Abstract

Initial studies have shown that fish lens protein can be modified in vitro to acquire a specific affinity for selected molecules (e.g. human hemoglobins) and cells (e.g. human erythrocytes). These studies were extended by preparing lens reagents that can distinguish bovine serum albumin from ovalburnin, and major groups of human erythrocytes in the ABO system from each other. Lens reagents appear to be potentially useful tools for the identification of specific molecules, either in solution or on cell surfaces.

Introduction

Fish eye-lens protein, after in vitro modification, appears to provide a new class of reagents for detecting selected molecules. Initial experiments suggested that these reagents may be effective in identifying a variety of molecules and cells (Smith, unpublished observations). Unlike antisera, lens reagents do not require living animals or tissues for their production and may not have the usual limitations of antigen-antibody reactions.

The initial studies were carried out with fish lens reagents prepared against human hemoglobins A and S1 and against group A1 human erythrocytes of the ABO system. Objectives of the present research were to e xtend these studies by determining if lens reagents can be prepared which will have the capability of distinguishing between bovine serum albumin (BSA) and ovalburnin (OA), and between major erythrocyte groups in the ABO system.

Materials and Methods

Lenses, Collection and Extraction

Lenses were collected from skipjack tuna (Katsuwonus pelamis) on the basis of availability. The sample consisted of 21 fish with the following fork lengths (cm): 41 (5 fish), 42 (1 fish), 43 (2 fish), 44 (3 fish), 45 (3 fish), 46 (4 fish), 48 (1 fish), and 49 (2 fish).

The tuna had been captured on November 10, 1983, near Cedros Island, Baja California, Mexico, and stored on ice until the lenses were removed. The lenses were stored frozen (-10ºC for approximately 3 months in dry, sealed containers and then dissected to obtain whole cores (nuclei) (1). Only protein from the lens nucleus was used for the principal reasons that there it is concentrated and uncontaminated by proteins from other tissues, e.g., blood, aqueous humor, etc., and essentially not turned over (for brief review, see reference 2).

The lens nuclei were pooled and granulated with mortar and pestle. An extraction solution of 0.018 g/dl NaCl was added to the lens particles to solubilize both albumins and globulins (3,4). The volume (ml) of fluid added was twice that of the saline-saturated tissue. The mixture was extracted under gentle agitation on a laboratory rocker for 2 days and then centrifuged at 10,000 rpm for 3 minutes. Both the extraction and centrifugation were performed at ambient temperature (23ºC) to prevent precipitation of cold precipitable protein (CPP).

Preparation of Stock Solutions

CPP solution was prepared by refrigerating (5ºC) the lens extract for 9 days which yielded a large precipitate. The supernatant was decanted, and the precipitate was washed with cold distilled water and then dissolved at ambient temperature in a volume of distilled water equal to 5 times that of the precipitate.

Solutions of BSA and CA (Grade 5 and Fraction V, respectively, Sigma Chemical Co., St. Louis, M0 63178, USA) were prepared by mixing 10 mq of each in 2 ml of distilled water.

Stock 3% suspensions of Rh human erythrocytes (A1, A2 , B, and 0) were obtained from Ortho Diagnostic Systems Inc. (Raritain, NJ 08869, USA). Agglutinability was confirmed with Ortho anti-A, -B, and -0 blood grouping sera. Modification of CPP for Specific Reactivity with BSA and OA

Equal volumes of CPP solution were separately mixed with equal volumes of BSA or 0A solutions. The mixtures were placed in a water bath at ambient temperature and then warmed to 55ºC. After 4 minutes at 55C. they were refrigerated for 48 hours. The resulting precipitates were washed with cold distilled water and then dissolved at ambient temperature in a volume of distilled water equal to 5 times that of the precipitates by gently swirling the mixtures inanually for 5 minutes. When placed in an ice bath, each solution formed a precipitate, and the supernatant containing free BSA or OA was discarded. This procedure, starting from the wash with cold distilled water, was repeated to remove additional BSA and OA. Finally, the CPP precipitate with BSA or OA removed was dissolved at ambient temperature in a volume of distilled water equal to 5 times that of the precipitate. These solutions will henceforth be referred to as BSA-CPP and OA-CPP.

