25 amino acids from the C-terminus,
16 or 24, 45, or 65 amino acids from the N-terminus (Zhang Y, et al.
IOVS 2010;51:ARVO E-Abstract 4179) (numbering excludes the signal peptide) were generated in
E. coli as intein fusion proteins. Intein fusion proteins were captured on chitin beads, eluted with β-mercaptoethanol (removes the intein tag
30 ), and passed over a diethylaminoethyl (DEAE) cellulose column to remove bacterial impurities (Zimmerman K, et al.
IOVS 2012;53:ARVO E-Abstract 4231). Four male AJ mice were immunized with keyhole limpet hemocyanin-linked DPAQEAGTSKPNEEIS, corresponding to amino acids 11 to 26 of the mature lacritin N-terminus, and boosted three times. Fusions were screened first against BSA-DPAQEAGTSKPNEEIS, then to lacritin truncation mutant C-59, and finally to tear lacritin immunocaptured in wells coated with protein A and polyclonal rabbit ‘anti-Pep Lac N-term' lacritin antibody.
24 1F5-C9-F4 (‘1F5’) showed the highest titer and was expanded in a 500-mL bioreactor (Lampire Biological Products, Piperville, PA), purified (GammaBind Plus Sepharose; GE Healthcare Life Sciences, Piscataway, NJ), and validated with lacritin truncation mutants. 1F5 is an IgG1. Also employed was new rabbit “anti-N-65 Lac C-term” (here designated as “ab C-term”), directed against the lacritin N-65 truncation mutant (amino acids 66–119 of mature lacritin). Rabbit terminal bleeds were collected on day 70 and purified on protein A for subsequent use. Slight background to
E. coli proteins was depleted by passage over an
E. coli lysate column prepared by AminoLink (Thermo Fisher Scientific, Inc., Rockford, IL) immobilization of lysate from
E. coli expressing C-59 lacritin. Similarly, tear background of secondary anti-mouse antibodies was depleted by passage over beads (Thermo Fisher Scientific, Inc.) coupled to normal human tears. Normal human tears were eluted off of flash-frozen Schirmer strips with ice-cold PBS at 1 μL per mm. For blotting, lacritins, tears, or TCA-precipitated saliva were loaded onto 10% or 4% to 20% gradient SDS-PAGE gels using 10 μL per lane of tears (in an equal volume of ×2 sample buffer with β-mercaptoethanol and boiling) and 37.5 μL per lane of TCA precipitated saliva that had been solubilized in 125 μL of ×1 sample buffer with β-mercaptoethanol and boiling. All gels were transferred to nitrocellulose. Blocking with milk was avoided since lacritin appears to be a constituent of milk (McKown RL, et al.
IOVS 2012;53:ARVO E-Abstract 4235). Instead, blots were blocked with 5% BSA in Tris-buffered saline with a detergent solution (Tween-20; Thermo Fisher Scientific, Inc.); and incubated with 1F5 (0.6 μg/mL) or with anti-N-65 Lac C-term (1/2000), using peroxidase-labeled secondary and ECL (Thermo Fisher Scientific, Inc.). TGM2 was detected with mouse monoclonal antibody CUB 7402 (Abcam, Cambridge, MA). For immunohistochemistry, zinc formalin-fixed, paraffin-embedded human lacrimal gland was sectioned, epitope–exposed, and peroxidase-blocked, as per Sanghi et al.,
15 and incubated with 1F5 (0.03 μg/mL) or ab C-term (1/20,000), followed by detection.