Modification of CPP for Specific Reactivity with Erythrocytes

Equal volumes of CPP solution were mixed with equal volunies of suspension of each erythrocyte group (A1 , A2 , B, or D). The mixtures were warmed in a water bath at 40 C for 4 minutes, refrigerated. For 48 hours, warmed again for 4 minutes to redissolve the CPP precipitates, and then centrifuged at 3,400 rpm for 1 minute. The supernatants were decanted from the pellets, and the procedure was repeated as before, i.e., erythrocytes (of the same group) were added to the supernatants and the suspensions were warmed, refrigerated, and centrifuged again. The final supernatants were diluted by adding to each a 1/3 volume of normal saline (0.9 g/dl NaCl) solution and will henceforth be referred to generally as cell-CPP (C-CPP) solution or as specific solutions of A1-CPP, A2-CPP, B-CPP, and O-CPP.

Tests

Chemical fixation tests, patterned after the immunofixation procedure (5), were carried out in duplicate an 2.5- x 14-cm Cellogel membranes (Kalex Scientific Co., Manhasset, NY 11030, USA), a gel form of cellulose acetate. These were soaked for 10 minutes at ambient temperature in phosphate buffered saline solution, pH 7.4 (Sigma Chemical Co.) and then blotted lightly with filter paper.

To test the reactivity of BSA-CPP and OA-CPP, 15 ul of BSA and OA solutions were separately spotted on each of 3 membranes. After absorption of the solutions on the membrane (5-10 minutes), they were treated as follows: Membrane A (control)--50 ul of CPP solution was spotted over the BSA and OA spots. Membranes B and C--similarly treated, except for the use of BSA-CPP and OA-CPP solutions, respectively. After each application, the membrane was placed in a humidity chamber and allowed to incubate for 30 minutes at ambient temperature. All the membranes were then washed in 700 ml of distilled water with slow stirring for 5 minutes. This procedure was followed by immersion in 6% trichloracetic acid (5 minutes), Ponceau S solution (Kalex Scientific Co.) (5 minutes), 5% acetic acid (5 minutes, with gentle agitation), and finally absolute methanol (10 minutes, with gentle agitation). The membranes were preserved in 2% acetic acid.

To test the reactivity of C-CPP with erythrocytes, 15 ul of each Suspension of A1, A2, B and D erythrocytes were separately spotted on each of 5 Cellogel membranes. The membranes were then treated as follows: Membrane A (control)--each spot of erythrocyte suspension was overlain with 50 ul of CPP solution. Membranes B, C, and D--seperately received over their spots of erythrocyte suspension 50 ul of A1 -CPP A2 -CPP B-CPP, and D-CPP solutions, respectively. After application of these sdutions, each membrane was treated as described for the albumin solutions except that normal saline solution, rather than distilled water, was used to wash the membranes after incubation in the humidity chamber.

To further test the reactivity of C-CPP with erythrocytest agglutination tests were carried out in duplicate, as follows:

Experimental - Four drops of suspensions of A₁, A₂, B, and O erythrocytes were separately placed in each of 4 sets of 10 x 75 mm test tubes. In addition, all tubes in a set received 1 drop of A₁-, A₂-, B-, or O-CPP solution and 4 drops of normal saline solution.

Control - As in the experimental series, except that the CPP solution was substituted for C-CPP solutions.

All tubes were centrifuged for 60 seconds at 3,400 rpm, and then examined both macroscopically and microscopically for signs of agglutination.

Results

Albumin Reactions (Figure 1)

Membrane A (overlaid with CPP solution) showed medium, equally stained spots of BSA and DA. Membrane B (overlaid with BSA-CPP solution) showed a much darker spot for BSA than for OA. Membrane C (overlaid with OA-CPP solution) showed a much darker spot for DA than for BSA.

Erythrocyte Reactions

Chemical fixation tests (Figure 2) - Membrane A (overlaid with CPP solution, control) showed pale, equally stained spots of all erythrocyte groups. On the remaining membranes, the heaviest staining reactions occurred on those spots where C-CPP solution and erythrocyte group correspond. In other words, the darkest stained spots on membranes B (overlaid with A₁-CPP solution), C (overlaid with A₂- CPP solution), D (overlaid with B-CPP solution), and E (overlaid with O-CPP solution) correspond to cells of groups A₁, A₂, B, and O erythrocytes, respectively.

Agglutination tests (Table 1) - Clumps of medium size were observed where CCPP solution and erythrocyte group correspond. In other words, these clumps formed in mixtures of A₁-, A₂-, B-, or O-CPP solutions with A₁, A₂, B, or O erythrocytes, respectively. Smaller clumps developed in mixtures of A₁ or A₂ erythrocytes with CCPP solution of reciprocal type (A₂- and A₁-CPP, respectively). The tiniest clumps were seen in the remaining mixtures of cells with C-CPP or CPP (control) solutions. Preliminary studies indicate that agglutinatiun reactions can be strengthened to maximal (4+) by concentration of C-CPP solutions.

Figure 1 Chemical fixation reactions of BSA (left spots) and OA (right spots). Membrane A, CPP-fixed albumins. Membrane B, BSA-CPP-fixed albumins. Membrane B, BSA-CPP-fixed albumins. MembraneC, OA-CPP-fixed albumins (lines in left spot are artifacts from membrane creases.)

Figure 2 Chemical fixation reactions of A1, A2, B, and O erythrocytes (spots, left to rights). Membrane A, CPP-fixed erythrocytes. Membrane B, A1-CPP-fixed erythrocytes. Membrane C, A2-CPP-fixed erythrocytes. Membrane D, B-CPP-fixed erythrocytes. Membrane E, O-CPP-fixed erythrocytes.

Table 1. Agglutination Reaactions of Erythrocytes with Protein Solution

Reactions are graded 0 (no agglutination), w+ (weakly positive), or 1-4+ (maximal agglutination). They were read and interpreted according to recommendations of the American Association of Blood Banks (6).

Discussion

It is apparent that CPP bound nonspecifically to both BSA and OA, and to the cell membranes of at the various erythrocyte groups. It is also evident that CPP was modified by the contact/separation treatment with albumins and erythrocytes, making it more specifically reactive.

The pattern of agglutination reactions suggests that C-CPP operates by combining with surface molecules that distinguish erythrocytes of each group. The small degree of cross-reactivity of A 1- and A 2-CPP solutions with erythrocytes of reciprocal group would imply that these molecules are either (1) the same in both A1 and A2 erythrocyte groups, but present in different concentrations; or (2) although related, in the two erythrocyte groups. While the reason is not clear, the reactions are in accord with the known close relationship of the A1 and A2 erythrocyte groups (7) and indicate a high resolving power of C-CPP solutions.

A preliminary erythrocyte agglutination study (Smith, unpublished) with lens protein from yellowfin tuna (Thunnus albacares) confirmed the skipjack findings and suggested that lens protein from species other than skipjack can be modified to develop specific reactivity.

The mechanism whereby lens protein develops increased reactivity and specificity from exposure to selected molecules is not known. However, based on the well-known tendency of lens proteins to interact with each other (8-10), and apparently also with non-lens molecules (Smith, unpublished observations), it is hypothesized that lens protein develops and holds new conformations rather easily after contact with and separation from specific (template) molecules. The new conformations then provide a better fit In binding reactions. These interpretations are consistent with current concepts about changes in conformation, binding sites, and affinities occurring in interacting molecular systems (11).

It is conceivable that lane protein modified by template substances can be used as a reagent to detect molecules otherwise undetectable with available reagentat e.g., antisere and lectins. This possibility is suggested by the successful preparation of lens reagents against human hemoglobin (Smith, unpublished observations), a poor antigen in mammals (12).

Many other human proteins are also poor antigens. For example, in serum, non- or week antigens account in part for mammalian antisera being able to detect to date only about 45 (13) of the welt over 100 proteins (14) known to exist. Thus, the majority of serum proteins are undetected, and lens reagents may contribute toward their identification.

Acknowledgements

I wish to thank Mr. D.L. Scott and Ms. L.R. Kaufman for assistance in the. laboratory, Mr. H.B. Clemens for the skipjack and yellowfin tuna lenses, and Dr. C. Ian Hood for critical review of the manuscript.

References

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